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De, Rosa Maria Caterina. "Studio dell’espressione di geni coinvolti in pathways metabolici regolati da nutrienti." Doctoral thesis, Universita degli studi di Salerno, 2015. http://hdl.handle.net/10556/1864.
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2014-2015Il profilo sierico, con particolare riferimento ai livelli di biomarkers, rappresenta uno strumento efficace ed affidabile per la diagnosi di malattie metaboliche, come il diabete o le malattie cardiovascolari. La composizione del siero è influenzata sia dal metabolismo endogeno che dall’apporto nutrizionale. In effetti, lo stile alimentare, con particolare riferimento alla qualità e alla quantità dell’apporto nutrizionale, può fortemente influenzare il rischio e la progressione di malattia, poiché alcuni nutrienti agiscono come composti bioattivi. A questo proposito, la letteratura attuale indica un importante ruolo di specifiche molecole nutrizionali provenienti dalla dieta che interessano specifiche vie metaboliche. L'obiettivo del nostro progetto è quello di individuare pathways metabolici regolati da nutrienti, con lo scopo di identificare possibili taget terapeutici in stati patologici. [ a cura dell'autore]
XIII n.s.
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Gupta, Apoorv. "Dynamic regulation of bacterial metabolic pathways using autonomous, pathway-independent control strategies." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/112511.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 86-91).
Metabolic engineering efforts have so far focused on strain optimization through careful metabolic modeling and tinkering with host genomes, through gene knockouts or knockins, to direct flux in desired channels. These efforts have borne fruit with the development of large manufacturing processes for numerous chemicals. The next challenge for metabolic engineering, however, lies in tackling issues associated with construction of more complex pathways, such as those that directly interfere with host metabolism, have branchpoints with promiscuous enzymes, or synthesize toxic intermediates or products. Dynamic metabolic engineering has emerged as a new frontier for tool development to allow regulation and control of native and cellular pathways during the course of a production run. Advantages in dynamic strategies are especially apparent in the aforementioned examples where traditional static strategies of gene knockouts or knockins are not an option. Instead, it is necessary to be able to control when certain genes are expressed, such as to build biomass before switching on growth-limiting production pathways, or accumulating intermediates to drive the reaction of a promiscuous enzyme along a certain branch. In this thesis, we propose enzyme control strategies that are independent of any biosynthetic pathway of interest. Therefore, they can theoretically be applied to a wide variety of contexts in a "plug-and-play" fashion to control pathway enzyme expression. After initial work to understand the limitations of nutrient starvation strategies to induce genetic circuits, we decided to use quorum sensing circuitry to create circuits that can be autonomously induced. We used parts of the Esa QS system (derived from Pantoea stewartii) to create circuit variants in the Lscherichia cohi genome, which switch off expression of the targeted gene at various times and cell densities. Switching times were varied by modulating the expression of the AHL synthase, and therefore the production rate of AHL, the quorum sensing molecule. Switching dynamics were characterized and ranked for the entire library of circuit variants using fluorescent reporters. The characterized device was used to identify optimal switching times for redirection of glycolytic fluxes into heterologous pathways, resulting in a 5.5-fold boost in myo-inositol (MI) and increasing glucaric acid titers from unmeasurable quantities up to >0.8 g/L. With a focus on industrial application, consistency of device performance was verified in benchtop bioreactors, achieving nearly 10-fold and 5-fold boosts in specific titers of myoinositol and glucaric acid, respectively. To demonstrate broad utility and "off-the-shelf" applicability, the control module was applied to dynamic downregulation of flux into aromatic amino acid biosynthesis to accumulate the industrially-relevant intermediate, shikimate, resulting in an increase in titers from unmeasurable quantities to >100 mg/L. Finally, this QS device was coupled with a MI-biosensor circuit to institute two layers of dynamic regulation and further improve glucaric acid titers. Production trials in these composite strains resulted in the highest glucaric titers (-2 g/L) reported to date from E. coli K-strains. This work reports the first completely autonomous dynamic regulation module and its application in bioproduction of multiple products from different metabolic pathways. We envision that the strategy presented here may be adapted to any pathway context and gene of interest. With increased prevalence of dynamic regulation, the relevant strategies may become standardized for general use.
by Apoorv Gupta.
Ph. D.
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Lisowska, Beata. "Genomic analysis and metabolic modelling of Geobacillus thermoglucosidasius NCIMB 11955." Thesis, University of Bath, 2016. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.690738.
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Geobacillus thermoglucosidasius is a Gram-positive thermophilic eubacterium (45-70‰) that has the ability to convert pre-treated lignocellulosic material LCM into ethanol. This organism has been genetically engineered such that its yield of ethanol production is in excess of 90% of the theoretical maximum [38]. There remains considerable scope to develop G.thermoglucosidasius to produce alternative fuels and chemicals of industrial importance. For such a useful bacterium the understanding of the global metabolism remains poorly characterised. To gain a better insight into the metabolic pathways and capabilities of G. thermoglucosidasius a bottom-up approach to construct a comprehensive metabolic model of the organism was applied. The model was build from manually annotated genome and incorporates data from wet lab experiments for accurate in silico analyses. The model simulations has highlighted a potential experimental design for the in silico production of succinate and butane-2,3-diol. PathwayBooster is also introduced in this study as a tool for curating metabolic pathways. The methodology is based on the assumption that the core metabolic capabilities are shared among evolutionarily closely related species [80]. This approach led to the further analysis of members of the genus Geobacillus with respect to their core metabolic capabilities, genome re-arrangements and shared unique features. Theoretical route for the biosynthesis of Vitamin B12 is presented here, which is novel to the canonical aerobic and anaerobic pathways known to date and ubiquitous amongst Geobacillus spp. The analysis of the gene assignment for this bacterium has highlighted the presence of NADP-dependent GAPDH. The theoretical function of this novel and previously uncategorised enzyme in the genus Geobacillus has been confirmed through enzymatic assays.
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Leung, Shuen-yi, and 梁舜頤. "Predicting metabolic pathways from metabolic networks." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B42664317.
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Leung, Shuen-yi. "Predicting metabolic pathways from metabolic networks." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B42664317.
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Edwards-Hicks, Joy. "Metabolic remodelling driven by MYC overexpression regulates the p53 tumour suppressor response." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31223.
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The MYC onocogene is frequently overexpressed in human cancer due to its capacity to promote cell growth and cell proliferation. MYC overexpression activates the p53 tumour suppressor pathway, which resists the pro-tumourigeneic program elicited by MYC. How MYC overexpression engages p53 is yet to be elucidated, and in this study I carried out a large metabolic siRNA screen to determine whether p53 responds to a specific MYC-driven metabolic pathway. Two clear lipid metabolic pathways emerged from the siRNA screen: PPARγ/arachidonate metabolism and de novo sphingolipid synthesis. Knockdown or inhibition of PPARγ increased p53 levels, and PPARγ ligands decreased following MYC overexpression. Knockdown of ceramide synthesis depleted p53 levels, and MYC overexpression increased de novo ceramide synthesis. This demonstrated that MYC-driven ceramide synthesis positively regulates p53, and highlights the role of cell metabolism in the tumour suppressor response to MYC deregulation.
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Zumbaugh, Morgan Daughtry. "Signaling pathways regulating skeletal muscle metabolism and growth." Diss., Virginia Tech, 2021. http://hdl.handle.net/10919/101750.
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Skeletal muscle can perceive cellular energy status and substrate availability and demonstrates remarkable plasticity in response to environmental changes. Nonetheless, how skeletal muscle and its resident stem cells (satellite cells; SCs) sense and respond to nutrient flux remains largely undefined. The dynamic post-translational modification O-GlcNAcylation has been shown to serve as a cellular nutrient sensor in a wide range of cells and tissues, yet its role in skeletal muscle and SCs remains unexplored. Here, we ablated skeletal muscle O-GlcNAc transferase (OGT), and thus O-GlcNAcylation, and found the knockout mice exhibited enhanced glucose uptake, insulin sensitivity, and resistance to high-fat diet induced obesity. Additionally, mKO mice had a 3-fold increase in circulating levels of interleukin-15 (IL-15), a potent anti-obesity cytokine, potentially through epigenetic regulation of Il15 by OGT. To further investigate if there was a causal relationship between OGT ablation and the lean phenotype, we generated muscle specific OGT and interleukin-15 receptor alpha (IL-15ra) double knockout mice (mDKO). As a result, mDKO mice had blunted IL-15 secretion and minimal protection against HFD-induced obesity. Together, these data indicate the skeletal muscle OGT-IL15 axis plays an essential role in the maintenance of skeletal muscle and whole-body metabolic homeostasis.
As satellite cells (SCs) play an indispensable role in postnatal muscle growth and adult regenerative myogenesis, we investigated the role of O-GlcNAcylation in SC function. To this end, we conditionally ablated OGT in SCs (cKO) and found cKO mice had impaired SC proliferation, in vivo cycling properties, population stability, metabolic regulation, and adult regenerative myogenesis. Together these findings show that SCs require O-GlcNAcylation, presumably to gauge nutritional signals, for proper function and metabolic homeostasis.
Another critical yet often neglected player in myogenesis are mitochondria. Traditionally depicted as a power plant in cells, mitochondria are critical for numerous nonconventional, energy-independent cellular process. To investigate the role of both mitochondrial energy production and alternative mitochondrial functions in myogenic regulation, we ablated ATP synthase subunit beta (ATP5b) and ubiquinol-cytochrome c reductase (UQCRFS1) in C2C12 myoblasts to disrupt mitochondrial ATP production and mitochondrial membrane potential, respectively. Ablation of UQCRFS1, but not ATP5b, impaired myoblast proliferation, although lack of either gene compromised myoblast fusion. Interestingly, addition of the potent myogenic stimulator IGF-1 rescued ATP5b fusion but could not override UQCRFS1 knockout effects on proliferation or differentiation. These data demonstrate mitochondrial ATP production is not the "metabolic switch" that governs myogenic progression but rather an alternative mitochondrial function.
In summary, skeletal muscle and their resident stem cell population (SCs) both use O-GlcNAcylation, feasibly to sense and respond to nutritional cues, for the maintenance of metabolic homeostasis and normal physiology. A deeper understand of both muscle and SC metabolic regulation may provide therapeutic targets to improve global metabolism and muscle growth.Doctor of Philosophy
Skeletal muscle is responsible for approximately 20% of basal energy expenditure and 70-90% of insulin-mediated glucose disposal, and as such changes in skeletal muscle metabolism and insulin sensitivity have profound impacts on whole body metabolism. Skeletal muscle is a plastic tissue that can perceive nutrient availability, which permits metabolic adaptations to environmental changes. Deletion of the nutrient sensing pathway O-GlcNAcylation in skeletal muscle (mKO) protected mice from high-fat diet induced obesity and ameliorates whole-body insulin sensitivity. Skeletal muscle can secrete myokines to elicit endocrine effects on other tissues in the body, and as such, we proposed perturbation of this nutrient sensing pathway in skeletal muscle alters myokine secretion to elicit responses in other metabolically active tissues to support its energy requirements. Indeed, circulating levels of interleukin-15, a potent anti-obesity myokine, increased 3-fold in mKO mice. To determine the contribution of IL-15 to the mKO phenotype, we used a genetic approach to blunt IL-15 secretion from skeletal muscle (mDKO), which partially negated the lean mKO phenotype. Our findings show the ability of skeletal muscle to "sense" changes in nutrients through O-GlcNAcylation is necessary for proper muscle and whole-body metabolism. Moreover, this nutrient sensing mechanism is also important for proper muscle stem cell function, also known as satellite cells (SCs). Loss of O-GlcNAcylation in SCs impairs their ability to regenerate muscle after injury, which can be attributed to a reduced capacity to proliferate and an inability to maintain a healthy SC population. Interestingly, SCs lacking O-GlcNAcylation have a greater mitochondrial content. Using a myoblast cell line, we investigated the contribution of mitochondria to myogenesis, the formation of muscle, and found mitochondrial energy production is dispensable in the myogenic process. Our studies show skeletal muscle and SCs rely on highly integrated signaling cascades that sense and respond to intrinsic metabolic changes and extrinsic nutritional cues to function properly.
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McArthur, George Howard IV. "Orthogonal Expression of Metabolic Pathways." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3087.
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Microbial metabolism can be tailored to meet human specifications, but the degree to which these living systems can be repurposed is still unknown. Artificial biological control strategies are being developed with the goal of enabling the predictable implementation of novel biological functions (e.g., engineered metabolism). This dissertation project contributes genetic tools useful for modulating gene expression levels (extending promoters with UP elements) and isolating transcription and translation of engineered DNA from the endogenous cellular network (expression by orthogonal cellular machinery), which have been demonstrated in Escherichia coli for the production of lycopene, a 40-carbon tetraterpene carotenoid with antioxidant activity and a number of other desirable properties.
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Bhargava, Prerna. "Immunomodulatory Pathways and Metabolism." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10696.
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Energy metabolism plays a vital role in normal physiology, adaptive responses and host defense mechanisms. Research throughout the last decade has shown evidence that immune pathways communicate with metabolic pathways to alter the metabolic status in response to physiological or pathological signals. In this thesis, I will explore how immunomodulatory molecules affect metabolic homeostasis and conversely, how metabolic sensing pathways modulate immune responses. The first part my work utilizes an immunomodulatory sugar motif to determine mechanisms by which immune cells influence metabolism. Specifically, I show in chapter 2 that lacto-N-fucopentaose III (LNFPIII), a motif used by pathogens to attenuate inflammation, is capable of improving systemic insulin sensitivity by increasing Il-10 production in macrophages and dendritic cells and subsequently improving white adipose tissue insulin sensitivity. Chapter 3 will address the observation that this same glycan is capable of directly activating Fxra in hepatocytes. This direct effect manifests as a reduction in high-fat-diet-induced hepatic triglyceride accumulation and improvement in liver function. Lastly, in chapter 4, I will discuss the role of metabolic regulators in the macrophage and how this affects the ability of the macrophage to kill bacteria. Specifically, I will show that lipid sensing nuclear receptors, such as Ppard and Pparg, are critical regulators of phagosomal function and bacterial killing. Macrophage-specific deletion of these receptors prevents efficient killing of Streptococcus pneumoniae, the causative bacterium in many cases of respiratory pneumonia. Ligand activation improves survival, suggesting a potential therapeutic role for Ppar activation during infection. Taken together, all the data suggest a critical role for the evolutionary interaction between metabolic and immune pathways. These interactions may be important when developing new therapeutics for complex metabolic and immunological dysfunctions.
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Johnston, Hannah. "The role of lipid metabolism in melanoma and identifying therapeutic targets in lipid metabolic pathways." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-lipid-metabolism-in-melanoma-and-identifying-therapeutic-targets-in-lipid-metabolic-pathways(44800322-0da3-4056-bc19-b947058ff203).html.
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There have been dramatic advances in melanoma therapy in the last 10 years, yet there is still a demand for effective and affordable therapies. To identify novel therapeutic pathways a transcriptome analysis was performed on zebrafish melanoma models representing the different stages of melanoma progression. Transcriptomic differences between pre-malignant and malignant conditions highlighted lipid metabolism as a potential mediator of progression. A mass spectrometry analysis confirmed multiple changes in lipid composition between wild type fish, pre-malignant and advanced melanoma models. To better investigate metabolism a positron emission tomography (PET) technique was developed in zebrafish. Tumours in the zebrafish were successfully scanned with FDG used to detect human tumours. A novel tracer of unconjugated FA was then developed and, consistent with inferences from the transcriptome and mass spectrometry, was shown to be incorporated into tumours. Demonstrating the feasibility of PET in zebrafish now opens the way to systematic use of this organism in tracer development with potential time-saving and cost benefits. One of the most significantly up-regulated genes exclusive to the malignant state encodes lipoprotein lipase (LPL). LPL is involved in the release and uptake of FA from circulating triglyceride. LPL was found to increase the rate of tumour appearance and tumour growth in a zebrafish tumour assay. LPL was expressed in human tumours and expression correlated with progression. Melanoma cell lines expressed LPL and knocking-down LPL resulted in reduced cell numbers. The effect was most dramatic in WM852 cells. A novel role for LPL in autophagy was identified. WM852 cells treated with LPL siRNA showed a stabilisation of p62/SQSTM and induction of LC3B II. Electron microscopy revealed large autolysosomal vacuoles in the cytoplasm. Additionally many cells showed damaged mitochondria with absent cristae. The dependency of cells on LPL seemed to be modified by the co-expression of fatty acid synthase (FASN) required for de novo FA synthesis, as the magnitude of the effect of LPL-knockdown was dependent on the levels of FASN expressed in melanoma cell lines. Moreover, combining LPL and FASN inhibitors synergised to kill cells previously less sensitive to LPL inhibitor. FASN and LPL co-inhibition could provide a unique combinatorial therapeutic strategy.
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Lee, Jonathan Patrick. "Use of [U-¹³C?]glycerol to delineate primary metabolic pathways involved in secondary metabolite formation /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487327695623261.
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Louca, Stilianos. "The ecology of microbial metabolic pathways." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/59313.
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Microbial metabolic activity drives biogeochemical cycling in virtually every ecosystem. Yet, microbial ecology and its role in ecosystem biochemistry remain poorly understood, partly because the enormous diversity found in microbial communities hinders their modeling. Despite this diversity, the bulk of global biogeochemical fluxes is driven by a few metabolic pathways encoded by a small set of genes, which through time have spread across microbial clades that can replace each other within metabolic niches. Hence, the question arises whether the dynamics of these pathways can be modeled regardless of the hosting organisms, for example based on environmental conditions. Such a pathway-centric paradigm would greatly simplify the modeling of microbial processes at ecosystem scales.
Here I investigate the applicability of a pathway-centric paradigm for microbial ecology. By examining microbial communities in replicate "miniature" aquatic environments, I show that similar ecosystems can exhibit similar metabolic functional community structure, despite highly variable taxonomic composition within individual functional groups. Further, using data from a recent ocean survey I show that environmental conditions strongly explain the distribution of microbial metabolic functional groups across the world's oceans, but only poorly explain the taxonomic composition within individual functional groups. Using statistical tools and mathematical models I conclude that biotic interactions, such as competition and predation, likely underlie much of the taxonomic variation within functional groups observed in the aforementioned studies. The above findings strongly support a pathway-centric paradigm, in which the distribution and activity of microbial metabolic pathways is strongly determined by energetic and stoichiometric constraints, whereas additional mechanisms shape the taxonomic composition within metabolic guilds.
These findings motivated me to explore concrete pathway-centric mathematical models for specific ecosystems. Notably, I constructed a biogeochemical model for Saanich Inlet, a seasonally anoxic fjord with biogeochemistry analogous to oxygen minimum zones. The model describes the dynamics of individual microbial metabolic pathways involved in carbon, nitrogen and sulfur cycling, and largely explains geochemical depth profiles as well as DNA, mRNA and protein sequence data. This work yields insight into ocean biogeochemistry and demonstrates the potential of pathway-centric models for microbial ecology.Science, Faculty of
Graduate
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Semjonous, Nina M. "Metabolic characterisation of hypothalamic appetite pathways." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486552.
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The Comprehensive Laboratory Animal Monitoring System (CLAMS) is an automated cage system that allows continuous and simultaneous measurements of food intake, activity, oxygen consumption, carbon dioxide production and respiratory exchange ratio. In this thesis, I have developed and optimised the CLAMS as a tool for measuring metabolic parameters within the laboratory. I have then used this system to characterise the effect of a single ICV injection of hypothalamic neuropeptides involved in energy balance. I examined the effect of the orexigenic neuropeptides neuropeptide Y (NPY), agouti-related protein (AgRP), melanin concentrating hormone (MCH), orexin-A and ghrelin in satiated male Wistar rats, whilst the anorexigenic neuropeptides a-melanocyte stimulating hormone (a-MSH), corticotrophin-releasing hormone (CRH) and neuromedin U (NMU) were given to overnight fasted animals. Some peptides exhibited a co-ordinated metabolic response. For example, NPY and orexin-A both stimulated food intake but had opposing effects on energy expenditure. Among the anorectic peptides examined, CRH and NMU showed contrasting temporal effects on feeding and activity, despite the effects of NMU being though to occur via the release of CRH. These studies demonstrate that the CLAMS are a useful tool in determining the roles of well characterised peptides in energy balance. I then went on to use the CLAMS to aid in the' characterisation of the recently discovered . hypothalamic neuropeptide neuromedin S (NMS). Centrally, NMS is specifically expressed in the hypothalamic suprachiasmatic nucleus and is structurally similar to the neuropeptide NMU. Both peptides bind to the NMU1R and NMU2R with comparable affinities. I have used the CLAMS to characterise the effects of intrahypothalamic injection of NMS on food intake, activity and energy expenditure. NMS dose dependently reduced food intake more potently than NMU. This inhibition of feeding was associated with an increase in stress-related behaviours, such as grooming. Microinjection of NMS directly into the PVN also activated the HPA axis at a similar magnitude to NMU, as demonstrated by elevated plasma ACTH and corticosterone. This work suggests that NMS may be involved in the regulation of energy homeostasis and the stress response.
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Favaro, Elena. "Cancer metabolic pathways regulated by hypoxia." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:a148c7a8-cb0c-4760-8073-678299fd837d.
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Metabolic reprogramming in cancer cells provides energy and important metabolites required to sustain tumour proliferation. Hypoxia represents a hostile environment that can encourage these transformations and other adaptive responses that contribute to poor prognosis and resistance to radiation and chemotherapy. Hypoxic signatures associated with worse prognosis were previously derived in different cancer types, and led to the selection of two candidates with potential metabolic implications, namely the mir210-putuative target iron-sulfur scaffold protein ISCU and glycogen phosphorylase (PYGL). Firstly, it was verified that the hypoxia-induced miR-210 targets ISCU. Iron-sulfur clusters represent cofactors for key enzymes involved in Krebs cycle and electron transport chain. Downregulation of ISCU was associated with the induction of reactive oxygen species (ROS) and reduced mitochondrial complex I and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. This indicates that the induction of a single microRNA, miR-210, can mediate a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation. Secondly, it was found that changes in PYGL expression reflect a characteristic upregulation of glycogen metabolism in hypoxia in both tumour xenografts and in cancer cell lines. More specifically, hypoxia stimulates glycogen accumulation and its utilisation, as well as the concurrent upregulation of several glycogen-metabolizing enzymes such as glycogen synthase (GYS1) and PYGL. PYGL depletion led to glycogen accumulation in hypoxic cells, increased intracellular levels of ROS, and a reduction in proliferation due to a p53-dependent induction of senescence. Furthermore, depletion of PYGL was associated with markedly impaired tumorigenesis in vivo. Finally, metabolic analyses indicated that glycogen degradation by PYGL is important for the optimal functioning of the pentose phosphate pathway. Collectively, this study shows the contribution of two important pathways to the metabolic adaptations induced by hypoxia.
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Regan, Lucy. "Metabolic pathway engineering of the toluene degradation pathway." Thesis, University College London (University of London), 1995. http://discovery.ucl.ac.uk/1317891/.
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This thesis addresses the problem of how to examine a metabolic pathway and identify what are the key elements, specifically with respect to rate-limitation. The aim is to be able to analyze a pathway, identify the bottlenecks and implement genetic modifications to remove these bottlenecks. This is done by defining the system of interest and developing a predictive model using kinetic data. The model predictions can then be verified using fermentation data and genetic techniques to make the appropriate changes for improved performance. The test system chosen for this study was the TOL meta-cleavage pathway for the degradation of benzoate. This system was chosen on the basis of the application of pathway engineering principles to other systems. The modelling strategy and software was developed using principles from metabolic control theory and biochemical systems theory. By applying this to the TOL pathway using kinetic data, the control coefficients for the pathway were obtained as well as the system parameters required for the optimization of the pathway. The simulated results obtained from this model must be validated by experiment. Errors can arise both from incorrect assumptions in the model and from the fact that the kinetic data taken from individual in vitro experiments may not be applicable to the in vivo system. The effect of the presence of the TOL pathway on the behaviour of E.coli JM107 during fermentation was investigated and the transient concentration data necessary to identify the bottlenecks in the pathway measured. This data is then used to calculate the flux control coefficients for the TOL pathway. The predictive results were verified by the fermentation data which identified the first two enzymes in the pathway as having significant flux control coefficients. This final chapter also addresses the issue of flux analysis, that is, the calculation of the fluxes in the system to determine where fluxes to unwanted by-products occur and to indicate points of control. A graphical user interface is used to provide a user-friendly and intuitive means of building and customising metabolic pathways which can then be interfaced with instrumentation to provide on-line flux analysis.
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Chou, I.-Chun. "Parameter estimation and network identification in metabolic pathway systems." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/26513.
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Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009.Committee Chair: Voit, Eberhard O.; Committee Member: Borodovsky, Mark; Committee Member: Butera, Robert; Committee Member: Kemp, Melissa; Committee Member: Park, Haesun. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Faust, Karoline. "Development, assessment and application of bioinformatics tools for the extraction of pathways from metabolic networks." Doctoral thesis, Universite Libre de Bruxelles, 2010. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210054.
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Genes can be associated in numerous ways, e.g. by co-expression in micro-arrays, co-regulation in operons and regulons or co-localization on the genome. Association of genes often indicates that they contribute to a common biological function, such as a pathway. The aim of this thesis is to predict metabolic pathways from associated enzyme-coding genes. The prediction approach developed in this work consists of two steps: First, the reactions are obtained that are carried out by the enzymes coded by the genes. Second, the gaps between these seed reactions are filled with intermediate compounds and reactions. In order to select these intermediates, metabolic data is needed. This work made use of metabolic data collected from the two major metabolic databases, KEGG and MetaCyc. The metabolic data is represented as a network (or graph) consisting of reaction nodes and compound nodes. Interme- diate compounds and reactions are then predicted by connecting the seed reactions obtained from the query genes in this metabolic network using a graph algorithm.In large metabolic networks, there are numerous ways to connect the seed reactions. The main problem of the graph-based prediction approach is to differentiate biochemically valid connections from others. Metabolic networks contain hub compounds, which are involved in a large number of reactions, such as ATP, NADPH, H2O or CO2. When a graph algorithm traverses the metabolic network via these hub compounds, the resulting metabolic pathway is often biochemically invalid.
In the first step of the thesis, an already existing approach to predict pathways from two seeds was improved. In the previous approach, the metabolic network was weighted to penalize hub compounds and an extensive evaluation was performed, which showed that the weighted network yielded higher prediction accuracies than either a raw or filtered network (where hub compounds are removed). In the improved approach, hub compounds are avoided using reaction-specific side/main compound an- notations from KEGG RPAIR. As an evaluation showed, this approach in combination with weights increases prediction accuracy with respect to the weighted, filtered and raw network.
In the second step of the thesis, path finding between two seeds was extended to pathway prediction given multiple seeds. Several multiple-seed pathay prediction approaches were evaluated, namely three Steiner tree solving heuristics and a random-walk based algorithm called kWalks. The evaluation showed that a combination of kWalks with a Steiner tree heuristic applied to a weighted graph yielded the highest prediction accuracy.
Finally, the best perfoming algorithm was applied to a microarray data set, which measured gene expression in S. cerevisiae cells growing on 21 different compounds as sole nitrogen source. For 20 nitrogen sources, gene groups were obtained that were significantly over-expressed or suppressed with respect to urea as reference nitrogen source. For each of these 40 gene groups, a metabolic pathway was predicted that represents the part of metabolism up- or down-regulated in the presence of the investigated nitrogen source.
The graph-based prediction of pathways is not restricted to metabolic networks. It may be applied to any biological network and to any data set yielding groups of associated genes, enzymes or compounds. Thus, multiple-end pathway prediction can serve to interpret various high-throughput data sets.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Davies, Andrew. "Investigating the selectivity and mechanism of allosteric regulation in α-IPMS enzymes." Thesis, University of Canterbury. Department of Chemistry, 2015. http://hdl.handle.net/10092/10849.
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Enzymes are nature’s wizards: balanced delicately on the margin of order and entropy, they perform chemical reactions and syntheses at rates and yields human chemists can only dream of. Many possess exquisite control mechanisms to keep the flow of metabolites through our cells precisely regulated. This work explores the regulation mechanism of α-isopropylmalate synthase (α-IPMS).
The branched-chain amino acid biosynthetic pathways in bacteria are of interest as novel antibiotic targets. α-IPMS catalyses the first committed step in the pathway to form leucine, an essential amino acid. It performs the Claisen condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Almost all previously characterised α-IPMS enzymes are feedback regulated by leucine, the end-product of this pathway.
This study uses the α-IPMS enzymes from two pathogenic species, Myco- bacterium tuberculosis and Neisseria meningitidis (MtuIPMS and NmeIPMS, respectively). These enzymes are homodimeric in solution, and have a catalytic dimer of (β/α)8 barrels. This is connected via two more subdomains to a dimerised C-terminal regulatory domain, where leucine binds. The crystal structures of MtuIPMS with and without leucine bound are almost identical. Thus, we do not yet fully understand the mechanisms by which leucine is
recognised, nor how the allosteric signal is conducted ̴ 50 Å from the regulatory domain to the active site, and how this disrupts catalysis.
Chapter 2 explores the residues responsible for recognising and binding leucine. We use insights from the partial crystal structure of a similar enzyme in Leptospira interrogans, citramalate synthase (CMS). CMS catalyses a similar reaction to α-IPMS: the condensation of AcCoA and α-ketobutyrate (α-KB) to form citramalate, as the first step in isoleucine production in this organism. CMS is feedback regulated by isoleucine just as α-IPMS is regulated by leucine. CMS also shares a very similar overall structure to α-IPMS, and four conserved residues in each enzyme were identified as being responsible for binding the allosteric effector. In previous work, Tyler Clarke1 mutated each of the four MtuIPMS residues to the corresponding residue from LiCMS in an attempt to make an isoleucine-regulated MtuIPMS. While one mutant did show an increased sensitivity to the related amino acid norvaline, none of these mutations by themselves were sufficient to create an isoleucine-sensitive MtuIPMS. This work found that by using certain combinations of these mutations, we were able to create isoleucine-inhibited α-IPMS enzymes.
Dr. Wanting Jiao has been using molecular dynamics simulations to identify the residues important for allosteric signal propagation and disrupting catalysis in NmeIPMS . Chapter 3 details several of these residues which we have mutated, and presents the preliminary results of activity and inhibition studies on the mutant enzymes.
Chapter 4 summarises our findings and outlines the work required to further our understanding of the allosteric control systems studied here. Adapting the power of enzymes to contribute to the development of green chemistry, biosensors, and new antibiotics may prove to be one of the greatest opportunities ahead of modern chemistry.
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D'Alessandro, Alba. "p53 regulates Mevalonate Metabolic pathway." Doctoral thesis, Universita degli studi di Salerno, 2014. http://hdl.handle.net/10556/1461.
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2012-2013The mevalonate pathway is an important metabolic pathway implicated in multiple aspects of tumorigenesis. In this study, I provided evidences about genetic and pharmacologic perturbation of p53, which directly influenced expression of mevalonate pathway enzymes, including 3’-Hydroxy-3’-Methylglutaryl - CoenzymeA Reductase, Mevalonate Kinase, Farnesyl Diphosphate Synthase, Farnesyl Diphosphate Farnesyl Transferase 1. Three different cell lines have been considered, U343 MG (U343) and U251 MG (U251) glioma cells, both classified as IV grade glioblastoma cell lines, with two different malignancy grade, and Normal Human Astrocytes (NHA),their normal counterpart. In particular, NHA and U343 cells have wild type p53 (wtp53) while U251bearing mutation (R273H)p53. This mutation affects p53 DNA binding site, preventing transcriptional function of the protein. Different basal expression level of the mevalonate pathway’s genes have found among the different cell lines considered and I hypothesized that this could be ascribable to p53 mutation status and function. Indeed, I observed that functional and active p53 recognized specific p53 Responsive Elements (p53REs) present in MVA enzymes gene-sequences. p53 bound to these regions correlated with increased transcription levels of mentioned genes and such effect has abolished in cells bearing mut(R273H)p53 or by site-directed mutagenesis of p53REs. These new findings expose another facet of p53 functions, unrelated to tumor suppression, and render it a novel regulator of mevalonate pathway providing insight into the role of this pathway in cancer progression. [edited by author]
XII n.s.
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Beveridge, Susan Elizabeth. "Metabolite transport pathways of Plasmodium falciparum." Thesis, University of Liverpool, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590055.
Full textAbstract:
Metabolite transport pathways of the malaria parasite, Plasmodium falciparum, are an important area for study in order to further the understanding of the parasite's biology. Identification and characterisation of the transporters involved in these pathways may also provide potential novel drug targets or drug delivery mechanisms. This is especially valuable as chemotherapy remains one of the main management strategies in the fight against malaria and the usefulness 0 f the current range 0 f antimalarial drugs is seriously threatened by the emergence and spread of resistance . .' In this thesis the Xenopus laevis oocyte heterologous expression system was used to functionally characterise a gene-specific cDNA library of 48 putative membrane proteins and the previously annotated putative amino acid transporter PFF1430c for the uptake of several amino acids. This screening failed to identify any definite amino acid transport by the cDNA library or PFF1430c, however this could have been due to the fact that uptake of a relatively narrow range of amino acids was tested and these were used at concentrations lower than found physiologically. Inherent issues with the X laevis expression system may also have been an issue, including the expression of endogenous transporters for the substrates being investigated.
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Chen, Yung-Pin. "Pathways of aromatic metabolism in Rhizobium." Thesis, Chen, Yung-Pin (1986) Pathways of aromatic metabolism in Rhizobium. PhD thesis, Murdoch University, 1986. https://researchrepository.murdoch.edu.au/id/eprint/51776/.
Full textAbstract:
Studies on the oxidation of a variety of aromatic substrates by Rhizobium trifolii TA1 and Rhizobium leguminosarum MNF3841 indicated that the former could oxidise aromatic substrates normally leading to the catechol or protocatechuate branches of the 3-oxoadipate pathway, while the latter could not use substrates like benzoate requiring the catechol branch. Assays for typical enzymes of the catechol and protocatechuate branches confirmed this conclusion. The detection of a catechol 2,3-dioxygenase only in R. trifolii TA1 further suggested the existence of a meta-cleavage pathway, though no further characterization of such a pathway was attempted.
The uptake systems for benzoate and 4-hydroxybenzoate were studied specifically in R. trifolii TA1 and for 4-hydroxybenzoate in R. leguminosarum. Both systems are active in nature, and both have a pattern of inhibition by aromatic substrates. The two transport systems were not cross-inducible in R. trifolii, but were induced by growth on the appropriate substrate. The transport systems for 4-hydroxybenzoate and protocatechuate also appeared to be distinct. A mutant defective in 4-hydroxybenzoate transport was also isolated.
Removal of periplasmic proteins by lysozyme-EDTA treatment of R. trifolii TA1 abolished 4-hydroxy-benzoate uptake activity in cells grown on 4-hydroxy-benzoate. Cells grown on succinate and treated similarly continued to transport succinate. The periplasmic protein fraction from 4-hydroxybenzoate-grown cells bound radioactive 4-hydroxybenzoate, but similar fractions from cells grown on glucose, succinate, protocatechuate or benzoate did not. A protein was purified from the hydroxybenzoate-grown cells which bound up to 1 mole of [l4C}-4-hydroxy-benzoate per mole of protein. It has been suggested that a binding protein may be required for the transport of 4-hydroxybenzoate.
The two dioxygenases involved in cleavage of the aromatic ring - protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase were purified from R. trifolii TA1, and some of their biochemical properties established. In general, they show considerable resemblance to comparable enzymes purified from Pseudomonas species. Both appeared to be dependent on Fe(III) for activity; the iron could be removed from them by 1,10-phenanthroline after reduction, and activity restored only with an EDTA chelate of Fe(III). Both were extremely sensitive to inhibition by the EDTA chelates of Ni(III) and Al(III) where EDTA itself was ineffective. It has been suggested that inhibition of the dioxygenases by soil aluminium in highly acid soils might well prevent utilization of aromatic compounds by rhizobia and lead to poor survival.
The regulation of the pathways from 4-hydroxy-mandelate and L-mandelate to 4-hydroxybenzoate and catechol, respectively, was studied. Unlike the situation in many Pseudomonas species, the pathways from mandelate and 4-hydroxymandelate appear to involve quite distinct enzymes in R. trifolii TA1.
Studies of enzyme induction in the 4-hydroxy-mandelate and mandelate pathways to protocatechuate and catechol, respectively, suggested that induction of enzymes was sequential for all enzymes, though only a limited range of substrates was available for growth.
When cells were grown on particular substrates, and another substrate downstream from it added, the enzymes upstream showed lowered activities. This finding was true for both the mandelate and hydroxymandelate pathways and for those segments of the 3-oxoadipate pathway which were tested.
Cells grown on aromatic substrates in the presence of other carbon sources showed lowered activities of the enzymes of aromatic degradation. These activities occurred whether the non-aromatic substrate lower were a product of aromatic degradation (succinate or acetate) or glucose. It was not clear whether this represented an end-product type of repression or a limited catabolite repression.
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Choi, Joonhyuk. "Sensing Inorganic Phosphate Starvation by the Phosphate-Responsive (PHO) Signaling Pathway of Saccharomyces cerevisiae." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10878.
Full textAbstract:
Inorganic phosphate \((P_i)\) is an essential nutrient whose intracellular levels are maintained by the PHO pathway in Saccharomyces cerevisiae. \(P_i\) limitation triggers upregulation of the PHO genes whose gene products primarily function to counterbalance the \(P_i\) deficiency. Despite a growing catalogue of genes that are involved in signaling of the PHO pathway, little is known about how cells actually sense \(P_i\) limitation. To better characterize the \(P_i\) sensing mechanism, I exploited two comprehensive and orthogonal approaches: 1) genome-wide genetic screening to identify novel genes involved in signaling \(P_i\) limitation through the PHO pathway and characterization of genetic interactions among these genes and 2) liquid chromatography /mass spectrometry (LC/MS)-based metabolic profiling to characterize the metabolomic response to changes in \(P_i\) availability. In genome-wide screening, I found that the aah1 mutant constitutively activated the PHO pathway and showed that AAH1 is involved in regulating PHO pathway activity. Moreover, I identified several novel genetic interactions of genes involved in inositol polyphosphate metabolism with those involved in purine metabolism and mitochondrial fatty acid biosynthesis.Through metabolomic profiling, I showed that all adenine nucleotides were downregulated in the constitutively induced ado1, adk1, and aah1 mutants in high \(P_i\) as well as in the wild type strain in low \(P_i\). These observations led to the hypothesis that downregulation of adenine nucleotides triggers activation of the PHO pathway. However, I find that decreases in adenine nucleotides appear to be the consequence of downregulation of glycolysis and of the pentose phosphate pathway rather than an activation signal for the PHO pathway.Among all the detected metabolites, S-adenosyl-L-homocysteine (SAH) responded the most quickly and significantly to changes in \(P_i\) concentration. It was known that SAH is an inhibitor of de novo synthesis of phosphatidylcholine (PC). I showed that overall PC levels were downregulated in low \(P_i\), suggesting that phospholipid metabolism is downregulated in low \(P_i\) conditions. Furthermore, I observed that exogenous SAH induces activation of the PHO pathway in high \(P_i\) implying a possible role of SAH as an initiating activation signal of the PHO pathway.Chemistry and Chemical Biology
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Sadhukhan, Sushabhan. "Metabolism & Signaling of 4-Hydroxyacids: Novel Metabolic Pathways and Insight into the Signaling of Lipid Peroxidation Products." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1339171892.
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May, Patrick, Jan-Ole Christian, Stefan Kempa, and Dirk Walther. "ChlamyCyc : an integrative systems biology database and web-portal for Chlamydomonas reinhardtii." Universität Potsdam, 2009. http://opus.kobv.de/ubp/volltexte/2010/4494/.
Full textAbstract:
Background:
The unicellular green alga Chlamydomonas reinhardtii is an important eukaryotic
model organism for the study of photosynthesis and plant growth. In the era of modern highthroughput technologies there is an imperative need to integrate large-scale data sets from highthroughput experimental techniques using computational methods and database resources to provide comprehensive information about the molecular and cellular organization of a single organism.
Results:
In the framework of the German Systems Biology initiative GoFORSYS, a pathway
database and web-portal for Chlamydomonas (ChlamyCyc) was established, which currently features about 250 metabolic pathways with associated genes, enzymes, and compound information. ChlamyCyc was assembled using an integrative approach combining the recently published genome sequence, bioinformatics methods, and experimental data from metabolomics and proteomics experiments. We analyzed and integrated a combination of primary and secondary database resources, such as existing genome annotations from JGI, EST collections, orthology information, and MapMan classification.
Conclusion:
ChlamyCyc provides a curated and integrated systems biology repository that will
enable and assist in systematic studies of fundamental cellular processes in Chlamydomonas. The ChlamyCyc database and web-portal is freely available under http://chlamycyc.mpimp-golm.mpg.de.
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Woods, John Henry. "OOMPF : an Object-Oriented Metabolic Programming Framework." Thesis, Oxford Brookes University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264472.
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Huda, Shahzya Shahnaz. "Metabolic pathways in normal and pre-eclamptic pregnancies." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2537/.
Full textAbstract:
Maternal metabolism undergoes dramatic changes in pregnancy in order to sustain and nourish the developing fetus. During healthy pregnancy the mother goes from an anabolic state in early pregnancy to a state of catabolism in late pregnancy with increased lipolysis together with a significant reduction in insulin sensitivity. Pre-eclampsia (PE) characterised by hypertension and proteinuria is a major cause of maternal and perinatal morbidity. There is acute ‘atherosis’ in PE placenta, and lipid accumulation within glomerular cells and liver. PE women have an early, excessive triglyceride and free fatty acid (FFA) rise and greater cardiovascular disease (CVD) risk in later life. The cause of these lipid abnormalities in PE is unknown but disordered adipocyte function including exaggerated lipolysis and aberrant release of adipokines (such as IL-6 and TNF alpha) is a major candidate pathway. Elevations in FFAs, and pro-inflammatory adipokines could underpin the oxidative stress, endothelial dysfunction, inflammation, and insulin resistance - characteristic features of PE. The aims of this thesis were to acquire a better understanding of lipid metabolism and function in normal pregnancy, to determine if adipocyte function was altered in PE and, if so, to establish mechanisms. In addition I planned to corroborate epidemiological evidence of increased future CVD risk and to establish which risk factors accounted for this increased risk. I collected subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) biopsies in non-labouring pregnant healthy (n=31) and PE (n=14) women who underwent caesarean section. Maternal blood was collected prior to delivery and phenotyping of the mother was performed including plasma assay for cholesterol, triglyceride, HDL-cholesterol, IL-6, TNF-α, leptin, adiponectin, high sensitivity CRP, glucose and insulin concentrations. Maternal BMI at booking, standardised blood pressure measurements and birth weight centile were also recorded. I determined ex vivo lipolytic activity (basal, isoprotenerol stimulated and insulin suppression of lipolysis) and adipokine production in response to lipopolysaccharide (LPS) stimulation from these biopsies. The gene expression of relevant target genes and macrophage densities in each adipose depot by immunocytochemistry (ICC) was also performed. In addition I performed carotid ultrasound assessment of women with a previous history of PE (n=31) and matched controls (n=29). Ethical approval was obtained from Glasgow Royal Infirmary LREC and all patients gave their informed consent. I found that in normal pregnancy, adipocyte lipolytic function is independent of maternal BMI. Adipocyte lipolytic function of SAT and VAT are also independent of each other. Adipose tissue is very metabolically flexible and the rate of whole body lipolysis is still insulin sensitive in late gestation. VAT is more closely related to markers of maternal insulin resistance (IR) and is more sensitive to catecholamine stimulation and less sensitive to insulin suppression of lipolysis than SAT, the basis of the “portal paradigm”. Increasing BMI is associated with an increase in VAT cell size, with increased lipolysis and an increase in pro-inflammatory adipokines, a potential mechanism through which increasing obesity could predispose to metabolic complications of pregnancy. In contrast SAT cell size is not closely related to BMI and this may reflect the adaptation of this depot to increasing fat mass through both hypertrophy and hyperplasia, a metabolically advantageous response. TNF alpha is an important correlate of basal lipolysis in SAT. In PE there is decreased insulin sensitivity of both SAT and VAT compared to controls as calculated by the fat cell insulin sensitivity index (or responsiveness to insulin once the tissue is stimulated by isoproterenol). This would potentially make a significant impact on total circulating FFA as almost 60% of circulating FFA are from these adipose depots. The rise in FFA in PE occurs early in pregnancy and contributes significantly to IR. Therefore the IR of adipose tissue could lead to a vicious cycle of increased lipolysis, increased FFA and further exacerbation of IR. In contrast to controls, SAT cell size is intimately related to BMI suggesting that adaptation to increasing fat mass is mainly through adipocyte hypertrophy which could lead to increased endoplasmic reticulum stress, increased IR and increased release of inflammatory adipokines. I have shown that SAT cell size does relate to adipokine release in PE, with increased release of leptin, CRP and PAI-1 and paradoxical increase in the anti-inflammatory IL-10. I had hypothesised that in addition to an inherent defect in adipocyte function there was an additional factor present in maternal serum of women with PE released from the placenta which excessively stimulated lipolysis. I failed to demonstrate any effect of maternal serum on adipocyte lipolysis in either controls or PE. I also found that after stimulation with LPS, there was increased release of TNF alpha and IL-6 in VAT in PE but not in controls, with higher gene expression of these adipokines. TNF alpha release also correlated negatively with the fat cell insulin sensitivity index (FCISI) of VAT implicating a paracrine effect in this tissue. I also demonstrated an increase in gene expression of cfms (activated macrophages) relative to control gene, and increased density of cfms+ macrophages/adipocytes in the VAT of PE women implicating activated adipose tissue macrophages as a potential source of the increased release of inflammatory adipokines. Lastly I attempted to corroborate epidemiological evidence for the increase future risk of CVD women with a history of PE by assessing two surrogate markers for atherosclerosis - carotid IMT and carotid plaque scores. Both were found to be increased, with plaque scores significantly so. Classic risk factors such as age, lipids, BP and smoking did not attenuate this effect and BMI only marginally attenuated it, therefore only partially explaining this increased risk. In summary the data presented in this thesis provides further evidence that PE is a “metabolic syndrome of pregnancy” with disordered adipocyte function and metabolism, with an increased future risk of CVD in later life. Further studies on adipose accumulation, function and composition in normal and complicated human pregnancy are warranted.
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Okonko, Darlington Obinnaya. "Anaemia and metabolic pathways in chronic heart failure." Thesis, Imperial College London, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.549708.
Full textAbstract:
Background: Anaemia is a common and adverse comorbidity in chronic heart failure (CHF), but critical aspects of its epidemiology, pathogenesis and treatment remain unclear. Objectives: This thesis tested the hypotheses that temporal trends in haemoglobin (Hb) relate to outcome in CHF, that anaemia might be subsequent to immune-mediated erythroid cell suppression, erythropoietin (Epo) resistance, cellular iron transport dysregulation, and altered adrenal steroidogenesis, and that low Hb levels could be amenable to recombinant Epo and intravenous iron therapies separately. Methods and Results: A post hoc analysis revealed that new onset anaemia occurred in 14% of patients at 1 year, developed more frequently on carvedilol than metoprolol, and was associated with increased mortality. Using flow cytomtry and cell culture techniques, CHF patients with anaemia of unknown origin were shown to exhibit markedly low reticulocyte production indices, and globally attenuated circulating erythroid lineage pools (C034+ stem cells, erythroid progenitors [BFU-E] and precursors). Only the depletion of anaemic monocytes from cultures enhanced erythroid colony growth. Only the addition of anaemic monocytes or sera to cultures blunted autologous and allogenic erythroid colony formation. Anti-TNFa neutralizing antibody abrogated the effects of anaemic sera on erythroid colonies. In additional assays, the ex-vivo responsiveness of erythroid cells to escalating doses of Epo was diminished in anaemic patients. This was not associated with Epo receptor downregulation but with a profound blunting of Epo-induced intracellular signalling. In biochemical analyses, the cortisol/dihydroepiandrosterone ratio, a marker of adrenal steroid hormone imbalance, was shown to inversely correlate to Hb levels. More importantly, disorded iron homeostasis was highly prevalent in CHF patients and independently predictive of anaemia, exercise intolerance and impaired survival. Sera from iron deficient subjects exhibited elevated pro-inflammatory cytokine and pro-hepcidin levels. Such sera downregulated ferroportin (iron export protein) and upregulated divalent metal transport-l (iron import protein) expression on monocytes ex-vivo, a pattern that facilitates inflammatory hypoferremia in vivo. Co-incubation with anti- TNFa neutralising antibodies abolished these effects. Finally, in separate randomised controlled trials, recombinant Epo escalated Hb levels but not exercise tolerance in anaemic CHF patients, whilst intravenous iron improved symptoms and exercise capacity but not Hb levels in anaemic and non-anaemic CHF patients.
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Williams, H. E. "Mathematical modelling of metabolic pathways in pig muscle." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/42536/.
Full textAbstract:
Improving efficiency within the agricultural industry is vital to maintain the food demands of the increasing population, as well the current preference for a more protein rich diet. One avenue for addressing these issues is to study animal-based growth to determine if the efficiency of the production system can be improved by increasing lean muscle mass. The aim of this thesis is to provide an alternative exploration to experimental work to provide an insight into how muscle metabolism in pigs is altered by the administration of a beta-agonist which induces muscle hypertrophy. This will be incorporated into a wider body of work to determine specific pathways to target for improving feed conversion efficiency, contributing to the necessary research into global food security. We begin by compiling a selection of statistical methods to analyse muscle microarray data, which enables the identification of a selection of genes whose expressions are altered by the exposure to a beta-agonist. These differentially expressed transcripts are then grouped via a k-means algorithm, with log likelihood and the Bayesian Inference Criterion calculations providing an optimal selection of clusters. This results in selecting a group of 51 transcripts and partitioning them into 9 clusters, and identifying several pathways which appear key to the regulation of muscle metabolism in the presence of beta-agonist. We have proceeded to incorporate this information into a mathematical model for glycolysis and the TCA cycle, in an effort to analyse biological hypotheses about how the promoters work. The equations describe the concentrations of metabolites within the cytosolic and mitochondrial compartments of a cell using mass balance ODEs. An initial model is presented, which is then increased in complexity, to keep up with developments in the experimental side of the overarching project. We make use of a selection of methods to analyse the model in an attempt to determine the effects that the different parameters cause. Through steady state analysis, we determine parameter ranges which permit positive steady states. In finding these regions, we also determine the existence of time dependent solutions, which occur when critical values of certain parameters are exceeded, and result in the build up of specific metabolites. We use asymptotic analysis to generate approximate solutions when steady states do not exist. The model parameters of most interest are those which were identified through the microarray work, namely the upregulated transcripts of PCK2 and those within the serine synthesis pathway, the control mechanism for the first half of the TCA cycle, the proportion of GTP producing enzyme from the second half of the TCA cycle, and the flux into the glycolytic pathway. We find that critical values for the glycolytic flux, and the GTP production parameter exist, determining whether the model lies within the steady state regime. In a large number of cases, the parameters we choose to represent the beta-agonist case push the system into the time dependent state. The model does not exhibit any interesting behaviour when the parameter controlling the PCK2 pathway is studied, indicating that initial intuition of the key controlling reaction mechanisms were incomplete. Whilst there are shortfalls in the model, which highlight areas for investigation, the system is set up for validation and parameter fitting when appropriate experimental data become available. We have been able to determine specific metabolic pathways within the cell which may be of significance to improving feed efficiency.
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Ehlde, Magnus. "Dynamic and steady-state models of metabolic pathways a theoretical evaluation /." Lund : Dept. of Chemical Engineering I, University of Lund, 1995. http://catalog.hathitrust.org/api/volumes/oclc/39065942.html.
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Lindgren, Kim. "Under which conditions is the C4metabolic pathway favored? : When does the C4 metabolic pathway become less costlythan the C3 metabolic pathway?" Thesis, Umeå universitet, Institutionen för ekologi, miljö och geovetenskap, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-53180.
Full textAbstract:
C4 photosynthesis is an advanced complement to the more ancestral and more commonpathway refereed to as C3. C4 metabolism has evolved in several taxa, and it is theorized thatit worked as an adaptation to the low CO 2 levels characteristic of late geological time. Theadaptation also carries with it some resistance to the negative effects brought on by hightemperatures and drought. C4 metabolism is, however, not free, meaning that underconditions of lower temperature and higher CO2-levels, C3 photosynthesis is still moreviable. This makes it interesting to study how C4-species might shift their ranges in responseto climate change, as it implies both elevated CO 2 levels and higher mean temperatures inmost parts of the world. In this report, I develop a model based on the CO 2/O2 specificity of Rubisco from Spinach(Spinacia oleracea) at different temperatures, using data found in literature on the subject.The resulting model has some success in describing the current distribution of C4 species,using temperature and CO2 concentration as explanatory variables.
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Kaur, Dipendra. "Mapping and Filling Metabolic Pathway Holes." Digital Archive @ GSU, 2008. http://digitalarchive.gsu.edu/biology_theses/14.
Full textAbstract:
The network-mapping tool integrated with protein database search can be used for filling pathway holes. A metabolic pathway under consideration (pattern) is mapped into a known metabolic pathway (text), to find pathway holes. Enzymes that do not show up in the pattern may be a hole in the pattern pathway or an indication of alternative pattern pathway. We present a data-mining framework for filling holes in the pattern metabolic pathway based on protein function, prosite scan and protein sequence homology. Using this framework we suggest several fillings found with the same EC notation, with group neighbors (enzymes with same EC number in first three positions, different in the fourth position), and instances where the function of an enzyme has been taken up by the left or right neighboring enzyme in the pathway. The percentile scores are better when closely related organisms are mapped as compared to mapping distantly related organisms.
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Meggiato, Alberto <1987>. "Comparing metabolic networks at pathway level." Master's Degree Thesis, Università Ca' Foscari Venezia, 2016. http://hdl.handle.net/10579/8501.
Full textAbstract:
Metabolic pathway comparison between different species is important to discover the differences in a metabolic function developed during the evolutionary process. This kind of analysis may allow the detection of important information useful also in drug engineering and medical science.
In this thesis we propose a method for metabolic pathways comparison based on their representation as sets and multisets of chemical reactions. The information is taken from the KEGG database because it has a standardised representation of each pathway in the different organisms.
The pathway comparison technique is then used in the context of metabolic networks comparison in order to solve the problems due to the size of the compared networks.
The proposed methods have been implemented in Java as part of a tool for metabolic networks comparison.
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Yuan, Wang. "Familiar Layouts Generation for Metabolic Pathway Graph Visualization." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1212764351.
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Haselden, John Neil. "The synthesis and metabolism of xenobiotic acylglycerols." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243552.
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Benito, Mauricio Adrián. "The study of RAS-induced metabolic reprogramming and the role of the pentose phosphate pathway in tumor metabolism." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/133025.
Full textAbstract:
The present doctoral thesis is focused on the metabolic adaptations induced by oncogene activation as well as the potential role of the metabolic network as antitumor therapy. Over the last years, it has emerged a renewed interest in the field of metabolism, particularly in cancer metabolism. Great efforts have been focused on the association of mutated oncogenes or tumor suppressor genes and tumor metabolic profiles, in the search of metabolic dependencies that offer new potential avenues for cancer treatment. The pursuit of discovering tumor metabolic alterations in which cancer cells rely on has represented the cornerstone of this interesting discipline. Thus, this thesis is part of this recent and promising scientific current and is intended to shed light on the metabolic alterations accompanying oncogene mutation and on potential metabolic pathways that might be of therapeutic interest in the future. Hence, the objectives of this thesis can be divided into two specific aims: i) analysis of the metabolic reprogramming of RAS oncogenic activation using stable transfected cell lines with mutated copies of K-RAS and H-RAS and ii) validation of the pentose phosphate pathway as a potential therapeutic target and exploration of its role within tumor metabolism in colon and breast cancer cell models. Thus, according to the proposed objectives, the main conclusions obtained are as follow:
1. The study of flux distribution in combination with metabolic control analysis performed by analyzing solely the sign of fixed-sign control coefficients, is a reliable approach to identify the key enzymes involved in metabolic reprogramming. The use of this methodology has allowed us to identify an increase in glycolysis and PPP fluxes as metabolic features of KRAS-induced metabolic reprogramming and to propose G6PD, PK and LDH as the key enzymes responsible for this metabolic transition.
2. H-RAS oncogenic activation reprograms glucose and glutamine metabolism by enhancing glycolytic and PPP fluxes as well as mitochondrial metabolism. Glutamine is responsible for sustaining the activated mitochondrial metabolism in BJ-HRasV12, while glucose-derived carbons in the mitochondria are primarily used to fuel lipogenesis. Moreover, lipogenesis is overactivated in BJ-HRasV12 cells, which are more sensitive to FAS inhibition than BJ cells.
3. G6PD enzyme is overactivated in colon cancer cells with oncogenic activation of the RAS signaling pathway. Nevertheless, G6PD seems to be dispensable for proliferation and survival in BRAF-mutated HT29 cell line. Furthermore, a new connection between PPP and glutamine metabolism has been unveiled, as G6PD is overexpressed in HT29 cells under glutamine-deprived conditions by a mechanism involving a concomitantly increase in ROS levels and NRF2 induction.
4. G6PD enzyme is important in proliferation, survival and regulation of ROS levels in breast cancer MCF7 cells. However, it exerts a low regulation over ribose synthesis flux through the oxidative branch of PPP. G6PD inhibition enhances glycolytic flux, promotes lactate secretion and increases glutamine consumption, which is used to maintain energy homeostasis, although it is not essential for cell proliferation.
5. TKT enzyme is dispensable for proliferation of breast cancer MCF7 cells, but it exerts a high control over ribose synthesis flux through the nonoxidative branch of PPP. TKT impairment reduces glycolytic flux and increases the consumption of glutamine, which is intended to maintain energy homeostasis but it is not essential for cell proliferation.La presente tesis doctoral se centra en las adaptaciones metabólicas inducidas por la activación de oncogenes así como en el potencial del entramado metabólico como diana antitumoral. A lo largo de los últimos años, ha resurgido un renovado interés en el estudio del metabolismo, particularmente en el metabolismo de las células tumorales, dando lugar a una nueva disciplina conocida como metabolismo tumoral. Numerosas investigaciones se han centrado en la asociación entre mutaciones en oncogenes o genes supresores de tumores con perfiles metabólicos característicos, en busca de dependencias metabólicas que ofrezcan nuevas posibilidades para el tratamiento de los tumores. La búsqueda de alteraciones metabólicas que constituyan vulnerabilidades de la célula tumoral representa la piedra angular de esta interesante disciplina. Así, esta tesis doctoral tiene como objetivo general elucidar las alteraciones metabólicas que acompañan a la mutación de oncogenes y explorar el potencial del entramado metabólico como diana antitumoral. Por tanto, los objetivos principales de este trabajo son los siguientes: i) análisis de la reprogramación metabólica inducida por la activación oncogénica de RAS empleando líneas celulares transfectadas de manera estable con copias mutadas de los oncogenes K-RAS y H-RAS y, ii) validación de la vía de las pentosas fosfato como potencial diana antitumoral y estudio de su papel en el metabolismo tumoral de modelos celulares de cáncer de colon y de mama. Así, en este trabajo de tesis doctoral hemos concluido que la activación oncogénica de RAS promueve una profunda reprogramación del metabolismo induciendo cambios significativos en la glucólisis, la vía de las pentosas fosfato, el metabolismo de la glutamina y la lipogénesis. Por otro lado, hemos determinado que la inhibición de la vía de las pentosas fosfato tiene distintos efectos según el tipo de tumor. La inhibición de la G6PD en la línea celular de cáncer colon HT29 no produjo efectos sobre la proliferación mientras que su inhibición en células de cáncer de mama MCF7 indujo una notable reducción de la proliferación y un incremento de la muerte celular. Por otra parte, en la inhibición en MCF7 del otro enzima clave de la vía de las pentosas fosfato, la TKT, no se observaron cambios significativos en términos de proliferación y viabilidad celular. Además, en este trabajo también se ha puesto de manifiesto una conexión funcional entre la vía de las pentosas fosfato y el metabolismo de la glutamina en ambos modelos celulares, sugiriendo un papel complementario de estas dos vías metabólicas.
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Yong, Carmen. "Enhancing adoptive immunotherapy : redirecting immune subsets and metabolic pathways." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT059.
Full textAbstract:
Le transfert adoptif de cellules T exprimant un récepteur chimérique reconnaissant un antigène (CAR), est un traitement qui génère des réponses impressionnantes dans les cancers hématologiques mais est beaucoup moins efficace pour le traitement de tumeurs solides. Les tumeurs solides modulent leur microenvironnement induisant des formes multiples d’immunosuppression qui inhibent l’efficacité des fonctions effectrices des cellules T ayant infiltrées la tumeur. Au cours de ma thèse, j’ai évalué le potentiel de deux stratégies pour améliorer les réponses anti-tumorales des cellules T CAR. La première se focalise sur l’étude du rôle potentiel des cellules immunes non T, exprimant un CAR sur la stimulation des fonctions et de la persistance de cellules T CAR+ dans le microenvironnement tumoral. Afin d’étudier la fonction des cellules CAR non T, nous avons généré un modèle de souris transgénique (vav-CAR) dans lequel les cellules immunes expriment un CAR reconnaissant l’antigène tumoral Her2 (ErbB2). Comme attendu, les cellules T CAR+ possèdent des fonctions anti-tumorales, mais nous avons aussi mis en évidence que les macrophages et les cellules NK exprimant le CAR montraient une réponse cytokinique, cytotoxique et phagocytiques spécifiques de l’antigène. De plus, en utilisant le modèle vav-CAR, nous avons démontré le potentiel des cellules immunes CAR+ dans le rejet des tumeurs et cela indépendamment des cellules T CD8+. Les cellules T CD4+ sont essentielles puisque leur élimination réduit considérablement les réponses anti-tumorales dans notre modèle vav-CAR. Il a été démontré que certaines sous-populations de cellules T auxiliaires participent aux réponses anti-tumorales avec les cellules Th1 et Th17 démontrant une efficacité plus robuste que les autres sous-populations. Notre deuxième stratégie s’est focalisée sur l’étude de l’impact du métabolisme au cours de la polarisation des cellules T CD4+ et plus particulièrement lors de la différenciation des cellules T CAR+ en cellules Th1. En effet, l’activation et différenciation des cellules T sont fortement associées à une augmentation des besoins métaboliques. Dans le microenvironnement tumoral, en raison de la forte demande en ressources de la tumeur, la déprivation en nutriments ainsi générée peut limiter l’accès aux nutriments d’autres types cellulaires et ainsi altérer le devenir et les fonctions des cellules immunes greffés infiltrant la tumeur. En conséquence, modifier les cellules immunes CAR+ afin qu’elles puissent résister à la compétition métabolique du microenvironnement tumoral pourrait leur permettre de conserver leurs fonctions effectrices. En étudiant l’impact de la déprivation en nutriments sur la différenciation des cellules T, nous avons trouvé que des concentrations limitantes en glutamine, l’acide aminé le plus abondant du plasma, inhibaient le potentiel des cellules T à se différencier vers la voie Th1 associée à la production d’IFNγ. Au contraire, cette condition favorisait la conversion de cellules T CD4 naïves en cellules régulatrices Foxp3+ ayant des fonctions suppressives (Tregs). De plus, nous avons montré que la présence d’un seul métabolite dérivé de la glutamine, l’α-ketoglutarate (αKG), suffisait à augmenter les fonctions effectrices anti-tumorales de plusieurs sous-types de cellules T auxiliaires CAR+, augmentant la production d’IFNγ et diminuant l’expression de FOXP3. Ainsi, durant ma thèse, j’ai développé un modèle murin vav-CAR, générant un outil permettant d’étudier et manipuler les fonctions de multiples populations de cellules immunitaires exprimant un CAR. Ce modèle permettra de promouvoir l’utilisation de cellules immunes optimisées exprimant un CAR dans le cadre d’immunothérapies dirigées contre des tumeurs solides. De plus, en utilisant ce modèle, nous avons identifié un métabolite de la glutamine, qui orchestre les réponses immunitaires au moyen d’une reprogrammation métabolique des cellules T CD4The adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) as a treatment for cancer has achieved impressive responses in haematological malignancies, but has been less successful in the treatment of solid tumors. The tumor microenvironment of solid tumors presents multiple forms of immunosuppression, inhibiting the efficient effector function of infiltrating anti-tumor T cells. During my PhD, we assessed the potential of two strategies to enhance the anti-tumor function of CAR T cells. The first focuses on the potential of other CAR-expressing immune subsets to stimulate CAR T cell function and persistence in the tumor microenvironment. To elucidate the function of CAR-expressing non-T lymphocytes, we generated a transgenic mouse model (vav-CAR) in which immune cells express a CAR against the Her2 (ErbB2) tumor antigen. As expected, CAR T cells harboured anti-tumor function but we also found that CAR-modified macrophages and natural killer cells (NKs) exhibited significant antigen specific cytokine secretion, cytotoxicity and phagocytosis. Moreover, using the vav-CAR model, we demonstrated the potential of CAR immune cells to mediate tumor rejection independently of CD8+ T cells. CD4+ T cells were critical for this response as their deletion severely abrogated the anti-tumor responses in our vav-CAR model. Distinct T helper subsets have been shown to participate to anti-tumor responses, with Th1 and Th17 cells demonstrating a more robust efficacy as compared to other T helper subsets. Our second strategy was focused on the impact of metabolism in the polarisation of CD4+ T cells, in particular the differentiation of CAR T cells to Th1 lineage. T cell activation and polarisation is highly associated with increased metabolic needs. Given that nutrient deprivation in the tumor microenvironment, due to a high demand of the tumor for resources, can limit the nutrients available for other cell types, the fate and function of adoptively transferred immune cells may be altered upon entering the tumor. Therefore, modifying CAR immune cells to resist metabolic suppression in the tumor microenvironment may help retain their effector functions. Upon assessing the effects of nutrient deprivation on T cell differentiation, we found that limiting concentrations of glutamine, the most abundant amino acid in the plasma, inhibited the potential of T cells to undergo Th1 differentiation with associated IFNγ secretion. Rather, this condition resulted in the conversion of naïve CD4+ T cells into suppressive Foxp3+ regulatory T cells (Tregs). Furthermore, we determined that a single glutamine-derived metabolite, α-ketoglutarate (αKG), enhanced the anti-tumor effector functions of multiple CAR T helper subsets, increasing the production of IFNγ and reducing FOXP3 expression.Thus, during my PhD, I generated a vav-CAR model, providing a platform in which the function of multiple CAR-bearing immune subsets can be studied and manipulated. This model will promote the utilisation of optimized CAR-bearing immune cells in adoptive immunotherapy for solid tumors. Furthermore, using the CAR model, we have identified a glutamine metabolite that orchestrates immune responses through the metabolic reprogramming of CD4 T cells
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Kalra, Paul Raj. "Natriuretic peptides and metabolic pathways in chronic heart failure." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503841.
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Richards, Jamie. "Investigation of metabolic pathways suitable for antimicrobial drug discovery." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424092.
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Onyiaodike, Christopher C. "A study of metabolic and inflammatory pathways throughout gestation." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/4979/.
Full textAbstract:
The effect of metabolic and inflammatory parameters on pregnancy success in terms of implantation, metabolic adaptation to pregnancy and fetal programming is yet to be fully understood. This thesis explores the activity of metabolic and inflammatory pathways in pregnancy, highlighting their importance throughout gestation. In a cell culture study, a model of in vivo blastocyst-uterine adhesion to study the effect of insulin during uterine implantation was explored. JAR spheroid-RL95-2 monolayer adhesion reached 98% by 24 hours in the absence of insulin. A low dose (0.03nM) of added insulin concentrations resulted in 26% adhesion, or 74% inhibition; a high level (0.24nM) inhibited the JAR spheroid-RL95-2 monolayer adhesion by 9%. Therefore insulin did not have a dose-dependent on JAR spheroid-RL95-2 monolayer adhesion in the cell culture model of implantation. Polymerase chain reaction (PCR) studies revealed laminin α1 RNA detection on JAR cells only, CD44 on RL95-2 cells only, no trophinin on both cell types, FBLN-1 and -2 on JAR and FBLN-1 on RL95-2 cells only and an insulin receptor in both cell types. Western blot and immunohistochemistry (IHC) studies showed laminin α1 detection and stains on the JAR cell extracellular matrix. In a prospective human study, the metabolites of lipid and carbohydrate metabolism and inflammatory mediators very early (between day 0 and day 45) in gestation and their link to successful pregnancy in women undergoing natural cycle frozen embryo transfer (FET) in assisted conception, was investigated. Plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), glucose, c-reactive protein (CRP) and non-esterified fatty acid (NEFA) were measured on routine biochemistry; insulin, interleukin (IL)-6, plasminogen activated inhibitor (PAI)-1 and PAI-2 on ELISA; IL-8 (CXCL8), CCL2, CCL3, CCL4 and CCL11 on BioPlex; and human chronic gonadotrophin (hCG) on an Immulite system. For all 196 FET cycles, participants' demographics and plasma parameters of pregnant (n=36) and non-pregnant (n=106) women were explored. Neither obesity, the plasma parameters nor insulin resistance were predictive of successful pregnancy, but ICSI (predominately associated with male factor infertility) was. Overall, the hCG, insulin, rebound TG and HDL-C (except TC), homeostasis model assessment (HOMA), CRP and PAI-2 levels were higher, whereas CXCL8, CCL2, CCL11 and PAI-1 were significantly lower by day 45. Baseline obesity related to positive changes in plasma insulin, HDL-C and HOMA and negative changes in CXCL8, CCL3 and CCL4. In a cross-sectional study in late pregnancy, offspring's reflection of parameters in women with preeclampsia (PE) (n=29) and intrauterine growth restriction (IUGR) (n=14), compared to BMI-matched healthy groups (n=87) and (n=42), respectively, was explored. Fetal cord was found to be hyperlipidaemic, normoglycaemic and had reduced inflammatory response, while mothers who suffered PE had altered plasma TG, TC, NEFA, glucose, leptin and IL-10 compared to controls. IUGR babies were dyslipidaemic. The role of cholesterol transporters was assessed in PE (n=20) and IUGR (n=9) BMI-matched controls (n=20 and n=9) respectively. Among fifteen steroidogenic acute regulatory protein (STAR)-related lipid transfer domains, only STARD6 and STARD15 were not detected in the placenta via PCR. IHC studies were also explored on the placentae. The real-time PCR (RT-PCR) of messenger RNA of low-density lipoprotein receptor (LDLR), STARD3 and ATP-binding cassette A1 (ABCA1) without protein were higher in PE compared to controls. LDLR, STARD3 and ABCA1 localisation and detection were consistent to placental lipid (cholesterol) transport systems. In summary, all this led to the conclusion of the importance of metabolic and inflammatory pathways in all stages of pregnancy in leading to pregnancy success; these pathways may influence implantation, adaptation to pregnancy and, potentially, fetal programming of offspring.
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Sakakibara, Norikazu. "Metabolic analysis of the cinnamate/monolignol and lignan pathways." Kyoto University, 2005. http://hdl.handle.net/2433/145058.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第11658号
農博第1514号
新制||農||911(附属図書館)
学位論文||H17||N4051(農学部図書室)
23301
UT51-2005-D407
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 島田 幹夫, 教授 關谷 次郎, 教授 坂田 完三
学位規則第4条第1項該当
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Zamora, Jorge. "Lipid Metabolic Pathways in the Midgut of Manduca sexta." Diss., The University of Arizona, 2006. http://hdl.handle.net/10150/195273.
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Insects such as Manduca sexta must be efficient in obtaining energy stores in order to survive. The main goal of the M. sexta larva is to eat and store enough energy to reach the adult stage, produce eggs (in the case of females), and reproduce. Triacylglycerol (TAG), the most important energy storage molecule, is stored mainly in adipose tissues in lipid droplets, although other tissues are also able to store TAG in similar organelle structures but to a lesser degree. The phosphatidic acid (PA) pathway and the 2-monoacylglycerol (MAG) pathway are both energy-dependant acyl-CoA processes and are the main synthetic pathways by which TAG is synthesized in adipose and other tissues. My research led to the discovery of an energy-independent pathway for the synthesis of TAG that was present in the M. sexta midgut. Based on partial purification, a transacylase/lipase enzyme is present and responsible for DAG and TAG synthesis in the M.sexta midgut. Lipogenesis and lipolysis in adipose tissue involves a series of enzymes. Adiponutrin and desnutrin, two proteins involved in fat homeostasis in humans and mice, have received a lot of attention since their activities are dependent on the fed or unfed state of the animal. In this study, bioinformatics analyses were performed, which allowed the identification of an insect gene that has an ortholog in human and mice that plays an important role in adipose tissue TAG hydrolysis and synthesis. Only one insect gene ortholog was found to be present in the Aedes aegypti (mosquito), Drosophila melanogaster (fruit fly), Anopheles gambiae (mosquito), Bombyx mori (silk worm), and Tribolium castaneum (red flour beetle) genomes corresponding to genes involved in the regulation of TAG metabolism in mice (adiponutrin, desnutrin) and humans (iPLA-epsilon, iPLA-zeta, and iPLA-eta). Expression of the M. sexta calcium-independent phospholipase A2 (iPLA) ortholog has demonstrated that the protein is able to transfer acyl groups between MAGs in an energy-independent manner, similar to that in human iPLAs. This is the first example of a transacylase identified in insects.
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Morrison, Erin S., and Alexander V. Badyaev. "Structuring evolution: biochemical networks and metabolic diversification in birds." BioMed Central, 2016. http://hdl.handle.net/10150/620926.
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Background
Recurrence and predictability of evolution are thought to reflect the correspondence between genomic and phenotypic dimensions of organisms, and the connectivity in deterministic networks within these dimensions. Direct examination of the correspondence between opportunities for diversification imbedded in such networks and realized diversity is illuminating, but is empirically challenging because both the deterministic networks and phenotypic diversity are modified in the course of evolution. Here we overcome this problem by directly comparing the structure of a “global” carotenoid network – comprising of all known enzymatic reactions among naturally occurring carotenoids – with the patterns of evolutionary diversification in carotenoid-producing metabolic networks utilized by birds.
Results
We found that phenotypic diversification in carotenoid networks across 250 species was closely associated with enzymatic connectivity of the underlying biochemical network – compounds with greater connectivity occurred the most frequently across species and were the hotspots of metabolic pathway diversification. In contrast, we found no evidence for diversification along the metabolic pathways, corroborating findings that the utilization of the global carotenoid network was not strongly influenced by history in avian evolution.
Conclusions
The finding that the diversification in species-specific carotenoid networks is qualitatively predictable from the connectivity of the underlying enzymatic network points to significant structural determinism in phenotypic evolution.
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Swati, Swati, Richard Sante, and Aruna Kilaru. "Characterization of Arachidonylethanolamide Metabolic Pathway in Moss." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/4803.
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Arachidonylethanolamide (AEA) is a bioactive lipid ligand for mammalian cannabinoid receptors (CB). Thus far, AEA was reported to occur only in animals and was shown to regulate a wide range of physiological responses. Our recent fi nding of the occurrence of AEA in moss has led us hypothesize that AEA might mediate stress responses in plants, similar to that in animals. In mammals, AEA is generated from hydrolysis of N-acylphosphatidylethanolamine (NAPE) by a NAPE-specifi c phospholipase D (NAPE-PLD), and degraded by a fatty acid amide hydrolase (FAAH) and this metabolic pathway is highly conserved among eukaryotes. Here, using in silico approach, putative genes encoding for AEA pathway enzymes, were identifi ed in moss. Full-length coding sequences for putative NAPE-PLD and FAAH were isolated from Physcomitrella patens and were cloned and expressed into a heterologous expression vector. Biochemical characterization of AEA pathway enzymes is underway and is expected to lead to generation of AEA metabolite mutants in moss. Such mutants will allow for elucidation of the role of AEA in development of moss and mediating stress responses. Overall, this study will provide novel insights into functional and evolutionary role of lipid-mediated signaling in plants.
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Swati, Swati, Richard Sante, Brent Kinser, and Aruna Kilaru. "Characterization of Anandamide Metabolic Pathway in Moss." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/4853.
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N-Acylethanolamines (NAEs) including anandamide (NAE 20:4) are fatty acid ethanolamides generated by the hydrolysis of N-acylphoshotidylethanolamine (NAPE) by phospholipase D (PLD) and degraded by fatty acid amide hydrolase (FAAH). In mammals, ligands such as NAE 20:4 act through cannabinoid receptors and regulate several physiological processes like neuroprotection, pain perception, mental depression, and appetite suppression. In plants, NAE with chain length C12 to C18 are common and affect physiological processes such as cytoskeletal organization, endomembrane trafficking, cell wall and cell shape formation, seedling growth and response to stress. However, our recent identification of NAE 20:4 in moss, Physcomitrella patens prompted us to elucidate its metabolic pathway and physiological implications. We hypothesize that unique NAE metabolites such as anandamide in moss might play a role in rendering moss its ability to tolerate temperature, dehydration, salt and osmotic stress. To address the above hypothesis, three main objectives are being pursued using P patens. 1)Biochemical and molecular characterization of NAE metabolic pathway, 2) Generation and phenotypic characterization of NAE metabolite mutants, and 3) Elucidation of the physiological role of NAEs in abscisic acid-mediated dehydration tolerance. A NAPE-PLD, known to synthesize NAE 20:4 has been identified in mammals and FAAH in several eukaryotes, including plants. Here, identification and cloning of putative NAPE-PLD and FAAH genes that are likely involved in NAE synthesis and degradation, respectively, in P patens is discussed. Our long-term objective is to understand lipid-mediated stress responses in plants.
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Planes, Francisco J. "Metabolic pathway analysis via integer linear programming." Thesis, Brunel University, 2008. http://bura.brunel.ac.uk/handle/2438/6134.
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The understanding of cellular metabolism has been an intriguing challenge in classical cellular biology for decades. Essentially, cellular metabolism can be viewed as a complex system of enzyme-catalysed biochemical reactions that produces the energy and material necessary for the maintenance of life. In modern biochemistry, it is well-known that these reactions group into metabolic pathways so as to accomplish a particular function in the cell. The identification of these metabolic pathways is a key step to fully understanding the metabolic capabilities of a given organism. Typically, metabolic pathways have been elucidated via experimentation on different organisms. However, experimental findings are generally limited and fail to provide a complete description of all pathways. For this reason it is important to have mathematical models that allow us to identify and analyze metabolic pathways in a computational fashion. This is precisely the main theme of this thesis. We firstly describe, review and discuss existent mathematical/computational approaches to metabolic pathways, namely stoichiometric and path finding approaches. Then, we present our initial mathematical model named the Beasley-Planes (BP) model, which significantly improves on previous stoichiometric approaches. We also illustrate a successful application of the BP model to optimally disrupt metabolic pathways. The main drawback of the BP model is that it needs as input extra pathway knowledge. This is especially inappropriate if we wish to detect unknown metabolic pathways. As opposed to the BP model and stoichoimetric approaches, this issue is not found in path finding approaches. For this reason a novel path finding approach is built and examined in detail. This analysis serves us as inspiration to build the Improved Beasley-Planes (IBP) model. The IBP model incorporates elements of both stoichometric and path finding approaches. Though somewhat less accurate than the BP model, the IBP model solves the issue of extra pathway knowledge. Our research clearly demonstrates that there is a significant chance of developing a mathematical optimisation model that underlies many/all metabolic pathways.
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Goel, Gautam. "Dynamic flux estimation a novel framework for metabolic pathway analysis /." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/31769.
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Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010.Committee Chair: Voit, Eberhard O.; Committee Member: Butera, Robert; Committee Member: Chen, Rachel; Committee Member: Kemp, Melissa; Committee Member: Neves, Ana Rute. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Hettne, Kristina. "Using nuclear receptor interactions as biomarkers for metabolic syndrome." Thesis, University of Skövde, Department of Computer Science, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-813.
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Metabolic syndrome is taking epidemic proportions, especially in developed countries. Each risk factor component of the syndrome independently increases the risk of developing coronary artery disease. The risk factors are obesity, dyslipidemia, hypertension, diabetes type 2, insulin resistance, and microalbuminuria. Nuclear receptors is a family of receptors that has recently received a lot of attention due to their possible involvement in metabolic syndrome. Putting the receptors into context with their co-factors and ligands may reveal therapeutic targets not found by studying the receptors alone. Therefore, in this thesis, interactions between genes in nuclear receptor pathways were analysed with the goal of investigating if these interactions can supply leads to biomarkers for metabolic syndrome. Metabolic syndrome donor gene expression data from the BioExpressä, database was analysed with the APRIORI algorithm (Agrawal et al. 1993) for generating and mining association rules. No association rules were found to function as biomarkers for metabolic syndrome, but the resulting rules show that the data mining technique successfully found associations between genes in signaling pathways.
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Hailes, Anne Maria. "The microbial degradation of the morphine alkaloids." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321432.
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Zhang, Rui. "Metabolic Disorder leads to Retinal degeneration: Function, morphology and metabolic pathway analysis." Thesis, The University of Sydney, 2020. https://hdl.handle.net/2123/25459.
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INTRODUCTION
The retina prefers to metabolize glucose through glycolysis rather than oxidative phosphorylation (OXPHOS) to meet their energy demand even when oxygen is abundant, known as the “Warburg effect”, despite having abundant functional mitochondria. We created transgenic mice with selective knockdowns of key enzymes in glycolysis and OXPHOS including hexokinase (HK) 2, lactate dehydrogenase (LDH) A and pyruvate dehydrogenase (PDH) E1α in rods to study the importance of glycolysis and OXPHOS in retinal metabolism and the role of metabolic derangement in the pathogenesis of retinal diseases.
MATERIALS AND METHODS
Immunohistochemistry and Western blots were performed to study the changes in protein expression and regulation. Scotopic electroretinography and optical coherence tomography were performed to study the retinal function and structure. Gas and liquid chromatography-mass spectrometry were employed to study the changes in13C-glucose-derived metabolites in the retina.
RESULTS
Knockdown of HK2 in rods led to photoreceptor degeneration, with reduction in the thickness of the retina and impaired retinal function. Knockdown of HK2 decreased pyruvate production but promoted the tricarboxylic acid (TCA) cycle in the retina.
Knockdown of LDHA and PDHE1α in rods also led to retinal degeneration, with thinning of the retina and impaired retinal function. Deletion of LDHA and PDHE1α suppressed glycolysis and the TCA cycle in the retina.
CONCLUSION
We found that HK2/LDHA-mediated glycolysis and PDHE1α mediated-OXPHOS in rods are indispensable for the maintenance of photoreceptor structure and function. Disturbance in aerobic glycolysis or OXPHOS leads to metabolic remodelling in the retina and photoreceptor degeneration.
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Homan, Edwin. "Discovery of Novel Lipid Pathways associated with the Metabolic Syndrome." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10343.
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The prevalence of obesity and type 2 diabetes has increased at alarming rates in recent decades. These diseases are prominent components of the metabolic syndrome, which is characterized by marked dyslipidemia. Adipose tissue contributes to the development of obesity-related diabetes through increased release of hormones and non-esterified fatty acids. The development of sensitive analytical tools for the broad detection of lipid biomolecules, such as liquid chromatographymass spectrometry (LC-MS), has spurred interest in the molecular determinants of the metabolic syndrome. The development of mature adipocytes from precursor fibroblasts—adipogenesis—plays a crucial role in the expansion of adipose tissue in obesity. We profiled differentiating 3T3-L1 pre-adipocytes by LC-MS and found that a class of monoglyceride lipids, monoalkylglycerol ethers (MAGEs), was transiently elevated early in adipogenesis. Upon addition to differentiating cells, MAGE specifically promoted adipocyte maturation and expression of adipogenic gene markers, indicating that MAGEs may be signaling molecules during adipogenesis. The insulin-sensitive glucose transporter, GLUT4, is downregulated during obesity and diabetes. In collaboration with Prof. Barbara Kahn, we studied a transgenic mouse model that overexpressed GLUT4 specifically in adipose tissue (AG4OX) and was protected from developing diabetes. We used LC-MS-based metabolomics to discover a previously undescribed class of bioactive lipids that was highly upregulated in AG4OX adipose tissue. We structurally characterized these lipids as fatty acyl hydroxy fatty acids (FAHFAs) and several positional isomers were chemically synthesized to confirm structural assignments via coelution studies. We discovered that individual FAHFAs, such as 5-palmitoyl-hydroxystearic acid (5-PAHSA), were differentially regulated by the transcription factor ChREBP. Circulating 5-PAHSA levels in mice and humans correlated with ChREBP expression and insulin resistance. In order to explore the biochemical regulation of FAHFAs, we developed an LCMS-based assay to measure FAHFA hydrolysis activity. We identified one enzyme, carboxyl ester lipase (CEL), as the major FAHFA hydrolase in pancreas, where the activity was highest. We confirmed its relevance in vivo by feeding labeled FAHFA to CEL inhibitor-treated mice. In this work we used LC-MS-based metabolomics to discover two lipids, MAGE and FAHFA, along with the CEL pathway, that may help us to better understand the pathogenesis of obesity and diabetes.Chemistry and Chemical Biology