Browsing by Subject "Bistability"

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    Analysis of central metabolic pathways in cultured mammalian cells
    (2014-10) Yongky, Andrew
    Recombinant therapeutic proteins have transformed the field of medicine since their advent more than twenty years ago, providing treatments for various refractory illnesses. Mammalian cells are the preferred hosts for the production of these therapeutics. However, a lack of understanding of the behavior of the cells results in numerous issues that affect the performance of process cultures including inefficient glucose metabolism and improper post-translational modification of the product proteins. With the advances in the knowledge of regulation of metabolism of mammalian cells and the availability of genomics and transcriptomics resources, systems biology approach combining kinetic model and "-omics" tools can now be used to address such issues. The work presented in this thesis employs such systems biology approach to better understand the metabolic behavior of the cells and devise strategies to enhance their performance in culture. Cultured mammalian cells consume huge amount of glucose and convert most of it towards lactate. The accumulation of lactate in culture adversely affects cell growth and productivity. The reliance of these cells on anaerobic glycolysis is also observed in proliferating cells such as cancer cells and embryonic stem cells. In contrast, quiescent cells metabolize glucose at slower rate and glucose is mostly oxidized to carbon dioxide. In the first part of this thesis, we attempt to unravel the regulation of glucose metabolism in proliferating and non-proliferating cells using a mechanistic model of glycolysis. We show that multiple allosteric regulations of glycolysis enzymes can act in synergy to confer bistable behavior to glycolysis activity: at a given glucose concentration, glycolysis can operate at either a high flux state or a low flux state. In proliferating cells, the default state of the cells is to operate at high glycolysis flux. However, it is possible to modulate factors extrinsic and intrinsic to the cells in order to make them switch to low flux state.In the late stages of fed-batch culture, mammalian cells have been observed to shift their metabolism from lactate production to lactate consumption. While it has been correlated with higher productivity, such metabolic shift is not a consistent occurrence as some cultures continue to produce lactate. The metabolic model is used to explain the underlying mechanism behind the metabolic shift to lactate consumption in fed-batch culture. We show that the bistable behavior in glycolysis differs somewhat due to lactate inhibition and growth rate regulation on metabolism. As a result, the cells in culture may or may not shift their metabolism to consume lactate depending on the glucose, lactate and growth rate of the cells. In continuous culture, a similar metabolic behavior has been observed. With the same operating conditions of dilution rate and feed glucose concentration, some continuous cultures reach steady state with high glycolysis flux, while others reach steady state with low glycolysis. The two steady states are marked by distinct steady state cell concentrations. Using a multi-scale reactor model that combines the intracellular metabolism and macroscopic cell growth, we show that multiple steady states exist in continuous culture. At high flux steady state the vast majority of glucose is converted to lactate, whereas at low flux steady state most of the glucose consumed is converted to biomass. The two types of steady states thus have different metabolic efficiency, conferring different cell concentrations.In the final part of the thesis, RNA-seq and microarray are employed to survey the variability in CHO cell lines. We observe a wide range of transcript levels of glycolysis enzymes in CHO cell lines, potentially contributing to distinct metabolic characteristics in different cell lines. The extent of genetic variation in the protein coding regions of the growth signaling pathway genes in CHO cells are discussed.
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    A bistable genetic switch controls antibiotic resistance transfer in Enterococcus faecalis.
    (2011-08) Chatterjee, Anushree
    The recent rise in microbial drug resistance is a growing challenge for future therapy of bacterial infections. Increased prevalence of antibiotic resistance in bacteria is an outcome of evolution via natural selection. However, the built-in design feature of bacteria to transfer DNA containing antibiotic resistance both within the same species and across species is the main culprit for the spread of drug resistance. One of the main factors driving the rise of drug resistant microbes is the transfer of antibiotic resistance genes present on mobile plasmids between donor and recipient cells via the mechanism of conjugation. In order to combat microbial drug resistance, novel strategies need to be developed to block such transmission of antibiotic resistance. In this work, the gene regulatory components involved in transfer of tetracycline resistance confers plasmid pCF10 between plasmid-carrying donor cells and plasmid-deficient recipient cells in bacterium Enterococcus faecalis is investigated. In the native state the donor cell exists in an OFF or conjugation-incompetent state. A pheromone released by the recipient cells serves as the chemical trigger for switching the donor cell from OFF to an ON or conjugation-competent state. The onset of conjugation is tightly regulated via multi-layered regulation offered by two-key genes prgQ and prgX present on pCF10 in response to the pheromone secreted by recipient cells. Using mathematical modeling and experimentation, we describe a novel mechanism of gene-regulation due to transcriptional interference and sense-antisense RNA interaction as a result of convergent transcription in the prgX/prgQ operon. We demonstrate that such a multi-layered gene-regulatory mechanism confers the system a bistable genetic switch controlling conjugative gene transfer between donor and recipient cells. A similar regulatory advantage offered by convergent transcription in attributing a bistable switch-like behavior in the scbA-scbR operon controlling antibiotic production in S.coelicolor is also investigated. Both mathematical model and experiments demonstrate that donor cells also control response to pheromone by changing the number of copies of pCF10 plasmid inside the cell. Cells with higher copies show increased robustness of the bistable switch and lower sensitivity to pheromone. Once bistable genetic-switch is ON, expression of genes encoding various proteins involved in the transfer of the plasmid are induced, however, this also causes production of an inhibitor of conjugation, thus giving rise to negative feedback loop which causes the donor to return to OFF state. Modeling and experimental analysis of dynamic response to induction indicate that this negative feed-back loop causes a brief surge of expression of the entire operon. We show that the inhibitor signaling peptide for pCF10 based system, acts as quorum-sensing signal with the role of turning-OFF conjugation at a population-wide scale. An interplay of positive and negative feedback loops allows the donor cell to quickly transition between ON and OFF states and is critical both for the transfer of plasmid and survival of the cell. Studying both the turning-ON and turning-OFF mechanisms of the switch allows identification of potential drug targets for blocking transmission of antibiotic resistance for use in future therapy.