Browsing by Subject "metabolism"

Now showing 1 - 7 of 7
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    Advancing Cell Culture Engineering Through Mechanistic Model Optimization
    (2020-04) O'Brien, Conor
    Over the past few decades, the emergence of new classes of treatments, including protein therapeutics, gene therapies, and cell therapies, has ushered in a new era of medicine. Unlike small molecule therapeutics, these treatments are produced in or consist of cells, typically mammalian in origin. Processes have been developed to produce many of these drugs at large scale, often in stirred tank bioreactors. Significant effort has driven staggering increases in the productivity of these processes, enabling economical manufacturing, and the potential to drive down costs and make drugs more widely available. However, the bioreactor is not a natural environment for cells isolated from a multicellular mammalian organism. Many biological regulations are carried over from the cells’ origin and can result in numerous undesirable behaviors manifesting in the dense, highly productive reactor environment. In certain culture stages, or in the case of excess nutrient supply, cells will secrete undesirable metabolites including lactate, ammonia, and many byproducts of amino acid metabolism. These compounds can retard cell growth, or otherwise alter the potency or productivity of the cultures. Traditional biologics process development employs the use of statistical design of experiments, often encompassing several reactors run in parallel for multiple rounds of experiments over a few months. There is thus substantial room for improvement for both the outcome of the development process, such as an increase in titer, and the time it takes to complete the development stage. Given that cell culture processes share intrinsic similarities in their underlying mechanistic behavior, there exists significant opportunity to reduce the overall number of experiments needed for process development, scaling, and diagnostics using models rather than treating cell culture processes as a black box. In this thesis, we present the case for the use of mathematical optimization of mechanistic models to accurately describe cell culture processes and augment their behavior. We first outline recent advances in understanding of metabolic regulation and homeostasis. Cell signaling and metabolic networks interact over multiple time-scales and through multiple means, resulting in cell metabolism with nonlinear behavior that is consequently context-dependent. In the following sections of this work, we then develop an optimization framework which can efficiently be used for the design of experiments to rewire cellular metabolism through metabolic engineering, or to otherwise understand the biological requirements of different metabolic phenomena. This framework is first applied to the Warburg effect, a century-old unsolved problem of rapid lactate production in proliferating cells to identify which enzymes may be altered to mitigate the lactate production. This framework in then applied to the problem of hepatic gluconeogenesis to study metabolic disease. As the expression of the enzymes specific to gluconeogenesis is not sufficient for glucose production, we explore what other requirements exist for the synthesis of glucose from different substrates. The next portion discusses the construction and optimization of a bioprocess model which includes metabolism, signaling, cell growth, and the reactor environment. This model is fit to a manufacturing-scale dataset to explore the origins of process variability and potential mitigation strategies. In the final segment of this thesis, we explore another aspect of protein therapeutics: product quality. A model of N-glycosylation is optimized in conjunction with successive rounds of experimentation with the goal of improving the galactose content on an antibody. This work highlights the benefits of feeding back experimental data to refine model parameters for better design and prediction of subsequent experiments.
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    Effects of Peripartum Recombinant Bovine Somatotropin Treatment and Prepartum Stocking Density on Immune Responses, Metabolism, Health, and Performance of Dairy Cows
    (2016-10) Basso Silva, Paula Regina
    Excessive fatty acids release from the adipose tissue of periparturient cows may result in hyperketonemia and hepatic lipidosis, which are known to compromise liver and damage immune cells. Immunosuppression in transition cows is a result of shortage in energy, nutrients and calcium impairing immune cells’ metabolism. Thereafter, immunosuppressed periparturient cows are at higher risks for developing infectious and non-infectious health disorders. Strategies that improve metabolism and immune function of periparturient dairy cows may reduce incidence of diseases. Six experiments were performed to test two main strategies: the use of recombinant bovine somatotropin (rbST) during the peripartum period and the reduction of prepartum pens’ stocking density (SD) from 100 to 80% of headlocks. The specific objectives of these experiments were to evaluate the effects of treating peripartum dairy cows with rbST on immune, inflammatory, and metabolic responses, incidences of postpartum diseases, performance, and hepatic and leukocyte gene expression; and to evaluate the effects of two prepartum SD (80 vs. 100%) on milk yield, concentration of metabolites, health and reproductive parameters, innate and adaptive leukocyte responses, and serum and hair cortisol concentrations. Results demonstrated that treatment of dairy cows with 125 mg of rbST improved innate immune responses and IgG concentration, with limited effects on metabolism, decreased the incidence of uterine disorders in Holstein and Jersey cows and increased yield of energy corrected milk during the first 30 DIM in Holstein cows. Administration of rbST during the periparturient period may improve liver function and health by increasing hepatic capacity for gluconeogenesis and lipid transport and by reducing inflammation and oxidative stress. Treatment of dairy cows with 125 mg of rbST during the periparturient period may improve leukocyte functions by upregulating mRNA expression of genes involved in glycolysis, pathogen recognition, phagocytosis and oxidative burst, antimicrobial peptides, and antibody production. Finally, in herds with weekly or twice weekly movement of new cows to the prepartum pen and separate housing of nulliparous and parous animals, 100% SD of headlocks on the day of movement does not affect health, metabolic, reproductive, and productive parameters and 80% did not improve leukocyte responses compared with 100% target SD.
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    Exploring the Universal Drivers of Glycosome Evolution in Kinetoplastea
    (2024-06) Durrani, Hina
    Subcellular compartmentalization is a defining feature of eukaryotic cells, an organizational complexity that traces back to the earliest common ancestor of all present-day eukaryotes. Understanding various organelles’ origins and early evolutionary stages remains a significant challenge. Organellar proteomes are shaped by controlled pathways that guide cytosol-produced proteins to their designated subcellular locales. The specific routing of these proteins can differ across varying physiological states, cell types, and evolutionary lineages. In eukaryotes, evolutionary retargeting—the process where the subcellular destination of a protein changes through evolutionary time—has been widespread and can involve any combination of organelles, complicating efforts to trace back and reconstruct the evolutionary history of organelles. This dissertation investigates the evolution of glycosomes, peroxisome-related organelles in kinetoplastids and diplonemids. Chapter 1 introduces the key concepts of kinetoplastids, glycosomes, and the test organisms used throughout the dissertation. Chapter 2 conducts an in-silico comparison of proteomic data from available organisms and transcriptomic data from other kinetoplastids, using glycosomal targeting sequences. Chapters 3 and 4 explore the factors driving protein compartmentalization within glycosomes, examining the effects of glucose and nutrient replenishment in Chapter 3, and hypoxia in Chapter 4. Chapter 5 presents a detailed study of PIP39, a glycosomally localized protein phosphatase in Leptomonas seymouri, highlighting its role in the oxidative stress response. The dissertation concludes with a summary of the major findings, a discussion of the limitations, and future research directions. This work significantly advances our understanding of metabolic pathway compartmentalization and elucidates specific molecular mechanisms that sustain enzymatic pathways within glycosomes. Furthermore, it establishes the glycosome as a uniquely defined and pedagogically useful system to demonstrate how selection influences the interplay between existing organizational structures and environmental stimuli, fostering adaptive evolution.
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    Gluconeogenic FBP1 plays a key metabolic role in activated T cells
    (2017-07) Bapat, Aditi
    Fructose bisphosphatase-1 (FBP1) is a rate limiting enzyme in gluconeogenesis that converts fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P). It is active in liver, kidney and skeletal muscle cells. This study suggests that FBP1 plays a novel non-gluconeogenic role in T cells. Targeted metabolomics using [13C]-6-glucose revealed a labeling pattern of F6P in stimulated CD3+ T cells that could only have resulted from FBP1 enzymatic activity. Following stimulation, T cells expressed a 27kD form of FBP1, in addition to the full-length 37kD protein. The hypothesis that T cells utilize an alternative translational start site to express a shorter, constitutively active, form is being tested. Future studies will also test the hypothesis that FBP1 activity increases carbon flux into the pentose phosphate pathway (PPP), to facilitate increased production of reducing agent and co-factor, NADPH, in preparation for proliferation. This research could contribute significantly to our understanding of T cell physiology and cancer cell metabolism.
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    Metabolic-response assessment of metastatic murine breast cancer in 2D and 3D cultures using intrinsic NADH as a natural biomarker
    (2019-08) Cong, Anh
    The majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture towards pathophysiological investigation, drug screenings, and cancer diagnostics. There is strong evidence, however, that suggests cellular behavior and metabolism in 2D cell culture is too simplistic of a model as compared with those in vivo tumor cells. In this project, we hypothesize that cancer cell metabolism and metabolic responses to external stimuli (e.g. drug treatments) are distinctly different in threedimensional (3D), tumor-like model as compared with that of the conventional 2D monolayer culture. To test this hypothesis, we employed two-photon (2P) fluorescence lifetime imaging microscopy (2P-FLIM) and time-resolved 2P-fluorescence anisotropy of the reduced nicotinamide adenine dinucleotide (NADH) in metastatic murine breast cancer cells 4T1. In addition, we investigated the cellular metabolic response of 4T1 cells in 2D monolayer and 3D collagen matrix cultures to drug treatment using two novel metabolic drugs, namely MD1 and TPPBr. Both 2P-FLIM and complementary time-resolved anisotropy approaches reveal significant differences between metabolic activities of 4T1 cells in 2D and 3D cultures. Our results suggest that these 4T1 cells in 3D culture adapt an oxidative shift but glycolysis dominances the metabolic state of 2D cells. In addition, 4T1 cells in 3D culture appear to adapt more quickly and exhibit enhanced metabolic activities in response to drug treatment. In contrast, 4T1 cells in 2D monolayer culture exhibit a mute response and are less sensitive to drug treatments. While the tumor-like 3D collagen matrix model may not be an exact replica of in vivo tumors, these studies represent a critical step towards the development of a fundamental understanding of cellular behaviors and metabolism in the more complex in vivo models. These studies would also help advance our understanding of how the cancer cell heterogeneity and microenvironmental conditions impact metabolism and metabolic plasticity in tumor growth and metastatic progression.
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    Regulation of Body Size by TGF-β Signaling
    (2018-09) Moss-Taylor, Lindsay
    Nearly all life history traits scale with body size, imparting incredible importance on control of growth and size during animal development. Nutrition, genetic and environmental inputs influence the growth rate during development to determine final body size. These inputs are processed by the insulin/insulin-like growth factor signaling pathway (IIS), which is the major regulator of growth, and other pathways, like TGF-β, which modulate tissue growth or IIS. Mutations in the gene coding for the Drosophila TGF-β ligand Activinβ (Actβ) cause reduced final body size and accelerated growth termination. Using the Gal4/UAS system, I show Actβ is expressed in distinct cell types in the nervous system. Using rescue experiments, I show the Actβ phenotypes can be rescued by overexpression of Actβ in some, but not all, of the cell types in which it is endogenously expressed. Additionally, the growth rate of Actβ mutants is reduced, demonstrating the size phenotype is not simply due to early growth termination from precocious timing. Muscle-specific knockdown of the TGF-β signaling transducer/transcription factor dSmad2 also reduces body size, identifying muscle is a target tissue of the Actβ signal. The change in body size is due to a reduction in the size of skeletal muscles, not a systemic reduction in size. Autophagy markers are upregulated in Actβ mutants but, surprisingly, overexpression of autophagy regulators does not rescue the Actβ size phenotype, indicating Actβ regulation of autophagy is TOR-independent. These results provide new insights into mechanisms of body size and muscle size control during animal development. This thesis details the functions of Actβ in the regulation of body size and developmental timing. Chapter 2 describes the study of how Actβ controls body size and developmental timing. Chapter 3 investigates the roles of Actβ in regulating metabolism and IIS.
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    Systems Analysis In Mammalian Cell Biomanufacturing - Linking Energy Metabolism And Glycosylation
    (2018-05) Le, Tung
    Mammalian cells have been the major workhorse to produce therapeutic proteins owing to their capability to perform complex post-translation modifications that are essential to the pharmacological activities of these proteins. The performance of mammalian cell culture is greatly affected by cell metabolism, while the robustness of glycosylation patterns of the product proteins still needs improvement. A meta-analysis of cell culture bioprocess data revealed a correlation between lactate metabolism, productivity, and glycosylation patterns. In this study, we develop an integrated platform for generation and visualization of the O-glycosylation network. The platform was used to explore the heterogeneity of O-glycans produced in mammalian cells. In addition, we use a kinetic model of energy metabolism to explain the mechanism behind the increase in lactate production during the late stage of fed-batch cultures. Such a metabolic behavior of lactate can have negative impacts on the culture productivity and potentially, product quality because it occurs during the main production phase. Insights from this study can be helpful in contriving strategies for more robust control of cell metabolism and obtaining consistent glycan patterns of biologics.