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Proteomics Analysis of Protein-Producing Chinese Hamster Ovary Cells during Apoptosis in Prolonged CultivationWei, Yi-Yun 05 1900 (has links)
Among the factors important for maintaining productivity of recombinant proteins in mammalian cells, culture lifetime, cell density and cell viability are three simple but essential aspects that can greatly influence the final yield. During cell culturing, the degradation of environmental conditions such as nutrient depletion and accumulation of toxic waste products, often lead to premature apoptotic cell death in cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. The work presented in this thesis is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells.
Flow cytometry analyses of the level of activated executioner caspases, caspase 3 and 7, demonstrated the onset of apoptosis in CHO batch cultures after the exponential phase. The monitoring of apoptotic cell death assesses the culture conditions at different time points and allows the association of changes in the protein abundances to the degradation of culture conditions, in particular the degree of apoptosis in the whole cultures. The detection of activated caspase 8 and caspase 9, which trigger the extrinsic and intrinsic pathway respectively, has shown that the onset of apoptosis in CHO cells during prolonged cultivation predominantly is via the intrinsic pathway.
To examine the proteomic changes in a monoclonal antibody-producing CHO cell culture at various phases of a batch culturing process, a differential in gel electrophoresis proteomic approach was employed in this study. CHO protein samples at four time points during cultivation were compared. A total of 40 differentially expressed protein spots were successfully identified by mass spectrometry sequencing, resulting in 28 unique protein identifications. These proteins include four structural proteins of the cytoskeleton, ten endoplasmic reticulum and cytosolic chaperones and folding proteins, seven metabolic enzymes and seven other proteins of varied function. The cleavage of cytoskeletal proteins is a known consequence of apoptosis and the activation of executioner caspases. On the other hand, the induction of seven ER chaperone and foldases is a solid indication for the onset of unfolded protein response, which is triggered by cellular and ER stresses, many of which are present during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes a change in the energy metabolism likely occurred when culture conditions degraded and apoptosis advanced. Interestingly, although a significant portion of proteins identified are well known housekeeping proteins, recent studies have shown that many of them exhibit a wide variety of other roles, including apoptosis regulation and execution. Overall, this study shows that the most drastic changes in aging CHO cultures, where apoptosis is known to be part of, involve the onset of UPR and upregulation of proteins catalyzing glucose metabolism.
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Characterization of the metabolic and secretory behavior of suspended free and entrapped cell laden microcarriers in fed-batch culturesArchibald, Petra A. 08 1900 (has links)
No description available.
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Cytoadherence of Plasmodium falciparum- and Plasmodium fragile-infected erythrocytes to human endothelial cells under shear conditionsLouis, Valerie 08 1900 (has links)
No description available.
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The effect of an in vitro mechanical environment on the proliferation and phenotype of bladder smooth muscle cellsVittur, Shannon Marlece 08 1900 (has links)
No description available.
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Extracellular polysaccharide in cell cultures of bush bean (Phaseolus vulgaris cv. Contender)Mante, Seth D. January 1974 (has links)
No description available.
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Control and measurement of oxygen in microfluidic bioreactors.Nock, Volker Michael January 2009 (has links)
Bioartificial Liver (BAL) is a term for medical devices designed to replace natural liver functions. The idea behind the use of artificial livers is to either externally support an injured liver to recovery or bridge a patient with a failing liver to transplantation. Central to all BAL systems is a bioreactor for culturing liver cells. The main function of this reactor is to provide a cell adhesion matrix and supply the necessary nutrient solution. A high cellular oxygen uptake rate combined with low solubility in aqueous media makes oxygen supply to the liver cells the most constraining factor in current reactor designs. Devices with parallel-plate channel geometry promise high efficiency for blood detoxification and liver metabolism. However, due to their specific flow regime oxygen depletion in the medium is a major problem in these devices.
This thesis explores a unique method of controlling and measuring dissolved oxygen in BAL cell-culture bioreactors and lab-on-a-chip devices. Testing is performed using simulations, prototype bioreactor devices and in-vitro measurement of dissolved oxygen. Several strategies developed to fabricate the bioreactors and integrate oxygen sensing are presented. Emphasis is placed on techniques that provide compatibility with commonly used microfabrication processes, while allowing for laterally-resolved measurement of oxygen in a re-usable, low-cost setup.
The most significant contribution presented is the development and assessment of the tapered cell-culture bioreactor with integrated PtOEPK/PS oxygen sensor. The combination adopts a unique approach to oxygen control. Bioreactor shape is used to modulate the oxygen supplied to cells via the resulting shear-stress function. By linearly increasing the shear-stress oxygen concentration can be maintained constant over the length of the reactor. Using the integrated oxygen sensor, the resulting concentration profile can be monitored in real-time with high lateral resolution. The advantage of the device over existing techniques is that no additional oxygenation inside the reactor chamber is required to maintain a certain concentration profile and that oxygen
concentration can be mapped in-situ without having to introduce further chemicals into the perfusion medium.
This thesis presents a number of other contributions: a grayscale mask process, development of the PtOEPK/PS sensor patterning method and signal optimization regime, demonstration of the multi-stream flow application, an experimental setup for sensor calibration and a process to pattern cell-adhesion proteins simultaneously with the oxygen sensor, a multi-layer BAL prototype and the results of a brief experiment to test an approach using vertically aligned carbon nanotube bundles as fluidic conduits for bile drainage.
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Knockdown of the ERK pathway using siRNA in cultured chicken cardiomyocytesOvrén, Caroline January 2014 (has links)
The ancient South American birds called tinamous (Tinamidae) have the smallest hearts known among birds and their cardiomyocytes have previously been shown to express significantly lower levels of the mitogen-activated protein kinase ERK compared to the more modern chicken (Gallus gallus). ERK is a well-known mediator of growth signalling in the heart, especially in hypertrophy. The aim of this project was to assess the effect of ERK knockdown on proliferation in cultured chicken cardiomyocytes. By transfecting these cells with a lipoplexed siRNA, ERK mRNA levels were knocked down to approximately half (45%, SD: 27%) compared to cells transfected with a negative control siRNA. The knockdown was coupled with a decreased proliferative response to insulin-like growth factor 1 (IGF-1) and foetal bovine serum (FBS). In conclusion, the ERK pathway was confirmed to be instrumental also in proliferative signalling. The results also support the notion that ERK itself is the rate-limiting step of this MAPK cascade. The low native expression of ERK in tinamou cardiomyocytes is expected to impose a strict limit on proliferative growth in response to various stimuli in these hearts. The genetic changes leading to higher expression levels, and with it the potential for larger hearts, in modern birds would have led to greatly increased evolutionary fitness by way of an increased aerobic scope and the ability to sustain flight.
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Studies on new tumour active compounds with one or more metal centresTayyem, Hasan Mohammad January 2006 (has links)
Doctor of Philosophy(PhD) / The present study deals with the synthesis, characterization, determination of anticancer activity of three mononuclear trans-planaraminepalladium(II) complexes code named TH5, TH6 and TH7 and three trinuclear complexes code named TH1, TH8 and TH14. The activity of the compounds against human cancer cell lines: A2780, A2780cisR and A2780ZD0473R, cell uptake, DNA-binding and nature of interaction with pBR322 plasmid DNA have been determined. Whereas cisplatin binds with DNA forming mainly intrastrand GG adduct that causes local bending of a DNA strand, TH5, TH6, TH7, TH1 and TH8 bind with DNA forming mainly interstrand GG adducts that causes more of a global change in DNA conformation. Although TH5, TH6 and TH7 each have two substituted pyridine ligands in a trans-geometry (3-hydroxypyridine in TH5, 2-hydroxypyridine in TH6 and 4-hydroxypyridine in TH7), the compounds differ in their activity against ovarian cancer cell lines, indicating that non-covalent interactions involving the hydroxyl group may be playing a significant role in activity of the compounds. Among trinuclear complexes TH1 is found to be significantly more active than cisplatin. It is actually twice as active as cisplatin against the parent cell line A2780, thirteen times as active as cisplatin against the cisplatin-resistant cell line A2780cisR and 11.5 times as active as cisplatin against the cell line A2780ZD0473R. Whereas the resistance factor for cisplatin as applied to the cell lines A2780 and A2780cisR cell lines is 12.9 that for TH1 is 1.98. The results suggest that TH1 has been able to significantly overcome resistance operating in A2780cisR cell line. The compound is soluble in water so that it may be taken orally. Provided it has favourable toxicity profile, TH1 has the potential to be developed into a highly active anticancer drug with a wider spectrum of activity than cisplatin. Although platinum drugs use a shot-gun approach to kill cancerous cells, widespread use in the clinic and increasing volume of their sale indicate that even in the genomic age, there is still need for shot-gun drugs in the clinic.
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A mechanistic analysis of mammalian cell metabolism in continuous culture /Dimasi, Don. January 1992 (has links)
Thesis (Ph.D.)--Tufts University, 1992. / Submitted to the Dept. of Chemical Engineering. Adviser: Randall W. Swartz. Includes bibliographical references (leaves 243-247). Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Optimization of cell culture procedures for growing neural networks on microelectrode arraysSanta Maria, Cara L. Gross, Guenter W., January 2007 (has links)
Thesis (M.S.)--University of North Texas, Dec., 2007. / Title from title page display. Includes bibliographical references.
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