No description available.

No description available.

No description available.

The profile of the inner-tubing gas pressure for a tubular membrane aeration system was quantified. The correlations among the overall volumetric oxygen transfer coefficient (k[subscript L]a), the inner-tubing pressure, the tubing tightness, and the gas throughput are experimentally analyzed. A mathematical model was developed to describe the underlying phenomena. The results established the base for comparison with other aeration techniques. A novel method employing in situ laser imaging technology to monitor bubbles and cells, and analyze bubble size distributions in a micro-sparged bioreactor was developed. The effects of bioreactor operations on bubble size distributions were determined with following results: 1) Spargers with larger pores produced larger bubbles in most cases 2) Higher sparging rates resulted in bubble size increases up to 10% 3) Pluronic F68 shrank bubbles up to 30%. When the concentration of Pluronic F68 exceeded 1 g/L, no additional impact was observed. 4) Emulsion silicone antifoam up to 25 ppm had no impact on bubbles 5) Cell density (up to 22x10��� cells/mL) or culture age has no effect on bubble sizes In multiple 1 5-L long-term high-density cultures of animal cells, the correlations between sparging rate and cell damage for using 0.5 ��m and 15 ��m-pore spargers were quantified. At cell density of 2x10��� cells/mL, sparging above 0.025 vvm using the 0.5-��m sparger was detrimental to cells, while 0.054 vvm was detrimental for the 15-��m sparger. A model was developed to predict the rate of cell death resulted from cell-bubble interactions for high-density industrial animal cell cultures. The effect of high superficial velocity of sparging gas on cells at the sparger surface proved insignificant. A new dissolved CO��� sensor proved to be reliable for long-term use in industrial perfusion cell cultures. A novel method for the control of dissolved CO��� while simultaneously maintaining DO��� and pH setpoints was developed. The continuous control of dissolved CO���, DO��� and pH is achieved by simultaneously adjusting the total sparging rate as well as the ratio of O���, N��� and CO��� gas contents. This control strategy enables optimization of dissolved CO��� in industrial culture processes and allows for improved cell growth and protein production. / Graduation date: 2002

Alzheimer’s disease is linked to the formation of amyloid fibrils, which are primarily composed of two Alzheimer amyloid peptides, Abeta40 and Abeta42. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques. Current detection methods utilize dyes or antibodies that bind specific conformations of the peptides. However, these observation tools are limited, as they fail to detect the whole ensemble of pre-amyloid structural conformations. We adopted the approach of covalently attaching a fluorescent molecule to the N-terminus of Abeta, which allows for following the aggregation profile and examination of the association of Abeta with neuronal cells lines in real time. With the aid of confocal microscopy and flow cytometry, the rate of Abeta association to neuronal cell lines was found to correlate directly with their aggregation propensities. A non-aggregating mutant of Abeta42 did not bind to live cells and none of these peptides were found to associate with a non-neuronal human lymphoma cell line, U937, which is resistant to Abeta toxicity. Aggregation of Abeta42 on the surface of cells was characterized over time using photobleaching Forster resonance energy transfer, fluorescence quenching, and photobleaching recovery. Furthermore, exposed regions of Abeta aggregates on the cell surface were identified with sequence-specific antibodies. Two populations of aggregates were revealed; the first population displayed reduced energy transfer, showed fluorescence quenching, and bound antibodies specific for the C-terminal of Abeta, whereas the second population of aggregates was capable of energy transfer, was resistant to quenching, and increased in number over time. Interestingly, neither population of aggregates displayed photobleaching recovery. Addition of Abeta monomers to neuronal cells lead to the formation of cell surface aggregates that were eventually internalized into endosomes and lysosomes. The rate of internalization was greatly enhanced when the peptides were partially aggregated by exposure to conditions similar to the lumen of endosomes. Highly aggregated Abeta did not bind to neuronal cells. These studies have mapped out the aggregation pathway of Abeta in the physiologically relevant milieu of neuronal cells in culture, and have potentially revealed information about the formation of the pathological hallmark of Alzheimer’s disease, the senile plaque.

Graduation date: 1992

Alzheimer’s disease is linked to the formation of amyloid fibrils, which are primarily composed of two Alzheimer amyloid peptides, Abeta40 and Abeta42. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques. Current detection methods utilize dyes or antibodies that bind specific conformations of the peptides. However, these observation tools are limited, as they fail to detect the whole ensemble of pre-amyloid structural conformations. We adopted the approach of covalently attaching a fluorescent molecule to the N-terminus of Abeta, which allows for following the aggregation profile and examination of the association of Abeta with neuronal cells lines in real time. With the aid of confocal microscopy and flow cytometry, the rate of Abeta association to neuronal cell lines was found to correlate directly with their aggregation propensities. A non-aggregating mutant of Abeta42 did not bind to live cells and none of these peptides were found to associate with a non-neuronal human lymphoma cell line, U937, which is resistant to Abeta toxicity. Aggregation of Abeta42 on the surface of cells was characterized over time using photobleaching Forster resonance energy transfer, fluorescence quenching, and photobleaching recovery. Furthermore, exposed regions of Abeta aggregates on the cell surface were identified with sequence-specific antibodies. Two populations of aggregates were revealed; the first population displayed reduced energy transfer, showed fluorescence quenching, and bound antibodies specific for the C-terminal of Abeta, whereas the second population of aggregates was capable of energy transfer, was resistant to quenching, and increased in number over time. Interestingly, neither population of aggregates displayed photobleaching recovery. Addition of Abeta monomers to neuronal cells lead to the formation of cell surface aggregates that were eventually internalized into endosomes and lysosomes. The rate of internalization was greatly enhanced when the peptides were partially aggregated by exposure to conditions similar to the lumen of endosomes. Highly aggregated Abeta did not bind to neuronal cells. These studies have mapped out the aggregation pathway of Abeta in the physiologically relevant milieu of neuronal cells in culture, and have potentially revealed information about the formation of the pathological hallmark of Alzheimer’s disease, the senile plaque.

In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispensable for the proper function of the epithelial tissue. In order to understand the way that the epithelial cells form highly specialized structure, an in vitro three-dimensional (3D) culture system was established. The Caco-2 cells were embedded in reconstituted basement membrane termed matrigel, whose biochemical constitution and physical properties were similar with the in vivo environment. The Caco-2 cells in matrigel spontaneously formed spherical multi-cell cysts, which could continuously expand. The confocal imaging and reconstruction technique helped understand the cyst structure and its formation process. The cysts developed central lumen surrounded by a layer of polarized cells. The apical domain of the cells faced the lumen, while the basal domain attached to the extracellular matrix. In the mature cysts, fluid was secreted by the cells around the lumen at the apical domain, and accumulated in the central lumen. The laser burning experiment showed that the intraluminal pressure was higher than the outer environment. The intact cell sheet was required to keep the engorged morphology of the cysts. The tension of the cell layer balanced with the intraluminal pressure. To investigate the effect of pressure on cyst development, the cysts were treated with cholera toxin, which could increase intraluminal pressure through promoting apical secretion. The time-lapse images showed that under cholera toxin treatment, the expansion of the cysts was accelerated. The high intraluminal pressure led to shape change of thecells, followed by increase in cell proliferation rate. Cholera toxin itself could not promote cell growth. In the3D cultured cysts, it was the increased intraluminal pressure that directly induced the acceleration of cell proliferation. It indicated that not only biochemical signals, but also mechanical force, contributed to epithelial morphogenesis. The mechanical stimulation could be converted into biochemical signals, further affect cell behavior. In response to mechanical stimulation, the focal adhesion kinase was activated in the cells around the cyst lumen. Furthermore, the microarray analysis suggested that multiple signaling pathways were altered under intraluminal pressure stimulation, including the pathways related to cytoskeleton organization, cell cycle and cell adhesion. Taken together, comparing with the conventional two-dimensional cell culture on rigid surface, the three-dimensional culture system provided the cells a more physiological environment. The 3D culture system allows the epithelial cells to form well-organized hollow structure. It is a convenient model for investigating the process and mechanism of epithelial morphogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Microscope optics have long been used to observe biological samples, when used in conjunction with a laser light source, they can also be a powerful means to probe and manipulate cells. This dissertation describes the development of methodologies for laser-based microfabrication in biological environments. These techniques use pulsed laser light at a wavelength transparent to the experimental medium except in a region with submicron dimensions defined by the focus of a high numerical aperture objective. At the focal region, high intensity light can modify sample material. The localized nature of this energetic event allows it to be accomplished in the vicinity of living cells, enabling microfabrication strategies that are used to probe and modify extracellular environments with high resolution. The basic principles of this process are explored and its use in several applications for cell culture manipulation are described. In one methodology the focused laser induces physical and chemical events that lead to the formation of a micron-scale solid from a precursor protein solution. By translating the relative position of the beam in the solution, arbitrary three-dimensional structures can be formed. The use of protein microstructures as a platform for probing and manipulating cellular microenvironments is investigated and an advanced method of rapidly patterning elaborate structures with a spatial light modulator is demonstrated. The high intensity laser focus is used in a second strategy to create microfluidic conduits in a device consisting of two stacked flow channels, one containing adherent cells in buffer and the other a reagent solution. With laser ablation of a pore, highly defined reagent plumes are directed into the cell-containing chamber where they can dose multiple specifically targeted regions with subcellular specificity. The unique microfabrication technologies described in this dissertation could prove to be of use for researchers developing diagnostic and therapeutic devices and could lead to more advanced tools for studying the basic biology of cells.

No description available.