Prolonged exposure of humans and experimental animals to microgravity is known to be associated with a variety of physiological and cellular disturbances. With advancements in aerospace technology and prolonged space flights, both organism and cellular level understanding of the effects of microgravity on cells will become increasingly important in order to ensure the safety of prolonged space travel. To understand these effects at the cellular level, on-line sensor technology for the measurement and control of cell culture processes is required. To do this measurement, multiple sensors must be implemented to monitor various parameters of the cell culture medium. The model analytes used in this study were pH and dissolved oxygen which have physiological importance in a bioreactor environment. In most bioprocesses, pH and dissolved oxygen need to be monitored and controlled to maintain ionic strength and avoid hypoxia or hyperoxia. Current techniques used to monitor the value of these parameters within cell culture media are invasive and cannot be used to make on-line measurements in a closed-loop system. In this research, a microfabricated hydrogel microarray sensor was developed to monitor each anlyte. Either a pH or an oxygen sensitive fluorescent agent was immobilized into a hydrogel structure via a soft lithography technique and the intensity image of the sensor varied from the target analyte concentration. A compact detection system was developed to quantify concentration of each analyte based on the fluorescence image of the sensor. The system included a blue LED as an illumination source, coupling optics, interference filters and a compact moisture resistant CCD camera. Various tests were performed for the sensor (sensitivity, reversibility, and temporal/spatial uniformity) and the detection system (temporal/spatial stability for the light source and the detector). The detection system and the sensor were tested with a buffer solution and cell culture media off-line. The standard error of prediction for oxygen and pH detection was 0.7% and 0.1, respectively, and comparable to that of commercial probes, well within the range necessary for cell culture monitoring. Lastly, the system was coupled to a bioreactor and tested over 2 weeks. The sensitivity and stability of the system was affordable to monitor pH and dissolved oxygen and shows potential to be used for monitoring those analytes in cell culture media noninvasively.

Fluorescence enhanced optical imaging and tomography studies involve the detection of weak fluorescent signals emanating from nano- to picomolar concentrations of exogenous or endogenously produced fluorophore concurrent with the rejection of an overwhelmingly large component of backscattered excitation light. The elimination of the back-reflected excitation light of the collected signal remains a major and often unrecognized challenge for further reducing the noise floor and increasing sensitivity of small animal fluorescence imaging. In this dissertation, we adapted collimating and gradient index (GRIN) lenses in an existing frequency-domain system to improve excitation light rejection and enhance planar and tomographic imaging. To achieve this goal, we developed planar and tomographic imaging systems based upon ray tracing calculations for improved rejection of excitation light. The “out-of-band (S (λx))” to “in-band (S (λm) - S (λx))” signal ratio assessing excitation leakage was acquired with and without collimating optics. The addition of collimating optics resulted in a 51 to 75% reduction in the transmission ratio of (S (λx))/ (S (λm) - S (λx)) for the phantom studies and an increase of target to background ratio (TBR) from 11% to 31% in animal studies. Additionally, we presented results demonstrating the improvement of model match between experiments and forward simulation models by adaptation of GRIN lens optics to a breast phantom study. In particular, 128 GRIN lenses on the fiber bundle face were employed to align the collected excitation and emission light normal to the filter surface in an existing frequency-domain system. As a result of GRIN lens collimation, we reduced the transmission ratio between 10 and 86 % and improved the model match for tomographic reconstruction of one (1 cm3) and two (0.1 cm3) targets in a 1087 cm3 of breast phantom. Ultimately, this work improves the sensitivity of NIR fluorescence imaging by enhancing the rejection of excitation light and shows that the current sensitivity challenges for translating fluorescence-enhanced optical imaging into the clinic can be overcome.

Perfringolysin O (PFO), a cytolytic toxin from by the pathogenic bacterium Clostridium perfringens, perforates mammalian cell membranes by forming large aqueous pores. Secreted as water-soluble monomers, the toxin molecules bind to cholesterol-containing membranes, form large, circular oligomeric complexes on the membrane surface and then insert into the bilayer to create pores with diameters near 300 à . Using multiple independent fluorescence techniques as primary tools, the mechanisms of PFO membrane binding and oligomerization have been identified. Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition. Remarkably, only the short hydrophobic loops at the tip of the D4 β-sandwich are exposed to the bilayer interior, while the remainder of D4 projects from the membrane surface. Thus, a very limited interaction of D4 with the bilayer core appears to be sufficient to accomplish cholesterol recognition and initial PFO binding to the membrane. Upon PFO membrane binding, a structural element in domain 3 (D3) of the molecule moves to expose the edge of a previously-hidden β-strand that forms the monomer-monomer interface. The β-strands that form the interface each contain a single aromatic residue, and these aromatics appear to stack to align the transmembrane β-hairpins of adjacent monomers in the proper register for insertion. Membrane-dependent structural rearrangements are thus required to initiate and regulate PFO oligomerization. Fluorescence resonance energy transfer measurements reveal that the elongated toxin monomer arrives at the membrane in an ‘end-on’ orientation, with its long axis oriented nearly perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form a prepore complex. In particular, the D3 polypeptide segments that form the transmembrane β-hairpins remain far above the membrane surface both at the membrane-bound monomer and prepore stages of pore formation. However, upon pore formation, the height of the oligomeric complex above the membrane surface is significantly reduced. The major topographical changes that occur during the prepore-to-pore transition of the PFO oligomer, therefore appears to result primarily from a collapsing of the extended domain 2 (D2) conformation in the monomer.

The need for overnight and continuous monitoring of glucose levels in diabetic patients is profound, especially among juveniles. Implantation of a chemical assay which responds optically to changes in glucose concentration shows promise as a technology capable of continuously monitoring blood sugar with little invasion into the body. Previous fluorescent chemical assays, based on the affinity binding reaction between Concanavalin A protein and dextran, performed well but suffered from limited dermal penetration. In this work, a novel replacement for the dextran molecule (glycosylated dendrimer) was fabricated and tested to determine if it would improve the overall response of the sensing chemistry to glucose. Experiments were carried out and it was found that the assay’s functionality was based on the controlled aggregation of the Con A protein and the modified dendrimer molecule. This new assay proved to be specific to glucose, reversible, and independent of fluorophore dye attached to the protein. This research was furthered by encapsulation of the new assay into a PEG hydrogel which showed response to glucose but, due to leeching, did not perform well under repeated exposures. A new method for encapsulation was proposed based on poration of the hydrogel to create micropores capable of holding the assay chemistry and allowing it to react to incoming glucose, while the surrounding polymer restricted leeching. Preliminary results with previous assays proved the potential of a mannitol based poration procedure, but unforeseen complications in lyophilization of the new sensor assay restricted its completion. Due to instability of Con A in solution, it was hypothesized that the immobilization of it onto the surface of an active substrate would increase its stability overtime as seen in previous works. The immobilization procedure was performed on Con A for both polystyrene spheres and gold (nanoshells and colloid). Both results showed an adequate amount of protein on the surface of the particles, but little binding activity was demonstrated. Overall, the improvements to the sensor chemistry response were notable and the potential for stabilization and enhancement of the response through the use of an active substrate is promising.

Secretory proteins that are unable to assemble into native proteins in the endoplasmic reticulum (ER) are transported back into the cytosol for degradation. Many cytosolic and ER resident proteins have been identified so far as being involved in this retrotranslocation process, but it is difficult to determine whether these proteins have a direct or indirect effect. Interpretations are further complicated if the loss of a specific protein is obscured by the presence of another protein that is partially or wholly redundant. To overcome these limitations, a mammalian in vitro system was developed that allowed to monitor retrotranslocation synchronously and in real time in the absence of concurrent translocation. To examine the roles of different components in ER-associated degradation (ERAD), well-defined and homogeneous mammalian ER microsomes were prepared biochemically by encapsulating a fluorescent-labeled ERAD substrate with specific lumenal components. After mixing ATP, specific cytosolic proteins, and specific fluorescence quenching agents with microsomes, substrate retrotranslocation was initiated. The rate of substrate efflux from microsomes was monitored spectroscopically and continuously in real time by the reduction in fluorescence intensity as the fluorescent substrates passed through the ER membrane and were exposed to the quenching agents. Retrotranslocation kinetics were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase, or all cytosolic proteins with only the 19S proteasome cap. Retrotranslocation was blocked by affinity-purified antibodies against Derlin1, but not by affinity-purified antibodies against Sec61α or by membrane-bound ribosomes. Since the substrate also photocrosslinked Derlin1, but not Sec61α or TRAM, retrotranslocation of this ERAD substrate apparently involves Derlin1, but not the translocon. By labeling either the C- or N-terminus, it was revealed that the N-terminus of one ERAD substrate leaves the ER lumen first. This finding suggests that the protein is retrotranslocated as a linear polymer in a preferred direction. When RRMs were reconstituted with a fluorescent-labeled ERAD substrate and various ions. Ca2+ ions in the ER lumen increased the rate and extent of retrotranslocation, while Ca2+ ions in the cytosol decreased retrotranslocation. This approach therefore provides the first direct evidence of the involvement and importance of specific ionic requirements for ERAD.

Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phosphoenolpyruvate (PEP), which act to activate or inhibit, respectively, by changing the substrate (Fru-6-P) affinity of the enzyme. Both ligands bind to the same allosteric site in EcPFK. Therefore, the questions we want to address are how these two molecules regulate EcPFK and how the allosteric signal is propagated throughout the enzyme. EcPFK has 28 potential site-site interactions. These interactions in turn derive from multiple copies of 6 potentially unique homotropic interactions and 4 potentially unique heterotropic interactions. Making hybrid tetramer of EcPFK is used to isolate a single heterotropic interaction. To improve the yield of the 1:3 hybrid, the in vivo hybrid formation method was developed. Four heterotropic interactions were isolated by this manner and re-evaluated. The same kinetics characteristics were obtained for each 1:3 hybrid from both the in vivo and in vitro method. To address the question of how the allosteric signal is transmitted throughout EcPFK, we identified residues (G184, Asp59 and S157) that are important for the allosteric regulation for both PEP inhibition and MgADP activation. The impact of each mutation on individual interaction is unique and also suggests that the structural basis for PEP inhibition is different from that for MgADP activation. Most importantly, since the sum of each heterotropic interaction with a modification in only one subunit is equal to the total heterotropic interaction with a modification in all four subunits, this result indicates that the heterotropic allosteric signal transmission is realized in a single subunit. The 23Ã heterotropic interaction, which contributes the most to the PEP inhibition, was chosen to study the dynamic properties. Fluorescence was used to study the dynamic perturbations of the 23Ã interaction upon ligand binding. Taking advantage of the hybrid formation strategy and the tryptophan-shift mutagenesis method, a tryptophan residue can be placed at different individual locations throughout the native subunit containing the 23Ã heterotropic interaction. The steady-state anisotropy and lifetime measurement at each tryptophan position indicate that the 23Ã allosteric interaction involves the perturbation of side-chain dynamics both near and quite far away from the respective ligand binding sites.

To reduce the image blurring which originates from contributions of a cylindrical array of photolysis events in a photo-induced experiment, a variant of fluorescence imaging techniques has been developed to study photodissociation dynamics and collisional relaxation processes in the bulk. The experimental arrangement utilizes sliced imaging techniques of photofragments by the laser-induced fluorescence detection scheme. An unconventional procedure is employed to guide the photolysis laser in the viewing direction of the imaging detector with a proper obstruction. The sliced image in the direction perpendicular to the photolysis laser is equivalent to a two-dimensional projection of the fluorescence image of photoproducts from a single photolysis center. Experimental images of state-selected CN photofragments from the ICN photodissociation are presented to illustrate the versatility of the present method.

Thesis (M.Med.Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 63-68).

Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaf 12). Also available via the World Wide Web.

Thesis (M.S. in chemical engineering)--Washington State University, December 2009. / Title from PDF title page (viewed on Feb. 12, 2010). "School of Chemical Engineering and Bioengineering." Includes bibliographical references.