Browsing by Author "Dr. Robert M. Kelly, Committee Member"

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Enzymatic Reactions in Water Soluable Polymer Solutions: Rheology and Kinetics
(2006-05-23) Mahammad, Shamsheer; Dr. Wendy E. Krause, Committee Member; Dr. Robert M. Kelly, Committee Member; Dr. George W. Roberts, Committee Member; Dr. Saad A. Khan, Committee Chair
The polysaccharides are being used in a variety of applications because of their biocompatibility, biodegradability and natural abundance. The enzymes, because of their specificity, offer a powerful method to modify the structure of the polysaccharides. The structural modifications alter the interaction of the polysaccharides with other synthetic and biopolymers that determine the physiochemical properties of the polymer solutions. In this regard, we explore the use of enzymes to modify the rheology of solutions containing polysaccharides and thereby, understand the interrelationship between the enzymatic modification and the resulting rheological consequences. In the first part of this research, we modify the structure of guar galactomannan using three glycosidase enzymes, β-mannanase, β-mannosidase and α-galactosidase, at different combinations and proportions. We investigate the effect of synergistic hydrolysis by multiple enzymes in terms of viscosity reduction patterns during the hydrolysis reactions. We develop a rheokinetic model combining a kinetic model with the viscosity-molecular weight relationship. The rheokinetic model is used to estimate the kinetic parameters by tracking changes in steady shear viscosity during the enzymatic reactions. The effects of the combined action of enzymes on degradation rates are quantified in terms of variation in rate constants and other model parameters. In the second part of this research, we focus on modulating the rheology of hydrophobically modified associative polymer that has a comblike structure with hydrophobic groups randomly attached to the polymer backbone. The intermolecular interaction between the hydrophobic groups forms a transient network resulting in the thickening of the polymer solution. Although the hydrophobic interactions are important from the rheological standpoint; it is often desirable to modulate these interactions. This is achieved by adding cyclodextrins that encapsulate the hydrophobes within their hydrophobic cavity, and causes reduction in viscoelastic properties of the polymer solution. Subsequent degradation of the cyclodextrin using an amylase enzyme enables complete recovery of the original rheological properties. We develop mathematical models to study the thermodynamics of cyclodextrin-hydrophobe complexation and the kinetics of the enzymatic reactions. We show that the model parameters can be estimated by measuring changes in the rheological properties during the cyclodextrin-hydrophobe complexation and subsequent enzymatic degradation process.
Functional Analysis of Adhesion Factors and Signaling Mechanisms in Lactobacillus acidophilus NCFM
(2006-08-02) Buck, Brock Logan; Dr. Hosni Hassan, Committee Member; Dr. Robert M. Kelly, Committee Member; Dr. Amy M. Grunden, Committee Member; Dr. Todd R. Klaenhammer, Committee Chair
The lactobacilli are traditionally used for the fermentation of food products, but have recently been investigated for their potential health promoting factors. One important property of these probiotic lactobacilli is their ability to adhere to the intestinal mucosa. Lactobacillus acidophilus NCFM is a probiotic organism which has the ability to adhere to intestinal cells in vitro, although the mechanisms for this adhesion are not clear. A functional genomic approach was utilized to identify specific genes related to the adhesion process of L. acidophilus. The Caco-2 cell model was employed for analysis of the adhesive properties of multiple isogenic mutant strains of L. acidophilus. Mutation of genes encoding a fibronectin-binding protein (FbpA, LBA1148), mucus-binding protein (Mub, LBA1392), and a surface layer protein (SlpA, LBA0169) resulted in a significant decrease in adhesion to Caco-2 cells compared to the control strain. It appears that multiple factors work together to contribute to the adhesive ability of L. acidophilus NCFM. When then environmental conditions of L. acidophilus were modified immediately before adhesion, an explosive adhesive state was induced, termed the Adhesion Adaptive Response (AAR). Microarray analysis was used to study the transcriptional response of the L. acidophilus population to AAR conditions. Interestingly, multiple stress-related genes were overexpressed under AAR conditions along with the quorum sensing related gene, luxS. LuxS acts to synthesize autoinducer-2 (AI-2) which acts as an interspecies signaling molecule. A mutant strain of L. acidophilus deficient in LuxS activity was constructed and analyzed for its adhesive ability. The LuxS- mutant strain exhibited a decrease in adhesion compared to the control when harvested directly from logarithmic phase, but still presented an explosive adhesive response following exposure to AAR conditions. Further analysis of the effect of LuxS on L. acidophilus was performed using microarray analysis and phenotypic confirmation studies. L. acidophilus was found to produce AI-2 throughout the logarithmic growth phase. As such, three points were selected for transcriptional analysis of both the wild type and LuxS- mutant strain. Following a mixed model statistical analysis we discovered that the majority of differential expression related to LuxS occurs in the early-log stages of growth. Multiple genes related to the stress response, host-microbe interactions, and general growth and metabolism were positively differentially expressed in the presence of AI-2. Multiple surface proteins of were identified that contributed to the ability of L. acidophilus NCFM to adhere to intestinal epithelial cells, in vitro. An explosive adhesive response was induced by altering the environmental conditions in a process called the Adhesion Adaptive Response. Finally, the impact of the autoinducer AI-2 was studied using a novel microarray loop design. We reported that AI-2 influenced the transcriptional response of L. acidophilus at the early stages of growth, suggesting that AI-2 acts as a unique quorum sensing molecule.
Signaling Pathways Activated by Interleukin-2 and Interleukin-4 Receptors Mediate T Lymphocyte Clonal Expansion
(2008-02-10) Comfort, Kristen Krupa; Dr. Carla Mattos, Committee Member; Dr. David F. Ollis, Committee Member; Dr. Jason M. Haugh, Committee Chair; Dr. Robert M. Kelly, Committee Member