Browsing by Author "Robert Kelly, Committee Member"

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Computer Simulation Studies of Self-Assembly of Fibril-Forming Peptides with an Intermediate Resolution Protein Model.
(2010-07-07) Wagoner, Victoria; Carol Hall, Committee Chair; Robert Kelly, Committee Member; Allan Clark, Committee Member; Keith Gubbins, Committee Member
Engineering Analysis of Spatial Gradient Sensing in Platelet-derived Growth Factor-stimulated Fibroblasts
(2006-09-08) Schneider, Ian Christopher; Robert Kelly, Committee Member; John van Zanten, Committee Member; Nina Allen, Committee Member; Jason Haugh, Committee Chair
Wound healing is a well-coordinated process in which different cell types invade the wound at different times, secreting different enzymes and factors. An important event in this process is the proliferation and directed migration of dermal fibroblasts. Evidence has shown that gradients of platelet-derived growth factor (PDGF) mediate the process of directed cell migration in fibroblasts. These cells use membrane lipids, specifically 3' phosphoinositides (PIs), to spatially sense the extracellular PDGF gradient by creating an intracellular gradient of 3' PIs. PDGF receptor stimulates 3' PI production by activating phosphoinositide 3-kinase (PI 3-kinase), a lipid kinase. However, the mechanism by which the cell generates intracellular 3' PI gradients is not well known in fibroblasts. A 3' PI specific binding domain fused to a fluorescent protein can be used as a 3' PI probe and can be imaged quantitatively using total internal reflection fluorescence (TIRF) microscopy. This technique allows for the selective illumination of the adherent portion of the cell membrane, providing a powerful technique to visualize protein translocation to the membrane in live cells. A mathematical model has been developed to quantitatively analyze the fluorescence data and we show here that these data are sufficient to set bounds on parameters that determine 3' PI production, diffusion and degradation. Under uniform PDGF stimulation we also find areas of differentially controlled 3' PI production, diffusion and degradation that can be linked to the specific morphology of the cell. This indicates that cell polarization may play an important role in PI 3-kinase signaling. Finally, we find that fibroblasts sense gradients in a manner distinct from other more common chemotactic models such as Dictyostelium discoideum and neutrophils. Fibroblasts are only sensitive to large relative PDGF gradients and intermediate PDGF concentrations. Consequently, the ability of these cells to spatially sense relies on the competition between the intrinsic bias caused by cell polarization and the external bias caused by the PDGF gradient, each of which may have different contributions depending on the extracellular gradient condition
Genomic Analysis of Lactobacillus gasseri Strains using Suppressive Subtractive Hybridization .
(2010-04-08) Schroeter, Joel; Todd Klaenhammer, Committee Chair; Robert Kelly, Committee Member; Trevor Phister, Committee Member
The Genomic Basis of Bile Tolerance in Lactobacillus acidophilus
(2009-09-25) Pfeiler, Erika Ann; Todd Klaenhammer, Committee Chair; Robert Kelly, Committee Member; Dahlia Nielsen, Committee Member; Jonathan Olson, Committee Member
Green Liquor Pretreatment for Hardwood chips to Ethanol conversion strategies
(2009-12-02) Diaz, Joscelin Teresita; Robert Kelly, Committee Member; Hasan Jameel, Committee Co-Chair; Lucian Lucia , Committee Chair
Modified Green Liquor pretreatment is a novel high sulfide pretreatment method that enhances carbohydrate stability. The influence of hardwood chips pretreatment with green liquor was investigated under alkaline conditions of 12% and 16% TTA, and sulfidities of 0%, 12.5%, 25%, and 37.5% to see its effect on the sugar recovery. Also, ethanol was added in the pretreatment conditions of 12%TTA and 25%S as an Organosolv pretreatment method. Quantitative analyses of glucose, xylose, mannose, and carbohydrates yield is discussed. The influence of pretreatment conditions such as alkalinity and sulfidity charge were the main variables controlled to analyze their influence in the carbohydrate yield. The results suggest that higher sulfidity promotes an increase in delignification, and also that this delignification is more notable in the samples pretreated with ethanol. The pretreated pulps were then hydrolyzed with cellulase, xylanase, and ÃŽÂ²-glucosidase enzymes. After enzymatic hydrolysis with these commercial cellulases enzymes, higher glucose yields were obtained at higher sulfidity and higher ethanol concentration in the liquor. The results also indicated that higher enzyme activity exhibited higher carbohydrates and glucan yield. The carbohydrates yield was 70.6% for hardwood chips pretreated with 16%TTA, 37.5%S, and hydrolyzed with cellulases at 20 FPU/g pulp. However, the higher carbohydrates yield obtained was 75.7% for the pulps pretreated at 20% ethanol and 20 FPU/g pulp. Different techniques were applied in the samples 16%TTA, 0%S and 37.5%S, and 12%TTA, 25%S, 20%ethanol to determine its pretreatment effect on lignin. ToF-SIMS technique was used to detect the changes in cellulose/lignin composition and then compare the results with ones the obtained from the sugar analysis using Ion Exchange Chromatography. ToF-SIMS suggested that the removal of lignin is mainly localized on the sample surface. The molecular weight distribution of lignin using Size Exclusion Chromatography technique was also studied. The results shown a decrease in the molecular weight when sulfidity was increased. It is hypothesized that this happened mainly due to cleavage of lignin in the ÃŽÂ²-O-4 linkage that decrease the chain length of the residual lignin. Green liquor has the advantage of altering the lignin structure, and preserving the celluloses and hemicelluloses present in the biomass. An economic analysis based in different assumptions was elaborated to compare all the study cases. In general the results shown that samples pretreated with higher sulfidity always shown better profit per liter compared with lower sulfidity. Also, in the case of the samples pretreated with ethanol, the profit was even better than without ethanol. But, variations in ethanol lost during the process shown that this method is not as attractive as the pretreatment at high sulfidity and high total titratable alkali.
Intracellular Signaling Networks in the Immune Response: Pathways Activated by Interleukin-2 and Interleukin-4 Receptors and their Roles in T Cell Proliferation
(2006-09-20) Comfort, Kristen Krupa; Robert Kelly, Committee Member; David Ollis, Committee Member; Jason M. Haugh, Committee Chair; Carla Mattos, Committee Member
Cells sense and respond to chemical and physical stimuli through signal transduction pathways, which mediate cell proliferation, differentiation, migration, and survival. The cytokines interleukin-2 (IL-2) and interleukin-4 (IL-4) are key regulators of the adaptive immune system, particularly influencing the clonal expansion and differentiation of T cells. At least in culture, both synergistic and antagonistic effects of IL-2 and -4 co-stimulation have been reported; the antagonism, when observed, is thought to arise from the utilization of a common subunit shared by IL-2 and IL-4 receptors. We have sought to characterize IL-2 and IL-4 signaling at the level of intracellular pathways activated by these receptors. IL-2 receptors are known to activate the Ras/extracellular signal-regulated kinase (Erk) and phosphoinositide (PI) 3-kinase pathways as well as the STAT5 transcription factor. IL-4 is unique among cytokines in that it does not activate Ras⁄Erk; it does activate PI 3-kinase⁄Akt as well as a distinct STAT, STAT6. The HT-2 mouse T cell line responds to both IL-2 and -4. We found that IL-4 initially antagonizes, and later synergizes with, IL-2-stimulated HT-2 cell proliferation in a dose-dependent manner. IL-4 also stimulates cell adhesion in static cultures. At the level of intracellular signaling, IL-4 antagonizes IL-2-stimulated activation of Akt, possibly through competition for limiting amounts of common receptor subunit and/or PI 3-kinase. Because IL-4 alone does not promote any Erk activation, we were surprised to find that IL-4 enhances IL-2-stimulated activation of Erk. IL-2⁄IL-4 co-stimulation provokes transient activation of STAT5 and prolonged activation of STAT6. This extended STAT6 activation may be critical in the IL-2⁄IL-4 induced synergy in T cell growth. Currently, we are investigating the crosstalk between these pathways and their functional roles in IL-2 and IL-4-stimulated T cell responses.
Molecular Characterization of Rickettsial Diseases in Dogs
(2006-07-26) Kidd, Linda Benjamin; Robert Kelly, Committee Member; Paul Orndorff, Committee Member; Ed Breitschwerdt, Committee Chair; Wayne Tompkins, Committee Member; Scott Laster, Committee Member
Spotted Fever Group Rickettsia are important causes of morbidity and mortality worldwide. These arthropod borne, obligately intracellular organisms are notoriously difficult to detect in blood samples from infected patients. Standard diagnostic techniques do not differentiate among SFG members, thus the species of infecting Rickettsia is presumed based on geographic location. With the advent of molecular biology, newly discovered and previously known pathogenic and "non-pathogenic" SFG Rickettsia have been associated with disease in people in expanding regions of the world. R. rickettsii, the cause of Rocky Mountain Spotted Fever, is arguably the most well known and well characterized SFG Rickettsia The manifestations of this disease are similar in dogs and people, and because infection can precede or occur simultaneously with infection in their human companions, dogs are considered sentinels for the disease. Despite this, the species of Rickettsia infecting dogs with RMSF had never been characterized using molecular techniques. Using PCR, we amplified and sequenced portions of three genes from SFG Rickettsia isolated from dogs and people with RMSF in an endemic region. Gene sequence based criteria were applied and the isolates were identified as R. rickettsii. This study provides support that naturally occurring RMSF in dogs is comparable to the disease in people. Rickettsia rickettsii has been declared a Select Agent by the CDC and a Category C priority pathogen by the NIAID due to concerns that it is amenable for use in a bioterrorist attack. As a result of this study, 15 consensus sequences of genes amplified from R. rickettsii naturally infecting dogs and people in North Carolina have been deposited in GENBANK (Accesion numbersDQ15680-DQ15694). Knowledge of sequences of naturally occurring isolates may help identify aberrant strains intentionally released as a result of an act of bioterrorism. Amplifying DNA from cultures of Rickettsia does not require a particularly sensitive PCR. Due to the nature of these organisms, sensitive assays are required in the clinical setting. No diagnostic tool that is sensitive, and can differentiate among species of infecting Rickettsia has been validated for use in infected dogs. We have created a PCR that amplifies a portion of the ompA gene from infected dog blood with a limit of detection of 1.5-30 copies of SFG Rickettsia. Sequencing of the product differentiates a number of different species. Using this tool we documented the presence of R. conorii ssp conorii DNA in the blood of three male Sicilian Yorkshire Terriers. These dogs had clinical illness compatible with acute rickettsiosis. This provides unique evidence that R. conorii may infect dogs and cause disease in this host. Future studies should investigate the role of R. conorii and other SFG Rickettsia as disease causing agents in dogs. Identifying SFG Rickettsia in naturally infected dogs may have implications for their human companions due to their potential role as sentinels for this group of illnesses.
Relating phosphoinositide 3-kinase (PI3K) signaling and cell motility dynamics during PDGF-stimulated chemotaxis
(2010-04-02) Melvin, Adam Thomas; Jason Haugh, Committee Chair; Robert Kelly, Committee Member; Balaji Rao, Committee Member; Sam Jones, Committee Member
Cell migration is essential for wound healing, the immune response, embryogenesis, and cancer metastasis. Chemotaxis, or cell migration directed by an external gradient of chemoattractant, is encountered in various physiological and natural settings and is a means by which cellular processes are coordinated in space and time. This cyclical process of protrusion, adhesion, and retraction is characterized by an asymmetric polarization of 3' phosphoinositides at the leading edge of a migrating cell and is common across multiple cell types. Activated by the phosphoinositide 3-kinase (PI3K) pathway, these 3' PIs act as membrane-bound second messengers which recruit additional signaling proteins, such as the Rho family GTPases, to initiate actin polymerization and membrane protrusion. These protrusions are capable of both cellular translocation as well as gradient sensing. The characteristics of chemotaxis depend on cell type. Amoeboid cells such as neutrophils and the slime mold, Dictyostelium discoideum, respond to shallow gradients of chemoattractants (~1% difference in concentration across cell dimensions) and migrate rapidly (~20 Âµm/min). Mesenchymal cells such as fibroblasts, on the other hand, require much steeper chemoattractant gradients to evoke similar polarization of PI3K signaling, but the sensitivity of chemotactic movement has yet to be quantitatively characterized. Using a 3' PI-specific, fluorescent biosensor and total internal reflection fluorescence (TIRF) microscopy, the spatial and temporal dynamics of PI3K signaling were monitored during chemotaxis of fibroblasts responding to gradients of the chemoattractant platelet-derived growth factor (PDGF). We show that the asymmetry of PI3K signaling positively correlates with cell directionality during chemotaxis and, consistent with previous work, that highly persistent movement aligned with the external gradient requires a steep gradient (> 10%) in PDGF receptor binding across the cell. Additionally, we present the development of novel microfluidic devices that generate linear gradients of chemoattractant and their integration with TIRF microscopy. In one particular device, fibroblasts exhibited similar characteristics of PI3K signaling asymmetry as compared with other methods. Finally, we evaluate the long-term viability of fibroblasts and methods to minimize toxicity in these devices.
Study of Particle Formation using Supercritical CO2 as an Antisolvent
(2007-04-03) Chang, Alan An-Lei; Michael Dykstra, Committee Member; George Roberts, Committee Member; Ruben G. Carbonell, Committee Chair; Richard Spontak, Committee Member; Robert Kelly, Committee Member
Particle design using supercritical CO2 has been of great interest in the pharmaceutical, microelectronic, catalytic, and related industries over the past 10 years. There have been numerous papers and patents published on the processes studied in this work. The solubility of most drug compounds in carbon dioxide is very low, making it a very attractive antisolvent for particle formation at suitable ranges of temperatures and pressures. This thesis explores the use of different CO2 antisolvent precipitation system designs for the formulation of small crystalline drug particles of a given size, morphology, and uniformity, using the precipitation of acetaminophen from ethanol as an example. In order to understand the precipitation process, the equilibrium concentration of acetaminophen in CO2 and CO2 plus ethanol were measured at a range of temperatures and pressures in a high-pressure extraction system. This information is important in understanding the supersaturation of the drug at various precipitation conditions. Several antisolvent processes were tested in order to determine their effectiveness in controlling the precipitation of acetaminophen from ethanol. The first system involved the use of Solvent Enhanced Dispersion by Supercritical Fluids (SEDS) patented by Hanna and York (WO9501221, 1994). This process uses a coaxial nozzle design where the solvent with the solute of interest is injected in the inner tube and the supercritical CO2 is injected in the outer tube. The two streams mix at nearly constant pressure and temperature in a small volume region of the nozzle before expanding through the nozzle tip into a chamber maintained at a fixed temperature and pressure. The fast mixing process rapidly expands the solvent with CO2 in order to induce phase split of the solid drug particles. The chamber pressure is maintained constant and nearly equal to the pressure in the nozzle. This process was studied because it was claimed that SEDS gave the best control of system parameters. However, the thermodynamic, hydrodynamic and kinetic mechanisms resulting in particle formation are still not well understood. The effects of the nozzle dimensions and vessel dimensions on system performance had not been studies previously. In addition, little work has been published on the effects of variables such as liquid solvent and CO2 flow rates, solute concentration, temperature, and pressure on particle size and morphology. A Design of Experiments (DOE) analysis was used to identify the more important process parameters that control particle size and morphology. DOE is a useful statistical tool to reduce the number of experiments necessary to find the most important variables at an early stage of experimentation. With DOE, a 512 full factorial run was reduced to 32 runs by confounding primary variables with higher order interactions (Example: concentration + temperature). The results of these experiments indicated that the most important factors in determining particle size and morphology are the concentration of acetaminophen in the solvent, the nozzle geometry (length of the mixing zone), pressure and temperature. These parameters were singled out for more detailed experiments aimed at determining the influence of these variables on particle size and morphology. A key feature of the experiments described in this thesis is the use of on-line monitoring of the acetaminophen concentration at the exit to the capture vessel in order to determine how the supersaturation of the solute varied with time during the process. In this way it was possible to determine the nozzle effectiveness in particle precipitation. In addition, the experiment performed in this thesis recognized that the SEDS process is in essence a batch process and it studied the effect of transients in co-solvent concentrations in the particle capture vessel on particle size and morphology. In addition to SEDS, the precipitation of acetaminophen from ethanol was carried out using a Precipitation with Compressed Antisolvent (PCA) process, which is very similar to SEDS without the coaxial configuration. This system is simple to install and has been widely studied. The parameters that were important from the SEDS experiments were studied in the PCA to characterize their effects on particle size and morphology for this system. These results were compared to those obtained using the SEDS process. Both SEDS and PCA yielded equal particle size and morphology if designed properly. The major feature of this work was the emphasis on the design of effective nozzles for the PCA application. Similar to the SEDS results, a good mixing volume along with adequate residence time for micromixing are the best nozzle designs.
Study of Protein Binding Sites on the GTPase RalA and the Sugar-binding Protein Hen Egg White Lysozyme
(2006-08-10) Nicely, Nathan; Robert Kelly, Committee Member; Carla Mattos, Committee Chair; Robert Rose, Committee Member; Dennis Brown, Committee Member; William L. Miller, Committee Member
Hen egg white lysozyme and simian RalA are two very different proteins by function and category. Lysozyme is an extracellular enzyme that catalyzes the hydrolysis of the β-linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) subunits in the peptidoglycan polymers that compose some Gram-positive bacterial cell walls. RalA is a Ras-related GTPase involved in multiple distinct signaling pathways. The structure and sequence of its core domain is similar to Ras and Rap (another Ras-related GTPase), but they have mutually exclusive sets of upstream activators and downstream effectors. Furthermore, RalA is activated by calcium-loaded Calmodulin through its carboxy-terminal domain, and it binds phospholipase D constitutively through its amino-terminal domain; both traits are unique within the Ras subfamily. Lysozyme has a deep active site cleft between two subdomains which is responsible for binding the saccharide substrate. This binding site is small in its surface area compared to the total accessible surface area of lysozyme. It is relatively well-ordered and pre-formed, with good shape complementarity to the substrate. We employ the Multiple Solvent Crystal Structures method which uses small organic solvent molecules as probes to map the functional surface of the protein. Of ten solvent-soaked crystal structures, 11 solvent molecules were identified as bound to lysozyme in a total of six sites. Nine of these 11 solvent molecules bound in the active site cleft in well defined clusters corresponding to the established subsites in which the NAM/NAG subunits of the natural substrates bind. Five of these nine bind in subsite C, which has the most favorable binding energy of the six subsites. Two bind in subsite D and one each in subsites E and F. The positions and orientations of the bound solvent molecules mimic the acetamido functional groups on the NAM/NAG subunits, especially in subsite C. Of the two organic solvent molecules which bound outside the active site cleft, one bound at a two-fold crystal contact and the other on the edge of the epitope for an anti-lysozyme antibody. RalA has two large segments, termed the switch regions (I & II), that experience disorder-to-order transitions upon complexation with binding partners. These regions are responsible for significant structural changes across a large patch of the protein's accessible surface. We have solved the crystal structures of RalA in both its GDP- ("off;" inactivated) and GTP analog-bound ("on;" activated) forms. Disorder-to-order transitions occur in both switch regions upon protein-protein interaction in the form of crystallographic and noncrystallographic symmetry contacts; however, in the absence of such protein-protein contacts, both switches are disordered. This indicates a departure from the behavior of Ras in which the presence of GTP analog alone is sufficient to order switch I. Also, we identify two possible sites for protein-protein interaction on the surface of RalA by comparing structural features of the protein with the available data regarding amino acid residues important for its biochemical functions and including the experimental functionality map for Ras generated by the Multiple Solvent Crystal Structures method. A thorough analysis of the binding sites on RalA and lysozyme reveal some trends which agree with recent hypotheses on the nature of protein-ligand interfaces. First, all the binding sites on both proteins tend to have centers which are relatively invariant in terms of structural plasticity. These cores are surrounded by residues which exhibit conformational flexibility. Second, the binding sites are sparsely hydrated; any bound water molecules at our binding sites can be displaced by solvent molecules. Conversely, the switch regions of RalA are well hydrated at protein-protein contacts, reflecting the ability of water molecules to contribute to the close packing of atoms and charge complementarity in protein-ligand interfaces.