Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 57-59). Also available on the World Wide Web.

Insight into the biochemical mechanisms utilized by retaining and inverting glycosyltransferase enzymes is an important stepping stone to the directed design of stereospecific inhibitor based drugs. The suitability of proposed mechanisms was probed using site directed mutagenesis of catalytically relevant residues as well as the use of catalytically inactive substrate analogs UMP-PO2-CH2-D-Gal and α-L-Fuc-(1→2)-β-D-(3-deoxy)-Gal-O(CH2)5CH3 with the retaining human enzyme a specific α-1,3-N-acetylglucosaminyltransferase (GTA) in conjunction with kinetic and structural approaches including two dozen high resolution X-ray structures and a 2.5 Å resolution neutron structure. The neutron structure depicts a remarkably non-polar active site which lacks suitably positioned hydrogen atoms to support a dissociative mechanism. Site directed mutagenesis of residues which should be essential to initiate and stabilize a dissociative oxocarbenium ion do not abolish enzyme activity. The catalytically inactive substrate analogs depict the acceptor nucleophile to lay very close to the anomeric carbon (2.5 Å), which is considerably closer than the closest observed enzymatic dipoles (4.8 Å). This is an indication that the active site architecture is more suited to facilitate a mechanism initiating with nucleophilic attack than dissociation. To ensure that these observations are applicable to other glycosyltransferases, in depth geometric analysis of all published liganded structures of GT-A fold glycosyltransferase enzymes are reported that display conserved architectures in which the acceptor nucleophile approach is closer than enzymatic dipoles required for dissociation for both inverting and retaining enzymes. Inverting and retaining enzymes present the donor sugar through different conserved geometries about the divalent cation cofactor: all inverting enzymes position the donor for inline nucleophilic attack by the acceptor, the retaining enzymes position the sugar to be attacked from an orthogonal angle. Such an orthogonal associative mechanism is the most direct proposed approach, and seems supported by all available evidence. / Graduate

ULK1 (UNC-51 like kinase 1) is a serine/threonine protein kinase that has been shown to play a crucial role in autophagy, a process of self digestion implicated in maintaining cellular homeostasis and in mediating type II programmed cell death. However, the exact mechanism by which ULK1 controls autophagy remains elusive, mostly because none of the known ULK1 targets have been directly linked to autophagy. To address this issue, I have employed a protein microarray screening approach to identify novel ULK1 substrates. I found five putative targets: MERTK (proto-oncogene tyrosine-protein kinase MER), B-RAF (v-raf murine sarcoma viral oncogene homologue B1), NOL4 (nucleolar protein 4), TBC1D22B (TBC1 domain family member 22B) and ACVRL1 (activin A receptor type II-like 1). My preliminary experiments have not confirmed that MERTK or B-RAF can be phosphorylated by ULK1 in vitro. However, further investigation will be required to firmly rule out MERTK and B-RAF as downstream targets of ULK1 and to test the ability of ULK1 to phosphorylate the other candidates. In addition, I have identified by in-gel kinase assay a ULK1 kinase at 34-kDa whose ability to phosphorylate the kinase domain of ULK1 was increased upon starvation. Using the genome information, I predicted this upstream kinase to be Pim1 (Proto-oncogene serine/threonine-protein kinase pim-1). I confirmed that Pim1 phosphorylated ULK1 in vitro at S147 and S224. Results of site directed mutagenesis suggest that phosphorylation at S224 correlates with increased ULK1 activity. This is consistent with observation that Pim1 is capable of activating ULK1 in vitro. Furthermore, I present preliminary data suggesting that Pim1 promotes autophagy in HeLa cells.

Glomerular podocytes are epithelial cells that are attached to outer glomerular basement memberane (GBM) by foot processes, and blood filtration occurs through podocytes, GBM, and endothelial cells. Podocytes are under constant mechanical stress due to their location around outside of glomerular capillaries, which can be associated with glomerular hypertension. It is important for podocytes to maintain their mechanical integrity, since podocyte adhesion to GBM is crucial to prevent podocyte loss, detachment, and associated alteration in cell adhesive properties, and further progression of glomerular disease. In this study, we examined the role of stiffness in podocyte function with hypothesis that increasing substrate stiffness would promote development of cell structural features that are associated with stronger adhesion. In order to test this, polyacrylamide substrates with different stiffness ranged from 3750 Pa to 152600 Pa were generated and immortalized mouse podocytes were cultured on these substrates. Then we measured how substrate stiffness affects cell morphology and several structural proteins distribution. We found that the size and the number of attached cells increased with longer actin filaments as stiffness of substrate increased. Since proteinuria or glomerulosclerosis can be associated with podocyte actin cytoskeleton defect, we suggest podocytes in a "softer" environment are vulnerable to glomerular diseases, since stress fibers were shorter and less organized as substrates decreased stiffness. Our results relating to the presence and distribution of certain proteins in cells were somewhat inconclusive, since intensity of synaptopodin and vinculin did not correspond to the changes of stiffness, due to the possibility of other underway mechanisms that interfere with podocyte adhesion. There was no clear relationship between YAP and the changes of substrate stiffness, and one possible explanation could be due to the optical irregularities in the substrate. Overall, this study was able to show that increased substrate stiffness promoted cell structural feature development in podocytes. However, further studies are needed to better understand how changes in substrate mechanical properties can affect structural protein distribution in these cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Calcium/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a multimeric holoenzyme composed of 8–14 subunits from four closely related isoforms (α, β, γ, δ). CaMKII plays a strategic, multifunctional role in coupling the universal second messenger calcium with diverse cellular processes including metabolism, cell cycle control, and synaptic plasticity. CaMKII exhibits broad substrate specificity, targeting numerous substrates with diverse phosphorylation motifs. Binding of the calcium sensor CaM to the autoregulatory domain (ARD) of CaMKII functions to couple kinase activation with calcium signaling. Important sites of autophosphorylation, namely T287 and T306/7 (δ isoform numbering), reside within the ARD and control either CaM dependence or ability to bind to CaMKII respectively, thus determining various activation states of the kinase. Because autophosphorylation is critical to the function of CaMKII in vivo, we sought to determine the relationship between the activation state of the kinase and substrate selectivity. We show that the ARD of activated CaMKII tunes substrate selectivity by competing for substrate binding to the catalytic domain, thus functioning as a selectivity filter. Specifically, in the absence of T287 autophosphorylation, substrate phosphorylation is limited to high-affinity, consensus substrates. T287 autophosphorylation restores maximal kinase activation and broad substrate selectivity by disengaging ARD filtering. The unique multimeric architecture of CaMKII is an ideal sensor which encodes calcium-spike frequency into graded levels of subunit activation/autophosphorylation within the holoenzyme. We find that differential activation states of the holoenzyme produce distinct substrate phosphorylation profiles. Maximal holoenzyme activation/autophosphorylation leads to further broadening of substrate specificity beyond the effect of autophosphorylation alone, which is consistent with multivalent avidity. Thus, the ability of calcium-spike frequency to regulate T287 autophosphorylation and holoenzyme activation permits cellular activity to dictate switch-like behavior in substrate selectivity that is required for diverse cellular responses by CaMKII.

Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS catalyzes the transfer of glucose from UDP-glucose to the non-reducing ends of glycogen and its activity is negatively regulated by phosphorylation and allosterically activated by glucose-6-phosphate (G6P). A highly conserved cluster of six arginine residues on the C-terminal domain controls the responses toward these opposing signals. Previous studies had shown that tetrameric enzyme exists in three conformational states which are linked to specific structural changes in the regulatory helices that carry the cluster of arginines. These helices are found opposite and anti-parallel to one another at one of the subunit interfaces. The binding of G6P beneath the regulatory helices induces large scale conformational changes which open up the catalytic cleft for better substrate access. We solved the crystal structure of the enzyme in its inhibited state and found that the tetrameric and regulatory interfaces are more compacted compared to other states. The structural consequence of the tighter interfaces within the inhibited state of the tetramer is to lower the ability of glycogen chains to access to the catalytic cleft. Based on these observations, we developed a novel regulatory feature in yeast GS by substituting two of its conserved arginine residues on the regulatory helix with cysteines that permits its activity to be controlled by reversible oxidation/reduction of the cysteine residues which mimics the effects of reversible phosphorylation. In addition to defining the structural changes that give rise to the inhibited states, we also used X-ray crystallography to define the mechanism by which the enzyme discriminates between different UDP-sugar donors to be used as substrates in the catalytic mechanism of yeast GS. We found that only donor substrates can adopt the catalytically favorable bent conformation for donor transfer to a growing glycogen chain.

This thesis investigates techniques for prototyping and evaluation of medium voltage (MV) power module packages. Specific focus will be given to the utilization of silver sintering as a bonding method for high temperature, high density power modules. Nano-silver paste and preform will be examined in detail as enabling technologies for a new generation of power electronics. To accomplish this task, analysis and characterization of the metal-ceramic substrate and its structure is performed. First, finite element models are created to evaluate the fatigue behavior of the large area bonds in the substrate structure. Prototypes of these multi-layer substrates have also been fabricated and will be subjected to thermal cycling tests for experimental verification of the efficacy of their sintered silver bonds. Stacked direct-bonded aluminum (DBA) substrates have been found to withstand up to 1000 thermal cycles of –40 °C to 200 °C when attached with low pressure-assisted silver sintering. The thermal performance of 10 kV SiC power module utilizing multi-layer DBA substrates bonded with a large-area, low pressure-assisted sintered silver bond will also be examined to ensure the sintered bond is viable for the harsh operating conditions of MV modules. A junction-to-case thermal resistance of 0.142 °C/W is measured on a module prototype utilizing stacked DBA substrates. Finally, analysis of a double-sided cooling scheme enabled by large area sintering is simulated and prototyped to demonstrate a 6.5 kV package for a MV power device. Residual stress failures induced by a highly rigid structure have been examined and mitigated through implementation of a 5 MPa pressure-assisted, double-sided silver sintering approach. / Master of Science / Power modules are the building blocks of the electrical grid of the future. As society transitions to renewable energy to fight the crisis presented by climate change, the structure of the energy grid will have to change to accommodate the increase in solar, wind, geothermal, and other renewable sources of energy generation. A clean energy grid structure will contain ubiquitous opportunities to use power modules for medium-voltage (MV) applications, like managing the flow of electricity from solar panels and wind turbines to neighborhoods and office buildings. However, these MV power modules will need to be resilient to extreme temperature and electrical stresses inherent to these applications. Current technology must be improved in both performance and reliability to match the needs of this future grid. This thesis investigates, through both experiment and computer simulation, techniques for improving the reliability of MV power modules without sacrificing thermal or electrical performance. Techniques presented in this work have the potential to transform power modules, so they may operate at higher temperatures and efficiencies for a longer lifetime than the current state-of-the-art.

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No abstract provided / Thesis / Master of Science (MSc)