Indiana University-Purdue University Indianapolis (IUPUI) / Glucose homeostasis is maintained through the coordinated actions of insulin secretion from pancreatic beta cells and insulin action in peripheral tissues. Dysfunction of insulin action yields insulin resistance, and when coupled with altered insulin secretion, results in type 2 diabetes (T2D). Exocytosis of intracellular vesicles, such as insulin granules and glucose transporter (GLUT4) vesicles is carried out by similar SNARE (soluble NSF attachment receptor) protein isoforms and Munc18 proteins. An additional regulatory protein, Doc2b, was implicated in the regulation of these particular exocytosis events in clonal cell lines, but relevance of Doc2b in the maintenance of whole body glucose homeostasis in vivo remained unknown. The objective of my doctoral work was to delineate the mechanisms underlying regulation of insulin secretion and glucose uptake by Doc2b in effort to identify new therapeutic targets within these processes for the prevention and/or treatment of T2D. Towards this, mice deficient in Doc2b (Doc2b-/- knockout mice) were assessed for in vivo alterations in glucose homeostasis. Doc2b knockout mice were highly susceptible to preclinical T2D, exhibiting significant whole-body glucose intolerance related to insulin secretion insufficiency as well as peripheral insulin resistance. These phenotypic defects were accounted for by defects in assembly of SNARE complexes. Having determined that Doc2b was required in the control over whole body glycemia in vivo, whether Doc2b is also limiting for these mechanisms in vivo was examined. To study this, novel Doc2b transgenic (Tg) mice were engineered to express ~3 fold more Doc2b exclusively in pancreas, skeletal muscle and fat tissues. Compared to normal littermate mice, Doc2b Tg mice had improved glucose tolerance, related to concurrent enhancements in insulin mumsecretion from beta cells and insulin-stimulated glucose uptake in the skeletal muscle. At the molecular level, Doc2b overexpression promoted SNARE complex assembly, increasing exocytotic capacities in both cellular processes. These results unveiled the concept that intentional elevation of Doc2b could provide a means of mitigating two primary aberrations underlying T2D development.

Indiana University-Purdue University Indianapolis (IUPUI) / Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.

Indiana University-Purdue University Indianapolis (IUPUI) / Safety researchers have devoted extensive attention to safety performance behaviors. However, current safety performance models have yet to differentiate between safety citizenship behaviors directed towards the organization and those directed towards individuals. This might be a potential oversight, considering that citizenship behaviors targeted at different beneficiaries might be associated with different antecedents. As such, the purpose of the present study was to develop and validate a new safety performance scale. Items from existing measures formed the item pool and those tapping into the proposed dimensions were selected. Next, items were pilot tested using an online panel of 333 employees from various safety-related industries. A 4-factor structure emerged after exploratory factor analysis and the scale was further refined using reliability analysis and item response theory analysis. Finally, confirmatory factor analysis was conducted to replicate the factor structure using data from 137 employees. Theoretically related variables were correlated with the safety performance dimensions to establish the nomological network. Results supported the 4-factor structure of the new safety performance scale and construct validation hypotheses were largely supported. Implications, study limitations, and directions for future research are discussed.

Indiana University-Purdue University Indianapolis (IUPUI) / Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in the second catalytic domain of KIT involving an aspartic acid to valine substitution is found in patients with systemic mastocytosis (SM) and acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. This mutation changes the conformation of the KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth. As there are currently no efficacious therapeutic agents against this mutation, this study sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells in diseases such as AML and SM. This study shows that oncogenic KITD814V (murine homolog) induced myeloproliferative neoplasms (MPN) occurs in the absence of ligand stimulation, and that intracellular tyrosines are important for KITD814V-induced MPN. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival. Residue tyrosine 719 is vital for activation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α, downstream from KITD814V. Downstream effectors of the PI3K signaling pathway, in of leukemic cells bearing KITD814V with an allosteric inhibitor of Pak or its genetic inactivation results in growth repression due to enhanced apoptosis. To assess the role of Rac GEFs in KITD814V induced transformation, EHop-016, an inhibitor of Rac, was used to specifically target Vav1, and found to be a potent inhibitor of human and murine leukemic cell growth. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of MPN and rescued the associated pathology in mice. These studies provide insight on mechanisms and potential novel therapeutic targets for hematological malignancies harboring an oncogenic KIT mutation.

Indiana University-Purdue University Indianapolis (IUPUI) / This paper presents a novel approach of particulate material (soot) measurement in a Diesel particulate filter using Electrical Capacitance Tomography. Modern Diesel Engines are equipped with Diesel Particulate Filters (DPF), as well as on-board technologies to evaluate the status of DPF because complete knowledge of DPF soot loading is very critical for robust efficient operation of the engine exhaust after treatment system. Emission regulations imposed upon all internal combustion engines including Diesel engines on gaseous as well as particulates (soot) emissions by Environment Regulatory Agencies. In course of time, soot will be deposited inside the DPFs which tend to clog the filter and hence generate a back pressure in the exhaust system, negatively impacting the fuel efficiency. To remove the soot build-up, regeneration of the DPF must be done as an engine exhaust after treatment process at pre-determined time intervals. Passive regeneration use exhaust heat and catalyst to burn the deposited soot but active regeneration use external energy in such as injection of diesel into an upstream DOC to burn the soot. Since the regeneration process consume fuel, a robust and efficient operation based on accurate knowledge of the particulate matter deposit (or soot load)becomes essential in order to keep the fuel consumption at a minimum. In this paper, we propose a sensing method for a DPF that can accurately measure in-situ soot load using Electrical Capacitance Tomography (ECT). Simulation results show that the proposed method offers an effective way to accurately estimate the soot load in DPF. The proposed method is expected to have a profound impact in improving overall PM filtering efficiency (and thereby fuel efficiency), and durability of a Diesel Particulate Filter (DPF) through appropriate closed loop regeneration operation.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydraulic wind power transfer systems deliver the captured energy by the blades to the generators differently. In the conventional systems this task is carried out by a gearbox or an intermediate medium. New generation of wind power systems transfer the captured energy by means of high-pressure hydraulic fluids. A hydraulic pump is connected to the blades shaft at a high distance from the ground, in nacelle, to pressurize a hydraulic flow down to ground level equipment through hoses. Multiple wind turbines can also pressurize a flow sending to a single hose toward the generator. The pressurized flow carries a large amount of energy which will be transferred to the mechanical energy by a hydraulic motor. Finally, a generator is connected to the hydraulic motor to generate electrical power. This hydraulic system runs under two main disturbances, wind speed fluctuations and load variations. Intermittent nature of the wind applies a fluctuating torque on the hydraulic pump shaft. Also, variations of the consumed electrical power by the grid cause a considerable load disturbance on the system. This thesis studies the hydraulic wind power transfer systems. To get a better understanding, a mathematical model of the system is developed and studied utilizing the governing equations for every single hydraulic component in the system. The mathematical model embodies nonlinearities which are inherited from the hydraulic components such as check valves, proportional valves, pressure relief valves, etc. An experimental prototype of the hydraulic wind power transfer systems is designed and implemented to study the dynamic behavior and operation of the system. The provided nonlinear mathematical model is then validated by experimental result from the prototype. Moreover, this thesis develops a control system for the hydraulic wind power transfer systems. To maintain a fixed frequency electrical voltage by the system, the generator should remain at a constant rotational speed. The fluctuating wind speed from the upstream, and the load variations from the downstream apply considerable disturbances on the system. A controller is designed and implemented to regulate the flow in the proportional valve and as a consequence the generator maintains its constant speed compensating for load and wind turbine disturbances. The control system is applied to the mathematical model as well as the experimental prototype by utilizing MATLAB/Simulink and dSPACE 1104 fast prototyping hardware and the results are compared.

Indiana University-Purdue University Indianapolis (IUPUI) / Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 h exposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, singleand multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatics analysis of proteins identified by LFQMS. Interestingly, after exposure to a high concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT, respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins common to both kinds of nanotubes are associated with the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The decrease in expression of the majority proteins suggests a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure. To examine the effect of nanotubes on the plasma membrane, we investigated the interaction of short purified MWCNT with model lipid membranes using a planar bilayer workstation. Bilayer lipid membranes were synthesized using neutral 1, 2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) in 1 M KCl. The ion channel model protein, Gramicidin A (gA), was incorporated into the bilayers and used to measure the effect of MWCNT on ion transport. The opening and closing of ion channels, amplitude of current, and open probability and lifetime of ion channels were measured and analyzed by Clampfit. The presence of an intermediate concentration of MWCNT (2 μg/ml) could be related to a statistically significant decrease of the open probability and lifetime of gA channels. The proteomic studies revealed changes in response to CNT exposure. An analysis of the changes using multiple databases revealed alterations in pathways, which were consistent with the physiological changes that were observed in cultured cells exposed to very low concentrations of CNT. The physiological changes included the break down of the barrier function and the inhibition of the mucocillary clearance, both of which could increase the risk of CNT’s toxicity to human health. The biophysical studies indicate MWCNTs have an effect on single channel kinetics of Gramicidin A model cation channel. These changes are consistent with the inhibitory effect of nanoparticles on hormone stimulated transepithelial ion flux, but additional experiments will be necessary to substantiate this correlation.

Indiana University-Purdue University Indianapolis (IUPUI) / Ignition of a combustible mixture by a transient jet of hot reactive gas is important for safety of mines, pre-chamber ignition in IC engines, detonation initiation, and in novel constant-volume combustors. The present work is a numerical study of the hot-jet ignition process in a long constant-volume combustor (CVC) that represents a wave-rotor channel. The mixing of hot jet with cold mixture in the main chamber is first studied using non-reacting simulations. The stationary and traversing hot jets of combustion products from a pre-chamber is injected through a converging nozzle into the main CVC chamber containing a premixed fuel-air mixture. Combustion in a two-dimensional analogue of the CVC chamber is modeled using global reaction mechanisms, skeletal mechanisms, and detailed reaction mechanisms for four hydrocarbon fuels: methane, propane, ethylene, and hydrogen. The jet and ignition behavior are compared with high-speed video images from a prior experiment. Hybrid turbulent-kinetic schemes using some skeletal reaction mechanisms and detailed mechanisms are good predictors of the experimental data. Shock-flame interaction is seen to significantly increase the overall reaction rate due to baroclinic vorticity generation, flame area increase, stirring of non-uniform density regions, the resulting mixing, and shock compression. The less easily ignitable methane mixture is found to show higher ignition delay time compared to slower initial reaction and greater dependence on shock interaction than propane and ethylene. The confined jet is observed to behave initially as a wall jet and later as a wall-impinging jet. The jet evolution, vortex structure and mixing behavior are significantly different for traversing jets, stationary centered jets, and near-wall jets. Production of unstable intermediate species like C2H4 and CH3 appears to depend significantly on the initial jet location while relatively stable species like OH are less sensitive. Inclusion of minor radical species in the hot-jet is observed to reduce the ignition delay by 0.2 ms for methane mixture in the main chamber. Reaction pathways analysis shows that ignition delay and combustion progress process are entirely different for hybrid turbulent-kinetic scheme and kinetics-only scheme.