I. Development of Rapid Conductance-Based Protocols for Measuring Ion Channel Activity; II. Expression, Characterization, and Purification of the ATP-Sensitive, Inwardly-Rectifying K+ Channel, Kir6.2, and Ion Channel-Coupled Receptors
AuthorAgasid, Mark Tadashi
AdvisorAspinwall, Craig A.
Saavedra, Steve S.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
EmbargoRelease after 15-Feb-2018
AbstractLigand-gated and ligand-modulated ion channel (IC) sensors have received increased attention for their ability to transduce ligand-binding events into a readily measurable electrical signal. Ligand-binding to an IC modulates the ion flux properties of the channel in label-free manner, often with single-molecule sensitivity and selectivity. As a result, ICs are attractive sensing elements in biosensoring platforms, especially for ligands lacking optical (e.g. fluorescent) or electrochemical properties. Despite the growing number of available ligand-gated and ligand-modulated ICs and artificial lipid bilayer platforms for IC reconstitution, significant work remains in defining the analytical performance capabilities of IC sensors. Particularly, few studies have described platforms for making measurements with rapid temporal resolution and high sensitivity. In this work, we describe an artificial lipid bilayer platform which enables rapid measurement of ion channel activity, a key parameter for developing IC sensors suitable for studying biological events, e.g. single cell exocytosis (Chapter 2 and 3). Additionally, we developed expression, purification, and reconstitution protocols for Kir6.2, a model ligand-gated ion channel, for use in sensor development (Chapter 4). The final goal is to reconstitute ion channel-coupled receptors (ICCRs), G protein-coupled receptor-Kir6.2 fusion proteins, into artificial lipid bilayers to detect small molecules and hormones targeting GPCRs. Towards this goal, we characterized the expression and function of two ICCRs, M2-Kir and D2-Kir, in HEK293 cells (Chapter 5).
Degree ProgramGraduate College
Degree GrantorUniversity of Arizona
Showing items related by title, author, creator and subject.
SINGLE CHANNEL ANALYSIS OF THE EFFECTS OF HALOTHANE ON THE NICOTINIC ACETYLCHOLINE RECEPTOR CHANNEL (CHOLESTEROL, CELL CULTURE, PATCH CLAMP, GENERAL ANESTHETIC).LECHLEITER, JAMES DONALD. (The University of Arizona., 1984)Anesthesia, a state of being absent of sensation and consciousness, has been recognized since antiquity. Even today anesthesia is still best characterized by the lack of consciousness and sensations. Since anesthetic potency is correlated with lipid solubility, the site of action of general anesthetics has been thought to be hydrophobic in nature and to involve excitable membranes critical for interneuronal communications. Thus, general anesthetics may interact directly with functionally-relevant membrane proteins (via hydrophobic pockets) or indirectly, with the lipids surrounding these proteins. To better understand the details of general anesthetic action, I examined how halothane interacts with a functional synaptic protein, the acetylcholine receptor channel embedded in the membranes of cultured Xenopus myocytes. Next, I examined how changing the lipid composition, of these membranes, affected this interaction. Using the extracellular patch-clamp technique, I found that halothane, at clinically-relevant concentrations, shortened the burst duration of single receptor channels without affecting their conductance. Moreover, the halothane-induced reduction of burst durations was significantly attenuated after pretreatment with cholesterol-rich lipsomes which increased significantly the cholesterol content of these cells. These findings provide the first direct support for the role of membrane lipids in the mechanism of GA action. In particular, I demonstrated that increases in membrane cholesterol antagonize the anesthetic action of halothane. Although direct action of cholesterol on synaptic proteins cannot be ruled out, my data strongly suggest that membrane lipids are involved at a critical, but as yet undefined, site with which GAs interact. The exact manner by which increases in membrane cholesterol antagonize GA action remains to be eludicated.
Secure and Spectrally-Efficient Channel Access in Multi-Channel Wireless NetworksZhang, Yan (The University of Arizona., 2015)Wireless services have become an indispensable part of our social, economic, and everyday activities. They have facilitated and continue to facilitate rapid access to information and have created a highly-interconnected web of users who are untethered to particular locations. In fact, it is expected that in the very near future, the number of users that access the Internet through their mobile devices will surpass those access the Internet from the fixed infrastructure. Aside from mobile Internet access, wireless technologies enable many critical applications such as emergency response, healthcare and implantable medical devices, industrial automation, tactical communications, transportation networks, smart grids, smart homes, navigation, and weather services. The proliferation and wealth of wireless applications has created a soaring demand for ubiquitous broadband wireless access. This demand is further fueled by the richness of the information accessed by users. Low-bit rate voice communications and text have been replaced with graphics, high-definition video, multi-player gaming, and social networking. Meeting the growing traffic demand poses many challenges due to the spectrum scarcity, the cost of deploying additional infrastructure, and the coexistence of several competing technologies. These challenges can be addressed by developing novel wireless technologies, which can efficiently and securely manage multi-user access to the wireless medium. The multi-user access problem deals with the sharing of the wireless resource among contending users in an efficient, secure, and scalable manner. To alleviate contention and interference among the multiple users, contemporary wireless technologies divide the available spectrum to orthogonal frequency bands (channels). The availability of multiple channels has been demonstrated to substantially improve the performance and reliability of wireless networks by alleviating contention and interference. Multi-channel networks, whether cellular, sensor, mesh, cognitive radio, or heterogeneous ones, can potentially achieve higher throughput and lower delay compared to single-channel networks. However, the gains from the existence of orthogonal channels are contingent upon the efficient and secure coordination of channel access. Typically, this coordination is implemented at the medium access control (MAC) layer using a multi-channel MAC (MMAC) protocol. MMAC protocols are significantly more sophisticated than their single-channel counterparts, due to the additional operations of destination discovery, contention management across channels, and load balancing. A significant body of research has been devoted to designing MMAC protocols. The majority of solutions negotiate channel assignment every few packet transmissions on a default control channel. This design has several critical limitations. First, it incurs significant overhead due to the use of in-band or out-of-band control channels. Second, from a security standpoint, operating over a default control channel constitutes a single point of failure. A DoS attack on the control channel(s) would render all channels inoperable. Moreover, MMAC protocols are vulnerable to misbehavior from malicious users who aim at monopolizing the network resources, or degrading the overall network performance. In this dissertation, we improve the security and spectral efficiency of channel access mechanisms in multi-channel wireless networks. In particular, we are concerned with MAC-layer misbehavior in multi-channel wireless networks. We show that selfish users can manipulate MAC-layer protocol parameters to gain an unfair share of network resources, while remaining undetected. We identify possible misbehavior at the MAC-layer, evaluate their impact on network performance, and develop corresponding detection and mitigation schemes that practically eliminate the misbehavior gains. We extend our misbehavior analysis to MAC protocols specifically designed for opportunistic access in cognitive radio networks. Such protocols implement additional tasks such as cooperative spectrum sensing and spectrum management. We then discuss corresponding countermeasures for detecting and mitigating these misbehavior. We further design a low-overhead multi-channel access protocol that enables the distributed coordination of channel access over orthogonal channels for devices using a single transceiver. Compared with prior art, our protocol eliminates inband and out-of-band control signaling, increases spatial channel reuse, and thus achieves significant higher throughput and lowers delay. Furthermore, we investigate DoS attacks launched against the channel access mechanism. We focus on reactive jamming attacks and show that most MMAC protocols are vulnerable to low-effort jamming due to the utilization of a default control channel. We extend our proposed MMAC protocol to combat jamming by implementing cryptographic interleaving at the PHY-layer, random channel switching, and switching according to cryptographically protected channel priority lists. Our results demonstrate that under high load conditions, the new protocol maintains communications despite the jammer's effort. Extensive simulations and experiments are conducted to evaluate the impact of the considered misbehaviors on network performance, and verify the validity of the proposed mechanisms.
ADVANCE PRACTICAL CHANNEL SIMULATORS FOR LEO SATELLITE CHANNELS WITH SELECTIVE FADING AND DOPPLER SHIFTSHaghdad, Mehdi; Feher, Kamilo; University of California Davis (International Foundation for Telemetering, 2001-10)Dynamic hardware and software schemes for trajectory based simulation of LEO satellite channel are presented and evaluated. The simulation models are based on the practical LEO satellite channels and change dynamically with the trajectory using the latitude and longitude of the LEO satellite as input. The hardware simulator is consisted of a trajectory based selective fade generator, a trajectory based Doppler shifter, trajectory based time shadowing simulator and a standard channel for addition of noise, ACI and CCI. A FQPSK modulated signal is passed through a trajectory based dynamic fade generator and the spectrum is distorted. Then the resulting signal is exposed to a trajectory based dynamic Doppler Shifter, simulating the passage of the satellite overhead. Then the proper AWGN, ACI or CCI is added to the signal. At the final stage the signal is passed through a trajectory based time Shadowing simulator. The software simulator is a dynamic real time simulator written in MatLab and its structure is similar to the hardware simulator.