Transient receptor potential vanilloid 2 channel (TRPV2) is a Ca2+-permeable ion channel that is highly expressed in leukocytes but is also present in skeletal and cardiac muscle and endocrine cells. The TRPV2 function is implicated in a number of physiological processes, including bacterial phagocytosis, pro-inflammatory cytokine production, cardiac hypertrophy, and cancer development. TRPV2 knockout mice exhibit a high incidence of perinatal mortality, arguing that the channel plays essential roles in physiology. Despite the importance of TRPV2 for normal homeostasis, the mechanisms that control TRPV2 gating in response to pharmacological agonists, heating, membrane stretch, bioactive lipids and reactive oxygen species (ROS) remain poorly understood. Here we demonstrate that TRPV2 is functionally expressed in microglia (i.e., ‘brain macrophages’) and the microglia-like BV-2 cell line, and demonstrate that the gating of an endogenous TRPV2-like conductance is positively modulated by the bacterial toxin lipopolysaccharide (LPS), which is known to cause pro-inflammatory (M1) activation and increase ROS production by NADPH oxidase. To determine how TRPV2 gating is modulated by ROS, we recorded single channel activity in inside-out patches excised from HEK-293 cells expressing GFP-rTRPV2. Unitary currents elicited by the TRPV2 agonist 2-aminophenyl borinate (2-APB) or cannabidiol (CBD) are linear in monovalent recording solutions and give rise to an estimated unitary conductance of ~100pS, which is similar to TRPV1 but significantly smaller than TRPV3. Intriguingly, we find that although TRPV2 is insensitive to ROS (in the form of exogenously applied H2O2) alone, apparent open probability is synergistically enhanced when H2O2 is applied together with CBD. We identify two intracellular Cys residues that are necessary for TRPV2 responses to H2O2 sensitivity and find that these residues are located close to one another, albeit in different subunits, in the TRPV2 structure, suggesting that ROS promote the formation of an inter-subunit disulfide bond that alters sensitivity to pharmacological agonists. We hypothesize that ROS-dependent modulation of TRPV2 activity may be an important contributor to pro-inflammatory activation of microglia underline central nervous system diseases and that TRPV2 antagonism could be a useful therapeutic strategy in the treatment of neuroinflammation.

Potassium ion channels play a key role in the generation and propagation of action potentials. The S4-S5 linker peptide (L45) is believed to be responsible for the anesthetic/alcohol response of voltage-gated K+ channels. We investigated this region to define the structural basis of 1-alkanol binding site in dShaw2 K+ channel. L45 peptides derived from dShaw2 and hKv3.4 K+ channel, which, if part of the complete channel, demonstrate different sensitivity to 1-alcohols. Specifically, dShaw2 is alcohol sensitive and hKv3.4 is alcohol resistant. Structural analysis of L45 with NMR and CD suggested a direct correlation between alpha-helicity and the inhibition of dShaw2 channel by 1-butanol. We used CD and NMR to determine the structure of L45 peptides in micelles and vesicles. We measured spin-lattice relaxation time (T1) and determined the location and surface accessibility of L45 in micelles. These experiments confirm that L45 of dShaw2 adopts an α-helical conformation, partially buried in the membrane and parallel to the surface. The binding and accumulation of rev proteins to an internal loop of RRE (rev responsive element) of unspliced mRNA precursors is a key step of propagation of human immunodeficiency (HIV) virus. Molecules that interfere with this process can be expected to show anti-HIV activity. Our work is based on an assumption that zinc fingers could compete with rev proteins, therefore impeding the life cycle of HIV and stopping its infection. We studied the influence of different cations, anions, and the concentration of salts and osmolytes on the binding affinity with Polyacrylamide Gel Electrophoresis (PAGE) and Isothermal Titration Calorimetry (ITC). We conclude that the types of anions and/or cations and their concentrations affect the enthalpy and entropy of the binding interacitons. Using a gel assay, we confirm that there are three products in RNA-Protein reaction, and both EDTA and salts (and their concentrations) in the gel or samples interfere with RNA-protein complex mobility.

The purpose of the present study is to present research data on the propagation of the southern spotted channel catfish.

The role of Ca++ in olfactory responses was investigated with inorganic and organic calcium channel antagonists. Electrophysiological responses to odorants were recorded from frog olfactory mucosa before and after aerosol application of different agents. Electroolfactogram responses were blocked by certain inorganic ions with the order of effectiveness Zn++ >Ln+++>Cd++>Ca++>Co++>Sr++>Mg++. Ba++ potentiated olfactory responses, and is known to potentiate calcium channel-mediated responses in other tissues. Certain local anesthetics which are thought to act through calcium channel blockade were inhibitory to olfactory responses, with the order of effectiveness being dibucaine>tetracaine>procaine. These data support the idea that Ca++ is involved in olfaction, perhaps acting as a current carrier and/or a second messenger. Preliminary experiments on channel localization were performed using a silicon-labeled amine. Attempts to localize the silicon label were inconclusive, although silicon was detected in the olfactory tissue.

Modern society relies heavily on communication networks that in turn rely on both wired and wireless infrastructure. This work pertains to scenarios where a group of people or robots need to communicate in an environment where there is no preexisting communications infrastructure. These include sites of emergencies and disasters (e.g., inside burning buildings, search and rescue operations) and unexplored areas on Earth and other planets. Wireless ad hoc or mesh networks offer the ability to keep such entities connected, but they falter when any single entity wishes to leave the developed coverage area. Utilizing mobile repeater nodes can help, but is costly and complicated. By eliminating the need for repeater nodes to traverse the environment, their size and cost can be vastly reduced. This work explores the use of static "breadcrumb" repeater nodes to increase the reach of such a network. Determining when and where to place a static repeater node can be difficult in an environment where radio propagation characteristics are unknown. In this work, several algorithms for node placement are compared under the constraint that placement of a static repeater node should not dictate the entity's movement. The algorithms investigated range from calculating rolling averages to modeling channel parameters on-the-fly. The placement algorithms were configured to run in real-time on TP-Link MR-3040 portable WiFi routers and the approach is demonstrated in an outdoor uncharacterized environment.

This thesis extensively analyses the performance of an energy detector which is widely employed to perform spectrum sensing in cognitive radio over different generalised channel models. In this analysis, both the average probability of detection and the average area under the receiver operating characteristic curve (AUC) are derived using the probability density function of the received instantaneous signal to noise ratio (SNR). The performance of energy detector over an ŋ --- µ fading, which is used to model the Non-line-of-sight (NLoS) communication scenarios is provided. Then, the behaviour of the energy detector over к --- µ shadowed fading channel, which is a composite of generalized multipath/shadowing fading channel to model the lineof- sight (LoS) communication medium is investigated. The analysis of the energy detector over both ŋ --- µ and к --- µ shadowed fading channels are then extended to include maximal ratio combining (MRC), square law combining (SLC) and square law selection (SLS) with independent and non-identically (i:n:d) diversity branches. To overcome the problem of mathematical intractability in analysing the energy detector over i:n:d composite fading channels with MRC and selection combining (SC), two different unified statistical properties models for the sum and the maximum of mixture gamma (MG) variates are derived. The first model is limited by the value of the shadowing severity index, which should be an integer number and has been employed to study the performance of energy detector over composite α --- µ /gamma fading channel. This channel is proposed to represent the non-linear prorogation environment. On the other side, the second model is general and has been utilised to analyse the behaviour of energy detector over composite ŋ --- µ /gamma fading channel. Finally, a special filter-bank transform which is called slantlet packet transform (SPT) is developed and used to estimate the uncertain noise power. Moreover, signal denoising based on hybrid slantlet transform (HST) is employed to reduce the noise impact on the performance of energy detector. The combined SPT-HST approach improves the detection capability of energy detector with 97% and reduces the total computational complexity by nearly 19% in comparison with previously implemented work using filter-bank transforms. The aforementioned percentages are measured at specific SNR, number of selected samples and levels of signal decomposition.

Biological and bio-inspired systems using ion transport across a membrane for energy conversion has inspired recent developments in smart materials. The active mechanism in bioderived materials is ion transport across an impermeable membrane that converts electrochemical gradients into electrical and mechanical work. In addition to bioderived materials, ion transport phenomenon in electroactive polymers such as ionomeric and conducting polymers produces electromechanical coupling in these materials. Inspired by the similarity in transduction mechanism, this thesis focuses on integrating the ion transport processes in a bioderived material and a conducting polymer for developing novel actuation systems. The integrated membrane has a bilayer lipid membrane (BLM) formed on a conducting polymer, and the proteins reconstituted in the BLM regulate ion transport into the conducting polymer. The properties of the polymer layer in the integrated device are regulated through a control signal applied to the bioderived layer and hence the hybrid membrane resembles an ionic transistor. Due to the bioderived nature of this device, it is referred to as a ‘bioderived ionic transistor’. The research carried out in this thesis will demonstrate the fabrication, characterization and design limitations for fabricating a chemoelectromechanical actuator using the BIT membrane. The BIT membrane has been fabricated using BLM (DPhPC) reconstituted with protein (alamethicin) to gate Na$^+$ transport into conducting polymer membrane (PPy(DBS)). In this membrane, the bioderived layer is fabricated with proteins by vesicle fusion method and conducting polymer is fabricated by electropolymerization. The bioderived layers, the conducting polymer layers and the hybrid membrane are characterized using electrochemical measurements such as cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy. The fabrication, characterization and design effort presented in this thesis focuses on the integration of ion transport through the bioderived membrane into volumetric expansion and bending actuation. The characterization efforts are supported by empirical and physics-based models to represent the input-output relationship for both PPy(DBS) actuator and bioderived membrane, and design rules for the proposed actuation platforms are specified. The electropolymerized PPy(DBS) actuator is anticipated to be used in a bicameral device with the chambers kept separated by the DPhPC-alamethicin bioderived membrane. The relationship between the gradient potential, ionic current through the gate, ion concentration, ion transport coefficient in the conducting polymer layer, and the induced tip displacement in the polymer has been concluded from experiments and fitted to the actuation system model. This thesis will also address future directions for this research and anticipated applications for this hybrid actuation concept, such as artificial muscle, drug delivery.

Activation of the intrinsic aqueous water-wire proton conductance (GAQ) in Hv1 channels is controlled by changes in membrane potential and the transmembrane pH gradient (ΔpH). The mechanism by which changes in ΔpH affect the apparent voltage dependence of GAQ activation is not understood. In order to measure voltage sensor (VS) activation in Hv1, we mutated a conserved Arg residue in the fourth helical segment (S4) to His and measured H+ currents under whole-cell voltage clamp in transfected HEK-293 cells. Consistent with previous studies in VS domain containing proteins, we find that Hv1 R205H mediates a robust resting-state H+ ‘shuttle’ conductance (GSH) at negative membrane potentials. Voltage-dependent GSH gating is measured at more negative voltages than the activation GAQ, indicating that VS activation is thermodynamically distinct from opening of the intrinsic H+ permeation pathway. A hallmark biophysical feature of Hv1 channels is a ~-40 mV/pH unit shift in the apparent voltage dependence of GAQ gating. We show here that changes pHO are sufficient to cause similar shifts in GSH gating, indicating that GAQ inherits its pH dependence from an early step in the Hv1 activation pathway. Furthermore, we show for the first time that Hv1 channels manifest a form of electromechanical coupling VS activation and GAQ pore opening. Second-site mutations of D185 markedly alter GAQ gating without affecting GSH gating, indicating that D185 is required for a late step in the activation pathway that controls opening of the aqueous H+ permeation pathway. In summary, this work demonstrates that the Hv1 activation pathway contains multiple transitions with distinct voltage and pH dependencies that have not been previously identified. The results reported here novel insight into the mechanism of VS activation in Hv1 and raise fundamental questions about the nature of pH-dependent gating and electromechanical coupling in related VS domain-containing ion channels and phosphatases.

Inwardly rectifying potassium (Kir) channels are critically important for regulating resting membrane potential in excitable cells, a job underscored by the severe pathophysiology associated with channel dysfunction. While all Kir channels require the activating lipid PIP2, many of these channels have diverse modulatory factors that couple to PIP2-dependent gating. Channels in the Kir3 (GIRK) family, in particular, have several co-activating elements, including G-protein betagamma subunits, ethanol, and sodium. During stimulation of Gq-coupled receptors, downstream activation of Protein Kinase C can phosphorylate and inhibit Kir3 channels, yet the mechanism of inhibition and phosphorylation sites are incompletely understood. We took a combined experimental and computational approach using neuronal Kir3.2 to investigate how phosphorylation at a putative PKC site identified in Kir3.1/3.4 could lead to channel inhibition. Kir3.2 inhibition was found to depend on the phosphorylation state of Ser-196, although mutagenesis data suggest it functions as an allosteric regulator of PKC inhibition. MD simulations identified a molecular switch whereby phosphorylation of Ser-196 recruits a critical gating residue, Arg-201, away from the sodium coordination site Asp-228. Neutralization of Ser-196 or Arg-201 resulted in less active channels which exhibited increased sensitivity to PKC inhibition. Additionally the interplay of PIP2 and PKC inhibition was examined in depth using homomeric Kir3.2, revealing that increases in channel-PIP2 interactions limit sensitivity to PKC inhibition, whereas low levels of PIP2 increase PKC sensitivity. Neutralization of Ser-196 uncoupled PKC inhibition from this PIP2 dependence. These studies suggest a model whereby PKC inhibition can occur along PIP2-dependent and PIP2-independent pathways, depending on the phosphorylation state of Ser-196.

During the traditional manufacturing route, there are large amount of titanium alloys wasted in the form of machining chips. The conventional recycling methods require high energy consumption and capital cost. Equal channel angular pressing (ECAP), one of the severe plastic deformation techniques, has been developed to recycle the metallic machining chips. The purpose of the PhD work is to realize the ECAP recycling of titanium alloys, in particular Ti-6Al-4V and Ti-15V-3Cr-3Al-3Sn, and investigate the effects of processing parameters on the resultant relative density, microstructure evolution, texture development and microhardness homogeneity. The microstructures of Ti-6Al-4V and Ti-15V-3Cr-3Al-3Sn machining chips obtained from conventional turning (CT) and ultrasonically assisted turning (UAT) were initially investigated. It was found that ultrafine grains were formed in the primary and secondary shear zones. For Ti-6Al-4V chips, the β phase in the shear zones was refined into nano-sized equiaxed grains and aligned up to form banded structures. For Ti-15V-3Cr-3Al-3Sn chips, the nano-crystalline grains were enveloped in the shear zones and have clear boundaries to the surrounding matrix. It was observed that in terms of microstructure, there is no significant difference between CT and UAT chips. Recycling of Ti-6Al-4V machining chips was carried out at moderate temperatures with various back-pressures. For single-pass samples, the relative density was increased with the applied back-pressure and operating temperature. It was found that after multiple passes, near fully dense recycled Ti-6Al-4V can be fabricated. The microstructure observations showed that the nano-sized equiaxed and elongated grains co-existed with relatively coarser lamellar structures which were initially refined after the first pass. In the subsequent passes, the fraction of equiaxed nano-grains increased with the number of passes. The original β phase banded structures were fragmented into individual nano-sized grains randomly distributed within α matrix. The chip boundaries were eliminated and nano-crystalline microstructure region was observed at the chip/chip interface after multiple passes. In the sample processed at 550 °C, < a →+c → > type dislocations were observed and oxide layer at chip/chip interface was detected. The texture evolution was investigated using electron backscatter diffraction. It was found that the recycled samples performed a strong basal texture along the normal to ECAP inclination direction after the first pass. After multiple passes, in addition to the normal to inclination direction, the recycled Ti-6Al-4V exhibits a basal texture towards the transverse direction. Microhardness mapping showed that the average hardness and degree of homogeneity were increased with number of passes, while the imposed back-pressure had little effect on the average value and homogeneity. Recycling of Ti-15V-3Cr-3Al-3Sn machining chips was implemented using similar ECAP conditions. The effects of processing parameters, such as back-pressure, operating temperature and number of passes, on the relative density were similar to those for Ti-6Al-4V. Microstructural characterization showed that equiaxed instead of needle shaped α precipitates formed in the β matrix due to the high dislocation density and sub-grain boundaries introduced during ECAP. In terms of microhardness, the maximum hardness was obtained at the specimen pressed at 450 °C. It was found that the applied back-pressure and number of passes enabled to improve the homogeneity, but had little effect on the average hardness.