Computational methods such as Molecular Dynamics (MD) simulations and Molecular Mechanics generalized Born surface area solvation (MM-GBSA) binding affinity calculations have been utilized to determine the binding modes and final binding affinities of small molecules that are known to interact with the heart sodium channel NaV1.5. Lidocaine, ranolazine, and flecainide are FDA approved arrhythmia drugs that are prescribed to patients in the event of heart disease. Here, we demonstrate the likely binding preferences and modes of action of all molecules with NaV1.5, the stability of the systems, and overall final binding affinities of the small molecules with the protein. To gain insights into the mechanisms of heart disease treatments, the MM-GBSA method was utilized to estimate the binding free energies of each molecule and pose to NaV1.5. The evaluation of the binding of small molecules to NaV1.5 contributes to enhancing our understanding of the underlying processes involved in heart disease treatments. The MM-GBSA approach provides a valuable tool for predicting and analyzing binding affinities, which can aid in the design and optimization of potential therapeutic compounds targeting NaV1.5.

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Voltage-dependent K+ channels can undergo a gating process known as C-type inactivation. This type of gating consists of entry into a nonconducting state that may involve conformational changes near the channel's selectivity filter. However, the details of the underlying mechanisms are not clear. Here, I report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K+ channel that lacks a canonical voltage sensor. In single-channel recordings, I observed that open probability (Po) decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca2+ or Ba2+, suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K+] (47 mV per 10-fold increase in [K+]), suggesting that K+ binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K+ ≈ Rb+ > Cs+ > Na+ > Li+ ≈ NMG+. Selectivity of the stabilizing site is somewhat weaker than that of sites that determine permeability of these ions, consistent with the idea that the site may lie toward the external end of the MthK selectivity filter. MthK gating was described over a wide range of positive voltages and external [K+] using kinetic schemes in which the open-conductive state is stabilized by K+ binding to a site that is not deep within the electric field, with the voltage-dependence of inactivation arising from both voltage-dependent K+ dissociation and transitions between nonconducting (inactivated) states. Studies of C-type inactivation in voltage-gated K+ channels have demonstrated that inactivation can be enhanced by quaternary ammonium (QA) derivatives, which block current through the channel by binding to a site at the cytoplasmic side of the pore. Enhancement of inactivation is thought to occur through a mechanism in which QA blockade leads to depletion of K+ ions in the pore, thus driving the channel toward the inactivated state. I tested this model by using divalent cations to block the current through the MthK channel, and then quantifying the effects on inactivation. I observed that the voltage-dependence of blockade by Ca2+, Mg2+, and Sr2+ was approximately equal (zδ ≈ 0.4 e0 for blockade by each of the divalent cations), suggesting a similar location for the site of blockade. However, Ca2+ and Sr2+ were found to enhance inactivation, whereas Mg2+ does not. Molecular dynamics (MD) simulations suggested that Ca2+ and Sr2+ bind to a site (S5) closer to the selectivity filter than Mg2+, consistent with the idea that binding of a divalent cation to S5 enhances inactivation; the bound cation may in turn electrostatically interact with K+ ions in the selectivity filter to break the K+ conduction cycle. Previous studies on inactivation in KcsA have identified a critical residue involved in the mechanism of C-type inactivation in this channel. This residue, E71, is located in a region known as the pore helix, and is involved in a hydrogen bonding network involving a tryptophan residue also in the pore helix, as well as an aspartic acid residue in the selectivity filter, which drives the channel toward the inactivated state. However, mutation to alanine breaks the hydrogen bonding network and effectively prevents inactivation. To determine whether a similar mechanism may enhance inactivation in MthK, I performed mutagenesis at the MthK residue analogous to KcsA E71 (V55). In single channel recordings, I observed that mutation to glutamate (V55E) destabilized the open state of the channel, consistent with the idea that a hydrogen bonding network that drives the channel toward the inactivated state may be formed in MthK to enhance inactivation, similar to the mechanism proposed for KcsA. These results, along with previous findings, suggest that inactivation gating is linked to the selectivity filter of the channel. In most K+ selective channels, the selectivity filter is composed of a sequence of highly-conserved residues (TVGYG). Within this sequence, the sidechain of the conserved threonine residue determines the entry to the selectivity filter, and may thus be a key regulator of the K+ conduction cycle. Interestingly, the rapidly inactivating voltage-gated K+ channel, HERG, contains a serine at this position instead of a threonine. To determine the impact of a change from threonine to serine, I quantified effects of the mutation T59S in MthK on conduction and inactivation, and further probed these effects using blockade by divalent cations. I observed that this mutation reduces channel conductance and enhances inactivation, compared to the wild type channel, and enhanced blockade by Sr2+. MD simulations suggested an increased energy barrier for K+ ions to enter the selectivity filter, which may account for the decreased conductance. In addition, the serine sidechain may effect a redistribution of K+ within the selectivity filter, which may impact stability of the conducting state. Overall, my results suggest that several mechanisms contribute to K+ channel inactivation, involving a combination of ion-ion interactions in the pore, structural interactions among residues in the selectivity filter that may affect the stability of the conducting state, and interactions between ions and a key sidechain at the entry to the selectivity filter. Further understanding of these components of the inactivation process may provide a clearer picture of the mechanisms that generate diversity in gating properties among K+ channels. / Biochemistry

<p> In this thesis we develop several approaches to the problem of blind channel equalization based on second-order statistics (808). We consider the single-input singleoutput (8180) system with minimum phase channel where the received signal is sampled at the symbol rate (T-spaced equalizer). We formulate the equalizer design criterion as a simple convex optimization problem, where the equalizer can be obtained efficiently avoiding the local minima problem. </p> <p> We also extend the problem to the single-input multiple-output (8IMO) systems where the received signal is sampled at an integer multiple of the symbol rate. We formulate the problem as a convex optimization problem using the features existing in the channel matrix structure. The problem can be solved efficiently to obtain the equalizer where a global minima is guaranteed. Moreover, we modify this formulation and deduce a closed form solution to the equalizer. Although both methods are sensitive to the channel order as well as existing subspace methods, they perform better than the subspace methods when the channel matrix is close to being singular. Furthermore, we propose an efficient direct minimum mean square error (MM8E) approach to estimate the equalizer. The method does not rely on the channel order and utilizes the channel matrix structure in SIMO systems. Therefore, it outperforms existing algorithms including the previously proposed methods. However, due to the large amount of computations involved in this method we present a new algorithm that belongs to the same class with moderate computational complexity and acceptable performance loss with respect to the latter algorithm. </p> / Thesis / Master of Applied Science (MASc)

The introduction of bio-molecules into cells and embryos is required in the fields of drug development, genetic engineering and in-vitro fertilization. It has been applied to create transgenic mammals and to improve pest and mold resistance in plants. However, the efficient transfection of materials still poses a problem, and a variety of techniques, broadly classified as biochemical and physical means, are actively being developed. One technique that is promising is capillary microinjection as it offers low cytotoxicity, targeted injections and high transfection efficiency. However, this process suffers from low throughput and variability as it is an operator mediated process. Other problems associated with capillary microinjection are limitations on the minimum needle size and variability in transfected volumes due to the use of pressure driven flow for injections. In this thesis we propose a device that employs microfluidic principles to enable cell microinjections in a 'lab on a chip' format and eliminates the problems associated with capillary microinjection. The device is fabricated using poly dimethylsiloxane (PDMS) rapid prototyping and features two separate channel structures-one to supply the targets and the other to supply the reagent. Integrated into the device are a microinjection capillary (10 μm tip diameter) and a suction capillary (0.5mm ID/1mm OD) which is used to immobilize the targets in the channel prior to injection. The actuation of the injection needle into the targets is achieved by the compliant deformation of the flexible PDMS substrate as a result of an externally applied displacement. This is made possible by the selective reinforcement of the PDMS substrate. From testing it was found that the effective needle actuation is 83.8% of the externally applied displacement. The injections occur in a planar configuration therefore providing precise control over the location of injection. Furthermore, the mechanism requires only one degree of freedom to perform injections, and therefore greatly simplifies existing injection techniques which require orientation in a three dimensional space. The limitations of the use of pressure driven flow for injections are overcome by performing reagent injection by electroosmotic flow, which is induced by applying a potential to electrodes embedded in the target and reagent supply channels. The applied potential induces electroosmotic flow through the embedded needle and into the injection target. This provides precise electrical dosage control. The flow rates were obtained by measuring the velocity of the interface between a neutral fluorescent marker and a clear pH 10 buffer solution. The obtained flow rates follow a predictable linear trend and correspond well to theory. The use of electroosmotic flow enables the use of smaller injection needles as it scales more favorably (r^-2) than pressure driven flow (r^-4) and becomes increasingly dominant in smaller dimensions. Present pressure microinjection systems are limited to injection needles with tip diameters larger than 0.2μm due to the high pressures required to dose at smaller dimensions. All components of the device are fully scalable and enable further miniaturization, multiple parallel injections and autonomous functionality. The device requires smaller volumes of samples and expensive reagents and also reduces the time required for performing injections. Overall, it device maintains the advantages of microinjection, while eliminating problems of low throughput, dosage control and restrictions on the injection needle size. The device was successfully used and characterized for the injection of single-cell Xenopus Laevis eggs and Zebrafish embryos. / Thesis / Master of Applied Science (MASc)

In 2005 an assessment of existing Total Maximum Daily Load studies by the U.S. Environmental Protection Agency showed sediment as the fourth leading cause of water quality impairment. A source assessment is important in developing a successful TMDL. Past research efforts have focused on controlling erosion sources in agricultural and urban land areas. New research suggests major contributions to overall sediment loads may be due to stream channel degradation. Monitoring and modeling techniques to assess the contribution of channel sediment to overall sediment load are needed to determine the reductions necessary to meet water quality standards. This research focused on testing the ability of watershed and reach-scale models to predict stream channel degradation. Model predictions were compared to estimates developed from a system of erosion pins and scour chains. A 500-m experimental reach in Blacksburg, VA, USA, was selected as the focus of channel degradation monitoring and modeling efforts. A series of over 250 erosion pins and seven scour chains were installed systematically throughout the experimental reach. A monthly monitoring program measured channel degradation for the period from July 2005 - June 2006. Point data were interpolated across individual bank segments to produce an estimate of soil erosion volume. Measured soil bulk densities were then used to calculate the estimated mass loading to Stroubles Creek from channel degradation. Two watershed models and one reach-scale model were developed to predict sediment loading to the stream channel from channel degradation. The Generalized Watershed Loading Function (GWLF) was selected to represent watershed models with limited channel degradation process detail; the Soil and Water Assessment Tool (SWAT) represented the level of channel degradation detail seen in the majority of watershed models; and the CONservation Channel Evolution and Pollutant Transport System (CONCEPTS) reach-scale model was used to evaluate the effectiveness of a detailed process model. Monthly model predictions were compared to retreat rates measured using the erosion pin network. Sediment loading to the stream from bank retreat was estimated as 41 tonnes/yr, based on erosion pin measurements. GWLF, SWAT, and CONCEPTS predicted stream channel sediment contributions of 8 tonnes/yr, 1500 tonnes/yr and 4 tonnes/yr, respectively. Theil-Sen non-parametric simple linear regression was used to test agreement between monthly model predictions and erosion pin estimates. No significant agreement was found between any model predictions and measured retreat, using a conservative a-value of 0.2. GWLF model predictions underpredicted measured channel degradation, but most closely approximated observed data. This result is likely due to similarities in climate and watershed characteristics for the Stroubles Creek watershed and the Pennsylvania watershed used in the empirical model development. SWAT predicted retreat rates exceeded measured values by two orders of magnitude. This result is explained by the inability of SWAT to predict daily flow and sediment discharge. Highly sensitive channel degradation parameters and the lack of calibration data also contributed to SWAT simulation error. CONCEPTS simulation predicted monthly retreat rates slightly less than GWLF. The lack of agreement between CONCEPTS simulation and observed data was mainly the result of limited input data availability. SWAT daily discharge predictions were used as CONCEPTS input data and likely contributed to poor model agreement. Poor estimation of sensitive sediment input parameters may have also contributed to underpredictions by CONCEPTS. Results showed the potential of screening-level watershed models in channel degradation prediction and the importance of flow and sediment time series discharge data in detailed process-based simulation. The limited flexibility of the GWLF channel degradation algorithm makes it unsuitable for evaluating the effects of stream restoration. SWAT and CONCEPTS should only be used for evaluation if appropriate input data are available. Future research will focus on the development of a long-term flow and sediment monitoring data set. Few long-term data sets of this nature exist, making channel degradation modeling difficult. Development of long-term data will allow more accurate modeling and better assessment of channel restoration impacts on channel degradation. Further modeling with GWLF in geographic regions outside the Eastern United States is also needed to determine the scope of applicability of the GWLF channel degradation empirical relationship. Additional research should also focus on the significance of subaerial processes for watersheds of various sizes and on the development of algorithms to simulate these processes. / Master of Science

In this thesis, an Interactive Video Data Service (IVDS) is developed. This service provides a mechanism for television viewers to interact with the program they are watching. Possible interactions include purchasing products from home shopping programs and requesting information from advertisers. Within the project, two areas were focused upon: channel simulation and the Repeater Unit. Additionally, the overall system was discussed along with its background. The purpose of channel simulation was to demonstrate the viability of the unique communication channel model proposed for the IVDS system. This channel was implemented using uni-directional transmissions, without acknowledgments. The Repeater Unit was designed to be a message processing system, intended to relay messages from system users to the home office. The design entailed both hardware and software. The hardware requirements were for a high level design, while the software required not only design, but implementation. / Master of Science

No / Lymphocytes have many ion channels. These ion channels contribute to T cell-mediated autoimmune and/or inflammatory responses and therefore are targets for pharmacological immune modulation [1]. Lymphocytes are also suitable surrogate cells for cancer [2] and other diseases states [3] where inflammation is associated with increasing disease incidence. Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, have been associated with anti-tumour effects in cancers [4]. We recently compared DNA damage caused by the nanoparticle forms (NPs) of the NSAIDs, aspirin and ibuprofen and their bulk forms in peripheral blood lymphocytes of patients with respiratory diseases and healthy individuals in the Comet and micronucleus assays [5]. In this present study, we investigate electrophysiological changes from lymphocytes after NP exposure and compare these results with their DNA damage. 10 ml peripheral blood was collected from patients and healthy control individuals. Ethical permission was obtained from the Bradford Ethics Committee REC ref no: 09/H1313/37, ReDA no: 1202, and the University of Bradford ref no: 0405/8. Ibuprofen USP was purchased from Albermarle Europe sprl (Belgium). Pharmcoat 606 (HPMC) was kindly donated by Shinetsu (Japan). Aspirin and sodium lauryl sulphate were purchased from Sigma. Kollidon 30 (PVP K-30) was purchased from BASF (UK). Bulk and nano compound suspensions of aspirin and ibuprofen (IBU) were kindly prepared by Lena Nanoceutics (Bradford, UK). Whole blood collected from healthy individuals and cancer patients were treated for 30 mins with 500µg/ml of IBU bulk and nano forms separately. Whole-cell currents were recorded with normal patch clamping technique. The extracellular solution contained (in mM) the following: NaCl 125; KCl 5; MgCl2 1; CaCl2 2.5; HEPES 10; pH 7.4. The electrode internal solution contained (in mM) the following: KF 120; MgCl2 2; HEPES 10; EGTA 10; and CaCl2 1, pH 7.4. All experiments were carried out at room temperature. Compared with untreated cells, lymphocytes treated with IBU in NP form had lower whole-cell currents and the activities of ion channels were inhibited by 20% compared to those in bulk form. This result is mirrored by the DNA damage which occurred in lymphocytes after exposure to nanoparticles [5]. Although the intracellular biochemical mechanisms and ion channels involved in our nanoparticle toxicity remain to be determined, this study provides direct evidence that 500 μg/ml IBU in nano form can cause membrane damage to lymphocytes after a relatively short exposure. Such cytotoxicity of nanoparticles in lymphocytes may be associated with early membrane damage. Further detailed investigation is needed to explain the changes of lymphocytes in response to different concentrations of NPs in real time. / Poster communications