This dissertation presents the development of a modeling scheme that is developed to model the membrane potentials and ion currents through a bilayer network system. The modeling platform builds off of work performed by Hodgkin and Huxley in modeling cell membrane potentials and ion currents with electrical circuits. This modeling platform is built specifically for cell mimics where individual aqueous volumes are separated by single bilayers like the droplet-interface-bilayer. Applied potentials in one of the aqueous volumes will propagate through the system creating membrane potentials across the bilayers of the system and ion currents through the membranes when proteins are incorporated to form pores or channels within the bilayers. The model design allows the system to be divided into individual nodes of single bilayers. The conductance properties of the proteins embedded within these bilayers are modeled and a finite element analysis scheme is used to form the system equations for all of the nodes. The system equation can be solved for the membrane potentials through the network and then solve for the ion currents through individual membranes in the system. A major part of this work is modeling the conductance of the proteins embedded within the bilayers. Some proteins embedded in bilayers open pores and channels through the bilayer in response to specific stimuli and allow ion currents to flow from one aqueous volume to an adjacent volume. Modeling examples of the conductance behavior of specific proteins are presented. The examples demonstrate aggregate conductance behavior of multiple embedded proteins in a single bilayer, and at examples where few proteins are embedded in the bilayer and the conductance comes from a single-channel or pore. The effect of ion gradients on the single channel conductance example is explored and those effects are included in the single-channel conductance model. Ultimately these conductance models are used with the system model to predict ion currents through a bilayer or through part of a bilayer network system. These modeling efforts provide a modeling tool that will assist engineers in designing bilayer network systems. / Ph. D.

Isoflavones have antioxidant activities in vivo, however, their antioxidative potential against oxidative stress initiated by exercise was not explored. The first study investigated the effect of high-genistin isoflavone (HGI) supplementation on erythrocyte antioxidant enzymes and tissues' thiobarbituric reactive substances (TBARS) in acutely exercised one-year old rats. All tissue genistein concentrations increased after exercise. Ingestion of HGI seemingly enhanced running time to exhaustion, and maintained glutathione peroxidase (GPx) and catalase (CAT) activities decreased due to exercise. The second study investigated the dose effect of HGI supplementation. Genistein concentrations were significantly higher (P<0.05) in tissues of rats fed the 1045 PPM HGI diet than in rats fed 522 or 209 PPM HGI diets and increased the glutathione (GSH)/total glutathione (TGSH) ratio (P<0.03). Reductions of the in vivo MDA concentrations (P<0.05) were observed only in the plasma of rats fed 522 and 1045 PPM HGI diets compared to those fed 0 PPM (-1.08, -0.82, and 0.03 mM, respectively). Therefore, isoflavones at 522-1045 PPM HGI diet have antioxidative effects in rats. The last two studies investigated the effect of isoflavone supplementation on the modulation of erythrocyte antioxidant enzyme activities, glutathione homeostasis, and other oxidative biomolecules in healthy young men undergoing 80%VO2pk exercise. In Study 3 exercise at 80%VO2pk increased oxidative stress which was best demonstrated by increased superoxide dismutase (SOD) activity (16.5%), GSH/TGSH ratio, in vivo MDA (12.6%), plasma uric acid (4.9%) and ferric reducing/antioxidant ability (FRAP) ( 7.8%). Therefore, 30 minutes 80% VO2pk exercise induced oxidative stress in moderately active college men. In study 4, four-week HGI supplementation produced plasma genistein and daidzein concentrations of 499 and 415 ng/ml, which were significantly increased to 633 and 539 ng/ml by exercise (P=0.04 and P=0.05). Isoflavones significantly decreased in vivo pre-exercise plasma MDA (P<0.05), increased pre-exercise blood TGSH (P=0.01) and pre-exercise erythrocyte SOD activity (P=0.0006), and maintained the decreased activities of GPx due to exercise at pre-exercise levels. Results demonstrated that isoflavones had antioxidant activity in vivo under normal physiological conditions in healthy young men. They also maintaining GPx activity which was decreased due to exercise, however, isoflavones may not overcome all oxidative stress initiated by intense exercise. / Ph. D.

This dissertation presents the development of a new form of biomolecular material system which features interface lipid bilayers capable of hosting a wide variety of natural and engineered proteins. This research builds on the droplet interface bilayer (DIB) platform which first demonstrated that, through self-assembly, lipid-encased water droplets submersed in oil can be physically connected to form a liquid-supported lipid bilayer at the droplet interface. Key advantages of the DIB method over previous bilayer formation techniques include the lack of a supporting substrate which simplifies bilayer formation and the ability to connect many droplets to form `cell-inspired' networks which can provide a collective utility based on the compositions and arrangement of the droplets. The research present herein specifically seeks to overcome three limitations of the original droplet interface bilayer: limited portability due to lack of droplet support, the use of externally supported electrodes to electrically probe the network, and the requirement that in order to form DIB networks, aqueous volumes must be individually dispensed and arranged. The approach presented in this document is to provide increased interactions between the contained liquid phases and a supporting substrate in order to achieve both increased usability through refined methods of packaging and in situ interface formation which eliminates the need to create individual droplets. Physical encapsulation is defined as the the use of a solid substrate to contain both liquid phases such that the aqueous volumes are physically supported on one length scale (10-1000µm) while not inhibiting the self-assembly of phospholipids at the oil/water interface occurring on a much smaller length scale (1-10nm). Physically-encapsulated droplet interface bilayers are achieved by connecting lipid-encased droplets within a substrate that tightly confines the positions of neighboring droplets. A term called the packing factor is introduced to quantify the ratio of the aqueous volumes per the total compartment volume. Physically-encapsulated droplet interface bilayers formed in high packing factor substrate (30%) that also features integrated electrodes demonstrate all of the properties that unencapsulated DIBs exhibit (electrical resistances greater than 1GΩ, failure potentials between |200-300|mV, and the ability to host transmembrane proteins) but these confined assemblies can be moved, shaken, and even completely inverted. Additionally, a structured experiment to quantify the durability of interface bilayers shows that encapsulated and unencapsulated droplet interface bilayers can both survive 3-7g of lateral acceleration prior to bilayer failure, but have different modes of failure. Encapsulated DIBs tend to rupture, while unencapsulated DIBs completely separate. Physical encapsulation is also shown to permit the in situ formation of durable interface bilayers when the substrate is made from a flexible material. The importance of this approach stems from the fact that, by using the substrate to locally partition a single aqueous volume into multiple volumes, there is no need to arrange individual droplets. This method of bilayer formation is termed the regulated attachment method (RAM), since the separation and subsequent reattachment of the aqueous volumes is regulated by the opening and closing of an aperture within the flexible substrate. In this dissertation, a mechanical force is used to directly modulate the aperture dimension for controlling both the initial formation and final size of the interface. With the demonstrated advantages of portability and controlled attachment offered by physical encapsulation, encapsulated lipid bilayers are formed within a completely sealed flexible substrate. A key aspect of this final work is to demonstrate that both the organic and aqueous phases can be stabilized internally, creating a complete material system that features tailorable interface bilayers. / Ph. D.

The decline of many freshwater mussel populations in the United States has brought about the need for facilities in which mussels can be held for purposes of relocation, research, and propagation. The Aquatic Wildlife Conservation Center (AWCC) of the Virginia Department of Game and Inland Fisheries (VDGIF) serves as a freshwater mussel conservation facility in southwest Virginia. The goals of this study were: (1) to determine whether adult freshwater mussels could maintain energy reserves at AWCC (2) to determine whether adults could produce mature gametes at AWCC and (3) to establish suitable rearing conditions for juvenile mussels at the AWCC. In fall 2002, four species of mussels, Villosa iris, V. vanuxemensis, Amblema plicata, and Pleurobema oviforme, served as surrogates for endangered species and were relocated to the AWCC. Three energy reserves (glycogen, protein, and lipid) were measured seasonally (fall 2002 to summer 2004) from mantle tissue and compared between AWCC specimens and those from their wild source populations. The gametogenic stage of each species was also compared to determine whether gametogenesis was occurring in captivity. In summer 2003, the first of two juvenile experiments tested the effects of three rates of water flow (1 L/min, 3 L/min, and 7 L/min) on the survival and growth of V. iris and Epioblasma capsaeformis reared in flow-through troughs. In summer 2004, round flow-through tanks were used to assess the effects of three sizes of substrate (fine sediment, fine sand, and coarse sand) and sampling frequency on the survival and growth of V. iris. Gut content analyses also were conducted at the end of each experiment to determine which algal species were being consumed. Overall survival rates were as follows: A. plicata, 100 %; V. vanuxemensis, 86 %; V. iris, 79 %; P. oviforme (2002 collection), 53 %; and P. oviforme (2003 collection), 50 %. All energy reserves varied among seasons, but every species except P. oviforme (2003 collection) had levels higher than those in source populations at the end of this experiment. Glycogen appeared to be the best indicator of condition in these species, with protein also being important in the 2003 collection of P. oviforme. Mature gametes were found in all four captive species in 2003 and 2004, with lipids appearing to fuel gametogenesis. Additionally, gametogenesis was occurring earlier in captive long-term brooders than in the wild, possibly due to warmer water temperatures at AWCC. The first juvenile experiment resulted in 15 % overall survival, with 1 L/min having the greatest survival (18 %), and the 3 L/min having the greatest growth (656 μm). In the second experiment, dishes left unsampled had significantly greater survival (40 %) (P<0.05) of juveniles than those which were sampled (27 %). The unsampled fine sand treatment had significantly greater survival than the other two unsampled treatments (52 %) (P<0.001). Sampled juveniles in fine sediment had the greatest growth (887 μm). Also, juveniles from Experiment 1 were consuming primarily Navicula, with Coelastrum and Chlorella consumed in greatest abundance in Experiment 2. Results indicate that most adult mussels maintained energy reserves and produced mature gametes, and that juveniles of V. iris had good survival and growth. Only P. oviforme had survival rates lower than expected and did not appear to maintain condition at AWCC. Based on results of the species tested, environmental conditions at AWCC appear suitable for the survival of most adult and juvenile freshwater mussels. / Master of Science

Anyone who has eaten spicy foods has experienced the adverse effects of capsaicin, the pungent chemical found in hot chili that causes a burning sensation. The specific action of capsaicin occurs by the activation of receptors in sensory neurons. This thesis investigates the interaction of capsaicin with model cell membranes representing the structure of neurons. In particular, we are interested in the changes induced by capsaicin to the structure and dynamics of membranes. Molecular dynamics simulations are used to study the molecular interactions. The first part of this study evaluates different molecular representations for capsaicin in an 1-octanol/water system. This inhomogeneous system is commonly used to determine the partition of compounds between hydrophilic and hydrophobic environments, as that found in biological membranes. The results of these simulations validate the OPLS united-atom force field as a reasonable molecular representation of capsaicin, as it describes the behavior of capsaicin both quantitatively and qualitatively in 1-octanol/water mixtures. In the second part, simulations are performed for capsaicin and model cell membranes consisting of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine, two of the most commonly found lipids. Simulations investigated capsaicin in the aqueous and lipid phases. The results provide insight into the changes to the bilayers caused by capsaicin. Bilayers containing dipalmitoylphosphatidylethanolamine showed lower permeabilities to capsaicin than those composed of pure dipalmitoylphosphatidylcholine. Temperature is found to be an important factor in the permeability of capsaicin in the bilayer. Capsaicin in the bilayer concentrated in a region beneath the lipid/water interface, in which favorable hydrophilic and lipophilic interactions occur. The structure of the bilayer is not significantly changed at the concentrations of capsaicin considered. One important result from the simulations indicates that the interfacial density decreases with increasing capsaicin concentration in the bilayer, supporting the experimental observations of increased permeability in bilayers exposed to capsaicin. / Master of Science

Active lipid metabolism is a key process required for the intra-erythrocytic development of the malaria parasite, Plasmodium falciparum. Enzymes that hydrolyze host-derived lipids play key roles in parasite growth, virulence, differentiation, cell-signaling and hemozoin formation. Therefore, investigating enzymes involved in lipid degradation could uncover novel drug targets. We have identified in P. falciparum, a glycerophosphodiester phosphodiesterase (PfGDPD), involved in the downstream pathway of phosphatidylcholine degradation. PfGDPD hydrolyzes deacylated phospholipids, glycerophosphodiesters to glycerol-3-phosphate and choline. In this study, we have characterized PfGDPD using bioinformatics, biochemical and genetic approaches. Knockout experiments showed a requirement for PfGDPD for parasite survival. Sequence analysis revealed PfGDPD possesses the unique GDPD insertion domain sharing a cluster of conserved residues present in other GDPD homologues. We generated yellow fluorescent fusion proteins that revealed a complex distribution of PfGDPD within the parasite cytosol, parasitophorous vacuole and food vacuole. To gain insight into the role of PfGDPD, sub-cellular localization was modulated and resulted in a shift in protein distribution, which elicited no growth phenotype. Kinetic analyses suggest PfGDPD activity is Mg₂⁺ dependent and catalytically efficient at the neutral pH environment of the parasitophorous vacuole. Next, our aim was to determine the upstream pathway that provides deacylated glycerophosphodiesters as substrate for PfGDPD. We identified via bioinformatics, a P. falciparum lysophospholipase (PfLPL1) that directly generates the substrate. Knockout clones were generated and genotyped by Southern and PCR analysis. The effects of PfLPL1 knockouts on parasite fitness were studied, and the results showed that PfLPL1was not required for parasite survival and proliferation. / Ph. D.

Alzheimer's disease is the leading cause of senile dementia in the elderly, and as life expectancy increases across the globe, incidence of the disease is continually increasing. Current estimates place the number of cases at 25-30 million worldwide, with more than 5.4 million of these occurring in the United States. While the exact cause of the disease remains a mystery, it has become clear that the amyloid β-peptide (Aβ) is central to disease pathogenesis. The aggregation and deposition of this peptide in the brain is known to give rise to the hallmark lesions associated with Alzheimer's disease, but its exact mechanism of toxicity remains largely uncharacterized. Molecular dynamics (MD) simulations have achieved great success in exploring molecular events with atomic resolution, predicting and explaining phenomena that are otherwise obscured from even the most sensitive experimental techniques. Due to the difficulty of obtaining high-quality structural data of Aβ and its toxic assemblies, MD simulations can be an especially useful tool in understanding the progression of Alzheimer's disease on a molecular level. The work contained herein describes the interactions of Aβ monomers and oligomers with lipid bilayers to understand the mechanism by which Aβ exerts its toxicity. Also explored is the mechanism by which flavonoid antioxidants may prevent Aβ self-association and destabilize toxic aggregates, providing insight into the chemical features that give rise to this therapeutic effect. / Ph. D.

Within the vestibular system are otolith organs, both the utricle and saccule. The primary function of these organs is to transduce linear head accelerations and static head tilts into afferent signals that are sent to the central nervous system for the utilization of image fixation, muscle posture control, and the coordination of musculoskeletal movement in dynamic body motion. The utricle of the red ear slider turtle was studied in this dissertation. The turtle's utricle is composed of several layers. The base layer contains a set of neural receptor cells, called hair cells, and supporting cells. The three layers above the base layer compose the utricle's otoconial membrane (OM) and are: 1.) a saccharide gelatinous layer, 2.) a column filament layer, and 3.) a calcite and aragonite otoconial crystal layer. The primary goal of this research was to study the dynamic response of the turtle's OM to a variety of natural inertial stimuli in order to characterize its inherent mechanical properties of natural frequency ("n), damping ("), and shear modulus (G). The medial-lateral (ML) and anterior-posterior (AP) anatomical axes parameters were measured for the utricle. The ML axis median with 95% confidence intervals was found to be "n = 374 (353, 396) Hz, " = 0.50 (0.47, 0.53), and G = 9.42 (8.36, 10.49) Pa. The AP axis median with 95% confidence intervals was found to be "n = 409 (390, 430) Hz, " = 0.53 (0.48, 0.57), and G = 11.31 (10.21, 12.41). Nonlinearites were not found to occur in the OM for the tested inertial stimuli and no significant difference was found between the mechanical properties for the ML and AP axes. Additionally, this research presents the initial steps to form a novel bio-inspired accelerometer based on the morphology of the utricle. The primary transducer element for this possible otolith organ inspired accelerometer design is a droplet interface bilayer (DIB). A DIB is a lipid bilayer that is formed when the interface of two aqueous droplets, that contain free-floating lipids, are joined. The aqueous droplets are suspended in a nonpolar environment (oil) and the oil/water interface forms a lipid monolayer. This research developed and used an experimental test setup to characterize the mechanoelectrical characteristics of a micron-sized DIB. This information, along with examples in the text, could be used to further design the aforementioned accelerometer. / Ph. D.

Heat stress (HS), a stress response in homeotherms mainly due to elevated ambient temperature and failure of effective heat dissipation, causes a substantial negative economic impact to livestock industry worldwide. Reduced feed intake, a typical phenomenon observed during HS, was thought to be the primary driver for the milk production loss. However, accumulating evidence indicates that HS influences animal metabolism and endocrine profiles independent of reduced feed intake. Previous studies comparing heat-stressed lactating cows with control group pair-fed (PF) to the intake of HS group but housed in thermoneutral conditions, in order to eliminate the confounding factors result from differentiated feed intakes, showed that HS increased circulating insulin and decreased plasma non-esterified fatty acid (NEFA) in lactating cow, the opposite responses typical of PF cohorts. Therefore, the present studies were performed in order to elucidate the mechanism(s) underlying these counterintuitive changes. In response to a glucose tolerance test (GTT), both HS and PF decreased whole body glucose disposal rate, a sign of insulin resistance. Only PF decreased skeletal muscle insulin sensitivity in terms of reduced protein kinase B (PKB/AKT) phosphorylation, a downstream protein of insulin receptor (IR), while HS group maintained similar intact insulin responsiveness in the liver and skeletal muscle as thermoneutral conditions. There was a global reduction in gene expression of the enzymes related to lipid metabolism in adipose tissue of heat-stressed cows. Similarly, β-adrenergic signaling, a major stimulator of lipid mobilization, was suppressed in terms of NEFA release response during a chronic epinephrine challenge in HS group. After the challenge, phosphorylations of cAMP-response element binding protein (CREB) and hormone sensitive lipase, both located downstream of β-adrenergic receptor, were decreased in HS, but not in thermoneutral conditions, another indicator of impaired adrenergic signaling. In contrast, IR and AKT phosphorylation were increased in HS conditions indicating insulin signaling may be elevated during HS in adipose. Collectively, HS reduces lipid mobilization and appears to favor glucose utilization via alterations of lipid metabolism and hormones signaling pathways. These unique alterations in HS might shed some light on developing counter-HS approaches in the future. / Ph. D.

This dissertation investigates the behavior of the Escherichia Coli mechanosensitive (MS) channel MscL, when incorporated within a droplet interface bilayer (DIB). The activity of MscL channels in an artificial DIB system is demonstrated for the first time in this document. The DIB represents a building block whose repetition can form the basis to a new class of smart materials. The corresponding stimuli-responsive properties can be controlled by the type of biomolecule incorporated into the lipid bilayer, which is in the heart of this material. In the past decade, many research groups have proven the capability of the DIB to host a wide collection of natural and engineered functional biomolecules. However, very little is known about the mechano-electrical transduction capabilities of the DIB. The research present herein specifically seeks to achieve three direct goals: 1) exploring the capabilities of the DIB to serve as a platform for mechano-electrical transduction through the incorporation of bacterial MscL channels, 2) understanding the physics of mechano-electrical transduction in the DIB through the development of theoretical models, and 3) using the developed science to regulate the response of the DIB to a mechanical stimulus. MscL channels, widely known as osmolyte release valves and fundamental elements of the bacterial cytoplasmic membrane, react to increased tension in the membrane. In the event of hypo-osmotic shocks, several channels residing in the membrane of a small cell can generate a massive permeability response to quickly release ions and small molecules, saving bacteria from lysis. Biophysically, MscL is well studied and characterized primarily through the prominent patch clamp technique. Reliable structural models explaining MscL's gating mechanism are proposed based on its homolog's crystal structure modeling, which lead to extensive experimentation. Under an applied tension of ~10 mN/m, the closed channel which consists of a tight bundle of transmembrane helices, transforms into a ring of greatly tilted helices forming an ~8 A water-filled conductive pore. It has also been established that the hydrophobicity of the tight gate, positioned at the intersection of the inner TM1 domains, determines the activation threshold of the channel. Correspondingly, it was found that by decreasing the hydrophobicity of the gate, the tension threshold could be lowered. This property of MscL made possible the design of various controllable valves, primarily for drug delivery purposes. For all the aforementioned properties and based on its fundamental role of translating cell membrane excessive tensions into electrophysiological activities, MscL makes a great fit as a mechanoelectrical transducer in DIBs. The approach presented in this document consists of increasing the tension in the lipid bilayer interface through the application of a dynamic mechanical stimulus. Therefore, a novel and simple experimental apparatus is assembled on an inverted microscope, consisting of two micropipettes (filled with PEG-DMA hydrogel) containing Ag/AgCl wires, a cylindrical oil reservoir glued on top of a thin acrylic sheet, and a piezoelectric oscillator actuator. By using this technique, dynamic tension can be applied by oscillating one droplet, producing deformation of both droplets and area changes of the DIB interface. The tension in the artificial membrane will cause the MS channels to gate, resulting in an increase in the conductance levels of the membrane. The increase in bilayer tension is found to be equal to the sum of increase in tensions in both contributing monolayers. Tension increase in the monolayers occurs due to an increase in surface area of the constant volume aqueous droplets supporting the bilayer. The results show that MS channels are able to gate under an applied dynamic tension. Interestingly, this work has demonstrated that both electrical potential and surface tension need to be controlled to initiate mechanoelectric coupling, a property previously not known for ion channels of this type. Gating events occur consistently at the peak compression, where the tension in the bilayer is maximal. In addition, the experiments show that no activity occurred at low amplitude oscillations (< 62.5um). These two findings basically present an initial proof that gating is occurring and is due to the mechanical excitation, not just a random artifact. The role of the applied potential is also highlighted in this study, where the results show that no gating happens at potentials lower that 80 mV. The third important observation is that the frequency of oscillation has an important impact of the gating probability, where no gating is seen at frequencies higher than 1 Hz or lower than 0.1 Hz. Each of the previous observations is addressed separately in this research. It was found that the range of frequencies to which MscL would respond to in a DIB could be widened by using asymmetrical sinusoidal signals to stimulate the droplets. By increasing the relaxation time and shorting the compression time, a change in the monolayer's surface area is achieved, thus higher tension increase in the bilayer. It was also found that a high membrane potential assists in the opening of MscL as the droplets are stimulated. This is due to the sensitivity of MscL to the polarity of the signal. By using the right polarity the channel could be regulated to become more susceptible to opening, even at tensions lower than the threshold. Finally, it was demonstrated, for the first time, that MscL would gate in asymmetric bilayers without the need to apply a high external potential. Asymmetric bilayers, which are usually composed from different lipids in each leaflet, generate an asymmetric potential at the membrane. This asymmetric potential is proven to be enough to cause MscL to gate in DIBs upon stimulation. / Ph. D.