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.

Due to the increasing need to provide refugia for freshwater mussels impacted by anthropogenic activities and exotic species, facilities should be identified and protocols developed for holding mussels in captivity. White Sulphur Springs National Fish Hatchery (WSSNFH), White Sulphur Springs, WV, has held freshwater mussels for nearly eight years, and has the potential to become an important refugium and propagation facility for conservation of mussels in the Ohio River Basin and elsewhere. The goal of this study was to determine the feasibility of holding adult freshwater mussels in long-term captivity at WSSNFH by evaluating survival, energy reserves, and gametogenesis of captive mussels in a recirculating pond system. I relocated three mussel species in the summer of 2001 and 10 mussel species in the summer of 2002 to a recirculating pond system (reservoir and raceway) at the hatchery. Water quality parameters of pH, alkalinity, hardness, temperature, and dissolved oxygen; and algal concentrations were measured periodically from summer 2001 to summer 2003. Annual survival rates of 10 species were estimated (August 2002 to August 2003) using the program MARK. Glycogen, protein, and lipid concentrations in mantle tissue of three captive species (Actinonaias ligamentina, Cyclonaias tuberculata, and Tritogonia verrucosa) were compared to those of wild mussels in the New River. Gametogenic activity and synchrony in A. ligamentina and C. tuberculata were compared between captive and wild mussels. Water quality parameters, with the exception of temperature, were within desirable ranges for most of the study. Temperatures of > 28° C were observed for several days during summers 2002 and 2003. Algal concentrations averaged 1903 cells ml-1 in the raceway (range: 300 to 4658 cells ml-1), which is comparable to algal concentrations reported for nearby rivers. The overall survival rate for 10 freshwater mussel species held in the raceway for one year was 77%. Villosa vanuxemensis had the highest survival rate (96%), and Lampsilis cardium had the lowest survival rate (31%). Although there were fluctuations in glycogen, protein, and lipid levels over 2 yr, there were no overall differences in energy substrates between captive and wild mussels at the end of the study. Captivity did not appear to have a negative affect on gametogenesis. Captive C. tuberculata spawned within the expected time frame between January and June, but slightly earlier than their wild counterparts in the New River. Due to the infestation of the gonads of both captive and wild A. ligamentina by digenean trematodes, little gametogenesis was observed. However, captive holding did not appear to have an effect on trematode infestation rates. From these results, I conclude that captive holding conditions in the recirculating pond system at WSSNFH were adequate for long-term holding of a wide range of mussel taxa. I recommend that WSSNFH continue to be used as an adult holding facility. Further research should be conducted to determine food and habitat preferences of freshwater mussel species in captivity so that optimal holding conditions can be provided for each species. / Master of Science

This research details the creation and characterization of a new biomimetic artificial inner hair cell sensor in an artificial cochlear environment. Designed to mimic the fluid flows around the inner hair cells of the human cochlea, the artificial cochlear environment produces controlled, linear sinusoidal fluid flows with frequencies between 25 and 400 Hz. The lipid bilayer-based artificial inner hair cell generates current through changes in the bilayer's capacitance. This capacitance change occurs as the sensor's artificial stereocilium transfers the force in the fluid flow to the bilayer. Frequency tuning tests are performed to characterize the artificial inner hair cell's response to a linear chirp signal from 1 to 400 Hz. The artificial inner hair cell's response peaks at a resonant frequency of approximately 83 Hz throughout most of the tests. Modelling the artificial stereocilium as a pinned free beam with a rotational spring at the pinned end yields a rotational spring stiffness of 177*10^-6 Nm/rad. Results with 0 mV potential applied across the bilayer indicate that current generation at 0 mV likely comes from other sources besides the bilayer. Increasing the voltage potential increases the broadband power output of the system, with an approximately linear relationship. A final test keeps the fluid flow frequency constant and varies the fluid velocity and applied voltage potential. Manipulation of the applied voltage potential results in a fluid velocity to RMS current relationship reminiscent of the variable sensitivity of the human cochlea. / Master of Science

No / Chemical means of manipulating the distribution of lipids across biological membranes is of considerable interest for many biomedical applications as a characteristic lipid distribution is vital for numerous cellular functions. Here we employ atomic-scale molecular simulations to shed light on the ability of certain amphiphilic compounds to promote lipid translocation (flip-flops) across membranes. We show that chemically induced lipid flip-flops are most likely pore-mediated: the actual flip-flop event is a very fast process (time scales of tens of nanoseconds) once a transient water defect has been induced by the amphiphilic chemical (dimethylsulfoxide in this instance). Our findings are consistent with available experimental observations and further emphasize the importance of transient membrane defects for chemical control of lipid distribution across cell membranes

No / Because of confounding factors, the effects of dietary n-3 polyunsaturated fatty acids (PUFA) on type 1 diabetes remain to be clarified. We therefore evaluated whether fat-1 transgenic mice, a well-controlled experimental model endogenously synthesizing n-3 PUFA, were protected against streptozotocin (STZ)-induced diabetes. We then aimed to elucidate the in vivo response at the pancreatic level. Beta-Cell destruction was produced by multiple low-doses STZ (MLD-STZ). Blood glucose level, plasma insulin level, and plasma lipid analysis were then performed. Pancreatic mRNA expression of cytokines, the monocyte chemoattractant protein, and GLUT2 were evaluated as well as pancreas nuclear factor (NF)-kB p65 and inhibitor of kB (IkB) protein expression. Insulin and cleaved caspase-3 immunostaining and lipidomic analysis were performed in the pancreas. STZ-induced fat-1 mice did not develop hyperglycemia compared with wild-type mice, and Beta-cell destruction was prevented as evidenced by lack of histological pancreatic damage or reduced insulin level. The prevention of Beta-cell destruction was associated with no proinflammatory cytokine induction (tumor necrosis factor-alpha, interleukin-1Beta, inducible nitric oxide synthase) in the pancreas, a decreased NF-kB, and increased IkB pancreatic protein expression. In the fat-1-treated mice, proinflammatory arachidonic-derived mediators as prostaglandin E2 and 12-hydroxyeicosatetraenoic acid were decreased and the anti-inflammatory lipoxin A4 was detected. Moreover, the 18-hydroxyeicosapentaenoic acid, precursor of the anti-inflammatory resolvin E1, was highly increased. Collectively, these findings indicate that fat-1 mice were protected against MLD-STZ-induced diabetes and pointed out for the first time in vivo the beneficial effects of n-3 PUFA at the pancreatic level, on each step of the development of the pathology-inflammation, Beta-cell damage-through cytokine response and lipid mediator production.

Yes / The current study was designed to investigate the influence of monofloral Indian mustard bee pollen (MIMBP) and processed monofloral Indian mustard bee pollen (PMIMBP) supplementation on chronic swimming exercise-induced oxidative stress implications in the gastrocnemius muscle of Wistar rats. MIMBP was processed with an edible lipid-surfactant mixture (Captex 355:Tween 80) to increase the extraction of polyphenols and flavonoid aglycones as analyzed by UV spectroscopy and high performance liquid chromatography-photo diode array. Wistar rats in different groups were fed with MIMBP or PMIMBP supplements at a dose of 100 mg/kg, 200 mg/kg and 300 mg/kg individually, while being subjected to chronic swimming exercise for 4 weeks (5 d/wk). Various biochemical [superoxide dismutase (SOD), glutathione (GSH), malonaldehyde (MDA), nitric oxide (NO), and total protein content], mitochondrial (Complex I, II, III, and IV enzyme activity), and molecular (myostatin mRNA expression) parameters were monitored in the gastrocnemius muscle of each group. Administration of both MIMBP (300 mg/kg) and PMIMBP (100 mg/kg, 200 mg/kg, and 300 mg/kg) wielded an antioxidant effect by significantly improving SOD, GSH, MDA, NO, and total protein levels. Further MIMBP (300 mg/kg) and PMIMBP (200 mg/kg and 300 mg/kg) significantly improved impaired mitochondrial Complex I, II, III, and IV enzyme activity. Significant down-regulation of myostatin mRNA expression by MIMBP (300 mg/kg) and PMIMBP (200 mg/kg and 300 mg/kg) indicates a muscle protectant role in oxidative stress conditions. The study establishes the antioxidant, mitochondrial upregulatory, and myostatin inhibitory effects of both MIMBP and PMIMBP in exercise-induced oxidative stress conditions, suggesting their usefulness in effective management of exercise-induced muscular stress. Further, processing of MIMBP with an edible lipid-surfactant mixture was found to improve the therapeutic efficiency of pollen.

Yes / The hydrophobicity of a drug can be a major challenge in its development and prevents the clinical translation of highly potent anti-cancer agents. We have used a lipid-based nanoemulsion termed Lipid-Oil-Nanodroplets (LONDs) for the encapsulation and in vivo delivery of the poorly bioavailable Combretastatin A4 (CA4). Drug delivery with CA4 LONDs was assessed in a xenograft model of colorectal cancer. LC-MS/MS analysis revealed that CA4 LONDs, administered at a drug dose four times lower than drug control, achieved equivalent concentrations of CA4 intratumorally. We then attached CA4 LONDs to microbubbles (MBs) and targeted this construct to VEGFR2. A reduction in tumor perfusion was observed in CA4 LONDs-MBs treated tumors. A combination study with irinotecan demonstrated a greater reduction in tumor growth and perfusion (P = 0.01) compared to irinotecan alone. This study suggests that LONDs, either alone or attached to targeted MBs, have the potential to significantly enhance tumor-specific hydrophobic drug delivery. / The work was funded by the Medical Research Council (grant number: MR/L01629X MRC Medical Bioinformatics Centre) and the EPSRC (grant number EP/P023266/1 Health Impact Partnership). EPSRC (EP/I000623/1, EP/K023845/1). Laura E. McVeigh was funded by an EPSRC PhD Studentship (EP/L504993/1).

Three types of dried milk (whole, nonfat, and buttermilk) were packaged in a modified atmosphere with a novel palladium-based oxygen removing catalyst and stored for eight weeks at 50°C. Powders stored in air with no catalyst and powders stored with the catalyst in an atmosphere modified to contain 5.7% hydrogen in nitrogen were evaluated by instrumental, chemical, and sensory methods. Hexanal concentrations were measured weekly using solid phase microextraction (SPME) and gas chromatography (GC) to compare the degrees of oxidation in the powders stored with the catalyst to those stored without it. Color changes were also monitored weekly using Hunter's L-, a-, and b-values. At the end of the eight-week period, a paired comparison sensory test was used to ascertain if the catalyst had an effect on odor. Anisidine values were also measured at this point to determine levels of oxidation in the powders. No significant difference was found in levels of oxidation between samples packaged with and without the catalyst in the modified atmosphere. At the end of eight weeks, the average hexanal concentration in the whole milk powder stored with the oxygen scavenger was 1.19 ± 0.20 ppm, while the average hexanal concentration in the air-packed whole milk powder was 1.06 ± 0.08 ppm. The average hexanal concentrations for the buttermilk stored with the catalyst and without were 0.84 ± 0.18 and 0.79 ± 0.15 ppm, respectively. In the nonfat milk powder, the sample stored with the catalyst had an average hexanal concentration of 0.91 ± 0.14 ppm and the sample stored in air without the catalyst had an average hexanal concentration of 0.83 ±0.20 ppm. Difference testing by volunteer sensory panelists also revealed no significant differences. It was expected that the milk powders stored with the catalyst in the modified atmosphere would have lower levels of oxidation and off-odors at the end of the eight weeks. However, the treatment ultimately resulted in no chemical or sensory differences. Thus, the catalyst proved ineffective in the given conditions. This could be due to a loss of the hydrogen required for the catalyst to function as time progressed or a lack of significant oxidation under the conditions employed. / Master of Science in Life Sciences

The formation of lipid bilayer membranes between droplets of ionic liquid is presented as a means of forming functional bimolecular networks for use in sensor applications. Ionic liquids are salts that have a number of useful properties, such as low melting points making them liquid at room temperature and exceedingly low vapor pressure. Ionic liquids have seen recent popularity as environmentally friendly industrial solvent alternatives. Our research demonstrates that it is possible to consistently form lipid bilayers between droplets of ionic liquid solutions. Analysis shows that the ionic liquids have negligible effects on the physical stability and electrical properties of the bilayer. It is also shown that the magnitude of the conductance levels of Alamethicin peptide are altered by some ionic liquids. / Master of Science

Triglycerides are the majority lipid component of most biochemical mixtures and are virtually water insoluble. Lipid removal is desired prior to protein purification processing to decrease nonspecific fouling of downstream chromatographic matrices. Transgenic pig milk was used as a model system to study delipidation from therapeutic protein sources. The majority of triglycerides was extracted from stable lipid micelles and removed with a method that can be incorporated in downstream protein purification processing without denaturing the target protein. An efficient delipidation treatment used TNBP, a non-polar solvent, to extract lipid micelles and then phase transfer milk lipids into a TNBP-swelled dextran particulate. A batch incubation of a whey/TNBP mixture with pre-swollen Sephadex LH-20 or hydroxyalkoxypropyl dextran (HAPD) beads at 4 C for 24 hours removed 67 + 2 % (0.645 mg triglycerides/ml Sephadex LH-20) and 71 o + 1 % (0.628 mg triglycerides/ml HAPD) of the triglycerides present in the skimmed transgenic whey, respectively. Fully swollen beads removed 20% more triglycerides than beads which were wetted but not swollen in TNBP, indicating that a larger phase volume and internal adsorption of the lipids onto the Sephadex matrix dominates over surface adsorption. Polyclonal ELISAs indicated that 89 + 6% of the recombinant human Protein C was still present in the transgenic whey after this delipidation treatment, indicating this treatment did not denature or harm the target protein. / Master of Science