Micro/nano-patterning of supported lipid bilayers (SLBs) has shown considerable potential for addressing fundamental biophysical questions about cell membrane behavior and the creation of a new generation of biosensors. Herein are presented several novel lithographic methods for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, chemically distinct types of phospholipid bilayers and/or Escherichia Coli (E. Coli) membranes can be spatially addressed on a single microchip. These arrays can, in turn, be employed in the studies of multivalent ligand-receptor interactions, enzyme kinetics, SLBs size limitation, and membrane-associated species separation. The investigations performed in the Laboratory for Biological Surface Science include the following projects. Chapters II and III describe the creation of lab-on-a-chip based platforms by patterning SLBs in microfluidic devices, which were employed in high throughput binding assays for multivalent ligand-receptor interactions between cholera toxin B subunits (CTB) and ganglioside GM1. The studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Chapter IV characterizes the enzymatic activity of phosphatase tethered to SLBs in a microfluidic device. Higher turnover rate and catalytic efficiency were observed at low enzyme surface densities, ascribing to the low steric crowding hindrance and high enzyme fluidity, as well as the resulting improvement of substrate accessibility and affinity of enzyme catalytic sites. Chapter V presents sub-100 nm patterning of supported biomembranes by atomic force microscopy (AFM) based nanoshaving lithography. Stable SLBs formed by this method have a lower size limit of ~ 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy. Finally, chapter VI demonstrates the electrophoretic separation of membrane-associated fluorophores in polymer-cushioned lipid bilayers. This electrophoretic method was applied to the separation of membrane proteins in E. Coli ghost membranes.

This dissertation focuses on the study of inverse temperature transition processes of the poly(N-isopropylacrylamide) (PNIPAM) and the elastin-like polypeptides (ELPs). A novel temperature jump microfluidic system is introduced and this system shows the ability to measure the kinetics of the PNIPAM and the ELPs collapse without a heat transfer problem. The conformational change of the ELPs during the phase transition process is utilized as a nanoscale protein filter to modulate ligandreceptor binding events on supported lipid bilayers (SLBs). This research study is divided into three main parts. The first part is the development of the temperature jump microfluidics. The kinetics of PNIPAM collapse is used as a model system to show the capability of this new device to measure millisecond time scale phase transition processes. The effects of salts on the kinetics of PNIPAM collapse are also shown in this part. To our knowledge, this is the first study which shows the effects of salts on PNIPAM collapse kinetics. The second part of this research is the application of the novel temperature jump microfluidics. The hydrophobic collapse of ELPs composed of identical sequence but different chain length is investigated. By controlling the molecular weight of the ELPs, the thermodynamic contributions from intermolecular hydrophobic interactions, and intramolecular hydrophobic interactions could be calculated individually for this unique system. The third part is the application of the phase transition property of ELPs. The ELPs are conjugated on the surface of the SLBs as a nanoscale protein filter. The conformation of the ELPs can be modulated by ionic strength of the buffer solution or ambient temperature. The ELPs conjugated SLBs platform showed the ability to block IgG binding to biotin conjugated on the SLBs when the ELPs were in the extended coil state and open the access for protein to bind to biotin in compact globule conformation.

The açai palm fruit has recently become the focus of numerous research endeavors due to its extraordinary antioxidant content. However, little is known about the fruit’s phytochemical rich oil, which is a by-product of the açai pulp. Therefore, the aim of this study was to investigate the phytochemical content of açai oil and its relation to oxidative stability. A total of 206 mg/kg of chlorophylls, which included chlorophyll a as well as four chlorophyll derivatives were tentatively identified by HPLC in crude açai oil (CAO). Two predominant carotenoids (216 mg/kg β-carotene and 177 mg/kg lutein) were also characterized in addition to α-tocopherol (645 mg/kg). Initial investigations into oil stability focused upon the photooxidation of açai oil due to its significant chlorophyll content and findings demonstrated that the increases of nonanal were observed when phospholipids and polyphenolics were reduced. Subsequently, investigations into the interactions between phospholipids and lipophilic antioxidants and their contributions to the stability of açai oil were also assessed by isolating phytochemicals and selectively reconstituting the oil. Findings demonstrated a potential relationship between phospholipids and lipophilic antioxidants, but this was not conclusive. Advances in the processing of the açai pulp by-product created from the clarification process yielded a partially refined açai oil (RAO). The stability of both RAO and CAO as a result of autoxidation were compared to other common food oils (olive, canola, and soybean) and primary and secondary oxidation data suggested CAO was most stable. The difference in the stability of the two açai oils was also investigated by blending these oils and assessing oxidation. An increased stability was demonstrated in the blended RAO, which suggested a significant antioxidant contribution from the CAO. With such similar lipophilic compositions in CAO and RAO, it is theorized that the driving factor behind the stability of CAO can be attributed to its water soluble antioxidant content. While further investigations are required to fully comprehend the interactions of açai oil phytochemicals, these experiments provide insight into the phytochemical content and stability of açai oil. The understanding and information obtained in these studies is geared at increasing the marketability of açai oil as a food ingredient.

Natural, plant-based tannin antioxidants are capable of inhibiting lipid oxidation in ground beef and may be possible alternatives to industry synthetic and natural standards of BHA/BHT and rosemary extract, respectively. Ground beef was purchased on three different days, each defining a batch during study 1. Treatments, added based on meat weight, included a control, 0.2 percent rosemary (RM), 0.02 percent BHA/BHT, 0.5 percent Chardonnay grape seed flour (CG), 0.1 percent and 0.25 percent chestnut wood flour (CN), and 0.25 percent and 0.5 percent of four sorghum bran varieties: black (BS), black with tannin (BTS), white (WS), and high tannin (TS). Patties, formed in duplicate, were randomly designated as cooked or raw and by 0 to 5 day storage, and were aerobically stored at 4 degrees C. Cooked patties were analyzed using the TBARS method. Raw patties were analyzed for subjective and objective color, number of ingredient specks, and pH. In study 2, six treatments were chosen for sensory evaluation including a control, 0.2 percent RM, 0.02 percent BHA/BHT, 0.5 percent CG, 0.1 percent CN, and 0.5 percent BTS. Preparation, and raw and cooked analysis occurred similar to study 1. Additional patties were made for day 1 consumption by consumer panelists. In study 1, all treatments except 0.25 percent WS reduced TBARS values over time compared to 0.2 percent RM. Four treatments (0.5 percent BTS, 0.5 percent CG, 0.25 percent CN, and 0.1 percent CN) showed no significant increase in TBARS values over storage. BS and BTS yielded the lowest color space values (CIE L*, a*, and b*; P<0.0001). Ingredient specks were possible color measurement influences. In both studies antioxidant addition reduced TBARS values over time compared to the control (P<0.0001), and percent discoloration was highest in patties containing a sorghum treatment (P<0.0001). Patties containing 0.1 percent CN were significantly favored in terms of overall like (P<0.0002) and flavor like (P<0.0001). Patties containing 0.2 percent RM were ranked lowest in overall and flavor like, and ground beef-like bite. Patties containing 0.5 perent CG were least liked according to tenderness level (P<0.005). These results indicate that CG, CN, and certain varieties of sorghum bran can be added to pre-cooked ground beef products and provide better antioxidant protection than currently used ingredients of BHA/BHT and rosemary extract.

Abstract The phospholipid PI(3,4,5)P3 works as a second messenger in PI3K signaling pathway. The PI3K signaling pathway is involved in insulin stimulated nitric oxide (NO) production in vascular endothelium, leading to vasodilation and increased blood flow. However, the production of NO also has been reported in neurons as a neurotransmitter and in nucleus tractus solitarii (NTS), NO plays a role in central cardiovascular regulation. Previously, microinjection of insulin into the NTS of rats produces prominent depressor and bradycardic and activates the PI3K downstream Akt. Therefore, to investigate the detail downstream signaling of insulin stimulated NO production in NTS, the effects of PI(3,4,5)P3 on NO production were determined in neuronal cell lines PC12 and GH3 and in NTS of SD rats. The GH3 and differentiated PC12 exposed to 10

No description available.

Heart-type Fatty Acid-Binding Protein (H-FABP) is required for high rates of skeletal muscle long chain fatty acid (LCFA) oxidation and esterification. Here we assessed whether H-FABP affects soleus muscle glucose uptake when measured in vitro in the absence of LCFA. Wild type and H-FABP null mice were fed a standard chow or high fat diet before muscle isolation. With the chow, the mutation increased insulin-dependent deoxyglucose uptake by 141% (P<0.01) at 0.02 mU/ml of insulin, but did not cause a significant effect at 2 mU/ml insulin; skeletal muscle triglyceride and long chain acyl-CoA (LCACoA) levels remained normal. With the fat diet, the mutation increased insulin-dependent deoxyglucose uptake by 190% (P<0.01) at 2 mU/ml insulin, thus partially preventing insulin resistance, and completely prevented the threefold (P<0.001) diet-induced increase of muscle triglyceride levels; however, muscle LCACoA levels showed little or no reduction. With both diets, the mutation reduced the basal (insulinindependent) soleus muscle deoxyglucose uptake by 28% (P<0.05). These results establish a close relationship of FABP-dependent lipid pools with insulin sensitivity, and indicate the existence of a non-acute, antagonistic, and H-FABP-dependent fatty acid regulation of basal and insulin-dependent muscle glucose uptake. Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of chain LCFA for oxidation and for peroxisome proliferator-activated receptor (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced &#946;-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism.

Oxidation of lipids influences the color and sensory qualities of meat products. Meat with a high fat content, such as ground meat, is susceptible to lipid oxidation that leads to the development of negative flavor and color changes. Antioxidants, such as butylated hydroxanisole (BHA), butylated hydroxytolune (BHT) and extracts of rosemary, are used in meat products to control the effects of lipid oxidation. Awika (2000, 2003) found that sorghum bran phytochemicals have high antioxidant properties. Our objective is to evaluate the pH, color, sensory and antioxidant effect of 10, 20 and 30% ground beef patties containing rosemary, BHA/BHT, and three levels of sorghum bran during 5 d of aerobic storage at 4°C. Beef trimmings containing either 50% or 90% lean were formulated into three meat blocks containing either 10, 20 or 30% lipid. Within a fat content, ground beef was equally divided into one of six treatments: 1) control-no added ingredients; 2) BHA and BHT at .01% of the meat weight; 3) rosemary at 0.2% of the meat weight; 4) high level of sorghum at 1.0% of the meat weight; 5) medium level of sorghum at 0.5% of the meat weight; and, 6) a low level of sorghum at 0.25% of the meat weight. The ground beef was aerobically packaged and stored for 0, 1, 3, or 5 days at 4°C. pH, thiobarbituric acid reactive substances (TBARS), fatty acid methyl esters (FAME), sensory color, Minolta color space values and descriptive sensory evaluations were determined. Antioxidant addition reduced TBARS values and increased hardness (P<0.020) and springiness (P=0.002) over time compared to controls. The addition of the high sorghum bran level resulted in lower raw color scores (2.0 vs. 2.9) (P<0.0001) and slightly increased bitter basic taste (2.47 vs. 2.65) (P=0.0069) when compared to control patties. The high sorghum level slightly increased pH (6.33 vs. 6.41) (P<0.0001) and resulted in darker (P<0.0001) and less yellow colored (P<0.0001) patties. With storage, patties had higher pH (P<0.0001) and color space values decreased (P<0.0001). Sensory properties of the patties differed across fat levels (P<0.05); however, interactions between fat level and antioxidant treatment were not significant (P>0.05). Moreover, the addition of sorghum bran at low levels can retard oxidative rancidity in ground beef patties without causing detrimental color changes and negatively affecting sensory attributes.

This study was conducted to examine the antioxidative effect of conjugated linoleic acid (CLA) in irradiated, cooked ground beef patties. The hypothesis was that CLA would be retained during irradiation and would reduce lipid oxidation that is caused by irradiation. The objective was to evaluate the effects of CLA alone and in combination with irradiation on lipid oxidation, fatty acid composition, cooking loss, moisture and fat content, and trained panel sensory evaluations of beef patties. CLA was added at 0, 1, 2, or 4% level during the grinding process. Addition of CLA during the grinding process increased CLA cis-9,trans-11 and CLA trans-10,cis-12 isomers in both irradiated and non-irradiated cooked ground beef patties (irradiated at 1.6 kGy) (P = 0.0001). Weight loss during cooking was greater in irradiated beef patties than in non-irradiated patties (P = 0.004). Irradiation reduced the serumy/bloody aromatic attribute and increased browned aromatic attribute, browned aftertaste, and wet dog/hairy aromatic attribute (P < 0.05). There was no significant main effect of irradiation on the basic tastes. The linoleic acid, CLA cis-9,trans-11, and CLA trans-10,cis-12 were decreased by irradiation (P < 0.05). Although irradiation decreased the CLA isomers, higher percentages of CLA isomers were retained in irradiated patties containing a 4% free fatty acid preparation of CLA (FFA-CLA), reflecting the ability of the FFA preparation to reduce lipid oxidation that is caused by irradiation. The thiobarbituric acid reactive substances (TBARS) values were significantly higher in irradiated, cooked ground beef patties than in non-irradiated ground beef patties (P = 0.004). Although the FFA-CLA was effective in reducing lipid oxidation that is caused by irradiation, it increased painty aromatic attribute, bitter taste, and astringent aftertaste due to the soapy flavor of the free fatty acid (all P < 0.05). The FFA-CLA decreased cooked beef/brothy and serumy/bloody aromatic attribute and browned aftertaste (all P < 0.05). The 1% triacylglycerol (TAG) preparation of CLA reduced TBARS in irradiated, cooked patties to levels seen in control, non-irradiated patties. The 1% TAG concentration also provided good retention of CLA in the cooked ground beef.

Lipid bi-layers are ubiquitous components of biological cells and are found in a variety of cell components. In biological membranes, lipid bi-layer membranes carry membrane proteins, which control transport of material and communication of signals in and out the cell. There are several disadvantages involved with patch clamping method as a way of studying biological membranes and protein interactions. Hence, artificial synthesis of bi-layer has been of great interest in basic biophysical studies, drug discoveries in pharmaceutical studies and study of protein nanopores for precise engineering applications. However, conventional lipid bi-layer synthesis techniques require skilled operators, have low repeatability (reliability), have portability restrictions and result in unstable bi-layers having a short lifetime. In this investigation a novel microfluidic device and a method for artificial synthesis of lipid bi-layer in-situ are explored. In the proposed method, lipid trapped at an aperture on a Teflon sheet, is thinned to form a lipid bi-layer by a continuous flow of buffer solution on both sides of the aperture in the microfluidic device. The microfluidic device is expected to have advantages from its compact design. Further, the new approach is expected to be repetitive and good for automation removing the requirement of a skilled operator. The microfluidic device was fabricated using two glass substrates. Two channels of ? ? shape were etched and through holes were fabricated at all four terminal ends of the microchannels on each glass substrate. A thin Teflon sheet carrying a 100?m diameter hole was sandwiched between the two glass wafers forming two sets of microchannels on both sides of the aperture. An analytical microfluid model of the microchannels was developed to investigate the nature of the flow and to select microchannel parameters. Experiments using the proposed device were performed to verify the feasibility of the novel approach for lipid bi-layer synthesis. Experimental results suggest formation of a lipid bi-layer at an aperture on the Teflon sheet but further investigation might be necessary for verification. Life time of the bi-layer is short mainly due to low quality of the used aperture.