Approximately 60% of drugs on the market today target members of the G protein-coupled receptor family.  GPCRs are a family of proteins that mediate signaling events in cells throughout the body.  These proteins are characterized by seven hydrophobic transmembrane helices connected by alternating intra- and extracellular loops.  Standard structure determination techniques cannot be easily applied to GPCRs, due to the hydrophobic nature of the transmembrane regions.  Determination of the three-dimensional structure of GPCR ligand recognition sites would result in more effective drug design.  Our goal is to use computational methods to generate models for GPCR ligand binding pockets, and to use protein engineering to produce variants of the receptors corresponding to these models.  Computational three-dimensional representations of two GPCRs, the Neurokinin 1 and 2 receptors, were utilized to generate models of the proteins’ ligand binding pockets.  It was determined that the majority of contact points with the ligand occurred along the extracellular loops of the GPCRs, which is in accordance with previous experimental evidence.  These models will assist in the generation of water-soluble GPCR homologues.  We will use genetic engineering to produce these homologues and purify them for structural and ligand binding studies.  Their low hydrophobicity will make them more tractable targets for structural studies and high-throughput drug screening.

Secretory proteins, which include important proteins such as receptors, hormones, and membrane proteins, are translocated into the endoplasmic reticulum (ER) following translation.  In the ER, they fold with the assistance of chaperone proteins.  One important chaperone protein is BiP, which is present within the ER lumen and binds to Ire1, preventing activation of the unfolded protein response (UPR).  As more unfolded proteins accumulate in the ER, BiP releases Ire1, initiating the UPR.  A fluorescent stress sensor has been constructed to easily monitor the UPR.  The green fluorescent protein (GFP) is linked to the unfolded protein response element (UPRE), and the fluorescence of GFP can be measured with a fluorescent spectrophotometer, giving a simple readout of cell stress.  Saccharomyces cerevisiae, budding or bakers' yeast, was studied under treatment of a reducing agent, dithiothreitol (DTT), which quickly unfolds proteins and triggers the UPR.  The effects of varying levels of BiP being produced within the cell have been studied; experiments included studying the effects of DTT concentration curves and time courses, as well as a recovery following removal of DTT.  This research was funded by the Howard Hughes Medical Institute Scholars Program and by NIH grant 1R01GM065501-01

This research compares the Sobel, Canny and Robert edge detectors with newly invented DarkSide and BrightSide edge detectors. The aim is to find edge detectors which preserve fine details.  All the detectors were implemented and tested on digital images generated by a computer program and on natural images; further testing were done after adding different amounts of Gaussian and Salt and Pepper noise to find out the detector’s performance on noisy images. Evaluation focused mostly on the fine details present on the original test images. Results for natural images show that all the detectors were able to detect the fine details in the original images. Results for digital images shows that the BrightSide and DarkSide edge detectors performed very well, while Roberts, Sobel and Canny detectors produced output that was very different from the original images.  Testing the detectors after adding noise to the input images shows varied results depending on the kind of noise added.  We conclude that for detection of fine details, the DarkSide and BrightSide edge detectors did as well as or better than the other edge detector under many conditions.

p53 is a tumor suppressor protein that binds to damaged DNA, arresting the cell cycle until damaged DNA is repaired.  If repair is not possible, p53 initiates apoptotic pathways.  However, in over 50% of all cancers, mutations in the p53 gene inhibit this important function.  Known mutations to p53 affect the protein in many ways, including affecting aggregation of the protein.  Our goals are to purify and to characterize the aggregation behavior of p53.  Expression of soluble full-length human p53 in E. coli was found to be maximal at induction temperatures of 30^oC, while the expression of insoluble protein is maximized at 37^oC.  Purification trials with the protein have led to a partial purification scheme for full-length p53.  The p53 tetramer is stable in salt solutions above 300mM NaCl, and is stable at 37^oC in about 500mM NaCl for up to four hours.  In contrast, the aggregation of the core domain of p53 at 37^oC as measured using size exclusion chromatography is much faster.  Future work will be focused on further improving expression and purification of p53 and on better understanding the stability of the protein. EJO funding provided by HHMI Undergraduate Science Education Program.

Organic photovoltaic cells are a promising alternative to their inorganic counterparts. They offer several advantages, including the ability to conveniently produce lightweight, flexible and transparent devices. However, several challenges such as low stability, limited charge mobility and a narrow match with the solar spectrum still need to be overcome to realize devices with enhanced properties. Ideally, materials with exciton dissociating heterointerfaces—which form free electron-hole pairs—are necessary for optimal device performance. The polymers investigated in this work were polyhexylthiophene (P3HT), polyphenylenevinylene (PPV), and oxadiazole PPV (3:1 and 5:1). We found that the blends of P3HT with 3:1 and P3HT with 5:1 displayed efficient quenching of photoluminescence, indicating exciton-dissociating. The effect of solvents on blend properties was also investigated. The use of toluene showed a dramatic enhancement in photoluminescence quenching and a broader absorption spectrum in comparison with using tetrahydrofuran. Optical microscopy, X-Ray diffraction, and Atomic Force Microscopy data will also be presented. 

Jean Baptiste Joseph Fourier showed that all periodic signals can be represented as an infinite sum of sinusoids, which became known as the Fourier series. Discrete or continuous linear time invariant input signals have Fourier series representation. Discrete time signals can be represented by Fourier series but this is not the case for all continuous time signals. When a continuous time input signal contains a discontinuity, the Fourier series representation of the values at the discontinuity converges to the average values on either side of the discontinuity. This is known as the Gibbs phenomenon. The purpose of my research is to find an approach that will represent a discontinuous continuous time signal with its exact Fourier series representation at the discontinuity, thereby alleviating the effects of Gibbs phenomenon. CQ supported by the Ronald McNair Program.

Pyrococcus furiosus, or “the rushing fireball,” thrives in environments near 100°C.   This organism is an extremophilic member of the Archaea group.  Extremophiles typically grow in harsh environments such as high or low temperatures (0-100°C), high pressure (280 atm), or extreme pH (2 or 10).  They produce proteins that are similar structurally and functionally to those of Eucarya and Bacteria but extremeophilic proteins do not denature under harsh conditions.  Because of the protein’s stability under these conditions, they could ultimately be used for industrial processes that run optimally under similar conditions. The specific protein being studied is beta-glucosidase from P.furiosus.  We would like to optimize its production for protein stability studies.  However, Archaea have low protein yields and they are very difficult to grow in laboratory environments.  Eukaryotes and prokaryotes are better systems for over expressing beta-glucosidase.  Using the yeast expression system developed in our lab, we studied the effects of temperature and folding chaperones. We also expressed beta-glucosidase in the bacteria, Escherichia coli, and constructed a fluorescence denatration curve to compare the stability of beta-glucosidase produced in bacteria to beta-glucosidase produced in yeast.  We found that expressing the protein in yeast at 37°C was the most effective. JP funded by the BRIN Program at the Delaware Biotechnology Institute.

This project takes the current program slicing implementation for OpenMP shared memory parallel programs to a level that will make it usable by a parallel programmer who knows little about the underlying infrastructure. Static interprocedural slicing for sequential codes is well understood and used in a variety of applications. A static program slice is defined as follows: Let P be a program, p be a point in P, and v be a variable that is defined or used at point p. Then a static slice relative to the slicing critierion (p,v) is the set of all statements in the program P that might affect the value of v defined or used at point p. Slicing is used for software development and maintenance activities such as program understanding, software testing, and debugging. By extending and modifying an interprocedural slicing algorithm for sequential programs, and an intraprocedural slicing algorithm for parallel programs, we have developed a technique for static interprocedural slicing of shared memory parallel programs, written using OpenMP explicitly parallel constructs. OpenMP is the standard for explicitly parallel shared memory programming.  This project consists of designing and implementing a user-friendly slicing tool for OpenMP based on our slicing algorithm. A demo can be found at   <http://128.4.133.79/slicer/slicer.html>. Funding from Computing Research Association - Distributed Mentor Program.

Expression of the anti-fluorescein single chain antibody (scFv), 4m5.3, involves the insertion of 4m5.3 DNA into BL21 competent Escherichia coli. The scFv is then expressed as inclusion bodies, which are in turn lysed from the cells, spun out, and solubilized in a 6M gdn HCl buffer solution with beta mercaptoethanol added to reduce the disulfide bonds. The first step in purification involves running the solubilized inclusion bodies through a sephacryl s-200 column. The fractions representing the protein of interest are then folded using a stepwise redox dialysis method. This involves successively transferring the protein first into gdn HCl buffer lacking the BME, then into a urea solution, and finally into a salt solution in which the active protein is stored. The folded protein is then run through a superdex 75 column to separate the various forms of the scFv from the others. These forms include aggregate, inactive monomer, and two active forms (A1 and A2). The two forms of most interest are the two active forms. Fluorescein quenching assays were performed using a Hitachi spectrophotometer. Other characterization work includes the crystallization of the two forms separately, so as structural differences can be readily determined. Finally, protein NMR was also employed as a device to help in the differentiation between the two forms. Funded by the Delaware Biotechnology Institute.

A G-protein coupled receptor (GPCR) is an integral membrane protein that helps to regulate a cell’s response to signals (or molecules).  GPCRs are believed to play a role in heart disease and cancer, so research about these proteins could lead to improved treatment of these conditions. The main goal of our research with GPCRs is to produce large amounts of functional protein, using the host system yeast, so detailed structural information of the proteins can be obtained.  In particular, I have been using the human A2a adenosine receptor (A2a).  A problem faced in previous research with the A2a protein is that there is a slow down in the production of A2a over time.  To understand the mechanism for changes in A2a production, we investigated changes in cell regulation.  The investigation focused on the potential effects of cytosolic stress due to the expression of the foreign protein.  This was tested by using a lac Z reporter gene fused to a heat shock protein promoter in the yeast cells that are expressing A2a.  If the heat shock protein promoter is activated, the lac Z gene will be turned on, which will produce beta-galactosidase in the cell.  A beta-galactosidase assay was performed to determine the relative amounts present in the cells over time during A2a expression.  Preliminary results indicate that a typical stress response pathway is being activated during A2a expression.  This research was supported in part by HHMI undergraduate research funding (ALV) and NSF BES59984312.