New Orleans, LA   APRIL 18-22, 2009

For the past nine years, the University of Delaware Howard Hughes Medical Institute’s (HHMI) Undergraduate Science Education program has sent undergraduate students to the Experimental Biology Meetings to present their research. As part of this conference, the American Society for Biochemistry and Molecular Biology sponsored its 13th Undergraduate Poster Competition in which 13 UD students participated. Since 2001, students from the University have received more awards in this competition than students from any other college or university.  They continued that tradition this year winning one of the four First Place awards and two Honorable Mentions. See article in November 2008 ASBMB Today and website for last year's meeting in San Diego.

The University of Delaware group included four faculty and 14 undergraduates.

Prof. Hal White  Chem & Biochem
Prof. Seung Hong, Biol Sci
Prof. Gary Laverty Biol Sci
Kathleen Cornely, Providence College
Christina Antonopoulos
Sander Frank
Allison Kasmari

Tyler Larsen
Amber Majid
Obinna Mmagu
Stephanie Myrick

Matthew Richards
Ritika Samant
Laura Sloofman

Katy Teixeira
Patricia Timothee
Ryan Wilson
Meghan Woods

The University of Delaware Students Received Awards in the ASBMB Undergradate Poster Competition

Amber Majid (left) received a first place award in the American Society for Biochemistry and Molecular Biology Undergraduate Poster Competition on Sunday, April 19 in New Orleans. Sander Frank (middle) and Stephanie Myrick (right) received honorable mention awards in the same competition.  Since 2001 when University of Delaware students first participated in this competition, more students from UD have received awards for their research posters than any other participating school.

University of Delaware students who participated in the American Society for Biochemistry and Molecular Biology Undergraduate Poster Competition on Saturday, April 18, 2009, in New Orleans, LA. From left to right: Obinna Mmagu, Laura Sloofman, Sander Frank, Christina Antonopoulos,  Matthew Richards, Meghan Woods, Ryan Wilson, Ritika Samant, Allison Kasmari, Stephanie Myrick, Tyler Larsen, Kathryn Teixeira, and Amber Majid. Not shown is Patricia Timothee who competed in the American Physiological Society's Undergradate Poster Competition.

Delaware students with their posters and abstracts.

Characterization and Hybridization of
Disease-Associated Human Adenylosuccinate Lyase Mutants

Christina H. Antonopoulos, Lushanti De Zoysa Ariyananda,

Peychii Lee, Jenna M. Currier, and Roberta F. Colman.
Department of Chemistry & Biochemistry

Adenylosuccinate lyase (ASL), a catalyst of key reactions in purine biosynthesis, is a homotetramer in which different regions of three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have constructed, expressed, purified and characterized two disease-associated ASL mutants (L311V and R396H) that are located in different regions of the enzyme. L311 is in the central helical region away from the active site, whereas R396 is in close proximity to the entrance to the active site. The Vmax for L311V and R396H are 8.6 and ~2.0 μmol/min/mg, respectively, as compared to 11.9 for wild type; however, all three enzymes have comparable affinities for adenylosuccinate. L311V has ~25 % less tetramer than wild type while R396H has similar amounts of tetramer as wild type. At 37 °C the specific activities of these enzymes decrease with time and reach a limiting specific activity without significantly changing the amount of tetramer. Most ASL deficient patients are compound heterozygotes (e.g. L311V/R396H). In vitro generation of hybrid tetramers from L311V and R396H, only one of which has a His6- tag, followed by their purification on a Ni-NTA column and characterization, is in progress in this laboratory and yields insights into the function of ASL in compound heterozygous patients. (Supp. by Autism Speaks and HHMI Undergraduate Science Education grant.)

Recipient of an ASBMB Undergraduate CompetitiveTravel Award.

Localizing Compartmental Gene Expression in Developing Mouse Prostate
Sander Frank, Qian Chen, and Robert Sikes
Department of Biological Sciences 

The prostate gland is a significant source of male genitourina<>ry tract morbidity.  As developmental processes share several features in common with metastatic cancer, we hypothesize that a better understanding of genes involved in prostate morphogenesis may identify possible targets for therapeutic intervention. The prostate is derived from the urogenital sinus (UGS) which is composed of 4 subdomains: epithelium/mesenchyme (UGE/UGM) and dorsal/ventral halves (UGD/UGV).  Each region develops into specific lobes of the prostate with unique properties. This study sought to validate prostate developmental cDNA arrays that showed about 530 UGE/UGM and 35 UGD/UGV differentiated genes.  Male UGS were separated either into UGE/UGM or UGD/UGV.  Total RNA was extracted and used to synthesize cDNA templates for each subdomain.  Quantitative-polymerase chain reaction (Q-PCR) was performed to observe relative mRNA levels and confirmed localization for 10/12 primary and 6/10 secondary compartmental genes.  Immunofluorescence (IF) localized 6 genes in different predicted compartments.  With this research, we have established the basis for a map of regional gene expression in the murine UGS. This work identifies candidate genes for further study and functional analysis by other developmental laboratories.  Funding: NIH DK63919 and P20RR016472.  Individual support: UD science and engineering and Stetson awards.

Recipient of an Honorable Mention Award in the ASBMB Undergraduate Poster Competition

Identifying the Potential Gene Targets of microRNAs Involved in the Metastasis of Prostate Cancer to Bone

Allison Jane Kasmari, Chu Zhang, and Mary C. Farach-Carson

Preferential metastasis of human prostate cancer cells to bone marrow contributes to disease mortality and morbidity.  MicroRNAs (miRNAs) are endogenous ~22nt RNAs that play important regulatory roles in animals and plants by binding to specific messenger RNA (mRNA) targets to either block their translation or trigger their degradation. Accumulating evidence indicates that miRNAs are involved in human cancer. Here we used the human prostate cancer cell line, LNCaP, and its derivative bone metastatic subline, C4-2B, as a model system to study the roles of miRNAs in prostate cancer progression. We sequenced more than two million small RNAs from LNCaP and C4-2B cell lines using a 454 library. Known miRNAs and new small candidate miRNAs with expression differences between these two cell lines were selected for analysis. Various bioinformatics tools and gene databases were used to identify potential targets for both novel and known microRNAs. Prostate cancer gene expression microarray data were used to further narrow the prospective target genes for known microRNAs. Biobase (http://bkl.biobase.de/cgi-bin/bkl/idb/1.0/searchengine/start.cgi) software was used to place these gene targets in relevant cellular pathways. Ongoing studies seek to explore the potential targets of miRNAs involved in these pathways. These data provide us with important information about how miRNAs are involved in prostate cancer including identification of new pathways for modulation of gene expression during progression to bone. Understanding the targets and functions of these miRNAs will translate into new means to inhibit prostate cancer metastasis and growth in bone. (Supported by NCI P01 CA098912 and University of Delaware Science and Engineering Scholars Cancer and Genetics Fellowship.)

Inherently Antibacterial Hydrogels:
Altering Activity via Tryptophan/Arginine Interactions

Tyler Larsen, Daphne A. Salick, Radhika Nagarkar, and Joel P. Schneider
Department of Chemistry and Biochemistry

Hydrogels are heavily hydrated materials that show considerable promise as artificial extracellular matrices for use in tissue regenerative therapies. The development antibacterial hydrogels has been of great interest to the hydrogel research community as a means to combat the threat of infection during material implantation.  We have developed MAX1, a self-assembling b-hairpin peptide hydrogel whose surface exhibits inherent antibacterial activity against several pathogens prevalent in hospital settings.  Under physiological conditions, MAX1 self-assembles into a highly crosslinked, mechanically rigid hydrogel whose solvent-exposed fibrils display positive charge, which is thought to be important for antibacterial activity.  This study aims to investigate the contributions of a cation-p interaction to the antibacterial activity of a newly designed peptide hydrogel.  Cation-p interactions are a common feature of many antibacterial peptides, where they assist in the disruption of bacterial membranes.  Thus, a new b-hairpin peptide (RWMAX1) was designed, incorporating a cross-strand Tryptophan/Arginine pair, in the hope of creating a more potent antibacterial hydrogel.  The folding and self-assembly properties were assessed using circular dichroism and rheology and the antibacterial activity was investigated against E. coli and S. aureus.  Supported by the Beckman Foundation.

Structure of Voltage Sensitive Calcium Channels
in Mechanosensitive Osteocytes

Amber Sobia Majid1, William R. Thompson2, Kirk J. Czymmek1, Randall L. Duncan1,2, Mary C. Farach-Carson1,2.
1Department of Biological Sciences, 2Biomechanics and Movement Science Program

Regulation of skeletal remodeling is a major focus of attention in translational osteoporosis research. A recent association study including 200 single-nucleotide polymorphisms on human chromosome 3p21 revealed CACNA2D2, (one of four α2δ subunits of the voltage sensitive calcium channel (VSCC) complex), as a novel susceptibility marker for bone mineral density (BMD) variation. VSCCs are a complex of polypeptides that influence osteoblast mechanosensitivity and consist of a pore forming α1 subunit, an intracellular β subunit, a dimer of α2 and δ subunits, and a γ subunit in some tissues. This study characterized the structure of the VSCC complex in an osteocyte-like cell line, MLO-Y4, using RT-PCR, Western blot, and immunostaining. We demonstrated that the T-type Cav3.2 (α1H) subunit is the predominant α1 subunit expressed in osteocytic cells in vitro and in cortical bone.  Additionally, MLO-Y4 cells express α2δ1, γ7, and β14 subunits. These findings represent a shift in the expression of VSCC from long lasting (L-type) in osteoblasts to transient (Ttype) channels in osteocytes. An association of the α2δ subunit with T-type channels in osteocytes could stabilize the functional channel and provide a mechanism for interaction with the extracellular environment. This key protein complex may provide a crucial function for mechanosensitive osteocytes in bone, having implications in skeletal remodeling and BMD.  (Supported in part  by HHMI Undergraduate Science Education Grant)

Recipient of First Place Award in the ASBMB Undergraduate Poster Competition

Modulation of MUC1 Protein Expression by PPAR-gamma
in Pancreatic Cancer Cells

Obinna Mmagu, Peng Wang, and Daniel Carson. 
Department of Biological Sciences

The survival rate of pancreatic cancer is approximately four months. Most pancreatic carcinomas overexpress MUC1. MUC1 is a glycoprotein that serves as a barrier to infection in normal cells.  However, MUC1 protects cancer cells from immune responses and cell death. Therefore, reducing MUC1 protein expression is expected to increase the sensitivity of tumor cells to killing by cells of the immune system. PPARs are transcription factors of the nuclear receptor family. PPARγ heterodimerizes with retinoid X receptor and binds to PPAR response elements.  Recent studies in our laboratory have shown that rosiglitazone reduces cytokine-stimulated MUC1 expression in various cancer cell lines.  Therefore, we hypothesized that rosiglitazone will reduce MUC1 protein expression in pancreatic cancer cells. Pancreatic cancer cell lines were cultured and serum starved for several days. The cell lines were treated with and without rosiglitazone. Western blotting and autoradiography were used to separate and detect the MUC1 proteins synthesized by the cells. Our results indicate that rosiglitazone significantly reduces MUC1 protein expression in Capan2 and HPAF2 cell lines. PPARγ agonists in combination with tumoricidal agents offer a promising avenue to increase pancreatic tumor cell sensitivity to killing by decreasing MUC1 levels. Funding provided by the HHMI Science Education Grant and the McNair Scholars Program.

Recipient of a FASEB MARC Program Poster/Oral Presentation Travel Award.

Design of a Cell-responsive de-PEGylation DNA Delivery System

Stephanie L. Myrick, Peter G. Millili, and Millicent O. Sullivan
Department of Chemical Engineering

We are interested in formulating efficient non-viral DNA delivery vehicles by incorporating multiple functional layers that can be sequentially cleaved off during the delivery process to expose new features. We have established a simplified model vehicle consisting of a polycation-condensed DNA core attached via linker peptides to a layer of poly(ethylene) glycol (PEG). PEG has been shown to protect biomaterials from salt-induced aggregation and immune recognition, but PEG coatings can inhibit DNA delivery at the target cell. This project is focused on validating cell-responsive de-PEGylation of the vehicle prior to delivery via the cleavage of linker peptides sensitive to matrix metalloproteinase-1 (MMP-1), a collagen-degrading enzyme upregulated by fibroblasts as they migrate through the extracellular matrix.  This migration is observed in tumor stroma and at sites of injury, making MMP-1 secretion a useful targeting signal. Having established the conditions that stimulate MMP-1 expression by human dermal fibroblasts via Western blotting and immunostaining, we are now investigating the efficiency of vehicle cleavage using light scattering in combination with salt aggregation assays (Figure 1) to detect PEG layer release. Further experiments in this area and cell studies are ongoing. Supported by HHMI, NSF, and UDRF.

Recipient of an Honorable Mention Award in the ASBMB Undergraduate Poster Competition

Optimizing Polymerase Chain Reaction Conditions
for the Human Perlecan (HSPG2) Promoter

Matthew Richards, Benjamin Rohe, and Mary C. Farach-Carson 
Department of Biological Sciences
Perlecan, also called HSPG2, is a heparan sulfate proteoglycan predominantly located in basement membranes and the matrix surrounding endothelial, mesenchymal and stromal cells.  The reactive stroma surrounding prostate cancer cell lines produces high levels of the protein which may play a role in delivery of growth and angiogenic factors, aiding survival and growth of metastatic tumors.  The overall goal of this project was to study the promoter in order to understand the up-regulation of perlecan in the tumor reactive stroma which occurs via transcriptional increases in perlecan biosynthesis.  The sequence for the human HSPG2 promoter was found using public databases and compared to a published human sequence (Iozzo et al., 1997) and a mouse promoter sequence found in online databases.  Several transcription factor binding sites of interest were identified for further study including NFkB, CREB, Smad3, Elk-1, c-Jun and TCF/LEF-1.  Currently we are working to build a promoter-reporter construct by isolating the promoter region using polymerase chain reaction (PCR) amplification.  Our current strategy seeks to optimize the PCR conditions with different sets of primers.  The next step will be creating a fluorescence protein reporter construct of the full promoter sequence in order to test the effects of the identified pathways.  This research was funded by the HHMI program and NIH/ NCI P01 CA098912.
Recipient of an ASBMB Undergraduate Travel Award.

The Screening of PDZ Domain Array
to Identify JAM-B Interacting Proteins

Ritika Samant and Ulhas Naik
Department of Biological Sciences

Tight junctions (TJs) host complexes that include PDZ domain-containing proteins. PDZ proteins participate in transmembrane molecule organization and TJ formation. Transmembrane protein Junctional Adhesion Molecule-A (JAM-A) is found in TJs and interacts with PDZ proteins AF-6 and ZO-1 via its PDZ binding motif. Another JAM family member, JAM-B displays such a motif but PDZ binding partners are unknown. Because of the similar roles and cytoplasmic regions of JAM-A and JAM-B, we hypothesize that JAM-B also binds to PDZ proteins. To identify these interactions, HA-tagged JAM-B will be used to probe a protein microarray spotted with PDZ domains. A cDNA construct encoding a HA-tagged GST JAM-B protein was created. E. coli BL21 cells transformed with this construct were induced by IPTG, and recombinant protein expression and solubility was analyzed by SDS-PAGE. JAM-B – PDZ domain interactions on the array will be detected by chemiluminescence. This analysis of JAM-B and PDZ protein interactions will further our understanding of signaling pathways particularly pertaining to TJ formation and regulation. Supported by an HHMI Undergraduate Science Education Grant, Beckman Foundation and the NIH.

Effects of Diminished Protein Synthesis on Bone Anabolic Response
to Load in RPL29-deficient Mice
Laura G. Sloofman1, David Chen2, Xiaozhou Zhou2, Christopher Price2, John E. Novotny2,
Liyun Wang2, and Catherine B. Kirn-Safran1 

Departments of 1Biological Sciences and 2Mechanical Engineering

Deletion of a single ribosomal protein, RPL29, increases bone fragility due to diminished protein synthesis and poor tissue quality, suggesting an important link between skeletal tissue growth and efficient protein production.  The goal of this study is to determine if reduced capacity to synthesize large volumes of proteins modifies bone anabolic response to mechanical load.  To test this idea, tibiae of RPL29-null mice and age-matched wild type (WT) controls were loaded at 20% maximal load, 0.5 Hz for 50 cycles/day, with a five-second rest period inserted between two cycles for two weeks. The cortical bone microstructure and mechanical properties of the non-loaded and loaded tibiae of both RPL29-null and WT mice were analyzed using micro-computed tomography (microCT) and three-point-bending tests, respectively.  Results indicate that following loading minor changes occur at the structural and material property levels in both control and null tibiae.  Ongoing studies are using dynamic histomorphometry to quantify differences in bone formation capacity between loaded and non-loaded conditions.  These studies will establish the importance of high volume protein synthesis for the regulation of bone formation. This work is supported by NIH P20 RR016458-06 and an HHMI Undergraduate Science Education Grant.

Selection of a Brain-Seeking Subline of Breast Cancer Cells

Kathryn Teixeira and Deni S. Galileo
Department of Biological Sciences

Our lab has shown that MDA-MB-231 human breast cancer cells can be isolated from the chick embryo brain after injection into the extra-embryonic vasculature.  Others have demonstrated with nude mice that re-injection of these cells results in sublines with enhanced capability to metastasize to the brain.  It remained unknown whether this was the result of cells specifically targeting the brain, or increased survival in the brain compared to other organs.  We transfected MDA-MB-231 cells with neomycin resistance and lacZ genes to enable drug-selection and visualization.  This allowed us to quantitate cells which extravasated into the brain and grew as colonies after dissection, dissociation, and drug selection following injection.  To determine the sensitivity of the chick embryo system for detecting brain metastases, cells were injected at high and low concentrations.  Colonies were detected following injection of as few as 5,000 cells (average of 32.2 colonies/brain).  Sublines of cells which had been through the brain up to five times were created.  The number of colonies isolated from the brain and liver after re-injection was compared to that of previously uninjected cells.  Re-injected cells produced a greater number of colonies, suggesting that these cells specifically target the brain.  This project was supported by NIH grant 2 P20 RR016472-07 under the INBRE Program of the National Center for Research Resources.
Recipient of an ASBMB Undergraduate Competitive Travel Award.

Protein Kinase C (PKC) Mediates Purinergic Receptor Induced Contraction
in MC3T3-E1 Osteoblasts.

Patricia Timothee, Victor Fomin, Kirk Czymmek, and Randall L. Duncan
Department of Biological Sciences

P2X7 receptor activation mediates the load-induced increase in bone formation, in vivo.  We find that activation of this receptor produces a rapid contraction of the osteoblast that we hypothesize is modulated by two distinct pathways; RhoA GTPase and PKC.  To test this hypothesis, we measured MC3T3-E1 preosteoblast contractions using the Zeiss 5LIVE rapid confocal microscope during activation of the P2X7 receptor in the presence or absence of specific inhibitors of RhoA GTPase and PKC pathways.  BzATP, a known agonist of the P2X7 receptor, was added to the cells and changes in cell area following BzATP stimulation were quantified using Differential Interphase Contrast (DIC) microscopy.  Addition of 0.5mM BzATP to MC3T3-E1 cells resulted in a 34.14% reduction in cell area.  Similar results were seen using a PKC activator, PMA.  Non-specific inhibition of PKC with, GF109203X, significantly attenuated the BzATP-induced contraction.  Specific inhibition of PKCα, a Ca2+ dependent isoform of PKC, amplified the contraction in response to BzATP, suggesting that PKCα is not responsible for the P2X7 induced contraction. Inhibition of myosin light chain kinase and Rho kinase (ROCK) also failed to block BzATP-induced contractions. These studies suggest that PKC mediates the response of P2X7 receptor activation that may be important in skeletal remodeling. (Supported by INBRE2 P20 RR016472-08 and NIH/NIDDK R01 DK058246)

Peroxiredoxin VI in Complex with a Transition Inhibitor

Ryan Wilson, Akhil Khanal, and Brian Bahnson
Department of Chemistry and Biochemistry

Peroxiredoxin VI (Prdx6) is an antioxidant enzyme highly expressed in the lungs.  Its antioxidant properties are due to its ability to reduce hydroperoxides found in lung surfactants and thus prevent the toxicity associated with hyperoxia.  Prdx6 is a bifunctional protein that contains two distinct active sites.  One active site catalyzes a phospholipase A2 (PLA2) type hydrolysis of phospholipids, and the second active site catalyzes the reduction of lipid hydroperoxides typical of 1-cys peroxiredoxins.  The structure of Prdx6 has been solved; however, there is no structural evidence to elucidate its catalytic mechanism.  This study is designed to solve the structure of Prdx6 in complex with MJ33, a PLA2 transition-state inhibitor, in an effort to understand the mechanism and conformational change Prdx6 undergoes during its catalytic cycle.  Here we report that Prdx6 has been successfully purified to homogeneity.  Our crystallization screens have produced protein crystals that have diffracted to a resolution of 2.8 Å.  This research is supported by the Chemistry Alumni Scholars.

Recipient of an ASBMB Undergraduate Travel Award.

Hybrid Hydrogels for Use in Vocal Fold Tissue Engineering

Meghan Woods1, Sarah Grieshaber2, and Xinqiao Jia2
Departments of
1Chemistry and Biochemistry and 2Materials Science and Engineering

Elastin is an extracellular matrix (ECM) protein abundant in vocal folds and other mechanically active tissues.  This protein provides these tissues with elastic recoil and strength.  Due to difficulties with natural elastin, there is a need to develop scaffolds for vocal fold tissue regeneration that mimics this elasticity and also provides tunability in a range of morphological, biological and mechanical properties.  Hybrid polymer-peptide materials are attractive candidates for tissue engineering scaffolds because their synthesis allows for this tunability.  In this work, synthetic polymers of poly(ethylene glycol) (PEG) and peptides with the amino acid repeat unit AKAAAKA found in natural elastin were synthesized and cross-linked to form hybrid hydrogels.  PEG was functionalized with azide end groups and cross-linked through a copper-catalyzed azide-alkyne cycloaddition reaction with peptides functionalized with multiple alkyne groups.  Both linear and 4-arm star PEG polymers with varying molecular weights were used in these trials, and the peptide length and functionality was varied by the number of repeat units.  It was found that the swelling ratio and mechanical properties of the hybrid hydrogels can be tuned according to the molecular weight and geometry of each component.  This project was funded by HHMI, NIH/NIDCD (1R01DC008965-01), and NSF/DMR (Career: 0643226).
Recipient of an ASBMB Undergraduate Travel Award.

The trip to the Experimental Biology Meetings in New Orleans was organized by the University of Delaware HHMI Undergraduate Science Education Program with additional support from travel grants from the American Society for Biochemistry and Molecular Biology, the FASEB MARC Program, the Beckman Scholars Program,  and the Women Scholars Program. The HHMI Undergradaute Science Education Program, the Arnold and Mabel Beckman Scholars Program, The Ronald McNair Program, Charles Peter White Fund, The Chemistry Alumni Scholars Program,  and the Undergraduate Research Program supported research by the students.

The annual dinner with UD alumni and friends was held at the Bourbon House Resrtaurant on Bourbon Street on Sunday, April 19.  A record number of alumni and friends joined us this year.

Clockwise from middle front: Allison Kasmari,
Stephanie Myrick, Laura Sloofman, Patricia Timothee, Kathryn Teixeira, Matthew Richards, and Ryan Wilson.

Clockwise from left front: Jens Hemmingsen,
Christina Antonopoulos, Evan Lebois, Obinna Mmagu, Ronald Ogbonna, Meghan Woods, Amber Majid, and Ritika Samant.

Clockwise from left front: Michael Cox, Luis Ralat, Damien Thevenin, Anastasia Fuzaylova, Judith Voet, Don Voet, and Andrew Hollenbach.

Clockwise from left front: Michelle Lazarus, Gary Laverty, Seung Hong, Tyler Larsen, Roberta Colman, Robert Colman, and Sander Frank.

Waiting for the bus to PHL.

Our bus to PHL.

Christina and Meghan with biochemistry textbook authors, Judy and Don Voet.

A small section of the exhibit floor at EB2009.

Liang Kang looking at Ritika's poster.

Obi being interviewed by Gregory Petsko, president of ASBMB.

Ryan (VP) and Meghan (Pres) receive the outstanding ASBMB-UAN Chapter award for the Northeast US from Ann Aguanno, Regional Director..

In the Ambassador Hotel lobby waiting for the Airport Shuttle.

Christina, Stephanie, and Matt at the Undergraduate Poster Competition.

A night out at Cafe du Monde. (A picture Dr. White didn't take.)

Ryan being interviewed by Gregory Petsko, president of ASBMB.

Return to  University of Delaware HHMI Home Page
Created 6 November 2008,  last revised 27 April 2009 by Hal White [halwhite at udel.edu]
Copyright 2008, 2009 Harold B. White, Department of Chemistry and Biochemistry, University of Delaware