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Cloning and Characterization of Two Unique Flavin and NAD(P)H-dependent Sulfur Reducing Enzymes

Nicholas Choe (2014); Mentor(s): E.J. Crane

Abstract: NADH Dependent Persulfide Reductase (NPSR) enzymes from Archeoglobius fulgidus and Shewanella loihica PV-4 were analyzed for their efficiency in reducing sulfur with the purpose of further elucidating mechanistic information on these enzymes. Archeoglobus is not able to respire sulfur despite the presence of the NPSR gene. Although we have had a significant success in a small scale overexpression and purification of the enzyme, we have not been successful in reproducing those results on the large scale. We are currently characterizing the stability of this protein in order to optimize its overexpression. The NPSR from Shewanella loihica PV-4 is a homodimer that shows a characteristic called “subunit asymmetry” in which only one of the two subunits appears to be operational at a given time, with the subunits “firing” in an alternating fashion. It has been proposed that this characteristic is catalytically necessary for some enzymes, although this has never been tested. We are constructing a homodimeric form of the enzyme in which one subunit has been inactivated and the other subunit is the fully active wild-type form. A “Gateway” method will be utilized to construct a co-expression plasmid to produce the mutant protein and wild-type subunits together in the cell, with each subunit labeled with a unique affinity tag that will allow us to purify the enzyme, and further test the role of subunit asymmetry in catalysis.
Funding Provided by: Sherman Fairchild Foundation; Pomona College Chemistry Department

Metagenomic Study of Biological Sulfur Respiration in the Salton Sea Mud Volcanoes and the Deep Subsurface

Tyler Hill (2014); Student Collaborator(s): Danny Lawrence (2011); Mentor(s): E.J. Crane

Abstract: The receding waters of the Salton Sea in Southern California have recently revealed new active geothermic hot mud volcanoes likely to host unique microorganisms. The volcanoes are continuously flushed by carbon dioxide resulting in anaerobic discharge of hot mud (60-100°C) at neutral pH and salinity of 0.8%. Microorganisms residing in the mud play a significant role in reducing sulfur-containing compounds to sulfide. Quantification of sulfur reducing enzymes present in the environment provides insight into sulfide production in the mud volcanoes. This study looks into the prevalence and identity of coenzyme-A disulfide reductase and sulfide dehydrogenase (I & II) in Salton Sea mud volcano samples. Adapting CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) design, amplification of coenzyme-A disulfide in isolated cultures; Thermococcus F761, Pyrococcus EC1, Pyrobaculum 521 has provided metagenomic relevant sequences for analysis. Sulfide dehydrogenase II subunit A was amplified from cultures of Pyrococcus EC1 and Thermococcus F761 and sulfide dehydrogenase I subunit A genetic sequence was isolated from Pyrococcus EC1. Evolution analyses of these enzymes correlate to the previous 16s ribosomal RNA phylogenetic studies. Future studies will expand the breadth of sulfur reducing enzymes detected by optimizing the sequences of primers and by amplifying the beta subunits of Sulfide Dehydrogenase as well NADH-dependent persulfide/persulfide reductase.
Funding Provided by: Pomona Alumni SURP Fund; Pomona College Chemistry Department

Random Walk Lengths on Finite Regular Lattices

Luis Díaz-Ruiz (2015); Student Collaborator(s): Samuel Young (2015); Kevin Guan (2015); Gianni Graham (2015); Briton Lee (2015); Additional Collaborator(s): Brianna Gomez*; Pablo Loria*; Mentor(s): Roberto Garza-Lopez
*Pomona College Academy for Youth Success (PAYS)

Abstract: Computational random walk calculations using Markovian chains can be used to predict the chemical properties, such as kinetics, diffusion, reaction rates and overall dynamics of a given chemical system. Using these calculations, we studied the efficiency of different families and growths of lattices which serve as catalytic surfaces that facilitate diffusion. Exact values for the absorption time, or average walk length, that a particle takes before it reaches the reaction center, or trap, of the lattice are calculated using Maple 15. Results are analyzed in terms of the type and dimensionality of the lattice, the valency of the sites, and the boundary conditions imposed. Investigating the properties of lattices and surface diffusion have implications for research pertaining to chemical and physical processes such as catalysis and crystal growth, as well as applications in nanotechnology.
Funding Provided by: Pomona College SURP (LDR); Evelyn B. Craddock McVicar Memorial Fund (SY); Frederic J. Robbins Endowed Fund (KG, GG, BL); Pomona College Chemistry Department

Tuning Optical and Morphological Properties of SILAR-deposited Quantum Dots on Crystalline Titanium Dioxide for Use in Gratzel-type Solar Cells

Andrew de Jong (2013); Mentor(s): Malkiat Johal

Abstract: A crystalline titanium dioxide thin film is fabricated from aqueous solution under ambient conditions on a polyelectrolyte multilayer-functionalized glass substrate. This liquid-phase deposition (LPD) substrate synthesis and modification procedure was chosen for its utility in providing the relevant surface for QCM and optical measurements. SILAR deposition of CdS quantum dot precursors on these crystalline thin films demonstrate optical properties of light-absorbing quantum dots acceptable for use in Gratzel-type solar cells.
Funding Provided by: Kenneth T. and Eileen L. Norris Foundation

Glycation of siderocalin and its effects on immune response and iron homeostasis in diabetics

Michael Haber (2013); Student Collaborator(s): Andrea Diaz (2015); Mentor(s): Malkiat Johal; Matthew Sazinsky

Abstract withheld upon request

Accounting for Unintended Interactions in the Analysis of Quartz Crystal Microbalance Kinetic Data

Gabriella Heller (2014); Student Collaborator(s): Theodore Zwang (2011); Elizabeth Sarapata (2013 HMC); Mentor(s): Malkiat Johal; Matthew Sazinsky; Ami Radunskaya

Abstract withheld upon request

pH Effects on Solid Support Lipid Bilayers

Marco Lobba (2013); Student Collaborator(s): Jenny Stewart (2015); Mentor(s): Malkiat Johal

Abstract: Lipid bilayers provide the exciting possibility of monitoring the behavior of cell membranes in controlled conditions. One of the most promising methods for studying membrane behavior is using the solid supported lipid bilayer which has been shown to behave similarly to biological membranes. Using a Quartz Crystal Microbalance (QCM) coupled with Dual Polarization Interferometry (DPI) we are investigating the effects of varying pH on the interaction between lipid bilayers and sensor surfaces. We have found a strong correlation between the pH and the electrostatic interaction between the bilayer and the surface, confirming that our system is capable of measuring changes in pH. These results will be vital to our understanding of protein-lipid systems which is the next target of our research. In additional research, we have found a linear correlation between vesicle rupture and the free energy of solution in high osmotic stress environments. All of this research has exciting implications for bio implants such as artificial heart valves and upcoming drug delivery systems, as well as the potential to provide a novel method to measure flux across ion channels.
Funding Provided by: Pomona College SURP; Frederic J. Robbins Endowed Fund (JS); Pomona College Chemistry Department; Gordon and Betty Moore Foundation

The Effect of Glycation on HSA's Affinity for Hemin

Alison Mercer-Smith (2015); Student Collaborator(s): Gabriella Heller (2014); Mentor(s): Malkiat Johal; Matthew Sazinsky; Kevin Sea

Abstract withheld upon request

Synthesis of the C-C disulfide-mimetic derivative of Malformin A1

Noll Sarah (2013); Mentor(s): Daniel O'Leary; Greg Copeland (1996)

Abstract: This research aims to synthesize and explore the biological activity and structure of the C-C disulfidemimetic derivative of Malformin A1, a bicyclic pentapeptide isolated from Aspergillus niger. The 8-membered ring of Malformin A1 is closed by a disulfide bridge, which is believed to be key to the peptide's bioactivity. To achieve a synthesis of the peptidomimetic, we will use a solid phase peptide synthesis and ring closing metathesis strategy, focusing on the substitution of the disulfide functionality with two carbon atoms. Once synthesized, the bioactivity and structure of the carbon analog will be compared to that of the parent compound, Malformin A1, and its derivatives to determine the roles played by the disulfide bridge and the 8-membered ring.
Funding Provided by: Pomona College Chemistry Department

Stabilization of the ?-Helicity of Temporin-SHf through a Hydrogen Bond Surrogate

Marvin Vega (2013); Mentor(s): Daniel O'Leary; Katy Muzikar

Abstract: Temporin-SHf (FFFLSRIF-NH2) is an antimicrobial peptide of interest because of its highly hydrophobic sequence and its broad microbicidal activity, even if it is only eight residues long. In order to further enhance the biomolecular activity of Temporin-SHf its secondary structure should be stabilized into a secondary structure that is prevalent with protein interaction. The α-helix structure is one that has a significant role on the surface of proteins for protein interactions. In this approach, a hydrogen bond surrogate was used to stabilize Temporin-SHf as an α-helix, where an actual carbon-carbon bond takes the place of a hydrogen bond. The peptide was built up using solid-phase peptide synthesis (SPPS), where the third residue from the C-terminus (Phe) had an N-allyl group coupled on and 4-pentenoic acid was coupled on to the end of the peptide at the C-terminus. A ring closing metathesis was then performed to couple the allyl group to the 4-pentenoic acid at the end and create a carbon-carbon bond, thus completing the hydrogen bond surrogate. The coupling of one amino acid to the secondary amine on the allylated residue proved to be difficult and would only go to partial completion, but the final product was synthesized based on Liquid Chromatography-Mass Spectrometry. The next step will be to scale up the reaction sequences to obtain more product and to cleave the product off the resin for further analysis.
Funding Provided by: Paul K. Richter and Evalyn E. Cook Richter Memorial Funds

Synthetic Efforts to Modify the N-terminal Phenylalanine Residues in the Antimicrobial Peptide Temporin-SHf

Shannon Washington (2013); Mentor(s): Daniel O'Leary; Katy Muzikar

Abstract: Temporin Shf (Phe-Ile-Arg-Ser-Leu-Phe-Phe-Phe) is an eight amino acid long, hydrophobic antimicrobial peptide isolated from the skin of a frog Pelophylax saharica. This peptided was built using solid phase peptide synthesis on Knorr resin. The process included using HBTU as an activating agent with the specific amino acid in order to load the amino acid onto the resin or add one to the residue creating peptide bonds. After each amino acid addition or coupling, an acetylation process was performed that would prevent the continued growth of the wrong amino acid on the resin. While making the peptide, confirmation that the coupling went complete through with the use HPLC and LCMS. After completing Temporin Shf, I then purified it. This whole process was repeated with 7 more peptides with various substitutions. Temporin Shf was used as a base for 3 of the variations. L-Phenylalanine tert butyl was substitued for Phenylalanine in one of the last 3 positions, creating 3 of the peptide variations. The another variation included substiting the Serine with another Arginine. This peptide, like Temporin Shf, was also used as a base for the 3 final variations with the same L-Phenylalanine subsitution.
Funding Provided by: Sherman Fairchild Foundation

Expression for Future Characterization of Fatty Acid 2-Hydroxylase in Pichia Pastoris

Hannah Hudson (2013); Student Collaborator(s): Roger Sheu (2014); Mentor(s): Matthew Sazinsky

Abstract withheld upon request

Cloning and expression of histidine-tagged FeoB in Escheridia coli.

Benjamin Smith (2013); Mentor(s): Matthew Sazinsky

Abstract withheld upon request

Optimization of Ferritin and Scara5 Expression in Escherichia coli: Progress and Future Work

Erica Storm (2013); Mentor(s): Matthew Sazinsky

Abstract withheld upon request

Quantifying Desolvation of Varying Protein-Drug Complexes via the Quartz Crystal Microbalance and Dual Polarization Interferometer

Albert Chang (2014); Student Collaborator(s): Andrea Diaz (2015); Mentor(s): Cynthia Selassie; Malkiat Johal

Abstract: Utilizing both the dual polarization interferometer (DPI) and quartz crystal microbalance with dissipation monitoring (QCM-D), we sought to demonstrate that it is possible to quantify the degree of desolvation involved when small drug molecules bind to a bovine albumin serum (BSA) film in real time by exploiting the difference in hydration sensitivity between the two instruments. On QCM, the mass measured includes both the drug and water molecules bound to an immobilized BSA layer, whereas on DPI, the mass measured ignores water molecules bound. Thus, in examining the bindings of drugs of varying hydrophobicities, we discovered that there is a significance difference in the mass bound to the BSA between the DPI and QCM-D measurements. By calculating the difference in masses, we believe we can measure the number of water molecules displaced - an important factor in determining the favorability of protein-drug complex formation. Consequently, we hope this method can be used to aid in the field of drug design, by accounting for the effects of desolvation in protein-drug complexes.
Funding Provided by: Sherman Fairchild Foundation (AC)

Synthesis of Analogs of the Flavonoid Catechin

David Pichardo (2013); Student Collaborator(s): Allison Wallingford (2012); Mentor(s): Cynthia Selassie

Abstract: Flavonoids are naturally occurring compounds found in plants. Recently, they have been thought to provide great health benefits: they have the ability to reduce the risk of many types of cancer and cardiovascular diseases. By capturing radical oxygen species (ROS), the radicals formed are less reactive; however, flavonoids may act as pro-oxidants, causing oxidative damage to DNA. Our synthesis project thus aims at identifying the structural features of flavonoids that determine whether it will act as an antioxidant or pro-oxidant. This long-term project involves the proposed synthesis of 4 Catechin derivatives where X=H, CH3, NO2, OH, as well as the biological evaluation of their radical quenching and metal-chelating abilities. Although our synthesis is not complete, we have been able to successfully synthesize several intermediates, with a hydroboration-reduction reaction and a reduction reaction now left. Once these analogs are synthesized their biological activity will be tested through a variety of methods.
Funding Provided by: Pomona College SURP (DP); Corwin Hansch and Bruce Telzer Undergraduate Research Fund (AW); Pomona College Chemistry Department

Synthesis of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3’-(3’’-X-anilinomethyl)phenyl)-s-triazines as potential Plasmodium falciparum DHFR inhibitors

Makeda Tekle-Smith (2014); Mentor(s): Cynthia Selassie

Abstract: Malaria is an infectious disease that infects an estimated 216 million people every year. Known antimalarial drugs have become less effective in combating the disease because mutations have increased malarial drug resistance. Therefore it is necessary for new antimalarial drugs to be synthesized in order to keep the disease under control. The enzyme dihydrofolate reductase (DHFR) is responsible for the cell growth of the protozoan parasite, Plasmodium falciparum. Plasmodium falciparum is the most virulent specie of the Plasmodium genus responsible for malaria. This project is focused on the synthesis of a series of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3’-(3’’-Xanilinomethyl)phenyl)-s-triazines as potential DHFR inhibitors. Each triazine derivative was synthesized through a four step process and structures at each step were confirmed by NMR spectroscopy. So far, CH3-, Br-, Cl-, SO2NH2- triazine derivatives have been successfully synthesized.
Funding Provided by: Linares Family SURP for Chemistry

Examining the Selective Bromination of Dimeric Resveratrol Derivatives

Dylan Zuckerman (2013); Mentor(s): Scott Snyder

Abstract: Previous studies have demonstrated that positionally-selective monohalogenation of complex, electron-rich resveratrol derivatives can be achieved by using different brominating agents. For instance, one specific brominating agent has been shown to reverse the selectivity observed with other bromonium sources on a compound known as ampelopsin F. This work has been focused on examining the basis for the selectivity observed between these different brominating agents by probing their effects on some closely related analogs of this material.
Funding Provided by: Frederic J. Robbins Endowed Fund; Pomona College Chemistry Department

Developing Bacterial Headspace Sampling Procedure to Maximize Detection of Produced VOCs

Alexandra Antonoplis (2014); Student Collaborator(s): Constance Wu (2014); Additional Collaborator(s): Angelika Niemz*; Peter Vandeventer*; Mentor(s): Charles Taylor
*Keck Graduate Institute

Abstract: Infectious bacteria, such as Pseudomonas aeruginosa, emit distinct combinations of volatile organic compounds (VOCs) as metabolic waste. These unique VOC combinations serve as disease biomarkers and can be detected using solid phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GCMS). To facilitate VOC detection by SPME/GCMS, a new sampling procedure was developed using E. coli as a model bacterium. The sampling procedure ensured the production of VOCs by the bacteria, utilizing aeration to supply the aerobic bacteria with oxygen and a stable, well-mixed 37-degree water bath to foster growth. Using 0.45 micron filters we were able to collect the VOCs produced (and not bacteria) using the SPME fiber inserted into the culture’s headspace. Preliminary results indicate that unique VOC compounds were produced by E. coli and readily detected by SPME/GCMS. Further research will be performed to determine an optimal headspace sampling time and utilize other strains of bacteria, such as the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureous.
Funding Provided by: The Rose Hills Foundation (AA); Paul K. Richter and Evalyn E. Cook Richter Memorial Funds (CW); Pomona College Chemistry Department; Collaboration with Keck Graduate Institute

Growth and Characterization of Vanadium Oxide Thin Films Prepared by Chemical Vapor Deposition

Ryan Dodson (2015); Mentor(s): Charles Taylor

Abstract: Vanadium oxide thin films may be used as the active element in chemical sensors. One approach to creating the thin films needed for such sensors is chemical vapor deposition (CVD). The sensing properties of these films can be enhanced by judicious choice of deposition conditions including substrate temperature and chemical composition of the precursor. Films were deposited via Low-Pressure CVD (LPCVD) on oxidized silicon wafers in the temperature range of 250°C – 625°C using vanadium (V) oxy-tri-isopropoxide (VOTIP) as the precursor. Select samples were then annealed in air at 600°C to examine the effect on morphology. Samples characterized by scanning electron microscopy, Raman spectroscopy and X-ray diffraction in order to determine the morphology and crystallinity of the deposited materials and determine the growth kinetics in the temperature range used. Correlations between deposition and annealing temperatures and grain morphology were observed.
Funding Provided by: Frederic J. Robbins Endowed Fund; Pomona College Chemistry Department

Developing Evanescent-Wave Raman Spectroscopy to Detect VOCs of Infectious Diseases

Constance Wu (2014); Student Collaborator(s): Alexandra Antonoplis (2014); Additional Collaborator(s): Hsiang-Wei Lu*; Angelika Niemz*; Mentor(s): Charles Taylor
*Keck Graduate Institute

Abstract: Volatile organic compounds (VOCs) are emitted by bacteria as metabolic waste in combinations unique to each strain. These VOCs are produced by normal and pathogenic bacteria alike. The overarching aim of this project is the development of a portable diagnostic system which employs evanescent-wave Raman scattering. This device will collect VOCs from a variety of sources up to and including human breath and permit the diagnosis of different infectious respiratory diseases. The optical configuration for evanescent-wave scattering enhances analytical sensitivity, requiring only a small amount of analyte. Prior to using this configuration with bacterial cultures, it must be characterized to ensure that we are employing the conditions best suited to the analysis. Toward this end, a modified Raman system with separated excitation and collection optics was used with benzene in order to track the intensity of a Raman peak as a function of collection angle; the resulting plots were compared to theoretical plots in order to assess the quality of alignment achieved. Preliminary results indicate that there is good agreement between experimental and theoretical plots, but further refinements are necessary to improve alignment. Once completed the system will be coupled with VOC collection devices used for gathering headspace samples from a variety of bacterial cultures.
Funding Provided by: Paul K. Richter and Evalyn E. Cook Richter Memorial Funds (CW); The Rose Hills Foundation (AA); Pomona College Chemistry Department; Collaboration with Keck Graduate Institute

Assessing Water Quality and Sediment Metal Mobility in a River Impacted by Acid Mine Drainage, SW Peru

Allison Sherris (2013); Mentor(s): Heather Williams; Charles Taylor; Jade Star Lackey

Abstract: The Choquene River in SW Peru drains the Sillustani tungsten mine, where oxidation of exposed sulfide tailings has caused acidification of surface water and resulted in injury to local livestock. During a one-month trip to SW Peru this summer, I sought to characterize the physicochemical conditions of the Choquene River from its origin at the mine site to its confluence with the larger Tocotoco River 11 km downstream. I also collected ~50 sediment samples and ~30 water samples for chemical analysis at Pomona College. On-site data collection showed that the pH of the river remains at ~2.5 for the first 5 km of the Choquene River before rising steadily to neutral; this pH rise coincides with inflow from three smaller alkaline tributaries. The redox potential and conductivity of the stream both decrease by at least 75% over the length of the stream. Preliminary analysis of metal concentrations in sediment samples by XRF show elevated levels of some heavy metals--including Co, Cu, Pb, and As--relative to USEPA Freshwater Sediment Screening Benchmarks. Thus, in situ measurements and preliminary data suggest impaired water and sediment quality downstream from the Sillustani mine. Further study throughout the 2012-13 academic year will include continued XRF analysis of metal content of sediment samples, XRD analysis of sediment mineralogy, and ICP-MS analysis of dissolved and total metal concentrations in water samples.
Funding Provided by: Schulz Fund for Environmental Studies