Summer 2013 Research Project: Jasper Werby '14

Jasper Werby '14 discusses his 2013 chemistry Summer Undergraduate Research Project, which he undertook under the mentorship of Professor Cynthia Selassie. He focused on the development and synthesis of a new bifunctional, antimalarial drug.

Hands-on lab research is an important part of being a chemistry major at Pomona College. Students have the opportunity to use modern instrumentation, computational facilities and sophisticated software in the classroom and on independent research projects as early as their first year. Below is a list of recent summer research projects conducted by chemistry students.

Characterizing the Elemental Composition of Coffee Using Flame Atomic Absorption Spectroscopy

Emily Chittick (2017); Mentor(s): Charles Taylor

Abstract: Research has demonstrated that the elemental composition of coffee can be used to pinpoint its location of origin (an important factor in its commercial value). Previous research in our lab focused trace elements in coffee samples acquired using wavelength-dispersive X-ray fluorescence combined with principle component analysis (PCA). Important findings included that rubidium and manganese were key in describing the majority of samples but are insufficient for characterizing growth location. This investigation adds major elements (i.e. potassium, sodium, magnesium, etc.) analyzed via flame atomic absorption spectroscopy (FAAS). FAAS has the advantage of higher throughput after sample preparation. Results were analyzed via PCA to determine which of the eight major elements investigated are most important in distinguishing samples’ origins. In addition, this work confirmed the suspicion that elevated rubidium correlates with decreased sodium in the coffees studied.
Funding Provided by: Schulz Fund for Environmental Studies

First Steps Towards Reverse Engineering a Polymeric Lens Coating Material

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

Abstract: Ventilator-associated pneumonia (VAP) is a serious health concern. Current VAP detection methods are time-consuming and costly, so it is of clinical interest to construct an instrument that cheaply and quickly detects VAP in a patient. Towards this end, we are developing an instrument for rapid diagnosis of VAP that uses Raman spectroscopy to analyze volatile organic compounds (VOC) in a patient’s breath. In order to achieve relevant detection limits for known disease biomarker VOCs, our system employs a commercially available polymeric lens coating material to concentrate VOCs on the sensing element surface. This polymer has thus far outperformed other trial polymers but the structure of the polymer is unpublished. A thorough understanding of the polymer would permit the design of more useful polymers, and thus optimization of this aspect of the VOC detection system. In this study, I attempt to elucidate the polymer’s structure using a number of analytical methods, including elemental analysis, IR spectroscopy, Raman spectroscopy, and NMR spectroscopy. Preliminary results are promising but additional work is necessary in order to confirm the proposed structure. Next steps include synthesis and analysis of the proposed polymer.
Funding Provided by: R. Nelson Smith ’38 & Corwin H. Hansch Fund for Summer Undergraduate Research in Chemistry & Biochemistry

Bacterial Biofilm VOC Analysis via GC-MS/SPME

Michael Etzel (2015); Student Collaborator(s): Paige Oliver (2016), Daniel Pak (KGI); Additional Collaborator(s): Angelika Niemz (KGI), Ilya Tolstorukov (KGI); Mentor(s): Charles Taylor

Abstract: Bacterial attachment to implanted medical devices and subsequent development of biofilms has been found to have profound effects on hospitalized patients. Ventilator associated pneumonia (VAP) is a common occurring nosocomial infection among patients whose immune systems are already compromised. Current methods of diagnosis are too slow (culture growth takes 2-3 days), and presumptive diagnosis leads to the overuse of antibiotics, which may increase the proliferation of antibiotic resistant strains. By analyzing the volatile organic compounds (VOCs) that are given off by bacterial biofilms grown in medically relevant conditions, new biomarker VOCs may be found and used to diagnose patients in a quicker, more accurate manor. In order to identify these biomarkers, we must grow bacterial biofilms in a system analogous to the tracheal tube of a hospital patient. Using silicone tubing, we were able to grow a uniform E. coli biofilm, helping us determine VOCs associated with E. coli including, indole and benzothiazole.
Funding Provided by: Pomona College SURP (ME); Fred J. Robbins Chemistry Fund (PO)

Measuring Uptake of Volatile Organic Compounds into Polymer Thin Films Using QCM-D

Alison Mercer-Smith (2015); Student Collaborator(s): Ryan Dodson (2015), Kelly Park (2012); Additional Collaborator(s): Angelika Niemz (KGI); Mentor(s): Charles Taylor

Abstract: Many pathogenic bacteria emit characteristic volatile organic compounds (VOCs) as metabolic waste. These VOCs and patterns in their abundances may be used to identify the bacteria. We are developing a spectroscopic platform for early infection detection based on VOCs produced by the pathogenic bacteria. We are exploring VOC sorption behavior for several polymers of interest using quartz crystal microbalance with dissipation monitoring (QCM-D). Our goal is to use these QCM-D measurements to build a library of polymers that will enable us to coat our lens with a polymer that has good kinetics and selectivity towards analytes of interest.
Funding Provided by: Howard Hughes Medical Institute (AMS); R. Nelson Smith ’38 & Corwin H. Hansch Fund for Summer Undergraduate Research in Chemistry & Biochemistry (RD); Paul K. Richter and Evalyn E. Cook Richter Memorial Fund (KP)

Synthesis of an Anti-Malarial DHFR/HDAC Hybrid Inhibitor for Treatment Against Plasmodium falciparum

Jerry Jiang (2015); Mentor(s): Cynthia Selassie

Abstract: In the 2013 World Malaria Report compiled by the World Health Organization, there were an estimated 207 million cases and 627,000 malaria-related deaths, 90% of which occurred in sub-Saharan Africa. The lethality and preventability of this disease highlight the necessity to develop effective forms of treatment. Histone deacetylases (HDAC) play a pivotal role in gene regulation. These enzymes catalyze the removal of acetyl groups from ε-N-acetyl lysine residues on histone units upon which DNA is wound. The acetylation status of histones modulates chromatin packing which affects the accessibility of transcription factors to DNA, thereby regulating gene expression. Recent studies have demonstrated HDACIs to inhibit growth of Plasmodium falciparum in vitro, purportedly through transcriptional deregulation. An additional protein system important for malarial progression is the dihydrofolate reductase (DHFR) and thymidate synthase (TS) system. In Plasmodia, DHFR and TS cooperatively drive DNA formation via nucleotides biosynthesis. As dihydrofolate reductase inhibitors have been shown to possess significant anti-malarial activity, they are a promising moiety for the further development of a novel anti-malaria drug. Here, we outline the synthesis of a novel anti-Malaria compound. By combining HDAC and DHFR inhibitor moieties onto a single molecular backbone, we create a dual HDAC/DHFR inhibitor hybrid that we hope to display exceptional anti-malarial qualities.
Funding Provided by: Corwin Hansch and Bruce Telzer Fund

Synthesis of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3’-(4”-N-hydroxyacetamidebenzyloxy)phenyl)-s-triazine as a potential dual DHFR/HDAC Inhibitor

Briton Lee (2015); Mentor(s): Cynthia Selassie

Abstract: Malaria disease is caused by several species of Plasmodium, with one of the more lethal species being P. falciparum. Though there are different ways to treat malaria, malaria become increasingly resistant to current forms of medicine, such as artemisinin. Since dihydrofolate reductase (DHFR) is essential in the synthesis of essential biological molecules, the inhibition of this enzyme has been researched for anti-malarial properties. Multiple derivatives have been previously synthesized in the Selassie lab in order to develop quantitative structure activity relationship (QSAR) models to optimize antimalarial activity while minimizing cytotoxicity to human cells. In addition to these DHFR inhibitors, there are histone deacetylase (HDAC) inhibitors that, among other properties, have promising, potent antimalarial activity. HDACs are integral in gene regulation, as they promote the coiling of DNA by deacetylating histones and exposing positively charged lysine residues. The goal of this project is to synthesize a hybrid molecule that functions as both a DHFR and HDAC inhibitor by building off the familiar backbone of DHFR while adding critical moieties characteristic of HDAC inhibitors. We predict that having the functional sites of both DHFR and HDAC inhibitors on the same molecule will show greater antimalarial activity than either inhibitor alone, and work towards creating a more effective class of antimalarial drugs.
Funding Provided by: Howard Hughes Medical Institute

Effects of Phenolic Compounds on Cytotoxicity of L1210 Cells

Samantha Morrison (2016); Mentor(s): Cynthia Selassie

Abstract: Phenolic compounds have been found to have protective, antioxidant properties, and prooxidant, cytotoxic properties. Prior in vitro and in vivo studies have characterized the radical scavenging, metal chelating antioxidant properties of phenols as well as their cytotoxic effect in mammalian cell cultures. Using this data, QSAR models were derived and it was determined that the cytotoxicity of electron-withdrawing phenols is due to their overall hydrophobicity, while the cytotoxicity of electron-releasing phenols is attributed to their ability to form phenoxy radicals. In this project we cultured L1210 murine leukemia cells and ran cytotoxicity assays on the L1210 cells to determine the IC50 for four 4-X phenols and four estrogenic phenols. The cytotoxicity assays of the 4- X phenols resulted in dose response curves that indicated that the 4-X phenols caused an inhibition of cell growth; however, the estrogenic phenols seemed to have no effect on the L1210 cells. Future work on this project will involve a fluorescence/luminescence assay to assess the degree of mitotoxicity induced by the X-phenols in order to further elucidate the specific mechanism of action of phenolic compounds and the site of their critical toxic reactivity.
Funding Provided by: Neal W. Cornell Fund for Student-Faculty Research

Studying the Hydration of BSA-Ligand Complexes Using the Quartz Crystal Microbalance and Dual Polarization Interferometer

Noah Stanton (2015); Mentor(s): Cynthia Selassie; Malkiat Johal

Abstract: Rational drug design requires understanding the properties of drug-like molecules, as well as the interaction between the ligand and its binding site. However, predicting interactions between drugs and binding sites is complicated by the involvement of water. Accounting for the movement of water within binding pockets can allow for a more complete understanding of binding interactions of ligands. Our methodology takes advantage of the different hydration sensitivities of two instrumental techniques — dual polarization interferometery (DPI) and quartz-crystal microbalance with dissipation monitoring (QCM-D) — in order to track the movement of water molecules upon drugs binding to immobilized bovine serum albumin (BSA), a common bloodstream protein. We employed this methodology to determine the approximate number of water molecules involved in desolvation ligand–protein complexes upon binding. We examined eight drugs with different hydrophobicities in this manner in search for a relationship between the ligands’ physicochemical properties and its desolvation behavior with BSA. We aim to use this data and quantitative structure activity relationship (QSAR) models to relate these desolvating properties to the structure of the drugs. If such a relationship exists, it would be of great importance in using rational drug design techniques to develop drugs with higher affinity for their targets.
Funding Provided by: Sherman Fairchild Foundation

Synthesis of a dual inhibitor prodrug for inhibition of enzymes vital to the life cycle of P. falciparum

Elise Wolf (2016); Mentor(s): Cynthia Selassie

Abstract: Plasmodium falciparum is the prevalent strain of malaria-inducing protozoa responsible for most malarial related deaths worldwide. High occurrence of resistance in P. falciparum combined with its prevalence in third world countries, particularly sub-Saharan Africa, make it an area of high importance in drug development. The enzymes dihydrofolate reductase (DHFR) and falcipain-2 /3 (FP 2/3) have been highlighted as crucial to various aspects of P. falciparum survival. A dual inhibiting pro-drug targeting both enzymes, connected by an amide linkage that would be cleaved in thebloodstream, would be valuable in treatment. Treatment has previously included multiple drug therapies that are hard to monitor and, when not taken to completion, often result in further drug resistance. The first of the two drugs is a pyrimidine derivative of trimethoprim, an antimalarial and DHFR inhibitor that is decreasing in effectiveness due to mutation that confers resistance. The derivative was successfully synthesized and nitrated to give one side of the amide linkage. The second drug, a proposed FP 2/3 inhibitor, is a larger molecule also containing a pyrimidine moiety as well as other optimized functional groups, and has proven more difficult to synthesize due to the reactivity of various groups within the overall molecule. Synthesis of the pro-drug will hopefully result in two drugs that independently show antimalarial activity, and together create a two- pronged attack on the malaria protozoa.
Funding Provided by: Neal W. Cornell Student-Faculty Chemistry Research Fund

Synthesis of Peptidomimetic Analogues of Temporin-SHf

Nathaniel Ash (2015); Mentor(s): Daniel O'Leary

Abstract: The previously studied antimicrobial property of Temporin-SHf (FFFLSRIF), a hydrophobic octapeptide isolated from the skin of the Pelophylax saharica, was the impetus for the synthesis of various analogues of this molecule. Because naturally occurring Temporin-SHf consists of all L- stereoisomer amino acid residues, we were interested in creating peptidomemetic molecules using R-stereoisomer amino acid residues instead. The three Temporin analogues we synthesized were the D-stereoisomer, the reverse L-stereoisomer (FIRSLFFF), and the reverse D-stereoisomer (FIRSLFFF). One of the main reasons for creating these analogues was to test their antimicrobial activities relative to Temporin. These analogues of Temporin are of interest because of the potential antimicrobial activity they could share with Temporin, and also because D-peptides are generally less susceptible to proteolysis in the stomach and in cells. In the future we would like to test the antimicrobial properties of these newly synthesized analogues as well as their proteolytic stabilities.
Funding Provided by: Fred J. Robbins Chemistry Fund

Synthesis of Hydrogen Bond Surrogate Analogs of Temporin-SHf

Phillip Clayman (2016); Mentor(s): Daniel O'Leary

Abstract: All species produce antimicrobial peptides (AMPs) as a non-specific response to infection. Increased resistance to current antibiotics necessitates the development of new therapies and AMPs could serve as a novel class of antibiotics. Temporin-SHf, an AMP isolated from a frog, is of particular interest due to its small size, high ratio of hydrophobic and aromatic residues, and broad spectrum antimicrobial activity. The α-helix structure that Temporin-SHf forms in a micellar environment is thought to be an important factor in its antimicrobial activity. A hydrogen bond surrogate (HBS) which replaces a hydrogen bond with a carbon-carbon bond was used to enhance this α-helix structure. Building the entire peptide using solid phase peptide synthesis (SPPS) proved extremely difficult as coupling to an allylated residue resulted in minimal product formation. To avoid a difficult secondary coupling, an allyl dipeptide was synthesized in solution phase then used in SPPS. Although the dipeptide coupling went to completion under standard conditions, significant racemization was observed. Racemization was minimized using propylphosphonic anhydride and pyridine in ethyl acetate though the yield was not ideal. Future work requires the synthesis of more allyl dipeptide, further optimization of the dipeptide coupling, followed by full synthesis of the HBS Temporin-SHf.
Funding Provided by: Dale N. Robertson Fund

A Chemoenzymatic and Organic Synthesis of Cytidine Diphosphate Glucose for the Characterization of PERM-1 in Ascarylose Biosynthesis in C. Elegans

Christian Gomez (2015); Additional Collaborator(s): Katy Muzikar; Mentor(s): Daniel O'Leary, Sara Olson

Abstract: Ascarylose biosynthesis in the model organism C. elegans has received little attention in the past. However, ascarylose plays a crucial role in the formation of the impermeable eggshell during C. elegans development. Furthermore, knowledge about this eggshell may aid in the prevention of infection by parasitic nematodes, which have similar shell composition. The goal of this project is to characterize the newly-identified gene product PERM-1, an enzyme which may catalyze the final transformation in ascarylose biosynthesis in C. elegans The current investigation utilized both organic synthesis and chemoenzymatic approaches to synthesize cytidine diphosphate glucose (CDP- glucose), the first product in the synthetic pathway towards CDP-4-keto-3,6-dideoxyhexulose, the putative substrate for PERM-1. Along with the synthesis, conditions were developed for monitoring and analyzing reaction products using a variety of analytical techinicques, including HPLC, TLC, NMR and LC-MS.
Funding Provided by: Fred J. Robbins Chemistry Fund

Synthesis of Malformin A1 and its ring-closed S-allyl-D-cysteine analog

David Kolin (2016); Mentor(s): Daniel O'Leary

Abstract: Malformin A1, a bicyclic pentapeptide isolated from Aspergillus niger, exhibits antibiotic properties believed to originate from its disulfide bridge. The purpose of this project is to synthesize Malformin A1 and a ring-closed S-allyl-D-cysteine analog of the peptide. Thus, comparing Malformin A1 to the allylcysteine analog will elucidate the importance of the disulfide bridge to the bioactivity of Malformin A1. Throughout the course of the summer, Fmoc-S-allyl-D-cysteine and Fmoc-S- benzyl-D-cysteine were synthesized on a large scale in preparation for solid-phase peptide synthesis. To synthesize the desired amino acids, first D- cysteine’s sulfur group was protected using either allyl or benzyl bromide. Then, the amino ends of the residues were protected by the Fmoc group. Solid- phase synthesis was used to form the remainder of the benzyl-protected peptide. The next step will be to remove the benzyl groups and oxidize the cysteine residues to form the desired Malformin A1 product. For the S-allyl-D-cysteine analog, ring closing metathesis (RCM) will be performed using the novel Grubbs’ catalyst to synthesize the bicyclic analog.
Funding Provided by: Rose Hills Foundation

Synthesis of a Ligand Precursor for Kinetic Isotope Effect Studies in a Metathesis-Relevant Ruthenium Complex

Cristina Saldaña (2015); Mentor(s): Daniel O'Leary

Abstract: The C-H activation in a metathesis- relevant ruthenium complex has garnered attention since it is unprecedented for ruthenium metal centers. To better understand it, nuclear magnetic spectroscopy (NMR) techniques have been employed. Specifically, they have been applied to study the kinetic isotope effects (KIE) of the C-H activation step. As part of the larger goal, this study focused on efficiently synthesizing the ligand precursor for the KIE studies. To be able to observe the activation step, several markers, carbon 13 and deuterons, need to be strategically placed on the ligand precursor. Thus, cost and reaction conditions were kept in mind during the synthesis. Once yield and purity are optimized, collaboration with Caltech will bring the two components of the complex together for the KIE studies. With these studies, we hope to lower the uncertainties found in a previous study.
Funding Provided by: Fred J. Robbins Chemistry Fund

Laboratory Study of Polymerization Reactions in Titan’s Lakes

Reina Buenconsejo (2015); Additional Collaborator(s): Robert Hodyss (Jet Propulsion Laboratory), Jeffrey Hein (Jet Propulsion Laboratory); Mentor(s): Frederick Grieman

Abstract: Laboratory studies of polymerization reactions in Titan's lakes were conducted. Titan is the largest Saturnian moon and the only natural satellite in our Solar System that has a dense atmosphere. This atmosphere is made mostly of molecular nitrogen and methane which react via photolysis from solar ultraviolet (UV) light to produce a variety of other species including HCN, butadiene, ethylene, acetylene, and acrylonitrile. These products eventually reach the surface and can dissolve in Titan’s hydrocarbon lakes and seas. Once dissolved, these products may react with free radicals (such as the cyanoethynyl radical formed from photolysis of dicyanoacetylene) or each other to form larger molecular weight polymers. Before studying the radical reaction involving the products, the formation of the radical must first be understood. Dicyanoacetylene is difficult to synthesize in the laboratory so azobis (isobutyronitrile) (AIBN) was used as the free radical source. We used matrix isolation infrared and ultraviolet spectroscopy to study the photochemistry of AIBN at low temperatures (12K). A variety of microwave discharge lamps were used and irradiation times were varied to determine the effect of wavelength and irradiation time on the formation or radical AIBN. FTIR spectra taken before and after irradiation show changes occur after irradiation that may indicate the formation of the 2- cyanoprop-2-yl radicals radical. Matrix-isolated ethylene will be studied next after which AIBN and ethylene will be mixed in liquid ethane to simulate the reaction in Titan’s hydrocarbon lakes.
Funding Provided by: Linares Family SURP for Chemistry

Determination of the Temperature Dependence of the Rate Constants for HO2 /Acetonylperoxy Reaction and Acetonylperoxy Self- Reaction

Emily Darby (2015); Student Collaborator(s): Aileen Hui (California Institute of Technology); Additional Collaborator(s): Stanley Sander (Jet Propulsion Laboratory), Mitchio Okumura (Jet Propulsion Laboratory); Mentor(s): Frederick Grieman

Abstract: Using a high sensitivity Infrared Kinetic Spectroscopy (IRKS) method, we are able to quantitatively determine the kinetics of atmospherically important reactions of the hydroperoxy radical, HO2 . Reactions of HO 2 with small alkylperoxy radicals are of interest due to their impact on the oxidation chemistry of the troposphere. These reactions have the potential to have a significant impact on the HOX inventory, and ultimately the ozone budget of the troposphere. The acetonylperoxy radical is a particularly abundant alkylperoxy in the troposphere because it is formed from the degradation of various volatile organic compounds emitted in large amounts to the atmosphere. Because previous studies of the HO2 /acetonylperoxy reaction have resulted in conflicting values and have only been conducted at room temperature, we have studied the kinetics of the HO2 /acetonylperoxy reaction and the acetonylperoxy self-reaction. In the IRKS method, the reactions are studied using a temperature- controlled slow-flow tube apparatus where flash photolysis of Cl2 is used to rapidly produce the HO2 radicals from methanol. Direct observation of the rate of reaction is accomplished by simultaneously measuring the HO2 disappearance using near-IR diode laser wavelength modulation spectroscopy and the acetonylperoxy disappearance in the ultraviolet at 300 nm. By directly measuring the HO2 and acetonylperoxy disappearance over an atmospherically relevant temperature range, we accurately determined the temperature dependence of these two rate constants.
Funding Provided by: Sherman Fairchild Foundation (ED)

Infrared Kinetics Spectroscopy Studies on HO2 Radicals from Reactions of the Criegee Intermediate CH2OO

Erin Delaria (2015); Additional Collaborator(s): Linhan Shen (Caltech/JPL), Sander Stanley (Jet Propulsion Laboratory); Mentor(s): Frederick Grieman

Abstract: Ozonolysis, a major removal mechanism for unsaturated hydrocarbons in the atmosphere, proceeds through a highly reactive carbonyl oxide intermediate, known as the Criegee intermediate (CI). CIs are very reactive species with substantial internal energies. The rapid isomerization and decomposition of CIs results in the formation of OH radicals, which represents a significant nighttime source of HOX. The simplest Criegee intermediate, CH2OO, has previously been shown to undergo unimolecular dissociation into OH and HCO radicals. The subsequent reaction of HCO with O2 also results in the formation of HO2 To study the kinetics of CI decay, we performed laboratory measurements of HO2 formed from CH2OO dissociation. In our laboratory studies, CH2OO was created through pulsed laser photolysis of a CH2I2/O2/N2 gas mixture using a 351nm XeF excimer laser. The formation and decay of HO2 was monitored in the near IR using Infrared Kinetics Spectroscopy (IRKS) with a frequency-modulated signal at 6628.6 cm-1. IO, a significant sink of HO 2 radicals and a major secondary product in this photochemical system, was simultaneously monitored in the UV at 427nm. The estimated overall unimolecular decay rate of CH2OO was 3000 s-1, equivalent to a lifetime of ~300 µs. The formation rate of HO2 was estimated to be 719 s-1 at 30 torr. The rate of CH2OO decay and HO2 formation shows significant pressure dependence in the range 10-60 torr.
Funding Provided by: R. Nelson Smith ’38 & Corwin H. Hansch Fund for Summer Undergraduate Research in Chemistry & Biochemistry

CRP Is a Regulator of the sRNA MtlS in Vibrio cholerae

Tina Solvik (2015); Mentor(s): Jane Liu

Abstract: In Vibrio cholerae, the small noncoding RNA MtlS represses expression of mtlA, encoding the mannitol-specific transporter. In contrast, cAMP receptor protein (CRP) is an activator of mtlA expression. Here, I investigated whether CRP has any affect on mtlS expression. I hypothesized that CRP would act as a repressor of mtlS, which would be consistent with CRP’s role in activating mtlA. To determine the effects of CRP on MtlS levels, I used northern blot analysis with wild-type and crp mutant strains. I consistently observed lower MtlS levels when CRP was absent, suggesting that CRP may be an activator of mtlS. I also studied the effects of excess CRP on MtlS levels through northern blot analysis. No significant difference in MtlS was observed between a strain that overexpressed crp and a control. To locate regulator-binding site(s) for mtlS, I used lacZ- reporter fusions with varying lengths of the mtlS promoter region fused to lacZ. I found increased LacZ activity in the 250+ bp strains, suggesting that a regulator of mtlS transcription may bind between the -100 and -250 region of the mtlS promoter. LacZ activity was also assayed in a lacZ-reporter strain lacking crp, and then compared to the wild-type. I found that the absence of CRP increased activity, suggesting that CRP is a repressor of mtlS. These results suggest that CRP is a regulator of mtlS, and that it may have multiple binding sites, allowing it to act as a repressor and as an activator.
Funding Provided by: National Science Foundation #CBET-1258307: PI J. Liu; American Society of Microbiology

Developing a BPA-Responsive Riboswitch Through Dual Genetic Selection

Erick Velasquez (2016); Mentor(s): Jane Liu

Abstract: Persistent organic pollutants (POPs) are non-biodegradable chemicals that can be toxic to living organisms in high concentrations. Bisphenol A (BPA) is a carbon based synthetic POP found in polycarbonated plastics, used in the production of plastic bottles. Small amounts may migrate into food resulting in health issues to both animals and humans. We set out to develop a riboswitch-based biosensor for BPA. Riboswitches are components of messenger RNAs that regulate the expression of genes based on the binding of specific small molecules. By fusing a gene that encodes for a reporter protein such as green fluorescent protein to the expression platform of a BPA-riboswitch we reasoned that we could qualify the presence of BPA in the environment of a cell harboring the BPA- riboswitch. Starting with a thiamine riboswitch, we generated a library of thirty thousand variants that were then subjected to dual genetic selection. After one round each of positive and negative selection, the genetic diversity of our library decreased, indicative that we may have successfully selected for BPA-responsive riboswitches.
Funding Provided by: National Science Foundation #CBET-1258307: PI J. Liu

Effects of Aluminum Ion on Membranes of Varying Phospholipid and Cholesterol Composition: A QCM-D Study

Fiker Bekele (2016); Student Collaborator(s): Hannah Wayment-Steele (2015); Mentor(s): Malkiat Johal

Abstract: Aluminum is the most abundant metal on earth; however, its ion Al3+ is a known neurotoxin. Even though the exact role of the aluminum ion in pathogenesis is not known, its disruption of normal cell function has been widely demonstrated. The detrimental effects of Al3+ have been studied in vivo, but less is known about the physical changes it induces in membranes. We use Quartz Crystal Microbalance with Dissipation (QCM-D) to analyze changes in adsorbed mass and viscoelastic properties of supported lipid bilayers. This work will present the effect of Al3+ on lipid bilayers of varying cholesterol content. Cholesterol is a key component of membranes that aids in membrane integrity and rigidity, and hence its presence is likely to influence the degree of liposome rupture observed when Al3+ is introduced. Understanding Al3+ interactions with the lipid components of cell membranes is important for a better understanding of Al3+ as a neurotoxin in biological systems.
Funding Provided by: Howard Hughes Medical Institute(FB); Arnold and Mabel Beckman Foundation (HWS)

Characterization of the Novel Nucleic Acid Dye DANPY 1

Conner Kummerlowe (2016); Student Collaborator(s): Marek Zorawski (2016); Additional Collaborator(s): Lewis Johnson (University of Washington); Mentor(s): Malkiat Johal

Abstract: DANPY-1 is a fluorescent dye with high affinity for DNA that has potential applications as an alternative to commonly used carcinogenic dyes, such as ethidium bromide. Using a Quartz Crystal Microbalance (QCM) and a Dual Polarized Interferometer (DPI) we characterized the binding of DANPY and ethidium bromide to a DNA surface supported by polyelectrolyte multilayers. We confirmed an intercalating binding mode between ethidium bromide and DNA, and found a surface binding mode between DANPY and DNA. Furthermore, we found that ethidium bromide binding to DNA causes a conformational change that leads to hydration of the DNA layer. On the other hand, the binding of DANPY to DNA causes a slight dehydration of the DNA surface. Lastly, we analyzed the binding kinetics of these two dyes to DNA, finding that both dyes displayed a similar magnitude of affinity to DNA and are both strong binders.
Funding Provided by: Sherman Fairchild Foundation (CK); Fred J. Robbins Chemistry Fund (MZ)

Investigating Effects of Al3+ on Lipid Membranes: FRAP and Molecular Dynamics

Hannah Wayment-Steele (2015); Additional Collaborator(s): Angelika Kunze (University of Goettingen), Sofia Svedhem (Chalmers University of Technology); Mentor(s): Malkiat Johal

Abstract: Aluminum is the most common metal in the earth's crust, yet the ion Al3+ is a well-known neurotoxin. It derives toxicity from the structural changes it induces in lipid membranes upon binding, including increasing bilayer rigidity, facilitating vesicle fusion, and inducing vesicle rupture. However, the mechanism for these processes is not well understood. We implement Fluorescence Recovery After Photobleaching (FRAP) and Molecular Dynamics (MD) simulations to better understand the mechanism of the system. We provide a quantitative analysis of the effect of increasing Al3+ concentration on membrane diffusion in both zwitterionic and anionic membranes, as measured by both FRAP and MD. We also investigate the coordination structure of Al3+ with phosphatidylcholine lipid head groups, and report Al3+ coordination to the phosphate groups and acyl chain carbonyl groups. Our results indicate that addition of Al3+ to membranes causes an overall increase in rigidity and decrease in diffusion for zwitterionic membranes, and possibly a transition to gel phase in anionic membranes. Understanding Al3+-membrane interactions is important for a better understanding of the mechanism of aluminum toxicity in biological systems.
Funding Provided by: Arnold and Mabel Beckman Foundation

Random Walks on Fractal Structures

Johnny Wang (2017); Student Collaborator(s): Aaron Tsai (2017), Devin Gladys (2017), Isaac Medina (2016); Mentor(s): Roberto Garza Lopez

Abstract: Computational random walk calculations using Markovian chains can be used to predict chemical properties such as kinetics, diffusion, reaction rates, and overall dynamics of a given chemical system. We studied the efficiency of diffusion-controlled reactions on different families of 2-dimensional and 3-dimensional lattices with reaction centers located at unique sites. We calculated numerically-exact values for the absorption time (or mean walk length) of a particle performing a nearest -neighbor random walk on finite, nth generation 2D and 3D Sierpinski lattices, binary fractals and cubic structures (Garza-López, et al., J Phys. Chem. B 1999). We obtained results demonstrating that the overall efficiency of different structures in terms of walk lengths is dependent on the size, growth of the system (N), valency of the active sites (v), dimensionality of the lattice (d), and boundary conditions.
Funding Provided by: R. Nelson Smith ’38 & Corwin H. Hansch Fund for Summer Undergraduate Research in Chemistry & Biochemistry (JW); Howard Hughes Medical Institute (IM); Fletcher Jones Fdtn. (DG); Fred J. Robbins Chemistry Fund (AT)

Synthesis and Electrospinning of Imidazolium Containing Polyesters with Applications in Drug Delivery

Neil Forsythe (2016)

Abstract: Synthesis of novel imidazolium containing monomers were investigated due the potential to create electrospun scaffolds for biomedical applications. Butyl derivatives of imidazolium polyesters have the potential to lyse endosomal membranes and provide a route for endosomal escape and DNA transfection. Both methyl and butyl derivatives of imidazolium containing diol monomers were synthesized via a 3 step organic synthesis. Poly (3-(3-hydroxy-2-(hydroxymethyl)-2- methylpropyl)-2-methyl-1H- imidazol adipate) (PHI) was synthesized from the methyl diol via step growth polymerization at 130° – 220°C. Gel binding studies indicated that the PHI bound DNA, which offers promise in DNA transfection and drug delivery. The butyl derivative was unable to be synthesized due to the diol’s low thermal stability. Thermogravimetric analysis (TGA) in N2 indicated a Td of 137 °C while isothermal TGA above 130 °C showed degradation within 30 minutes. Poly(neopentyl adipate) (PNPA), an uncharged analog of PHI, was synthesized and electrospinability was compared to the charged, imidazolium containing polyester. Electrospinning of PNPA did not result in fiber formation due to its low molecular weight. Solution rheology confirmed the lack of an entanglement concentration (C e ). Initial electrospinning of methyl imidazolium PHI resulted in droplets forming on the collector plate instead of fibers. In order to improve electrospinability, PHI was blended at various ratios with poly(ethylene oxide) (PEO) (200,000 g/mol). An optimal blend ratio of 80:20 PHI and PEO (respectively) resulted in uniform fibers.
Funding Provided by: National Science Foundation