2012 Trainees
2012 Student Testimonials |
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2012 REU Students |
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Bradley Baker
Abstract: The bacterium Staphylococcus aureus, a facultative anaerobe, is a significant threat to public health, partly due to its ability to cause chronic, antibiotic resistant infections. Strains of S. aureus can undergo mutations in genes that control menaquinone and heme biosynthesis, which prevent the bacterium from generating energy by respiration forcing it to ferment. These bacteria are called small colony variants (SCVs) and are difficult to treat with standard antibiotics. A high throughput screen for activators of the heme sensing system HssRS, which is responsible for sensing and alleviating toxic levels of heme within the bacterium, provided several small molecules that can also inhibit growth in S. aureus, in addition to activating HssRS. The lead molecule of this project ‘8740 is an N-acyl thiourea containing a benzo furan-2-yl and a 4-tert-butyl-2-aminophenol moiety. A library of derivatives was synthesized by reacting benzofuran 2-carbonyl chloride with ammonium thiocyanate to provide the isothiocyanate, which was then reacted with a variety of anilines and amines. These compounds will be tested in S. aureus for HssRS activation using the XylE reporter gene and for their ability to inhibit growth under fermentative conditions. The data collected in this project will be used to aid in the development of more potent small molecule inhibitors of growth in fermenting bacteria and the utilization of their antimicrobial properties in the treatment of SCVs. |
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Jennifer Benoy
Abstract: Smith Lemli Opitz Syndrome (SLOS) is an autosomal recessive disorder that afflicts 1 in 20,000 people and results in mental retardation, disfigurement of features, malformation of parts of the digestive system and infant mortality. The cause of the syndrome stems from the presence of elevated levels of 7-dehydrocholesterol (7-DHC) in the bloodstream when 7-dehydrocholesterol reductase is dysfunctional due to mutations in its genetic encoding sequence. 7-DHC is a naturally produced cholesterol precursor which readily undergoes free radical autoxidation to form many different oxysterols. The specific oxysterols being formed from 7-DHC are of interest in order to help understand their potential reactivity with proteins and implications in SLOS. To simplify the analysis of these protein adducts, an alkynyl 7-DHC was synthesized so that pull-down techniques specific to the alkyne could be used. Alkynyl 7-DHC was synthesized in ten steps using ergosterol as the starting material. The alkynyl group was added as a way to tag the molecule, allowing it to act as an in vitroprobe which simplifies the extraction of oxysterol products. The alkynyl 7-DHC was then incorporated into cells to insure that it behaves as the naturally occurring compound. Formation of cholesterol esters indicates that the alkynyl 7-DHC is taken up and metabolized by cells. Click chemistry was used to extract the alkynyl products from the cell mixture, a process in which the alkynyl group reacts with a solid-supported azide. (Scheme 1) After immobilization of the alkynyl products onto the solid-support, the non-alkynyl compounds were washed away. The pure alkynyl compounds were then released from the support and analyzed using High Performance Liquid Chromatography and Mass Spectrometry. |
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Hollie Black
Abstract: In a recent publication from our laboratory, a class of compounds, represented by 3-cyano-N- (1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), were found to show positive allosteric modulator (PAM) activity at the metabotropic glutamate receptor subtype 1(mGluR1) in rodent cell lines. MGluR1 is a class C GPCR that plays a key role in the fast, excitatory synaptic transmission in mammalian CNS. A recent study has shown that populations of schizophrenic patients feature deleterious mutations in the GRM1 gene, which encodes for mGluR1. As a result, the mGluR1 receptor, which has been shown to play a role in anxiety, learning and memory, is thought to function at reduced levels. In order to study the structure activity relationship (SAR) of this model pyrazole scaffold for mGluR1 positive allosteric modulator activity, a repetitious parallel synthesis campaign was undertaken. The first generation library was focused on the eastern amide, where we substituted various aryl rings. Heterocycles, such as pyridines, pyrazines, and furans were also incorporated to further evaluate that position. To date, a total of 45 compounds have been synthesized. While initial results have not shown any improvements over the model compound, further evaluation is in order. The second-generation library involved manipulating the substitution pattern around the central pyrazole core. Various hydrazines with analogues of benzoyl acetonitrile were combined in a matrix-based approach in order to quickly sample a range of different substitution patterns. This current effort has resulted in an additional 36 compounds to be tested for PAM activity. Although no definitive improvements over the initial hit have been made, this project is at the outset, and further progress will be reported in due course. |
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Reniesha Franklin
Abstract: Sulfolobus solfataricus DNA polymerase IV (Dpo4) is a Y-family translesion synthesis polymerase (TLS). Y-family polymerases have been observed to induce the majority of mutagenic events. These polymerases have a high proclivity to insert improper base pairs. When the replication polymerase stalls at the site of DNA damage, the TLS is recruited to insert a base opposite the lesion. The inserted base is usually elongated. After the insertion, the TLS dissociates and is replaced with the replication polymerase. The replicated DNA leads to genetic mutations if the incorrect base is incorporated. As a result of its low fidelity rate, Dpo4 is able to bypass certain aberrant bases, such as those produced by exposure to carcinogens like the heterocyclic amine (HCA), 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). Human exposure to HCAs is associated with different cancers. With the intention of characterizing its biochemical properties, Dpo4 was overexpressed in an E. coli strain, and then purified by FPLC. Crystallographic analysis of Dpo4 complexed with the IQ adduct will be used to gain greater insight into the base substitution and frameshift fidelity of DNA synthesis by Y-family polymerases. |
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Ross Havens
Abstract: The heat shock response is an important cell survival mechanism that is of interest as a potential cancer therapy target. This response is mediated by heat shock factor-1 (HSF1), which can be activated by heat, oxidative stress, and lipid electrophiles such as 4-hydroxynonenal (HNE). When activated, HSF1 trimerizes and translocates to the nucleus, where it binds to promoters containing heat shock elements (HSE) resulting in the synthesis of heat shock proteins (HSPs). Many phosphorylation and dephosphorylation steps are required for full HSF1 activation. In order to understand the involvement of different signaling pathways in HNE-induced HSF1 activation, we developed a high-throughput Luciferase assay to measure HSF1 activity. HNE treatment of RKO cells transfected with a pGL3-promoter construct bearing four repeats of HSP 70 HSEs resulted in a time-dependent increase in Luciferase activity. Pretreatment of cells with inhibitors of PI3K, MEK, and p38 followed by HNE resulted in a six-fold decrease in the Luciferase activation signal relative to the cells treated with HNE alone. Overall, this method appears to accurately assess HSF1 activity under multiple conditions, and implicates the PI3K/Akt /MAPK cascade in HNE-induced HSF1 activation. |
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Rachel Horness Abstract: The increasing need for rapid, accurate methods for bacterial identification and characterization arises from a wide range of applications including medical diagnostics, food safety, and chemical/biological warfare detection. Often, the most time-consuming and labor-intensive aspects of bacterial analysis are sample preparation and purification. Not only do these processes delay potentially time-sensitive results, but excessive handling often introduces experimental error. Use of the atmospheric solids analysis probe (ASAP) (Waters Corporation, Manchester, UK) in conjunction with ion mobility-mass spectrometry (IM-MS) eliminates several limitations associated with current methods of bacterial analysis. ASAP-IM-MS requires little or no sample preparation, and utilizes a simple and rapid procedure for detection of volatile and semi-volatile liquid and solid compounds. In addition, ion mobility-mass spectrometry allows for two-dimensional separation of complex mixtures based on structural variability and mass-to-charge ratios, respectively. In this work, ASAP-IM-MS parameters were optimized with solutions of standard metabolites. After initial optimization, several mixtures of standards were prepared to assess the efficacy of metabolite detection in complex solutions. We have shown that the optimized parameters are heavily dependent on the identity of the analytes; however, individual metabolites can be identified even in complex mixtures with little sample preparation. Further tailoring of the method for analysis of complex bacterial extracts will allow for high throughput metabolomic classification of known and newly discovered bacterial strains directly from complex mixtures. |
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Corinne Lariviere
Abstract: Aminoglycosides are a class of antibiotics which target the ribosome and are responsible for inhibiting mRNA and tRNA translocation. Hygromycin B, an aminoglycoside, is regularly added to animal feed to prevent the growth of both gram positive and negative bacteria, as well as worms. Despite hygromycin B’s regular use and presence in veterinary medicine, nothing is known of its mechanism of action. This antibiotic differs from the other aminoglycosides in that it contains a dual ether linkage (orthoester) instead of the typical glycosidic linkage. Our goal was to develop a substrate to feed to the hygromycin B enzyme to study formation of its unique orthoester linkage. The first approach required chemical degradation of hygromycin B by reduction of the orthoester to create the hypothetical orthoester forming enzyme substrate. We attempted to directly reduce the orthoester with sodium borohydride but then resorted to first add protecting groups to the amines and hydroxyls. The addition of protecting groups to the already large hygromycin B molecule equated to difficulties with resolving the molecule (which was too large for LCMS and too complex for NMR, including 2D techniques.) This degradation was also studied using an acid hydrolysis reaction to break the hygromycin B molecule into D-talose sugar and a disaccharide, both of which would be useful substrates once separated. Another synthetic approach was the creation of a substrate based on additions to a basic sugar molecule. Protecting groups were first added to the hydroxyls (either acetates or TBS groups), and various methods of opening the ring using 1,3-propanodithiol were then explored. Additionally, a microbiology approach using biocatalysts was also employed. Primers were created based on the gene sequence of hygromycin D, and the TOPO cloned DNA was then inserted into a pEt-28 A+ vector, eventually leading to the isolation of protein. The hygromycin B substrates could then be fed to the protein, in hopes of monitoring the formation of the orthoester. |
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Candace Marshall Abstract: Small molecule-protein binding plays an important role in biological processes, and can be used as therapeutics. Some examples include sequestering ligands, stimulating signaling pathways, delivering other molecules to sites of action, and serving as in vivo diagnostics. Although computational design of proteins that bind small molecules would be of great value, it has not been done yet. Small molecules that have an intrinsic binding affinity to the protein scaffold HisF were used as the starting point for design. This is because it is easier to design in activity when some of the desired activity is already present. Using the computational design program RosettaLigand, developed in the Meiler lab, these molecules were docked into the binding site while the binding site was being designed. This created an optimized small molecule-protein interface. The resulting models were scored based on RosettaLigand’s energy function, and the models with the best interface energy were chosen to be experimentally tested. |
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Jeremy Mason
Abstract: Highly diastereo- and enantioselective aza-Henry reactions between Boc aldimines and aryl nitromethanes were developed utilizing novel unsymmetrical chiral mono(amidine) catalysts. This technology provided enantioenriched material for the preparation of new analogs of (–)-Nutlin-3, a potent p53/MDM2 inhibitor developed by Hoffmann-La Roche. |
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Samantha Monk
Abstract: Solution-based syntheses of nanoparticles are common techniques that allow for effective control over the size, shape, and composition of the products. However, these procedures frequently require high boiling solvents and utilize capping ligands that render the particles insoluble in water. The inability to transfer these particles reliably into aqueous environments has limited the use of nanoparticles in biomedical and photocatalytic applications. For this reason, a facile method of transferring organic-capped nanoparticles to water was investigated. Common capping agents for solution-based syntheses include oleic acid and oleylamine, which both possess a double bond allowing for the opportunity to perform metatheses. Therefore, cross-metatheses were performed utilizing Grubbs catalyst in an attempt to incorporate a different functionality into the surfactant molecules. Metatheses were performed on oleic acid capped CuS, CuInS2, CuInS2:Pt hybrids, and Fe2O3 nanoparticles with styrene and allylamine. Preliminary results of this project indicate that the success of the metathesis is partially determined by how strongly the ligands are bound to the surface of the particles during the reaction. |
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Wesley Murphy
Abstract: Sequestration of zinc and manganese ions (Zn2+ and Mn2+) by the S100A8/S100A9 heterodimer, a neutrophil protein often referred to as calprotectin (CP), has been shown to play a vital role in vertebrate host defense against bacterial pathogens. Previous work with the pathogen Staphylococcus aureus has established that the chelation of extracellular Mn2+ and Zn2+ ions by CP inhibits S. aureus growth by depriving the bacterium of these essential metal ions. This type of metal chelation based innate immunity is termed nutritional immunity. Like other members of the S100 family of EF-hand calcium binding proteins, CP has two transition metal binding sites located at the dimer interface that can each bind one Zn2+ ion with high affinity. However, CP is the only member of the S100 family with the ability to tightly bind Mn2+, which it accomplishes at only one of its transition metal binding sites. Furthermore, a recently obtained high resolution X-ray crystal structure of CP with Mn2+ bound revealed that CP creates a novel Mn2+ binding site from six histidine residues, differentiating CP from all other Mn2+-binding proteins. In order to better understand the biophysical and antimicrobial properties conferred by this unique Mn2+ binding motif, two CP variants with amino acid substitutions for the two coordinating histidines found in the S100A9 C-terminal tail were created via site-directed mutagenesis: CP Tail HàN and CP Tail HàA. Isothermal titration calorimetry was used to measure the Mn2+ and Zn2+ binding affinities of these CP variants. S. aureus growth inhibition assays were performed to assess antimicrobial activity. Our data suggest that the elimination of the two coordinating histidines from the S100A9 C-terminal tail impairs Mn2+ binding capability, does not affect Zn2+ binding affinity, and decreases antimicrobial activity against S. aureus as hypothesized. Ongoing investigations of the structural basis for Mn2+ binding to CP will improve current understanding of its transition metal binding specificity. This information will be used in the design of innovative therapeutics that could augment nutritional immunity as an alternative to conventional antibiotics that are becoming increasingly resisted by many pathogenic bacteria. |
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Alec Pawlukiewicz
Abstract: Two major barriers to the proper diagnosis of malaria in developing countries are the cost of the diagnostic tests and the lack of necessary infrastructure to maintain and utilize the technology involved. The current method used to address these issues is the use of low-cost rapid diagnostic tests which make use of antibody-biomarker interactions to provide a diagnosis. The issue with this approach is that the higher temperatures present in many of these developing regions lead to a decrease in efficacy of these tests. Our solution to this issue is the use of a robust iron oxide nanoparticle platform which binds specifically to a pfHRP-II malarial biomarker, a histidine-rich protein found in the blood of an infected patient. The synthesis of these particles yields the formation of amine-terminated Fe3O4 nanoparticles, resulting from the conversion of FeCl3 to Fe3O4 with hexamethyldiamine used as a stabilizing ligand. These amine-terminated nanoparticles are then hydrothermally coated with glucose to produce a carboxy-rich shell surrounding the particles. Finally the nanoparticles are incubated in NiSO4 to coordinate Ni2+ to their carboxy-rich surface. TEM images not only reveal that the particles have an average diameter of approximately 50 nm, but also confirm the presence of the dielectric glucose shell encompassing the particles. Additionally, the Zeta potential measurements and ICP-OES results indicate the presence of nickel coordination to the dense glucose shell surrounding the particles. Aggregation studies were then conducted on these particles using DLS to analyze the change in size of the particles in solution with poly L-Histidine, a pfHRP-II mimic. These studies confirmed that the nickel-charged nanoparticles aggregate in the presence of the histidine-rich mimic of pfHRP-II with a limit of detection of approximately 80 nM poly L-Histidine. Furthermore, the negative controls of the aggregation study, L-Histidine and physiological levels of human serum albumin induce no significant aggregation of the particles. The aggregation activity of these particles indicates their potential use in a low-cost diagnostic assay due to their robust nature and the specificity with which they bind pfHRP-II mimics. |
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Bianca Ramirez
Abstract: Within a 24 hour period, the body undergoes many physiological and metabolic changes which are all controlled by an intrinsic circadian timing system, the circadian clock. On a cellular level, the circadian clock controls daily oscillations in gene expression, which in turn, oscillate biological processes such as sleeping patterns, body temperature, and hormone secretion. As the identification and understanding of the circadian clock has grown, so has the interest in altering its natural rhythm. Discovering a set of compounds that potently affect the function of the circadian clock will form a foundation for therapeutic strategies aimed at treating circadian disorders. A high throughput screen identified N-phenylphenazine-2-carboxamide as a lead circadian rhythm period elongating compound and two N-(2-fluorophenyl)-7,7-dimethyl-2,5-dioxo-1-(p-tolyl)-1,2,5,6,7,8-hexahydroquinoline-3-carboxamides as lead period shortening compounds. In order to identify structural analogs that could enhance the activity of these amide moieties, a compound library was synthesized by coupling various substituted anilines and amines to phenazine-2-carboxylic acid and hexahydroquinoline carboxylic acids to synthesize several phenazine and hexahydroquinoline amides. These derivatives will be screened for activity using the Rat1 fibroblast cell line to determine their effects on the circadian rhythm period. |
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Kevin Schwarz
Abstract: Collagen IV networks are proteins found in the basement membranes of the epithelia of all animals. The networks provide structural robustness to tissues and serve as scaffold in the production of other macromolecules. At the C terminus of Collagen IV, two protomers associate through trimeric non-collagenous domains, forming a hexamer structure. It has been postulated that the protomer-protomer interface is covalently cross-linked by a rare nitrogen-sulfur double bond between hydroxylysine and methionine of Collagen IV. The objective of this investigation is to replicate the sulflilimine cross-link between a lysine and methionine presented on a synthetic small molecule scaffold. Oxidative formation of the sulfilimine bond will provide an opportunity for the chemical and analytical characterization of the (S=N) sulfilimine bond. Furthermore, the formation of the lysine-methionine cross-link presents a potential hapten for production of an antibody for sulfilimine bond recognition in biological samples. |
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Lana Thomas
Abstract: Detection of pathogens can be conducted using nucleic acid-based diagnostics which are specific and highly sensitive. Today, these diagnostics can be performed by using commercially available Rapid Diagnostic Tests (RDT). RDT kits require sample preparation procedures prior to analysis to eliminate potential interferents. However, the procedures often require the use of specialized instrumentation and trained technicians. In a low resource environment, specialized instrumentation and trained technicians are not readily accessible. Therefore, we utilized a previously developed self-contained nucleic acid extraction cassette suitable for a low resource environment. The cassette contains preloaded solutions separated by air gaps within a continuous length of 1.6 mm inner diameter Tygon tubing. The mRNA binds to the dT sequence of the Dynabeads® Oligo (dT) 25. The beads are then transferred into each preloaded chamber of the Tygon tube using an external magnet to wash and elute into the final solution. The efficiency of the cassette extraction was evaluated by quantitatively measuring the mRNA recovery. mRNA was recovered from Lysis Binding Buffer using RNase- free tubes and the Tygon tubing with 62.75 and 88.00 % efficiency, respectively. Future studies will be conducted to isolate mRNA from complex samples. |
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