Festival of Science 2019 - Student Abstracts
Festival of Science was held Friday, April 26, 2019
David Bain, Chemistry, Faculty Sponsors: Samantha Glazier and Catherine Jahncke
“Kinetic Characterization of Chemotherapy Drugs”
Abstract: Certain flat, planar molecules can insert between DNA base pairs (intercalate) to disrupt replication and transcription upon mitosis in cells. This makes them ideal candidates for mitigation of tumor growth, a process which relies on uncontrolled cell division. To design improved drugs, an understanding of both thermodynamic and kinetic intercalative pictures is necessary. Currently, kinetic information is technique limited. Stopped-flow fluorimetry measurements have 10 ms time resolution, unfortunately many binding reactions occur on timescales orders of magnitude faster. A homebuilt temperature jump (T-jump) apparatus could achieve up to 1 μs resolution via capacitance discharge heating coupled with relaxation kinetics. This works by converting stored electrical energy from a capacitor to thermal energy in solution where binding occurs. A faster capacitor discharge leads to quicker solution heating and increased resolution in relaxation kinetics calculations.
An interesting case study is provided by the intercalating drug proflavine. Using molecular dynamics calculations, Mukherjee et al. found that the intercalation mechanism involves both the major or minor groove of DNA. This is the first report of major groove involvement. Systematically blocking each groove before measuring kinetics via T-jump will show, experimentally, whether both grooves are involved in the intercalative binding mechanism. This technique and investigation of major groove involvement can be applied to many chemotherapy agents and will pave the way for future drug synthesis for superior anti-tumor potency.
Lisa Kozodoy, Biochemistry, Faculty Sponsors: Nadia Marano and Lorraine Olendzenski
“Isolating amyloid fibers from Haloferax volcanii”
Abstract: Amyloids are proteins that when aggregated form large insoluble fibers characterized by their cross-beta sheet structure. While these proteins are often implicated in human neurodegenerative diseases, such as Alzheimer’s, many organisms make functional amyloids. These have been studied in bacterial biofilms and are known to aid in stability and structure. Archaea also form biofilms, however, they have not been researched as extensively. Functional amyloids have been identified in the archaeon Haloferax volcanii. The aim of this study is to isolate and purify amyloid fibers from H. volcanii to elucidate more about their composition and structure. Isolation is done through differential centrifugation and sonication, following previous research by Heather Raimer ‘17. Sonicating the cells releases the amyloid fibers from the cells, which are detected using Thioflavin T (ThT) assays. Low-speed centrifugation allows for the removal of cellular debris. Ultracentrifugation then isolates the larger fibers from suspension. Lastly, tube SDS-PAGE is used to purify the amyloids from contaminating proteins because they do not enter the gel. Current research is being done to deaggregate the fibers into monomers using formic acid. The depolymerized monomers can then be resuspended and sent off to a collaborator for sequencing.
Evan Ketcham, Biochemistry, Faculty Sponsor: Matthew Skeels
“Engineering Thermodynamically Stable Mutants of Human Basic Fibroblast Growth Factor Protein”
Abstract: Fibroblast growth factor 2, also known as FGF basic, is a signaling protein that plays a variety of physiological roles in the body. Of particular interest in recent studies is its ability to promote angiogenesis following blood vessel injury. However, it is a labile protein that tends to break down quickly under the stressors of the human body. Previous research has determined, in silico, several rationally-designed mutant proteins that are predicted to be more stable than the wild type. This project seeks to create these mutants and compare their stability in vitro to FGF2. A bacterial expression system is used to create the protein, which is then purified using chromatographic methods. After purification, the linear extrapolation method (Vivian and Callis 2001) is used to experimentally determine the change in Gibbs free energy of unfolding for the protein. In this method, the unfolding of a protein is followed by monitoring intrinsic tryptophan fluorescence as it is exposed to increasing concentrations of chaotrope. These data can be mathematically transformed into an experimental ΔG value. The data have demonstrated that the trend in stability matches the computational model, with the mutants known as FGFbase (N71E, E78K, K86E, T112E) and FGFrecp (N53E, K68E, R102E, K111E, K117E) being more stable under chemical denaturing conditions than the wild type FGF2. Further research is being conducted to probe more mutants for their stabilities and assess their relative biological activities using cell cultures.
Jiarui Zhang, Chemistry, Faculty Sponsor: Larry French
“Synthesis of Anthraquinone Derivatives by Editing DNA Binding Functional Groups”
Abstract: Anthraquinone is a polycyclic aromatic hydrocarbon, and its derivatives have appeared in many anticancer chemotherapeutic drugs, such as Doxorubicin, Mitomycin, and many other biologically active compounds. The anthraquinone moiety may act as a DNA intercalator and its derivatives can slide between nucleotide bases forming covalent bonds with DNA to act as cytotoxic molecules by stopping the cell cycle within various cell types. This project is to expand the variety of anthraquinone derivatives. The multistep reaction started with the Diels-Alder reaction forming the anthraquinone molecule. The Diels-Alder reaction allows the formation of a six-membered ring by creating two sigma bonds, and has been utilized in many reactions of organic synthesis. The double Diels-Alder reaction utilizing myrcene as diene and benzoquinone as dienophile will form two six-membered rings flanking quinone. This multi-step synthesis can be used to generate derivates based on the multiplicity and variability of the functional groups, be editing and expanding the variety of side functional groups may cause more interactions with DNA backbone or nucleotides for potential pharmaceutical interest. In particular, we are trying to synthesize an anthraquinone that is flanked by two piperazine moiety that we hypothesize may increase the affinity of DNA binding.
Dylan Babcock, Chemistry, Faculty Sponsor: Larry French
“Sustainable Chemistry Utilizing Terpenes Synthesis of Anthraquinone Analogs from Myrcene”
Abstract: The use of terpenes in synthesis of chemical products may bring about a more sustainable future. Currently, chemical procedures include bulk chemicals such as hydrogen, benzene, and propylene obtained from oil and gas. The movement towards a more sustainable future is promoting green alternatives in industrial synthesis. This research focuses on exploring the functionalization of myrcene towards known DNA intercalators. A myrcene derived anthraquinone analog is foundation of our work with DNA intercalating agents. The molecules synthesized in this research are useful in that they eliminate the need for some environmentally harmful precursors as well as have the potential to mimic some of the structural properties that important drugs have. Analysis of these compounds is being done through GC/MS as well as C13 and H1 NMR. Future experiments will focus on the continuation of functionalizing the myrcene arms to better interact with DNA base pairs as well as finding methodology for testing our molecules efficiency as intercalators.
Sarah Potter, Chemistry, Faculty Sponsor Matthew Skeels
“Nutritional Analysis of Honey”
Abstract: Bee populations around the globe have been decreasing for the last several decades. While many factors are contributing to this decline, several are related to nutrition. A diverse diet is critical for bee’s growth, reproduction, learning, and resistance to viruses and parasites. The nutrition available to bees can be investigated by analyzing the nutritional profile of one of their major food sources, honey. The goal of this work is to develop facile methods to analyze the nutritional profile of honey. Analytes include moisture content; minerals and metals; sugars, glycerol, and organic acids. Moisture content will be measured as a percentage of weight after honey is heated to a constant mass and through refractometric methods. Samples will be subjected to microwave digestion and minerals and metals will be determined with FAAS. An enzymatic assay will be applied for glycerol and glucose determination. Organic acids will be analyzed using solid phase extraction followed by HPLC. Techniques developed will be applied to the study of monofloral honeys to compare their respective nutritional profiles. Eventually, this information will be used to create environments with plants that provide appropriate nutrition for pollinators year-round.
Lillian Devereux, Biochemistry, Faculty Sponsor Nadia Marano
Detection of Amyloid Oligomers using ANS (1-Anilinophthalene-8-sulfonic acid), Bis-ANS (4,4’ –Dianilino- 1,1’ –binaphthyl -5,5’-disulfonic acid), and DCVJ (4-dicyanovinyl-julolidine)
Abstract: Amyloid fibers are a component of bacterial biofilms that can be isolated through various protocols for analysis. The standard assay uses Thioflavin T (ThT) to identify the presence and quantity of mature amyloid fibers. ThT is a molecular rotor meaning that it rotates around a single bond. When bound to amyloid fibers this rotation is inhibited and stabilizes the excited state, thus producing measurable fluorescence. However, ThT is not an effective measure of amyloid oligomer formation, the smaller building blocks of mature fibers. ANS, Bis-ANS, and DCVJ are three alternatives to ThT that are characterized by a multi-ring structure. DCVJ acts in a similar way to ThT in that, when bound, the molecule is locked into an excited state producing fluorescence. In contrast, ANS and Bis-ANS bind to the hydrophobic regions of proteins which are exposed during amyloid fiber formation and following binding, the molecules fluoresce. Little is currently known about the binding and consequent fluorescence of these molecules to oligomers. This research will examine the ability of these three fluorophores to bind to oligomers throughout the aggregation process, giving an indication of kinetics and method of amyloid fiber formation. Insulin will be used because it forms amyloid fibers under well-defined conditions. Preliminary research shows DCVJ and ANS follow the pattern of ThT, meaning that as mature fiber concentration increases, so does the fluorescence signal. Bis-ANS, however, displays the opposite pattern, indicating it is likely detecting varying concentrations of amyloid oligomers via binding to the exposed hydrophobic residues.
Heinrich Salzmann, Chemistry, Faculty Sponsor Adam Hill
“Hafnium Containing Heterobinuclear Light Absorber Units”
Abstract: Harnessing solar energy efficiently is one way to tackle climate change and our dependency on fossil fuels. Metal to metal charge transfer in heterobinuclear units matches the energy of visible light. Hence, heterobinuclear light absorbers are potentially key features in artificial photosynthesis systems. The excited electron state is unknown but expected to undergo a spin flip. Heterobinuclear units on silica surfaces make an effective system in which to investigate intersystem crossings in the context of electron transfer to a 2,2’-bipyridine chromophore. Hafnium is heavier than previously studies early transition metals in heterobinuclear units and will have 60 times stronger spin-orbit coupling. Hafnium-cobalt oxo-bridged on silica nanoparticles have been synthesized. Infrared (IR) and Raman spectroscopy were used to ensure successful synthesis steps. Samples have been probed with 2,2’ bipyridine and analyzed with time-correlated single photon counting (TCSPC) and time-resolved fluorescence, to investigate the lifetime of the excited state.
Alissa Stone, Chemistry, Faculty Sponsor Adam Hill
“Time-correlated single-photon counting measurements of electron transfer between heterobinuclear units and bipyridine”
Abstract: Understanding the excited metal-to-metal change transfer (MMCT) states of heterobinuclear units is vital to creating artificial photosystems. To learn more about the metal electron transfer and to model artificial photosystems, binuclear units on silica nanoparticles were synthesized using air free techniques and coupled to bipyridine chromophores. Particles were pressed into transparent pellets and were characterized using UV-visible and FT-IR spectroscopy. Time-correlated single-photo counting (TCSPC) and 2D fluorescence data were collected to measure the excitation spectrum, emission yield, and lifetime of each binuclear unit/chromophore charge transfer polyad. These data provide evidence that when the energy levels of the system are in the correct rectifying arrangement, the excited state persists to the microsecond timescale.
Abigail Enders, Chemistry, Faculty Sponsors Adam Hill and Samuel Tartakoff
“Kinetics of substituted Wagner-Jauregg reactions studied by 1H-NMR with an adaptive Python program for integration of poorly resolved spectra”
Abstract: Monitoring chemical kinetics by NMR requires reproducible and accurate integration for hundreds of 1H NMR spectra, but existing software packaged with many instruments is insufficient for the purpose. Kinetics studies of the Wagner-Jauregg reaction between para-substituted styrenes and maleic anhydride for determination of the mechanism have been previously limited by NMR instrumentation and its accompanying software. This class of reactions is an example of a challenging problem in which variability between substrates and other background signals make good integrations difficult using common techniques. Custom code, written in Python and using the lmfit library incorporates the reproducible integration of multiple peaks and is adaptable for a variety of spectral features. A graphical user interface makes the code accessible to new users. With this additional analysis, the Diels–Alder-type mechanism of Wagner-Jauregg was confirmed by measuring the effects of substitutions on rate.
Any changes need to be made please let Diane Chase know.