Graphene is a ground-breaking material with over twenty thousand patents involving its applications and modifications. It is the strongest, yet thinnest material known with semi-conducting properties. Extensive research has been performed on graphene to improve characteristics such as its conductivity, formation of holes and chemically manipulating its structure. Specifically, graphene is a two-dimensional, single atomic layer of graphite; which is an allotrope of carbon made up of tightly bonded carbon atoms organized into a hexagonal lattice. What makes graphene of interest is its sp2 hybridization and nanoscopic atomic thickness (of 0.345nm). An alternative structure comparative to graphene involves utilizing a self-assembly condensation between amine containing compounds and carbonyl groups, which form a graphene-like nitrogenated derivative. These materials have ordered pores with high crystallinity. The synthesis of such compounds is well established and have been characterized as Covalent Organic Frameworks (COFs). This amine/carbonyl condensation reaction can produce significant control of the lattice structures desired in the final 2-D material. This material can then be fabricated into membranes for applications in separations, batteries and more. The goal of my work utilizes this condensation method to chemically synthesize a framework with two chemically distinct pores in the lattice. The two pores are capable of chemical manipulation, which would potentially be superior to graphene. This gives rise to a variety of applications of the material and its derivatives, which we hope to improve on by using these nitrogenated frameworks.

Pseudomonas fluorescens strain L5.1-96 has been known to help combat the wheat take-all fungal pathogen, Gaeumannomyces graminis var. tritici (Ggt). Strain L5.1-96 is maintained in the soil longer than others, so it is known as a supercolonizer, which contributes to the phenomenon referred to as Take-All Decline (TAD). The strain L5.1-96 is a non-pathogenic bacterium which grows in the rhizosphere of plants and produces an antifungal agent known as 2.4-diacetylphloroglucinol (DAPG) causing TAD. The DNA fragment we amplified, sequenced, and analyzed was a perfect match to P. brassicacearum strain BS3663. Through bioinformatics, we also found our fragment matched the genetic code for pyridoxal 5'-phosphate (PLP)-dependent aminotransferase, a co-enzyme which catalyzes the transamination of amino acids to alpha-keto acids. We did not identify new proteins that are responsible for the advantages that this bacterium holds against wheat take-all; although, we did find the best phylogenetic fit for this strain. These findings contribute information to the broader study at Washington State University to investigate whether strain L5.1-96 can be used as a biocontrol agent against wheat take-all.​

Epigenetic modifications regulate chromatin structure and function, playing important roles in altering DNA transcription levels and subsequently cell fate decisions. Various next-generation sequencing (NGS) methods have been developed to detect these epigenetic changes in the genome, such as chromatin immunoprecipitation followed by sequencing (ChIP-seq). Even though ChIP-seq is extensively used to analyze DNA and histone modification levels, this method is limited to one histone marker at a time and requires significant amount of input cells, which masks the profiles of cell-to-cell variation and the complex interaction between the epigenome and gene expression. To overcome these limitations in current next-generation sequencing methods, we have been developing a multiplexed assay that could detect multiple epigenetic modifications in single cells. So far, I have developed a NGS data analysis pipeline to identify potential gene candidates that are highly differentially modified by histone markers. In the future, we hope to use these gene candidates as templates to design DNA-fluorescent in situ hybridization (DNA-FISH) probes and perform Expansion Microscopy and DNA-FISH with these probes to link histone modifications to specific gene loci at high resolution. After the assay is fully developed and validated, we plan to utilize the assay to take the epigenetic profiles of hematopoietic stem cells and study cell fate decisions in hematopoiesis.

Many biochemical processes rely on the conversion of electrical energy into chemical energy. Tryptophan is one aromatic amino acid that facilitates electron transport through proteins by means of reduction and oxidation reactions, allowing for the energy conversion to occur. The ability for a protein to tune the reduction potential of tryptophan is critical to its function in reactions within enzymes such as cryptochromes, which are involved in regulation of circadian rhythm, and photolyases, which are involved in DNA repair. This tuning of the reduction potential is achieved by modulation of the microenvironment surrounding tryptophan residues within a protein. In order to study the relationship between protein environment and tryptophan oxidation, we have designed and synthesized a series of model peptides to engineer specific environments which have the same geometry but differing charge states and levels of solvent exposure. The reduction potential of each peptide was determined via differential pulse voltammetry over a pH range from 1-7.5 in order to determine how the pH dependence of tryptophan's reduction potential responds to changes in its microenvironment. The results indicate that the reduction potential of tryptophan at the physiologically relevant pH=7.5 increases with increasing positive charge in the surrounding microenvironment. The results across the entire pH range indicate that the pKa of oxidized tryptophan increases with decreased solvent exposure. Finally, our entire series of peptides indicates that the tryptophan oxidation reaction does not directly follow the expected Nernstian behavior for pH dependence of a single proton-coupled electron transfer reaction and thus indicates that the oxidation process may have differing degrees of reversibility in difference environments.

Synthetic biology allows us to use an organism’s metabolic pathways to produce new, useful metabolites cost-effectively. Growing organisms in a culture allows the production of these metabolites on a commercially-viable scale. However, management of these cultures is time-consuming and labor-intensive, such as HPLC. Our project aims to provide a low-cost, automated system that analyzes the culture in real time at low cost. Our device, a turbidostat bioreactor called the Chromastat, uses images and other optical data to measure the current state of a culture. If these measurements show metabolite production outside specifications, the system modifies the culture’s metabolic expression by adding inducers. To make this system cost effective, we focused on using affordable materials, an open-source controller and analysis system, and visible color outputs instead of the traditional fluorescence. To test our system, we used the violacein metabolic pathway in yeast. By regulating gene expression with inducible promoters, this pathway yields up to four visually distinct pigments. In a production environment, these pigments could provide a proxy measurement to produce useful substances which are costlier to detect. Pigment production is measured by the popular low-cost and open-source Raspberry Pi computer running a program displayed in Java Swing. Optical sensors collect color and opacity information about the culture. Our software, installed on the Raspberry Pi, analyzes the color, and in response, the hardware introduces inducers to stabilize the culture’s production. Data recorded by the Pi over time reveals the relationship between gene expression and metabolite production rates. By combining biological, software, and hardware systems, our unique design can generate previously unavailable visual data in certain biosynthesis processes, such as those involved in antibiotic production or fermentation.

lncRNAs play a wide range of cellular functions, and the roles they play in cell maintenance are of increasing importance. An unusually conserved lncRNA, OIP5-AS1 (known as Cyrano), plays a crucial role in stem cell differentiation and development. It has also been shown to interact with the miRNA miR-7 and the protein HuR. Although there is growing information about interactions of Cyrano, truly understanding this lncRNA’s function is hindered by the lack of structural information. To gather information on the structure of the evolutionary conserved region of Cyrano, the secondary structure was elucidated using SHAPE and RNA prediction software. A 4-way junction was observed when assessing these data. The 2D predictions were used as constraints to generate 3D models of the 4-way junction. Overall, three separate prediction software generated topologically similar 3D models of Cyrano (RMSD range of 13.362 Å). These models produce a 4-way junction with 2 coaxial stacked helices (H1H2, H3H4). These modeled structures suggest a Family cH 4 way junction, where H1 and H3 interact forming an A-minor interaction between junction J(1,2) and Helix H3. This model is important as it aids in directing future experimental approaches for studying Cyrano, and also hint at a potentially important structural motif for Cyrano function.

My presentation reviews existing literature on the use of boron clusters in liquid crystals. Liquid crystalline substances are usually composed of long, thin carbon-based (organic) molecules and have applications in electronic display technology and chromatography. Many compounds with liquid crystalline phases contain rigid substituent groups. The rigidity of inorganic boron clusters and their ability to form linear compounds by attaching groups to atoms on opposite ends of the cluster make them an intriguing starting point for the synthesis of new liquid crystalline materials. Work conducted on the synthesis and properties of boron cluster-based liquid crystals in comparison with organic cyclohexane- or benzene-based analogues is discussed. In addition, new derivatives of a specific boron cluster, CB11­H12­-, synthesized in our lab using microwave and stardard Schlenk line techniques, are presented, along with an evaluation of their potential as precursors for new liquid crystals. We present a relaible pathway to a new difunctionalized carboxylic acid derivative.

Colloidal semiconductor quantum dots (QDs) have recently garnered attention for applications in nanoscale technologies including photovoltaics, displays, sensing, and energy storage. Radiative luminescence originating from recombination between one electron and one hole in the excited state is vital to the performance of these materials for these applications. One prominent relaxation pathway that competes with radiative luminescence in QDs is non-radiative Auger recombination, a process that can either be detrimental to device performance or in some cases harnessed to enhance various technologies. For this project, we use redox chemistry to controllably engineer negative trions (two electrons and one hole) in colloidal QDs, purposely introducing a negative-trion Auger recombination pathway. Previous results have demonstrated a size dependence of negative-trion Auger recombination rates in various QDs. This work uses time-resolved photoluminescence spectroscopy to explore the difference in size dependence for QDs with and without trap states. Preliminary findings indicate that negative-trion Auger recombination generally occurs more efficiently and exhibits a shallower size dependence when one of the carriers is trapped than when all carriers are delocalized. This work will allow for future scientists and engineers to design QDs that either suppress or make use of Auger recombination to optimize device performance.

In humans, CYP2J2 is involved in both drug metabolism and converting arachidonic acid to epoxyeicosatrienoic acids (EETs), which then act as signaling molecules. EETs are involved in angiogenesis, vasodilation, opening of ion channels, and general protection against cardiotoxicity. Currently, the Totah lab is testing the hypothesis that CYP2J2 has a critical role in the heart as one of the main contributors to EETs production. This research project seeks to determine whether CYP2J2 regulation is similar among different tissues, specifically between the heart and the liver, under oxidative stress conditions. A critical function in the heart would mean that under times of stress, CYP2J2 expression will increase. In contrast, CYP2J2 expression in the liver is expected to decrease similar to other CYP proteins under oxidative stress. To test this, reagents that increase reactive oxygen species (ROS) levels have been incubated with hepatic cells (HepG2) and the levels of CYP2J2 mRNA were measured and compared to untreated control cells. In the heart, CYP2J2 has a more important function in creating protective EETs and the benefits from increasing production of EETs have been shown in our lab to protect against the harmful effects of ROS. A few of the conditions being tested are: varying concentrations of hydrogen peroxide, cobalt chloride, and hypoxic conditions. MTT, a tetrazolium based dye, assays were used to quantify cell viability and rt-PCR was used to determine gene upregulation or downregulation of CYP2J2. Afterwards, the results were compared to similar treatments and conditions in cardiomyocytes. If transcription in HepG2 cells is downregulated but increased in cardiomyocytes, it supports the hypothesis that CYP2J2 has a critical role in the heart compared to the liver, specifically under times of ROS stress.

Protons take part in many important reactions such as redox, acid-base chemistry and biochemistry; however, the mechanisms for proton transfer and hydronium’s stabilization are less understood. Hydronium sequestered in 18-crown-6 ether (CE) has an unusually broad and intense vibrational absorbance (OH-feature) compared to bare hydronium. One environmental factor affecting this OH-feature is the range of hydrogen bonded geometries between the hydrogen atoms in hydronium and the oxygen atoms in the CE, which is sampled by the complex even in its ground vibrational state. The breadth of the OH-feature is attributed to changes of the OH stretch frequency of hydronium as it rattles within the CE, and to the CE distorting to accommodate the hydronium. Hydronium will donate a proton to diethyl ether in the binary complex, because the diethyl ether is a stronger base than water. However, when hydronium hydrogen bonds to three diethyl ether molecules in a ternary complex, all three hydrogen atoms in hydronium are pulled by the ether molecules preventing any one of the protons from transferring. The hydrogen bonded geometries we analyze involve complexes of hydronium with both simple ethers and cyclic CE molecules. The CE hydrogen bonds to all three of the hydrogen atoms in hydronium similarly to the ternary complex, thus the CE complex prevents proton transfer. We explore several other environmental factors, including increasing the rigidity of the CE by replacing carbon-carbon single bonds with carbon-carbon double bonds. This increase in CE rigidity reduces the rattling of the hydronium. We also explore the effects of reducing electron density on the oxygen atoms in the CE by replacing hydrogen atoms in the CE with fluorine atoms. Optimizations, frequencies and potential energy scans are made at the B3LYP level of theory using a 6-311G(2d,p) basis.

Human genetic variation is ubiquitous and its impacts are often not fully understood. The purpose of this study is to examine and catalog genetic variation in the specific context of the Fanconi Anemia disease family. Fanconi Anemia (FA) results from mutations in any of 22 FANC (Fanconi) genes. FA disease-associated pathogenic variants lead, in almost all instances, to loss of Fanconi protein expression and/or function: this is consistent with the recessive Mendelian inheritance of FA as a clinical disease phenotype. Recent work has identified aldehyde damage as an important driver of FA pathway function, and an important contributor to FA disease pathogenesis. Aldehydes are ubiquitous, potentially toxic molecules that are generated as part of normal cellular metabolism. Aldehyde damage is limited by the actions of aldehyde (ALDH) and alcohol (ADH) dehydrogenases, two gene families encoded by 26 different genes. To systematically characterize base pair-level pathogenic and human population-level genetic variation in the FANC, ADH and ALDH gene families, we built new databases of human genetic variation for clinically-ascertained FANC gene pathogenic variants (n = 1,015 variants), and for population genetic variants in the 22 FANC genes (n = 13,306 variants) and 26 ADH/ALDH genes (n = 8,388 variants). Population genetic variation was sourced from over 60,000 individuals included in the Exome Sequencing (ESP), the 1000 Genomes (1KGP) and the Exome Aggregation Consortium (ExAC) projects data. In order to interrogate potential pathogenicity we used CADD (Combined Annotation-Dependent Depletion) scoring, which provides a robust assessment of the predicted deleteriousness of base substitution and short indel variants by objectively integrating many diverse annotations into a single measure (C score) for each variant. This project tested the implementation of CADD scoring as a model and identified new potential pathogenic candidates in the ADH/ALDH gene families for future experimental confirmation.

The purpose of this study was to explore the interrelationship between two major DNA repair systems--photoreactivation, with blue light as an energy source, and excision repair, which uses ATP as an energy source--of Chlamydomonas reinhardtii, a single-celled algae, by studying the in vivo efficiency of photoreactivation when the excision repair system was suppressed. Working with a mutant which was completely lacking in excision repair, we found that this strain was also deficient in photoreactivation, as compared to wild-type, at low levels of post-UV visible light treatment, measured by killing on plates. We hypothesized that the level of light intensity might have an impact on the efficiency of the photoreactivating enzyme. Using alkaline agarose gel electrophoresis analysis, we quantitatively evaluated the extent of repair of DNA damage over 24 hours under two different light intensities. We found less damage removal in the mutant under low light intensity, suggesting that a positive correlation does indeed exist between the flux of visible light used in our experiments and the amount of photoreactivation for the mutant. This correlation was not observed in the wildtype. Our work is interesting in the context of human-caused changes to Earth’s atmosphere; both photoreactivation and excision repair of DNA damage are present in virtually all organisms studied, including bacteria, fungi, plants and most animals (although not in placental mammals). With the weakening of the stratospheric ozone layer, there is the possibility of increasing solar UV flux to the surface of Earth, with possible negative downstream effects on biological systems. Understanding the functional relationship between these two DNA repair systems could provide information of fundamental importance to ecological and agricultural problems arising from increased solar UV flux to Earth.

Utilizing protein-based systems, specifically nuclease-dead Cas9 variants, to alter mammalian gene expression is an emergent subfield in synthetic biology. Such methodologies offer means to study a vast diversity of genetic networks including but not limited to cell fate decisions, stem cell differentiation, cell signaling networks, and key genetic players in tumorigenesis. Here, we offer a novel chemical tool for which drug concentration can control gene expression within target cells, by using new chemically induced dimerization systems that can respond to multiple chemical agonists and antagonists. Within our system, we use a protease dead NS3a complex, which functions as a scaffolding complex, and an entirely synthetic protein complex, either a danoprevir-NS3a complex reader (DNCR) or a grazoprevir-NS3a complex reader (GNCR), which specifically bind NS3a only when danoprevir or grazoprevir, respectively, are introduced. Furthermore, nuclease-dead Cas9 offers a means for target sequence specific tethering of nuclear effectors, and may be directed using sgRNAs. The sgRNA sequence may be elongated to allow for a protein specific tethering complex. Here we use an orthogonal scaffold RNA/RNA-binding protein pair (MS2/MCP and PP7/PCP) to localize DNCR (bound to PCP) or GNCR (bound to MCP) to select DNA sequences. The corresponding NS3a complex is fused to enhancer protein VPR, which serves to upregulate gene expression when in sufficiently close proximity. Hence upon drug induction, NS3a-VPR will bind the DNCR-PCP (or GNCR-PCP) complex tethered to dCas9 localized ideally upstream of a target gene, and induce gene expression upon introduction of danoprevir (or grazoprevir). As a direct function of this research, we have demonstrated CID capacity for linearized fold increase of several endogenous and non-endogenous genes in correlation to drug concentration, and now seek to demonstrate the capacity of our tool by studying the concentration-dependence of scaffold function in the JNK pathway.

In scientific research labs, in general, experiments are generally treated as a black box: a prepared sample goes in, something happens, and one gets results that are then obtained via elaborate characterization steps. Several important dependent or correlated parameters are either discarded or ignored because of a lack of coherent dependency analyses that require critical thinking, linking, and pattern recognition. In this research we are working to stop treating experiments and computational simulations as black boxes, and create a cohesive platform where materials used, processes and parameters utilized and results achieved can be brought together as separate but related sets of databases. In the next step, the relationships between all the different parameters can then be connected, analyzed and visualized. Machine learning and AI techniques can then be used to predict results using these databases, thereby reducing experiment time, and taking away the traditional ‘trial and error’ method of experimentation. The research involves creation of a software interface, with numerous image and signal processing tools and applications running on libraries made customizable to research fields, types of experiments, etc. Assorted variety of services such as parallelization, compression, data analysis, and visualization, caching (among others) are also provided. We are improving the accuracy of time series data analysis and using fingerprinting to depict all parameters for improved predictability, flexibility and accuracy. When fully developed, we anticipate that the program will enable experimental and computational researchers to extensively use, customize and apply data analytics, machine learning and AI even in niche research in the hard sciences at the intersection of biology and genetics, materials science (physics, chemistry) and engineering, and computational modeling and informatics, enabling faster and accurate cross disciplinary innovation in technology and medicine. The research is supported by NSF-DMREF (DMR-1629071) program at GEMSEC-MSE, as part of National Materials Genome Initiative.

Conventional farming techniques involve large quantities of chemical fertilizers, which often leach into bodies of water causing eutrophication. The influx of excess nutrients from fertilizer results in rapid increase of aquatic algal populations followed by dissolved oxygen depletion. This process creates regions of low oxygen that negatively impact the water quality of many major lakes and coastal regions. To address this problem, the sensitivity of plant growth-promoting bacteria (PGPB) to excess fertilizer was investigated. We hypothesize that the addition of high amounts of fertilizer will result in smaller, less productive plants and diminished rhizosphere colonization of the PGPB, Bacillus subtilis. A modified Kirby-Bauer disk diffusion test was performed as an initial assessment of the effect of varied fertilizer concentrations on B. subtilis. Roots of romaine lettuce seedlings were then inoculated with B. subtilis and grown in soil treated with the same range of fertilizer concentrations. The effects of each treatment on plant growth were determined using total leaf area, quantification of the rhizosphere colonization by B. subtilis, and carbon assimilation measured with a LI-6800 Portable Photosynthesis System. These results are an important step towards establishing guidelines for appropriate application of agricultural fertilizer in order to mitigate the frequency and severity of eutrophication events in aquatic systems.

Perovskites are a class of material with the formula ABX₃ with lead-halide perovskites having promising photovoltaic properties. Over the past few years syntheses of colloidal inorganic lead-halide perovskites have been reported. Recently concerns over the toxicity and instability of lead-halide perovskites have driven research toward lead-free alternatives, including double perovskites with the elpasolite structure. Elpasolites are double perovskites where the divalent cation is replaced with a trivalent and a monovalent cation to get the formula A₂BCX₆. Despite this motivation towards less toxic alternatives, synthetic approaches remain limited with no examples of heterometallic elpasolite nanocrystals being reported. We report colloidal synthesis and characterization of nanocrystals of Cs₂AgBiX₆ (X = Cl, Br) elpasolites via hot-injection of TMS-halide under inert atmosphere and rapid quenching with an ice bath. We further show that through postsynthetic anion exchange or cation extraction the nanocrystals can be converted to new materials, such as the previously experimentally unknown Cs₂AgBiI₆. Nanocrystals of Cs₂AgBiI₆ were made via anion-exchange using trimethylsilyl iodide and have strong absorption throughout the visible region which confirms predictions that this material could be a good photovoltaic absorber. The synthetic methodologies presented are expected to be generalizable, with work already under way to optimize this for more well-known nanocrystals. This work also shows how ion-exchange reactivity of nanocrystals can lead to the discovery and development of lead-free halide perovskite materials which would be difficult, if not impossible, to make via direct synthesis.