Lck is a lymphocyte-specific tyrosine kinase involved in T cell activation, which is essential for the human immune response. Upon antigen engagement with the T Cell Receptor (TCR), Lck phosphorylates the CD3ζ chain of the TCR, transducing intercellular signaling that activates T cells. Recent studies have demonstrated that Lck’s phosphotransferase activity is not only important for T cell activation, but that Lck also plays a critical role in scaffolding the interaction between the phosphorylated CD3ζ chain of the TCR and the kinase ZAP70 using its regulatory domains. Lck’s phosphotransferase activity has been shown to be toxic to yeast, with increased activity correlating with decreased yeast-growth rates. Using a yeast growth-based deep mutational scan (DMS), we calculated the activity scores of ~5,000 single amino acid variants of Lck’s kinase domain. Through this DMS, we identified all positions on the kinase domain that are amenable to substitution without perturbing kinase activity. In particular, we focused on positions where we could install cysteine residues on the kinase domain without perturbing kinase activity. Currently, we are expressing these cysteine variants in primary T cells, and applying parallel chemoselective profiling methods to quantify changes in the electrophilic reactivity of the cysteine side chains upon TCR stimulation. The expected changes in alkylation of the cysteine side chains upon TCR stimulation will provide insight into changes in the conformational flexibility of Lck, accessibility of the substituted residue sites, and intramolecular protein-protein interactions (PPIs) of Lck upon TCR stimulation. Ultimately, this insight into the conformational dynamics of Lck can be applied to deepen our understanding of basic immunology and the T cell activation signaling cascade.

TKIs are a class of targeted chemotherapeutics preventing cancer cell proliferation by inhibiting the Human Epithelial Growth Factor (EGFR) phosphorylation signal cascade. However, the transport of TKI in tissue is poorly understood. In addition, studies of direct drug resistance are hindered by an inability to measure native intracellular drug concentration. Therefore, it is important to understand drug exposure and response at the single-cell level to gain insights into the single-cell drug response and resistance development. The project objective is to quantify the intracellular tyrosine-kinase inhibitors (TKI) drug concentration in human lung cancer tumors using stimulated Raman scattering (SRS) microscopy to better understand cellular drug resistance, drug response, and microenvironmental heterogeneity upon drug exposure. We use the single cell level in vitro model and the mouse dorsal skinfold chamber (DSC) in vivo model. We will use a novel chemical imaging tool, SRS microscopy, to directly quantify single-cell drug concentrations. We first study the cellular-level drug response and resistance in live cells. By comparing the data of MTT Assay and single cellular level image drug concentration, we will be able to study the resistance level of A549 cells.Next, we will move to an in vivo mice model. The dorsal skinfold chamber (DSC) model is well-established for studying tumor development and time-lapse analysis of tumor progression. Quantifying tumor concentration gradient allows for discovering optimal drug dosing conditions that penetrate inner layer tumor cells without harming the host. The SRS microscopy imaging technique enables the quantification of intracellular TKI concentrations within individual cells in the necrotic layer against the proliferation layer of the tumor. By comparing the results from in vitro single-cell image to the in vivo model, we are able to better understand the drug response of A549 to TKI treatments.

Computational chemistry can be used to guide experimental studies by giving insight into what could occur in experiments on the quantum level. Although valuable, the information from these simulations is stored in large datasets, which could be difficult to interpret. Thus, visualization of quantum solutions is important. This software development project focuses on the visualization of molecular orbitals for heavy-element systems and time-dependent electron density for systems undergoing an external field with the Chronus Quantum (CQ) computational chemistry program. We visualize molecular orbitals by taking molecular orbital (MO) coefficients from calculations with CQ, and running the data through an algorithm to reformat into a visualization file readable by the commonly used software, GaussView. Visualizations can be done for standard quantum mechanical methods such as DFT; however, we can also visualize complex MOs in the 2- or 4-component formalisms often needed for relativistic calculations, which can account for the spin-orbit coupling important for heavier elements. After transcribing data from CQ to a visualization file, the coefficients and basis functions are processed with the GaussView program, yielding 3D representations of MOs. These visualizations will aid our understanding of quantum phenomena involving heavy elements such as late-row transition metals, lanthanides, or actinides. Additionally, we want to provide a visualization tool for electronic density of time-dependent quantum systems such as those experiencing an electric field. The resulting movies of time-dependent electron density increases the qualitative understanding for researchers in the field, while providing insightful information that is simple to disseminate in the scientific community.

Organic mixed ionic electronic conductors (OMIECs) are a class of semiconducting compounds that have recently sparked major interest due to their unique ability to conduct both ions and electrons when electrochemically doped. This unique property of OMIECs make them great contenders for applications in biosensing and neuromorphic computing, since they are soft organic materials that can change their state of conductivity. The rate at which these polymers can undergo redox reactions is known as the polymer’s doping kinetics, which is an important parameter for understanding these materials. In this work, I measured the doping kinetics of the commercially available polymer, poly(3-hexylthiophene-2,5-diyl) (P3HT), using UV-Vis spectroelectrochemistry to measure the electrochemical oxidation rate in solutions of different anionic species and solution concentrations, as well as varying the film thickness. I predicted that anionic species of larger sizes, at higher concentrations, results in faster doping kinetics of P3HT. I also expect to see faster doping in thinner films of P3HT, when compared to thicker films. My results show that the choice and concentration of the electrolyte plays a large part on the kinetics of electrochemical doping. I showed that using electrolytes that have larger anions were able to generate a faster doping kinetics. Increasing the electrolyte concentrations also increased the kinetics of doping the polymer. I also found that the thickness of the polymer film, when decreased, resulted in a faster doping kinetics. Using P3HT as a model system, I have examined the effects of anion, electrolyte concentration, and polymer film thickness, which are important parameters to understanding the factors that go into making these materials good conductors for a range of applications.

As tool compounds aimed at profiling drug leads, covalent small molecule probes targeting amino acid residues present great value to the field of chemical proteomics. Recent progress has expanded the search for these irreversible inhibitors to those targeting lysine residues on select kinases. These small molecule probes are typically composed of an aminophilic group that binds lysine, a scaffold that directs the inhibitor to the residue of interest, and a reporter group that facilitates visualization of binding on a gel. Here we present the organic synthesis of numerous novel small molecule inhibitors, as well as their live cell labeling behavior. The inhibitors of choice are designed with a variety of electrophilic groups that serve as aminophiles targeting lysine. Each inhibitor is built around either a Foretinib or Xo44 scaffold, and uses a transcyclooctene (TCO) click handle as the reporter group. The TCO handle rapidly binds tetrazine, allowing for the linking of a fluorophore to the inhibitor, which reports labeling of lysines through SDS-PAGE. An initial live cell labeling assay reveals clear binding activity in a foretinib-based inhibitor with a squarate electrophile, as well as minor bands in select sulfonyl-fluoride based inhibitors. Through mass spec (MS) proteomics, the protein targets of these promising inhibitors will be identified. Assuming MS reveals selective, high affinity binding, these small molecule probes aid the profiling of drug leads, and expand the range of targeted covalent inhibitors available for chemical proteomics. 

Phase separation in phospholipid membranes occurs in living systems like yeast vacuole membranes and consists of domains enriched in specific lipid components. Phase separated domains coalesce and merge together into a singular phase as membranes are heated above the phase transition temperature, T_mix. The phase transition temperature depends on the lipid composition of a membrane. In the lab, it is useful to produce simple, model membranes to isolate phenomena like phase separation from the complexity of biological systems. Emulsion phase transfer is one such technique used to generate giant unilamellar vesicles (GUVs) by using a centrifuge to drive emulsion droplets coated in lipids through a lipid-oil solution and water interface. However, there are several specific challenges for emulsion phase transfer that require optimization: drying the lipids down with nitrogen gas into a lipid film, the time sensitive creation and layering of the lipid-oil emulsion, and finding the optimal centrifugation parameters. Here, we optimize emulsion phase transfer in three ways: 1) evenly coating lipids films via swirling, 2) creating the emulsion and depositing it as quickly as possible, and 3) tuning centrifugation to maximize vescile formation and minimize vesicle aggregation. Further, we measure the phase transition temperature for GUVs made of a ternary lipid mixture consisting of DPPC (16:0 PC), DOPC (18:1 PC), and Cholesterol in a 1:1:3 ratio. To visualize membrane phase separation, a fluorescent lipid that partitions preferentially to only one phase was added to lipid mixtures used to prepare GUVs. Due to the crucial role of cholesterol in membrane phase behavior, the phase transition temperature of GUVs generated through this technique will vary from those produced by other techniques due to poor cholesterol incorporation.

Moss is a bioindicator that can be useful in evaluating air quality in metropolitan areas. One study in Portland used a common moss, Orthotrichum lyellii, to locate sources of cadmium pollution, leading to regulations for glass factories in the area. The current study applied a similar methodology to locate sources of metal contamination in and around the South Puget Sound region. O. lyellii was collected from locations with a wide range of vehicular traffic. Additionally, collection sites were divided between industrial, commercial, residential, and forested areas. Moss samples were dried, digested with acid and peroxide, and analyzed for metal content using ICP-MS. Our study shows increased concentration of trace metals such as titanium and vanadium in industrial areas. Some major metals such as calcium may be more closely linked to vehicle traffic. These results may indicate human health hazards in industrial areas in the South Puget Sound area. 

The association of eukaryotic DNA with histone proteins serves not only to package entire genomes into the nucleus of a cell, but these histone-DNA functional units, called nucleosomes, are hubs for biochemical signaling that regulates gene expression. In the Chatterjee lab, we are fascinated by the transcriptional biology of the small ubiquitin-like modifier protein 3 (SUMO-3), a posttranslational modification that occurs on histones and has been correlated with reduced gene expression. Previous members have demonstrated that SUMO-3 stimulates the activity of transcriptionally repressive enzymes by binding with a scaffolding protein called CoREST1. Hence, my focus has been to understand the functional details of the SUMO-CoREST interaction, particularly how cancer-associated mutations in the SUMO-interacting motif (SIM) of CoREST1 affect its ability to bind SUMO-3. To answer this question, I started by using solid-phase peptide synthesis to prepare truncated CoREST SIM peptides bearing the mutations of interest. I then utilized these peptides, along with SUMO-3 enriched in nitrogen-15, for two-dimensional nuclear magnetic resonance spectroscopy. By comparing the chemical shifts of [¹⁵N]-SUMO-3 with and without the presence of each peptide, I could assess the effects of mutations on the proportion of bound and unbound species in solution. Of special interest was an acidic residue in the hydrophobic core of the CoREST SIM, which distinguishes it from canonical SIMs found in other proteins. Excitingly, my results indicate that substitution of this amino acid with lysine, a mutation found in gallbladder cancer, ablates binding. I observed a similar effect for other mutations in the hydrophobic core of the CoREST SIM. Using these results to guide studies with full-length CoREST in biochemical assays, my research will identify the effects of these mutations on downstream biochemical pathways that may be misregulated in human cancers.

Major Depressive Disorder (MDD) is one of the fastest growing causes of global disability and requires a variety of treatment options due to its multifactorial etiology. While it’s long been known that blood levels of the neurotransmitter GABA are predictive of MDD, recent studies have implicated the human microbiome as a potent GABA metabolizer that can affect circulating GABA levels. Our collaborators used bioinformatics to identify bacteria containing putative GABA-synthesizing genes (puuD) that are predictive of host GABA levels and depression scores. However, previous literature indicates that the puu operon is repressed under anaerobic conditions in E. coli and the putative PuuD proteins have low sequence similarity to the model PuuD enzyme. My project seeks to confirm the biochemical activity and substrate-specificity of these genes using in-vitro assays measuring both the putative PuuDs’ chemical reactivity and kinetics. Due to conservation in the active site structures between the putative proteins and the model PuuD, I expect that the novel PuuD-like proteins will have gamma-glutamyl hydrolase activity and substrate specificity for gamma-glutamylated-GABA. This work provides base-level evidence for the use of systems biology techniques to identify enzymatic function and lays the foundation for targeted therapeutics manipulating the microbiome for improved MDD outcomes.

Electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool for detecting unpaired electrons in molecular systems. By attaching two spin-labels which contain unpaired electrons to different regions of a protein, the distance between the spin labels can be measured, making this technique particularly useful for studying protein structure and dynamics. The signal sensitivity of these measurements is especially sensitive to protons on amino acids adjacent to the spin label. While it is known that the presence of neighboring protons to the spin label decreases the signal intensity, the magnitude in which specific amino acids affect the signal is not well understood. My research aim is to determine how specific neighboring amino acids affect the EPR signal. Here, I design model systems in which spin labels are placed on various parts of maltose-binding protein (MBP) to construct a sample set that contains the spin label in diverse amino acid environments. These spin labels are placed on MBP through a process called site-directed spin labeling. MBP is mutated through site-directed mutagenesis using specific primers, the final plasmid transformed into competent E. coli cells. Through collecting EPR data of these MBP mutants, we can gain insight to which amino acids neighboring the spin label most affect the signal. This project will help us understand how to determine spin-labeling sites to result in maximum EPR signal intensity. Maximizing this signal intensity will enable us to use EPR to study biological systems that could not previously be studied due to lack of sensitivity, such as membrane proteins.

Organometallic porous materials can be utilized as highly accurate, effective, and low-energy agents in gas separation. Metal centers bind selectively to gas molecules to fill the pores in the material. This project tests the ability of chromium metal-ligand clusters that are coordinated to one another through hydrogen-bonding networks — as opposed to traditional organic linkages — to create a long-range, high-density, highly selective, porous material. Multiple ditopic ligands have been synthesized using standard organic synthetic procedures, and their structure analyzed with 1H NMR. These ligands were reacted directly with chromium metal compounds, chromium (III) chloride hexahydrate and chromous acetate, to create metal-organic complexes. Analysis through UV-Visible and Infrared spectroscopy indicated successful synthesis of the desired chromium complexes. Preliminary results indicate that the use of bidentate carboxylate ligands with nitrogen-rich substituents is the most effective way to generate a stable metal cluster with a strong hydrogen-bonding ability to ensure long-range rigidity. A ligand synthesized from dicyandiamide and 4-cyano benzoic acid has proven to form stable chromium clusters in both inert and atmospheric conditions. Current research is focused on forming long-range hydrogen-bonding networks with the aforementioned clusters to create a porous material. When this material is synthesized, the metal sites remain bound to one solvent molecule, which is then removed through vacuum to open a highly selective binding site. It is expected that the low oxidation state of the metal center will lend itself to be readily oxidized by gas molecules such as O2; but, critically, will not react with reducing agents such as N2. This highly selective nature of the binding site promises successful use of the material to replace thermal gas separation processes and reduce energy consumption nationwide.

Additive manufacturing, also referred to as 3D printing, enables the fabrication of objects of any design based on a computer-aided design model. 3D printed structures comprising biodegradable protein-polymer networks have potential use for biomedical applications. The Nelson lab has developed a resin for vat photopolymerization 3D printing based on the protein bovine serum albumin (BSA). In my work, I investigated the response of these materials under different pH environments in order to simulate physiological conditions and gain an understanding of how these hydrogels respond to these different environments. I chose the protein-polymer network MABSA-PEGDA (Methacrylated Bovine Serum Albumin-Poly(ethylene glycol) diacrylate), a functionalized version of BSA that protects the globular structure of the protein. When altering the pH of given MABSA-PEGDA resins they retain their low viscosity, based on rheological measurements, and thus they retain printability. When printed, MABSA-PEGDA hydrogels have altered swelling and water holding capacities in pH 2 conditions as well as altered compressive moduli depending on the pH used to make the resin. Additionally, we performed CD spectroscopy and found that the alpha helicity of the protein was maintained, meaning secondary structure is not altered. The results suggested that there must be a change in the tertiary structure of the protein which induced changes in the protein-polymer matrix and altered the mechanical properties of the hydrogel. The next set of studies will include protein analysis techniques to understand the structure of BSA within the hydrogel constructs. The cumulative results of these studies will enable the use of these BSA-based materials for applications such as oral drug delivery that requires survival in harsh gastrointestinal environments.

Soluble factor signaling between immune cells and fibroblasts is critical in regulating biological processes. However, it is often dysregulated in diseases and leads to physiological changes, including airway inflammation in asthma and allergies. One immune cell type that can be attributed to airway inflammation is eosinophils (EOS). When activated by interleukin-3 and heat-aggregated immunoglobulin G, EOS release certain soluble factors associated with the activation of lung fibroblasts. To investigate the interactions between human lung fibroblasts (HLFs) and EOS, we used the open microfluidic coculture device. This device has two chambers, in which two types of cells can be cocultured in the shared media while being physically separated by a half wall. We found that HLFs in coculture with activated EOS had the highest levels of proinflammatory gene expressions and proinflammatory cytokines. However, the exact mediators responsible for promoting these biological processes are still uncertain. We hypothesize that EOS secrete a cytokine, interleukin-1 alpha (IL-1a), and a protein, transforming growth factor alpha (TGFa), to be consumed by HLFs, triggering proinflammatory responses of HLFs. The goal of this study was to elucidate the roles of IL-1a and TGFa in airway inflammation. HLF-EOS cocultures are seeded in the microfluidic coculture device, then IL-1a, TGFa, and their respective cellular receptors are neutralized using antibodies. Enzyme-linked immunosorbent assays are used to measure the level of EOS-derived neurotoxins after their activation. Then, reverse transcription quantitative-polymerase chain reactions are used to quantify gene expression levels relevant to proinflammatory responses of HLFs, in addition to multiplex immunoassays to analyze the secreted soluble factors from both cell types. We anticipate that HLF-EOS cocultures treated with neutralizing antibodies have lower expression levels of proinflammatory genes than cocultures without antibodies. Findings from this study will help us better understand the key regulators that promote proinflammatory behaviors of HLFs in airway inflammation.

With the ever-increasing interest in new photovoltaic materials, much attention is being given to the study of nanoparticles and their assembly. One of the primary goals in this field is the self-assembly of particles, such that they can be programmed to form a desired structure given only a template and a solution of particles. In my project, I investigate the effect of proteins (specially designed through de novo synthesis) on the aggregation of gold nanoparticles, with samples prepared in buffers of salt and Tris base. The particles used are nanospheres of sizes 100, 50 and 10 nm, as well as nanorods of different aspect ratios which can offer more information on the directionality of the assembly. To obtain the necessary data on these samples I use a number of spectroscopy techniques (ultraviolet-visible, dynamic light scattering and circular dichroism) and microscopy methods (hyperspectral and scanning electron). A stereospecific response is obtained from the protein-particle mixtures if the materials formed are chiral, that is, if they rotate plane polarized light. I have shown that the proteins stabilize the particles in a salt solution, which is an indication of protein-particle binding - similar results have been correlated in literature to the formation of a chiral organic-inorganic complex. Such complexes would potentially benefit from both the plasmonic properties of the nanomaterial by absorbing light at a particular wavelength in the visible range, as well as the stereospecificity imparted by the protein helix. Being able to achieve such a result is an important step towards understanding the optoelectronic properties of biotemplated nanostructures, which has a diverse array of applications, including materials for solar energy production, photodynamic cancer therapy in which tumor cells can be specifically targeted, and drug delivery systems. It would also be invaluable for the customizable design of catalysts, enzymes, probes, sensors and diagnostic tools.

Diaryliodonium salts have recently been shown to facilitate metal-free mechanoredox free radical polymerizations. Prior literature reports focus on the role of diaryliodoniums as photoinitiators; these salts have well established fragmentation mechanisms and kinetic profiles. However, their use in mechanochemistry has not been extensively investigated. Mechanochemistry is an emerging field of chemistry that uses force as a stimulus for chemical reactions. Compared to traditional stimuli such as light, heat, and electricity, mechanical force avoids the use of transitional metal additives and often has a lesser environmental impact. This report looks to explore functionalized (e.g., electron-rich versus electron-deficient) diaryliodoniums and to determine the impact of reactivity in a mechanoredox polymerization setting. Herein we synthesized a library of salts of diverse electronic structures and tested them within an established mechanoredox ball mill system. We report data on their initiation based on radical trapping as well as changes in polymers molecular weight. The hypothesis is that salts with functionalities that withdraw electron density such as alkyl halogens or cyano groups will initiate faster than salts with electron donating functionalities due to their lower reduction potential as demonstrated in literature. Exploration of these functionalized salts will provide kinetic insight and open new avenues of synthesizing commodity polymers. This is particularly applicable in 3D printing, where having control over the rate of initiation could be used to tune downstream physical properties.

Indium phosphide (InP) magic-size clusters (MSCs) are atomically-precise molecules that can be used as precursors to quantum dots (QDs). In a reaction to form InP MSCs, QDs are the thermodynamic product, whereas MSCs are a kinetic product, so there is a critical temperature below which a reaction will form MSCs but above which a reaction will form QDs. The goal of this project is to explore the effect of ligand identity on the formation and stabilization of InP MSCs and their subsequent conversion to QDs. For carboxylates, which bind weakly to InP surfaces, the critical temperature is about 120 ËšC. For phosphonic acids, which bind strongly to InP surfaces this temperature is so high that cannot be reached via a heating mantle – above about 400 ËšC. I am working to investigate the effects of native thiols/thiolate ligands on the synthesis of InP MSCs. Thiols are intermediate in their binding strength to and are commonly used with InP surfaces. I will probe the concentrations and temperatures at which thiolate-capped InP MSCs form. I hypothesize that the critical temperature for the synthesis of MSCs versus QDs reflects the ligand binding strength. If this is true, thiolate-capped InP MSCs should form readily at temperatures above 120 ËšC, but the temperature at which QDs are formed should be achievable via a heating mantle, opening up new parameter space for QD and cluster synthesis and study.

Metal–organic frameworks (MOFs) are a class of crystalline, porous extended solids that are formed through coordination between metal cations and bridging organic ligands. These materials have been a topic of acute interest in the scientific community due to their intrinsic porosity, high surface area, and precise tunability. However, MOFs are typically insulating, which limits the scope of their applications. The recent development of electrically conductive MOFs has opened the door to exciting multifunctional applications in electrocatalysis, advanced electrochemical energy storage, chemical sensing, and much more. However, a molecular-level understanding of charge transport in MOFs remains lacking. My research aims to address this knowledge gap through the investigation of one-dimensional (1D) metal–organic chains. In this presentation, I will introduce the synthesis of a series of highly-tunable 1D metal–organic chains that exhibit delocalized π systems and high electrical conductivity along with studies of how structural parameters such as metal/ligand identity and chain geometry influence their overall electrical and magnetic properties. My preliminary results demonstrate trends in these structure-property relationships that may inform how these materials can be rationally designed with specific magnetic and conductive properties. Ultimately, this work will contribute towards a molecular-level understanding of charge transport and magnetism in metal–organic frameworks, enabling the design of new conductive porous materials that can use electricity to drive chemical processes.

Many standard oxidants and reductants are non-reusable and toxic, so it is important to pursue cleaner alternatives. In this project, we have synthesized and characterized two metal-bipyridyl supramolecular cages and have studied their application as catalysts for the electrochemical reduction of organic substrates. Supramolecular cages are formed from the self-assembly of organic ligands and metal ions in solution, and they contain internal cavities with unique electronic microenvironments, similar to the interior of enzymes. While these polyhedral structures have been investigated as catalysts for traditional synthetic pathways, their role in electrosynthesis remains underexplored. Electrosynthesis involves the transfer of electrons to and from substrates using an applied potential, rather than chemical redox agents. This method is often hindered by a high kinetic barrier at the electrode-substrate interface, but catalysts can lower this barrier. We hypothesize that redox-active supramolecular cages – cages that can readily interconvert between charge states – can serve as effective electrocatalysts by encapsulating and transferring charge to substrates. To understand the effect of ligand geometry on electrocatalysis, I have synthesized two redox-active ligands with bipyridyl chelating groups. One contains a highly conjugated perylene core, while the other contains a compact core formed from pyromellitic dianhydride. We have metalated these ligands with iron ions to form two tetrahedral supramolecular cages. We then utilized cyclic voltammetry to assess cage-facilitated charge transfer to vicinal dihalide substrates. We observed that the reduction of multiple substrates, including 1,2-dibromo-1,2-diphenylethane, occurred at milder voltages in the presence of the cages, indicating a reduced kinetic barrier. For these substrates, we then performed bulk electrolysis, from which we determined that the percent conversion to the desired product was significantly higher when a cage was present, supporting our hypothesis. Ultimately, we aim to use these cages to enable electrosynthesis of organic feedstocks at lower voltages and with fewer byproducts.

Iron centers which feature metal ligand multiple bonds can be powerful group transfer agents, for example, terminal Fe-oxo intermediates in soluble methane monooxygenase can perform oxo-atom transfer for the selective oxidation of methane to methanol. Abiologically, ligand constructs which enforce desirable electronic and structural configurations have been shown to enhance group transfer to a range of organic substrates. We developed and studied an iron (Fe) molecular complex with two aminophosphine selenide ligands (Se=PPh₂NTol; Ph=Phenyl, Tol=4-Tolyl) that chelate the metal center via the selenium and nitrogen. The iron complex (FeL₂) was synthesized by a reaction between Fe(HMDS)₂ (HMDS = bis(trimethylsilyl)amide) and the aminophosphine selenide. Characterization shows a tetrahedral, high spin, symmetric compound. We hypothesized that FeL₂ can activate and transfer heteroatoms and explored the reactivity of FeL₂ with oxidants, oxo atom donors, and organic azides. Treatment with iodine (I₂) results in oxidation of the iron center (Fe(II) to Fe(III)) and coordination of the iodide counterion results in structural reorganization to a five-coordinate square pyramidal complex. Reactivity with oxo atom donors shows that either the ligand or Fe center are oxidized, and we identified a µ₂-oxo dimer, which is the first Fe-O-Fe dimer to have selenium in its first coordination sphere. We found that FeL₂ forms Fe-nitrenoid intermediates and can perform nitrene transfer to form diazos or do C-H amination, when treated with aromatic and aliphatic azides, respectively. The presented complexes are characterized by single crystal X-ray diffraction (XRD), Evan’s method, nuclear magnetic resonance (NMR), and Ultraviolet-Visible Spectroscopy (UV-Vis). This research builds upon the knowledge of transition metal complexes for heteroatom transformations.

Previous work takes inspiration from 2D metal–organic frameworks to synthesize 0D metal–organic macrocycles which maintain the conductivity of the original material while introducing solution processability. These macrocycles self-assemble into nanotubes through π-π stacking of the aromatic core but, the nanotubes do not have a preferred orientation when imaged by atomic force microscopy (AFM). We hypothesize that by adjusting solvent, drying conditions, and organic ligand functionality we can create a preferred orientation of the macrocycle nanotubes on common substrates which will improve charge carrier mobilities through the aromatic core. This work demonstrates the formation of large domains of nanotube alignment which can lead to greater charge carrier mobility. With unique ambipolar charge carrier transport, metal–organic macrocycles have applications in energy storage, chemical sensing, and electrocatalysis.

Adoptive T cell therapy is a promising therapeutic strategy for the treatment of many hematologic malignancies, however, its efficacy in solid tumors poses several challenges. Some of these challenges include the limited infiltration and activation of cytotoxic T cells due to the effects of a diverse immunosuppressive environment within the solid tumor. One of the main suppressive immune cells present in several solid tumors are regulatory T cells (Tregs) and high numbers of Tregs within the solid tumor have been correlated with poor prognosis. Therefore, there is a clinical need to develop strategies targeting the suppressive immune cells that limit the efficacy of adoptive T cell therapy. Tumors have highly dysregulated metabolism, which results in the secretion of multiple metabolites into the extracellular space. This allows a buildup within the tumor microenvironment, which may have an effect on the infiltrating immune cells. Our group has identified one metabolite, succinate, that enhances Treg numbers within the tumor microenvironment. Here we further explored the effect of succinate on the function of Tregs. To examine this, we identified tumor cell lines that produce succinate (lung and melanoma) and further altered these to modify the levels of succinate secreted by these cells. We then co-cultured high succinate secreting tumor lines with healthy donor CD4+ T cells that were isolated from PBMCs. We screened these cells and found that the higher levels of succinate resulted in higher numbers of Tregs and increased anti-inflammatory function, as evidenced by TGFb levels in Tregs. Future experiments will validate these findings in in vivo mouse models with the aim of developing synergistic approaches to enhance adoptive T cell therapy.

North America is experiencing more frequent and intense wildland fires. Most public health research into exposure of wildland fire smoke is retrospective (backward-looking) examining emergency room visits, hospitalization, and emergency medication usage. In 2021 the Theberge Lab enrolled participants (n=64) in a research opportunity to map and study the inflammatory response of wildland smoke exposure. Participants self-draw liquid whole blood for baseline, smoke events, and 3- and 6-month post season follow-ups. A key advantage of this prospective study is the ability to compare participants’ exposure samples and surveys back to their own pre-exposure baselines. Blood samples are taken by the participants in the comfort of their home using technology developed in our lab called the homeRNA kit. The single use kit allows for a small whole blood sample (100-500 μL) to be withdrawn using the Tasso-SST™, stabilized with RNAlater™ , and mailed to our lab for processing and analysis. While providing samples a series of survey questions are answered. As study coordinator, I assembled sample kits, triggered when to send exposure kits by tracking multiple maps for smoke, shipped and received sample packages, and interfaced with participants. After all samples and surveys are completed, I examined the survey answers in relation to self-reported symptoms, self-reported well-being, general usability, and willingness to use the homeRNA kit during a disaster event. This research intends to provide real-time symptom data from exposure to smoke and to test the feasibility of the homeRNA kit. In the future, the homeRNA kit could be deployed for disaster events, research that requires frequent blood samples, and as a mail-in diagnostic tool between clinics and patients.

Electrochemical water splitting, a promising green energy solution, produces O2 and energetically rich H2 gas molecules as products on the anode and cathode, respectively. Attachment of O2 and H2 bubbles on the surface can decrease the overall efficiency of electrochemical water splitting.Therefore, continued investigation of nanobubble characteristics is of utmost importance to improve the technologically relevant electrochemical generation of H2 gas. Nanobubbles can interfere with the efficiency and productivity of industrial processes by blocking electrode surfaces, however, they could also potentially be utilized to catalyze reactions themselves. To better understand these processes, it is important we create consistent samples of nanobubbles that can be formed individually and reproducibly, and gain a deeper understanding of their properties. We have been working to create a procedure that efficiently and reliably produces carbon nanoelectrodes for single nanobubble analysis. To accomplish this, we pull quartz capillaries to a nano-sized tip to create a nanopore, and heat the nanopores while applying a flow of methane in an oxygen-free environment to deposit carbon inside the capillaries, producing nanoelectrodes. We test the electrochemical properties of the nanoelectrodes by measuring the observed current when applying a potential and evaluating whether the cyclic voltammetry graph generated suggests that the nanoelectrode is capable of generating a nanobubble. Certain elements of our current procedure may need to be adjusted to improve the reliability of the nanoelectrodes, but so far our experimentation in nanoelectrode fabrication has allowed for a more reliable process in generating ideal hydrogen nanobubbles. This procedure has helped us gain a better understanding of the impact of nanobubbles on an electrochemical system and provide a better physicochemical description of the bubble. In the future, we plan to apply the knowledge gained through these experiments on theta nanoelectrode fabrication, which is an electrode with a partition in the middle.

The CandyCollect is a lollipop-inspired saliva collection device initially developed to aid in strep throat testing. The device utilizes open, plasma-treated microfluidic channels to collect pathogens in saliva in a noninvasive manner. The device’s isomalt candy coating aids with saliva production, and acts as a built-in timer to ensure adequate oral sampling time. In our previous studies, multiple institutional review board (IRB) approved and IRB exempt studies have been performed to test the functionality of the CandyCollect devices with human subjects. We have also demonstrated the device’s detection of oral bacteria such as Streptococcus pyogenes and Staphylococcus aureus. We noted throughout these studies that the candy’s intended 3 minute dissolving time was longer than expected due to the coating’s large mass. Thus, we are also developing a 1 and 2 minute version to more accurately match the necessary length of sampling times for various pathogens without being unnecessarily uncomfortable for the user. Our goal for this study is to identify the relationship between candy mass, dimensions, and dissolving time so that we can use the candy as a tunable “timer”. We are modifying the depth, diameter, and mass of the device to develop new CandyCollects targeted to take, on average, 1, 2, and 3 minutes to dissolve. These new CandyCollects are more suitable for specific, higher-concentration oral pathogen collections. By reducing the candy’s mass on the devices, we expect the modified devices will decrease the required dissolving time to make the user experience of oral pathogen collection more pleasant. We plan to recruit participants (>18 years) within the university to test these modified devices, and they will provide feedback on their experience and dissolving time. Using these results, we will be able to improve the CandyCollect device to provide a more comfortable sampling experience.

The COVID-19 pandemic demonstrated the importance of screening large numbers of individuals for respiratory pathogens. However, existing sample collection methods, including throat and nasal swabs are unpleasant and invasive, especially for younger patients. The CandyCollect device was designed to alleviate these barriers to efficient sample collection by utilizing specially engineered and surface-treated polystyrene lollipop sticks to capture pathogens from saliva. Prior studies by our team demonstrated the CandyCollect’s effectiveness in collecting commensal bacteria in samples kept stable through ambient temperature shipping, which were then eluted from the CandyCollect and detected and quantified with standard quantitative Polymerase Chain Reaction (qPCR) assays. Here we aim to augment this functionality by testing CandyCollect’s effectiveness with viruses in addition to bacteria. In our Institutional Review Board (IRB) approved clinical study STUDY00013842, we recruited 25 participants from the general public nationwide who were presenting symptoms of any respiratory disease, and sent each a testing kit containing CandyCollect devices in addition to a selection of the current gold standard sampling devices: an oral swab, nasal swab, and a spitting tube. Participants self-collected samples using provided devices and returned kits to our lab for qPCR analysis. Analysis revealed the detection of pathogens Streptococcus pneumoniae, respiratory syncytial virus (RSV A), and rhinovirus (the virus causing the common cold). Of the first five participants with positive signals on at least one of the conventional methods studied, all had concurrent positive CandyCollect signals, suggesting that the rate of successful sample retention of the CandyCollect for these common infection vectors is comparable to that of the currently employed sampling methods. Additional samples continue to be tested with qPCR, and further analysis of these results will be conducted. With this initial effectiveness of pathogen detection, we plan to further streamline the CandyCollect device for younger users, and improve efficiency in clinical settings.

The CandyCollect is a lollipop-inspired device that utilizes microfluidic channels to capture oral bacteria in saliva. The original intent of this device was to successfully collect oral Streptococcus pyogenes (S. pyogenes) from saliva samples and screen for strep throat. Using in vitro experiments, we were able to capture and elute S. pyogenes using the CandyCollect. To explore the functionality of the device, we performed in vitro experiments to capture and elute two different strains of commensal bacteria, Staphylococcus aureus (S. aureus) and Streptococcus mutans (S. mutans). Collection of bacteria using the CandyCollect would allow us to sample from the general population rather than strictly individuals infected with S. pyogenes, allowing the CandyCollect to be used in various applications. The method of bacteria elution we performed was compatible with polymerase chain reaction analysis. We conducted an IRB (institutional review board) approved study to compare the CandyCollect to two other standard saliva collection methods (ESwab™ and SpeciMAX Stabilized Saliva Collection Kit™). Our results showed that the CandyCollect was able to capture both of these commensal bacteria strains when present. We then performed a human subjects study to compare the detection of the CandyCollect to the other two methods. We focused on detection of S. mutans and S. aureus due to their high prevalence in healthy adults. Our results showed that for participants in which a given bacterium (S. mutans or S. aureus) was detected in one or both of the commercially available methods, CandyCollect devices had a 100% concordance with those results. Surveys were sent out to participants to assess the comfort of the sampling methods. The CandyCollect was the preferred method of sampling. Based on the results of this study, we are hoping to one day incorporate the CandyCollect into clinics for strep throat diagnosis to replace more invasive current methods.

Cells communicate with each other and their environment via signaling pathways. When a cell receives a signal, it is processed by several proteins. Oftentimes, the same proteins are used by multiple pathways that process distinct signals and produce distinct cellular outputs. I am investigating how the cell is able to correctly process signals using Wnt signaling as a model pathway. Wnt signaling helps regulate cell growth and differentiation and is therefore tightly associated with many diseases. Both Wnt and insulin signaling processing utilize the enzyme glycogen synthase kinase 3β (GSK3β). Activation of insulin signaling results in the phosphorylation of GKS3β. However, if phosphorylated GSK3β from insulin signaling interacts with Wnt proteins, Wnt signaling can be inappropriately activated in the absence of a Wnt signal. The mechanism by which cells prevent improper activation of Wnt signaling is unknown. I am researching the role scaffold proteins play in signaling pathway insulation. Traditionally, scaffold proteins facilitate reactions by binding enzymes and their substrates, bringing them in close proximity to each other. It has also been shown that scaffold proteins can assist in promoting chemical reactions through mechanisms other than binding. Because the scaffold protein Axin binds GSK3β as well as PP2A, an enzyme that dephosphorylates GSK3β, I propose that Axin promotes insulation of the Wnt pathway. Using in vivo human cell culture assays, I have determined that Axin promotes dephosphorylation of GSK3β by PP2A through a mechanism more complex than bringing the two proteins in close proximity with each other. I will use in vitro kinetic assays to determine the underlying kinetic mechanism of this effect. Determining the kinetics of scaffold-mediated insulation will produce a model that can be applied to other signaling pathways and is important in understanding how to specifically target Wnt signaling for disease treatment without affecting other pathways.