DNA-Binding Proteins (DBPs) hold a strong affinity to interact with the major grooves of DNA for the purposes of transcription, translation, and repair. Although DBPs are found in nature, their specificity is difficult to predict and their production expends excessive resources. Therefore, our project’s goal is to efficiently generate DBPs that allow us to enable exact processes to occur. This is promising for future use in treating genetic diseases. In our research, we studied point mutations in the hexosaminidase subunit alpha (HEXA) gene and adenosine deaminase (ADA) gene, which can lead to Tay-Sachs disease (TSD) and Severe Combined Immunodeficiency (SCID), respectively. TSD is a rare genetic disease that affects infants and ultimately leads to brain damage, resulting in these children not making it to grade school age. Similarly, SCID is genetic, where children lack a strong immune system. This increases their susceptibility to infections, especially during their first year of life. Targeting the HEXA and ADA genes, we first developed designs utilizing computational software like RosettaFold, RFdiffusion, x3DNA, and LigandMPNN, followed by rigorous filtering via RosettaFold Nucleic Acid. Afterwards, we tested the final designs using yeast cultures and fluorescence-activated cell sorting (FACS) in the laboratory to determine which bind best to their generated DNA template sequences. Overall, we expect to find a few specific DBPs that bind effectively as predicted during the computational pipeline. These successful designs can be utilized as genome-editing proteins, correcting their target DNA sequences and restoring normal function.

The lithium isotopic composition of metasomatized rocks preserves a history of fluid movement that may be related to large subduction zone earthquakes. High pressure and temperature conditions within subduction zones cause the dehydration of hydrous minerals, and the resulting fluid can raise the pore pressure and trigger slip along the plate interface. The role of fluid movement in subduction zone processes can be better understood by constraining the duration of these events. These relatively fast and periodic fluid increases are recorded by chemical diffusion between the fluid and rock. Fluid containing lithium within a subduction zone can drive the diffusion of lithium into the surrounding rock. Lithium has two stable isotopes that diffuse at different rates, ⁶Li diffusing faster than ⁷Li, creating spatial heterogeneity in the isotopic composition of the reacted rock. Metamorphic rocks from the Western Alps have a reacted rind structure where fluid interaction occurred within an extinct subduction zone. The period of this interaction was examined using lithium isotopes. I prepared these samples for isotopic analysis, first weighing rock powders, then digesting them in acid, and finally separating the lithium with cation exchange columns. Lithium isotopes were measured on a multi-collector inductively coupled mass spectrometer. The spatial distribution of lithium isotopes along the profile from the reaction rind to the unreacted core, together with a thermodynamic model of Li diffusion through the rock, constrain the duration of the fluid interaction and provide insight into the role of fluid in catalyzing slip along the plate interface. I expect the duration of this fluid contact to be short, consistent with pulsed fluid movement within subduction zones. Modern subduction zones, including the Cascadia Subduction Zone that underlies Seattle, pose seismic hazards that can be better understood by examining the relationship between fluid movement and slip in extinct subduction zones.

Dopamine (DA) producing neurons of the ventral tegmental area (VTA) in the midbrain regulate reward association learning and motivation. These DA neurons are modulated by neuropeptides and can be separated into distinct subpopulations based on differential gene expression, regulation of activity, and projection patterns. But how these different patterns are established and contribute to distinct functions of DA subpopulations remain poorly understood. One potential key component for these neuropeptides is the transient receptor potential canonical (TRPC) channels. Specifically, we identified the gene encoding TRPC type 6 channel (Trpc6) as having enriched expression in the VTA DA neurons. To determine whether Trpc6 is differentially expressed in VTA DA subpopulations, I utilized the quantitative, multiplexed in situ hybridization methods. Using wild-type mice, I probed for the expression of tyrosine hydroxylase (Th), a marker of all DA neurons, and Trpc6 as well as markers of two subpopulations, corticotropin releasing hormone receptor 1 (Crhr1) and cholecystokinin (Cck). The analysis showed that Trpc6 expression is significantly higher in the Crhr1 subpopulation, of 81%, than in the Cck subpopulation, of 66%. Because neuropeptides like neurotensin increase calcium concentration in DA neurons, we hypothesized that TRPC6 contributes to these neuropeptide-evoked calcium signals. To investigate the role of TRPC6 in DA signaling, I used a viral-based CRISPR/Cas9 approach to induce selective mutagenesis of TRPC6 in specific DA subpopulations. Then, I assessed the calcium responses of subpopulations to neurotensin by measuring the amplitude and neurotensin-evoked oscillations using acute brain slices. We expect the calcium responses to decrease more in the Crhr1 subpopulation than in the Cck subpopulation compared to the control as the Crhr1 population has higher Trpc6 expression. By elucidating the role of TRPC6, we hope to contribute to discovering pharmacological interventions for diseases caused by dopaminergic system dysfunctions such as Parkinson’s disease and substance use disorders.

The antibiotic penicillin is highly effective at treating the STI syphilis, caused by the bacterium T. pallidum. However, the United States has seen increases in syphilis cases every year for the past 20 years; congenital syphilis cases have risen more than 219% from 2017 to 2021 and overall syphilis cases have risen 32% from 2020 to 2021. This situation demonstrates the need for an effective vaccine as current approaches are not working. The aim of this project is to utilize phage immunoprecipitation sequencing (PhIP-Seq) techniques to assist in the development of an effective vaccine in rabbits and eventually humans. To this end I have been using PhIP-Seq techniques to systematically profile the immune responses to vaccine candidates and T. pallidum infections in rabbits. When rabbits are immunized with a cocktail of three strains of the protein TprC we saw a protective immune response against treponemes (resulting in no viable treponemes) whereas an immunization with TprD saw reduced immune protection. I used PhIP-Seq methods - informed by next-generation sequencing (NGS) and differential expression analysis - to determine the epitope-specificity of antibodies in polyclonal serum samples from rabbits immunized with these vaccine candidates. Epitope-specificity comparisons between the resulting antibodies of the two immunogens can shed light on regions of these proteins critical for protection against treponemes. In the next few months I plan to integrate alanine scanning mutagenesis into the project to assess amino acid binding specificity and accurately identify crucial residues for antibody-binding. The fusion of scanning mutagenesis with PhIP-Seq will allow me and the other research scientists assisting with the project to refine of the effectiveness of our existing vaccine candidates.

Phosphoribosyl Pyrophosphate Synthetase (PRPS1) is an enzyme in the nucleotide biosynthesis pathway that makes a molecule necessary for de novo nucleotide synthesis. It is known that PRPS1 protein hexamers can stack into linear filaments in the presence of ADP and phosphate. When these filaments are broken, catalytic activity is lost, and it is hypothesized that enzyme inhibition is lost as well. Mutations in PRPS1 lead to a wide spectrum of diseases in humans. In addition, changes in cell regulation of the enzyme have been linked to cancer. Motivated by research that connects PRPS1 phosphorylation to increased cancer proliferation, my project investigates the effects of phosphorylation on PRPS1 structure, enzyme activity, and inhibition properties. I have transformed plasmid DNA containing the PRPS1 phosphomimetic mutations S47E, S103D, and S308E into E. coli strains BL21 and pLysS. I then grew overnight bacterial cultures and induced protein expression using IPTG. After verifying protein expression with gel electrophoresis, I purified the protein from bacteria using nickel resin affinity and size exclusion chromatography. Having made and purified protein mutations that mimic phosphorylation, I conducted a negative stain screen to analyze filament formation trends. This has yielded preliminary findings that S47E and S103D phosphorylation mutations of PRPS1 break enzyme filament formation. Variation in filament formation between mutations points to the importance of phosphorylation location and its potential impact on enzyme activity and inhibition. To assess the catalysis of the phosphomimetic mutations in PRPS1, I will conduct biochemical assays which measure the activity and inhibition of the enzyme. Through these ongoing experiments we will learn how phosphorylation modifies PRPS assembly and activity and the implications of PRPS1 dysregulation in cancer proliferation.

The robotic exploration of Mars has found that the early atmosphere was similar to current day Earth, suggesting that life could have existed on Mars in its past. The former atmospheric conditions and potential ancient rivers and lakes are preserved in the sedimentary rocks found across the surface. Interpretation of the Martian sedimentary record in Gale Crater, a possible ancient lake, requires differentiating between a variety of processes that alter sediment chemistry. Our study will contribute to the reconstruction of source rock composition based on the sedimentary records in ancient river systems on Mars. Iceland is a useful analog to ancient Mars as it has a similar climate and geologic makeup as well as similar environmental features, such as glaciers, volcanoes, rivers, and lakes. Here we characterize the chemical composition of source rocks in a cold, basaltic Mars analog source-to-sink system in Iceland and compare them to adjacent sediments. If the source rocks are primarily composed of palagonite, a glassy product of hydrothermal alteration of volcanic glass that weathers easily, we hypothesize that palagonite is the dominant component of the sediments. We analyzed the samples using Micro X-ray Fluorescence (μXRF) to examine thin sections of rock for changes in the elemental composition, and X-ray Fluorescence (XRF) to measure the bulk chemical composition of rocks and sediments. We used thin section classification to quantify the percent proportion of altered rock (palagonite). In source rocks with relatively high amounts of palagonite (greater than 10%), there was no significant chemical difference. The sediment samples are higher in Al, Si, and Fe and have less Mg and Ca. The difference in sediment and source rock chemistry indicates that another process is occurring, such as chemical weathering, sediment sorting, or that palagonite is a major portion of the sediments.

The nucleolus is an essential subnuclear organelle that performs central regulatory roles in cellular metabolism, epigenetic programming, and stress signaling. In mammals, nucleoli are disassembled and rebuilt de novo with each cell division, through an elaborate assembly mechanism that has long eluded molecular characterization. This assembly process is spatiotemporally controlled by a long noncoding RNA termed the 47S pre-ribosomal RNA (47S pre-rRNA), which initiates nucleolar assembly at the site of its transcription, and for which continued expression is required to maintain nucleolar integrity. Yet, while the 47S’ roles in nucleating and scaffolding nucleolar architecture are well established cytologically (they were first observed nearly a century ago), the structural elements on the 47S that enable these architectural functions remain unknown. I hypothesize that an RNA domain within the 47S, termed the 5´–External Transcribed Spacer (5´–ETS), harbors the long-sought structural scaffolds of the nucleolus. To test this, I am implementing a live-cell reporter assay that will monitor, in real time, if transcripts derived from the 5´–ETS drive nucleolar localization and architecture. My approach leverages recent advancements in artificial gene synthesis and live-cell RNA imaging. A novel drug-inducible promoter will enable me to temporally control expression of 5´–ETS sequence variants in live cells. I will monitor the kinetics and efficiency with which these transcripts localize into the nucleolus by two-color live cell imaging, using the newly discovered fluorescent RNA aptamer RhoBAST, and a fluorescently tagged nucleolar marker protein. To design our negative controls, I implemented a bioinformatic pipeline that generates scrambles of long, low-complexity RNA sequences—ablating primary structure but preserving dinucleotide content. This allows us to investigate whether nucleotide composition or sequence affects nucleolar formation. We anticipate that this powerful system will set the stage for detailed molecular characterization studies, revealing the long-elusive molecular interactions that control nucleolar architecture in health and disease.

The catalytic ability of an industrial heterogeneous catalyst is determined by the interactions between the active sites, which are often transition metals, and the support. Insights into the interplay between the active sites and support during catalysis are difficult to gain because of the inherent complexity of heterogeneous surfaces. Alternatively, molecular catalysts are well-defined, and can be studied by a range of spectroscopic characterization techniques. To model multi-active site dynamics on a molecular scale, the Velian group has developed a system involving a cobalt selenide cluster with amido phosphine ligands that are used to tether transition metals that act as catalytically active sites onto the cluster surface. My project is probing the tri-metalated clusters’ (M₃Co₆Se₈L₆; M = Cr, Mn, Fe, Co, Cu, Zn; L = PPh₂N^-Tol, Ph = phenyl, Tol = 4-tolyl) ability to catalyze intramolecular carbon-hydrogen (C-H) amination. Previous work has shown that these clusters are remarkable catalysts for carbodiimide formation, but we have yet to compare reactivity among the tri-metalated clusters. I probed the transformation of aliphatic azides to pyrrolidines, a class of 5-membered-N-heterocycles with. This study seeks to understand how the reactivity of the clusters change as edge metal identity changes, and the role of the three active sites during catalysis. A substrate scope has shown how the steric and electronic profile of the azide affects the capability of the clusters for this reaction. This research provides insights into metal-support interactions that are important for heterogeneous catalysis. Development of next generation catalysts that can perform complex transformations benefits from the information these studies provide.

Solar energy is a promising form of renewable energy that will play a major role in reducing carbon emissions. Perovskite-based solar cells have attracted significant attention due to their high power conversion efficiency (PCE), which reached 26.1% this year, surpassing commercial silicon’s (23.3%). High PCE, low cost of materials, and ability to be solution processed make perovskite solar cells a prime candidate to replace silicon. However, efficiencies are still below the theoretical limit and these materials suffer from limited operational stability. To tackle these problems, scientists have focused on minimizing active layer and interfacial defects which act as barriers for charge extraction in a solar cell, lowering the device efficiencies. Defects also electronically dope the perovskite layer, changing the recombination kinetics in the sample. The goal of this project is to quantify how the electronic doping and defect concentration of the perovskite sample is affected by surface passivation treatements via fluence dependent photoluminescence (PL) and time resolved photoluminescence (TRPL) spectroscopy. By solving the kinetic equations at the basis of charge recombination, we can extract the rate constants that correspond to different charge recombination pathways. We pioneer a global fitting analysis to simultaneously fit TRPL and PL measurements for robust determination of these kinetic constants that are subsequently used to determine the doping density of films before and after passivation. We show that the electronic doping density is higher than previously reported in literature, and that this doping is reduced with a surface passivation treatment. We collaborate with the University of Arizona to correlate our measured electronic doping density to electrochemically measured defect densities on the same samples. This work will provide an implementable tool to quantitatively assess electronic doping and defect density values for various perovskite compositions which will be useful for optimizing future solar cell devices.

The nitrile imine produced by photolysis of 2,5 dimethyltetrazole undergoes a cross-linking reaction with the amide group in peptide-tetrazole conjugates and tetrapeptide-nucleotide complexes. In our work, we synthesized various peptide conjugates furnished with 2,5-diphenyltetrazole phototag. Upon laser pulses at 250 nm, nitrile imine intermediates can be generated by loss of N2 from tetrazoles. These nitrile imines can then crosslink with other parts of the molecule that contains amide groups. These crosslinking reactions are quite effective, achieving about 50% conversion with just two laser pulses at about 2 mJ. We could detect the formation of crosslinked products by tandem mass spectrometry. The UVPD-CID-MS3 spectra of these conjugates showed unique fragments including internal fragments of peptide sequence, indicating possible crosslinking. Moreover, we can confirm the structures and compositions of these crosslinked products using UV–Vis action spectroscopy and cyclic ion mobility mass spectrometry (c-IMS). By comparing experimental and calculated data, we confirmed the presence of nitrile imines and certain crosslinked products. We also explore thermal chemistry when nitrogen gas is lost from the peptide-tetrazole conjugates, and it seems to be a mildly energy-consuming process. The extra energy from breaking down tetrazoles is likely driving the reaction towards forming crosslinked structures involving peptide amide groups. Digging into the mechanism of this reaction, we found the proton transfer as the initial step, followed by a series of steps like cycloaddition and breaking of certain chemical bonds. Interestingly, other reactive groups, like cysteine thiol, do not interfere with this process. Within the complex of peptide conjugate and 2′-deoxycytidylguanosine, the intermolecular crosslinking efficiency is over 80%. The CID-MS3 and optimized structure showed the nitrile imine selectively targets guanine. In particular, the discovered reactivity of peptide amide groups toward nitrile imines appears promising as it provides potential clues to cross-link structure elucidation and conformational analysis.

Black phosphorus (bP), an allotrope of phosphorus, is a 2D Van der Waals material composed of corrugated layers of phosphorus atoms. Few-layer bP is a semiconductor with interesting physical properties, including relatively high carrier mobility and layer-dependent band gap. These properties may be harnessed for applications including nitrogen fixation photocatalysts, thin film transistors, and sensing devices. One limitation that must be overcome before bP can be used in devices is its degradation into phosphoric acid when exposed to oxygen, water, and/or light. Finding passivation methods is crucial for the future use of bP in electronics or photochemistry. As each passivation treatment changes bP’s electronic properties, it is important to find protection methods that are compatible with each use case. To investigate possible methods to reduce surface degradation, we exfoliate bP in solution and treat it with a passivation candidate. We then use ultraviolet-visible light (UV-Vis) spectroscopy to track the amount of unoxidized bP that remains in solution during ambient exposure. Since bP absorbs strongly across the UV-vis region, while the decomposition products, phosphorus oxides, do not, UV-vis is an ideal method for measuring degradation. Possible treatments include attaching alkoxy or thiolate groups via peroxides or disulfides to bP edges to protect the particularly reactive dangling bonds, treatment with radical scavengers such as butylated hydroxytoluene, or noncovalent protection with Tetracyanoquinodimethane (TCNQ).

We collected and compared the spectra of air plasma and argon plasma in a dirty and clean direct current (DC) plasma discharge device. After cleaning the plasma tube we hypothesize the measured plasma spectrum will have fewer lines because it wont have as many impurities. The fourth state of matter, plasma, is matter that has been superheated, causing the electrons to be ripped from the atoms. This forms an electrically charged gas that consists of negative electrons and positive ions. Our plasma was created using a DC plasma discharge device. This device creates a plasma between two electrodes inside of a vacuum chamber. A high DC voltage is applied across the two electrodes and a current flows between them. DC plasmas can be utilized as sputter sources to deposit thin films for solar panels and the purity of the plasma can affect performance. Our vacuum vessel was accidentally contaminated with oil and dirt. To evaluate the effectiveness of our cleaning practices, spectra was measured for plasmas in the vessel contaminated with oil and other dirt and then again after the vessel was cleaned. Spectra, the range of wavelength produced when light is dispersed, emitted by air plasma and argon plasma were measured between 645 nm and 1050 nm with an Ocean Optics ST-NIR spectrometer. Spectra before and after cleaning were compared to measure the effectiveness of the cleaning. Our research provides evidence for the best way to clean DC plasma discharge devices in order to remove impurities. The conclusion of this analysis is imperative for efficient thin film plating using DC plasma.

Upconversion (UC) is a non-linear optical process where a material absorbs two lower energy photons and subsequently emits one of higher energy. Currently, inorganic UC materials used in lasers and photovoltaics are primarily lanthanide-based. However, a few transition metals also exhibit UC, such as Re4+ , Os4+, Ti2+, Ni2+, and Mo3+, and due to their high oscillator strengths, d-d transitions, and a strong ligand field dependency, offer the potential for greater tunability and efficiency in upconverting optoelectronics than their than their lanthanide counterparts. The goal of this work is to increase Re4+’s PLQY by isovalently doping low-phonon vacancy-ordered double perovskites (A2BX6 : A = Cs+, NH4+; B = Ti4+, Zr4+; X = Cl-, Br-) with rhenium to minimize non-radiative decay that can occur through defects and lattice vibrations. This has been attempted via schlenck line synthesis of the host lattice and coprecipation and ion-exchange doping procedures. While [ReX6]2- has previously demonstrated near-IR to visible upconversion in the bulk, this work aims to characterize its upconversion mechanism on the nanoscale with variable temperature and time-resolved photoluminescence. If made successfully, the colloidal stability of Re4+:Cs2TiBr6 nanocrystals would allow for new post-synthetic processing avenues including electrohydrodynamic inkjet printing and core-shelling, and new applications in flexible electronics.

With the increasing use of lithium-ion batteries, the trajectory of the modern world’s energy needs calls for an improvement in their safety and functionality. Current lithium-ion batteries use a lithium salt dissolved in organic carbonates, which results in a liquid electrolyte with high lithium dissociation and fast conductivity. However, the use of organic solvents makes these electrolytes flammable and prone to combustion in the event of a dendritic formation causing a short. One approach to potentially improving these electrolytes is a solid single-ion polymer electrolyte where the anion is part of the polymer chain to potentially allow higher conductivity and lithium transference numbers. Our approach is to synthesize single-ion copolymers with varying weight percentages of a single-ion monomer (SIM) to control the ion concentration and a monomer with an oligomeric ethylene oxide sidechain (ONDI-12) to lower the glass transition temperature. The goal of my work is to identify the ratio of the two monomers that gives optimal conductivity, improving the potential use of our copolymer as a solid single-ion copolymer electrolyte in lithium-ion batteries. Initial electrochemical impedance spectroscopy measurements of our previous copolymers indicate that lower ion concentrations and lower glass transition temperatures resulted in increased conductivity with 20 wt.% SIM. Building from this work, I synthesized the single-ion monomer (SIM) with an attached anion incorporated into the structure to facilitate lithium cation motion. I anticipate copolymerizing this SIM with ONDI-12 at lower SIM percentages using ring-opening metathesis polymerization (ROMP) and subsequently measuring their conductivity. Identifying the SIM to ONDI-12 ratio that optimizes conductivity will improve our understanding of these materials and potentially advance future polymer electrolyte design.

In a recent breakthrough, bullvalene, renowned for its “shape-shifting” molecular nature with over 1.2 million degenerate isomers, has been successfully integrated into polymer backbones. This integration addresses challenges in solubility and thermal properties crucial for tailoring polymers used in manufacturing diverse products ranging from phone screens to organic solar cells. This project aims to deepen our understanding of the interplay between fluxionality and thermal properties by examining the thermal stability of small molecule bullvalene models. Through extrapolating insights for bullvalene-substituted polymers, our research seeks to contribute to the advancement of the development of advanced materials suited for varying thermal conditions. We synthesized small molecule bullvalenes to mimic polymer chains, subjecting them to diimide reduction to suppress fluxionality before comparison with their fluxional counterparts. Their thermal properties were characterized using Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). Key findings reveal a decrease in glass transition temperature upon reduction of bullvalene, highlighting the impact of fluxionality on thermal stability. Future work will delve deeper into exploring the thermal properties of small molecule models, providing insights into polymer behavior. We anticipate bullvalene as an internal plasticizer capable of modulating rigidity, solubility, and thermal properties within different classes of polymers, thus enabling a more efficient and cost-effective large-scale industrial production of a wide array of polymeric materials.

In recent years, there has been a stark decline in bee colony populations. Many studies believe neonicotinoids to be a significant contributor to this problem. Neonicotinoids are insecticides that are often used in farming or agricultural settings. One of the neonicotinoids suspected to play a role in the bee colony decline is Imidacloprid. Imidacloprid is believed to directly negatively affect the navigational skills of bees. It interrupts their homing abilities, making it difficult for them to relocate their hive after coming in contact with Imidacloprid. Previous research projects have been conducted to study the affect of Imidacloprid on bees, but the proportion of Imidacloprid to the bee itself is not realistic of what they would be affected by in a veritable agricultural setting. Here we demonstrate an extraction protocol based on sample clean-up with both ion-exchange and reversed phase separations prior to targeted gas chromatography-mass spectrometry (GC-MS) analysis. This protocol is similar to the QuEChERS (Sigma, St. Louis, IL) method, however, the extraction protocol presented here shows how capturing and concentrating the Imidacloprid while washing away the other matrix components can help improve sensitivity and selectivity for Imidacloprid. Our targeted detection strategy involves selected ion monitoring (SIM) to decrease limits of detection. Initial findings show that C-18 separation is optimized by adding a centrifugation step prior to SPE (solid phase extraction). As we continue to develop a targeted detection strategy for Imidacloprid, the hope is that the strategy will be used in future farming and agricultural environments to determine if the level of neonicotinoids being used in specific fields is detrimental to the bee colony population in the area. 

Recent progress in synthetic biology has focused on utilizing probiotics as therapeutic production factories in the gastrointestinal environment to treat GI-related diseases. Although oral administration of probiotics is a convenient method for patients, a key challenge lies in the poor survival rate of probiotics in gastric and intestinal areas. Engineered living materials (ELMs), which are comprised of genetically engineered microbes embedded in a polymer matrix, present a novel formulation for orally-administered probiotics. Herein, we developed ELMs containing probiotics in a protein-based polymer matrix, aiming to enhance their viability in the GI tract. The ELMs’ photocurable polymer matrix allows us to 3D print our formulation into oral tablets. To form our protein-based polymer matrix, we functionalized bovine serum albumin with polyethylene glycol diacrylate. We then added a photoinitiator and E. coli Nissle genetically engineered to produce tryptamine (an anti-inflammatory agent) and subsequently photopolymerized this resin to 3D print probiotic tablets. We placed these tablets through a simulated gastrointestinal tract and observed cell escape using optical density measurements and cell viability through live/dead staining and fluorescence imaging. Liquid-chromatography mass-spectrometry was used to quantify the extent of therapeutic bioproduction in vitro by our ELMs over time. Overall, we found that the ELMs successfully delivered viable probiotic cells able to perform in situ therapeutic bioproduction. Furthermore, we observed that encapsulation of probiotics in ELMs yielded a higher survival rate of cells in the GI tract, suggesting that our polymer matrix formulation protected cells and allowed for extended proliferation and colonization in the colon. These findings are also supported by our observations that ELMs produced significantly higher amounts of tryptamine in the GI tract compared with non-ELM, free cells. The findings from our study can be applied to further development of orally-administered probiotic therapeutics, and show promise for future directions in drug delivery.

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 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 TGFa in airway inflammation. HLF-EOS cocultures are seeded in the microfluidic coculture device, then TGFa and their respective cellular receptors are neutralized using antibodies. 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.

We have observed that conventional respiratory pathogen sampling methods, such as pharyngeal swabs, elicit unpleasant experiences for both adults and children. Particularly for pediatric patients, having a non-invasive, enjoyable sampling approach is crucial to facilitate prompt diagnosis and treatment. In prior research, we introduced a novel saliva sampling device, the CandyCollect. This lollipop-inspired device, with its isomalt candy coating, is specially produced and surface-treated for capturing pathogens from saliva, providing a pleasant sampling experience to child patients. Clinical studies approved by multiple Institutional Review Boards (IRB) revealed an average candy dissolving time for CandyCollect (with a mass of 0.90g~1.10g) of 3.51 minutes, with a minimum of 1.25 minutes. To compete with original sampling methods which take up to 10 seconds, it is desirable for CandyCollect to have a shorter sampling time around 15–20 seconds. Therefore, this study aims to decrease the dissolving time by introducing a new CandyCollect recipe and design. For the new candy recipe, we replaced isomalt with a mix of glucose and sucrose. Additionally, baking soda (sodium bicarbonate) was added to increase the candy’s contact area with the tongue. An ongoing experiment will assess if baking soda affects PCR results for pathogen samples, and this modified recipe will be employed in a new clinical study. Concurrently, we have modified the CandyCollect design by placing the candy on the same side as the spiral, with a small candy reservoir beneath the spiral to decrease the mass to 0.06g-0.1g. To validate the efficiency of this new design, we plan to conduct another clinical study recruiting 30 younger participants and obtaining feedback about the new design. This collaborative study will provide valuable insights towards achieving a faster dissolving time, ultimately enhancing the viability of CandyCollect as an improved and more efficient replacement for conventional sampling methods.

Alzheimer's Disease (AD) is clinically characterized as a predominantly amnestic (memory impairment) syndrome at presentation that progresses to affect other cognitive domains. AD is pathologically defined by the presence of amyloid plaques and neurofibrillary tangles of hyperphosphorylated tau (pTau) in stereotypical brain regions. AD shows clinical and pathological diversity, including non-amnestic subtypes, severity of tau pathology across brain regions, and co-pathologies such as aggregates of hyper-phosphorylated transactive response DNA-binding protein 43 (pTDP-43). This study aims to examine the association between pTau and pTDP-43 using new highly quantitative approaches. By examining the combined pathology, we hope to identify patterns of pTau related to pTDP-43 across the different clinical and pathologic subtypes. The University of Washington Alzheimer's Disease Research Center clinical core autopsy cohort was characterized and subdivided into amnestic and non-amnestic syndrome subtypes. The subjects were analyzed to identify the prevalence of pTDP-43 and its correlation to the subject's cognitive data and patterns of progression. This analysis was used to select a subset of 29 cases with non-amnestic dementia and a matched subset with an amnestic subtype for more in-depth neuropathological and molecular profiling of several brain regions. Using the HALO platform, I generated quantitative measures of pTau in the frontal, temporal, and parietal cortex, as well as the hippocampus. The integration of these findings aims to understand how pTDP-43 pathology influences tau distribution based on clinical presentation These results will allow us to select a small set of cases for further work that will include using NanoString GeoMx Digital Spatial Profiling to identify potential pathways relevant to the association between pTDP-43 and pTau severity concerning mechanisms of clinical and pathologic heterogeneity in AD. These insights will allow for further research of these pathways to determine their biological relevance and ways to mitigate their effects.