The life history traits of parasites are dependent on the host environment and host immune response. Heligmosomoides bakeri, is an intestinal nematode parasite that infects laboratory mice (Mus musculus). This investigation used rapamycin, an immunosuppressant drug, in the diet of the host. Rapamycin is currently being considered as an over the counter drug to extend life expectancy. The purpose of this experiment was to determine if inhibition of the host immune system by rapamycin had a significant effect on parasite life history traits during a secondary infection. Mice were fed either a rapamycin diet or control diet for two months. Mice were then infected with H. bakeri, drug cleared of the primary infection, then re-infected with a secondary H. bakeri infection. Starting eight days after the secondary infection fecal egg counts were done daily to determine worm reproduction in vivo. Eighteen days after the secondary infection the mice were euthanized and worms were removed, sexed and counted. Length of ten female and ten male worms from each mouse was then measured, and 10 females were cultured to measure ex vivo reproduction. After 24 hours of incubation in culture media, egg output and worm motility (an index of viability) were measured. For this experiment I was involved in recording the worm length and was responsible for performing the ex vivo egg count and motility measurements. Results will provide important information about potential over-the-counter use of rapamycin in humans to slow biological aging and how changes in immune function may affect susceptibility to parasite infection.

Tuberculosis is a major infectious disease with approximately 2 billion people infected worldwide. With the emergence of multi-drug resistant tuberculosis (MDR-Tb), efforts to develop efficient treatments with fewer cytotoxic effects are currently under investigation. Dual host and pathogen directed therapeutics hold promise towards more effective treatments against tuberculosis (Tb) through mechanisms that enhance bacterial clearance and reduce tissue damage and immunopathology. We investigate RGFP966, a drug that modulates the host's immune defenses and directly reduces Mtb growth (a histone deacetylase 3 (HDAC3) inhibitor). The aim of my project is to characterize the effect of RGFP966 on Mtb growth. My hypothesis is that the antimicrobial effects of RGFP966 are specific to Mycobacterial species, and the bacterial target of RGFP966 is required for its survival. Within the experiential growth period, I treated Mycobacterium avium and M. Smegmatis in 7H9 broth with a range of RGFP966 concentrations (0.5 uM, 5 uM, and 50 uM) and incubated in culture tubes and 96-well plates for 3-7 days. Within designated time intervals, I analyzed levels of dose dependent inhibition using optical density, luminometry, and fluorometry methods. Current results through optical density and luminometry demonstrate that RGFP966 controls M. Avium growth in 7H9 broth with an MIC50 of approximately 5 uM. Fluorometry and optical density results indivate no restriction of M. smegmatis growth. Comparing these results to previous findings in our laboratory, where RGFP966 lacked effects against common gram-positive and gram-negative bacteria, indicates possible specificity of the antimicrobial effects of RGFP966 to Mycobacterial species. We will further examine the effects of RGFP966 against mycobacterial growth. A comprehensie understanding of its pathogen directed effects combined with its host directed effects hold promise as an adjunctive therapeutic against Tb infection. 

Small Heat Shock Proteins (sHSPs) play crucial roles in protein homeostasis, the state of maintaining steady internal cellular conditions, despite changes to the cellular environment. As the “molecular life rafts” of the cell, sHSPs target partially misfolded proteins during stress measured as chaperone activity, and without consuming energy, prevent toxic aggregation. Mutations or malfunctioning of the sHSP, HSPB5 (B5), in humans are associated with neurodegenerative diseases such as Alzheimer's and Alexander's disease, as well as cancers, myopathies, and cataracts. As one of the ten sHSPs encoded in the human genome, B5 contains structural elements (the building block) common to all sHSPs: a highly conserved alpha-crystallin domain (ACD), that is flanked by variable, less conserved, N- and C-terminal regions (NTR and CTR, respectively). Previous studies determined that interactions between building blocks occur between a three amino acid region known as the I-X-I motif (a “knob”) in the CTR, and a hydrophobic groove (“hole”) in the neighboring block, similar to how two pieces of Lego come together to build a larger structure. Interestingly, in B5, there is an additional I-X-I motif in the unfolded NTR, but its role and structure are unknown. I hypothesize that the CTR and NTR motifs compete for binding into the hydrophobic groove, indicating the “knob” into “hole” interaction is loose, where the other “knob” can bind the “hole” when one leaves. To test this hypothesis, I prepared mutants containing CTR, NTR, and double motif deletions. I aim to uncover the effects of these mutations on chaperone activity and oligomeric size, by optimizing protein purification and using a combination of in vitro biochemical assays, and native (non-denaturing) gel electrophoresis on purified B5 mutants. These results will assist in demystifying the role of the NTR, direct future sHSP studies, and provide important insight for development of future therapeutic strategies.

The abuse of opioids has become an epidemic in the United States. In 2017 the mortality attributed to opioid overdose was 70,237 individuals which is more than the death toll from the Iraq and Vietnam wars combined. Microdirect laryngoscopy (MDL) is a very common surgery performed among otolaryngologists with varying post-operative analgesia regimens. We aimed to characterize current opioid-prescribing patterns among otolaryngologists performing MDL by using a cross-sectional survey of otolaryngologists at a national meeting. Our eventual goal was to have a standard post-operative opioid prescribing protocol to improve safety and allow early identification of individuals that may be at risk of opioid misuse. We surveyed 57 practicing otolaryngologists: we found that 91% of surgeons routinely prescribed opioids, 84% of surgeons recommended non-opioid analgesics as adjunctive medications, and only 5% of surgeons report never prescribing opioids. When opioids were prescribed, patient preference, difficult exposure and a history of opioid use were the most influential patient factors. Concerns of opioid abuse, the physician role in the opioid crisis, and literature about post-operative non-opioid analgesia were also underlying themes in influencing opioid prescription patterns after MDL. Opioid stewardship should be a consideration for MDL. It appears that over 90% of physicians are prescribing opioids after MDLs, although many are also prescribing non-opioid analgesia. Armed with the knowledge of current practice and influences, otolaryngologists can work to decrease opioid prescribing patterns appropriately to improve patient safety and mitigate the contribution of post-operative prescribing patterns on the opioid epidemic.

With regards to most tissues, humans lack the ability to regenerate, instead scarring in response to injury. This often leads to poor patient outcomes, especially in the event of spinal cord damage. Xenopus tropicalis are capable of avoiding this scarring response as tadpoles but not as adults. They instead fully regenerate tail tissue and are thus an excellent model system for the investigation of how regenerative and non-regenerative organisms differ in their response to injury. Due to the complexity of this process, many transcription factors have been implicated to have a role in regeneration, though the precise roles of many such transcription factors remain unknown. Here, we focus on the transcription factor Hif1α, which is canonically involved in responses to hypoxia and oxidative stress. Using an Assay for Transposase-Accessible Chromatin (ATAC-Seq) we have found that over the course of regeneration, Hif1α response elements (HREs) increase in accessibility. To understand the role of Hif1α during regeneration, I have used echinomycin, a small molecule known to inhibit binding of Hif1α to HREs. Tadpoles treated with echinomycin fail to regenerate, indicating the necessity of Hif1α in regeneration. In order to determine the effects of Hif1α on gene expression, I have queried several genes known to be differentially expressed during regeneration through the use of quantitative polymerase chain reaction. I have shown that inhibition of Hif1α transcriptional activity via echinomycin significantly alters Wnt target gene expression, indicating that Hif1α regulates Wnt target genes. This provides an improved understanding of the regulatory processes that enable regeneration.

Bloodborne pathogens are wide spread and can pose risks to health care workers and vulnerable patient populations alike. Conventional diagnostic tests for bloodborne pathogens are costly and time intensive, so fast, affordable, and sensitive diagnostic methods are needed that can be performed under low-resource conditions by untrained personnel. Microfluidic self-digitization technology, developed in the Chiu Laboratory, provides the foundation for such low-cost diagnostics. As undergraduates, we work on the fabrication and optimization of cheap, robust devices used to load samples, as well as dPCR of samples required to detect diseases. As part of a larger research endeavor, we are developing a portable instrument that, in combination with our proprietary microfluidic chip technology, will expand rapid diagnostics to low-resource settings around the world. Expanding diagnostics for bloodborne pathogens will both help prevention as well as monitoring treatment of patients already infected.

Inflammasomes are multimeric protein complexes involved in innate immune responses. Inflammasomes include a protein sensor, such as NLRP3 or pyrin, linked to the enzyme caspase-1 via the adaptor protein, ASC. Active caspase-1 is responsible for release of the cytokine interleukin (IL)-1ß and triggering inflammatory cell death. Inflammasomes are crucial in defense against pathogens. However, excess inflammasome activation is linked to diseases such as Alzheimer's, atherosclerosis, and other inflammatory conditions. Although inflammasomes are linked to several diseases, we do not fully understand how inflammasomes are activated. We are researching the role that potassium plays in inflammasome activation. To detect inflammasome activation, I measured released IL-1ß using enzyme-linked immunosorbent assays (ELISAs). I determined whether inflammasome activation requires potassium efflux by measuring IL-1ß released from cells stimulated in high extracellular potassium, which prevents potassium efflux. I found that IL-1ß release triggered by NLRP3 inflammasome activators is prevented when cells are stimulated in high extracellular potassium. However, IL-1ß release triggered by the pyrin inflammasome was not affected by high extracellular potassium. From these results, we conclude that the NLRP3 inflammasome is dependent on potassium efflux from the cell, whereas the pryin inflammasome is not. The implications of our research are two-fold. First, our findings argue against a long-standing hypothesis that high extracellular potassium blocks ASC binding. Both NLRP3 and pyrin need ASC, but our data show that only NLRP3 is affected by potassium concentration. This suggests that potassium affects NLRP3 activation at an unknown point. Second, understanding the role of potassium in regulating inflammasome activity provides a potential therapeutic target. There are drugs that regulate ion concentrations by controlling ion channel activity. Knowing whether an inflammasome pathway is potassium efflux dependent could be beneficial in limiting excess inflammasome activation that is linked to a variety of human diseases.

Despite the yearly development of a vaccine, influenza (flu) still causes annual epidemics and is responsible for tens of thousands of deaths in the United States. Current flu vaccines are not fully effective in preventing viral infection because they protect people against a finite number of flu strains, which people may or may not be exposed to during a particular flu season. To address this challenge, we are engineering new protein therapeutics to increase the efficacy of flu vaccines. The two major proteins that flu viruses use to infect host cells, hemagglutinin (HA) and neuraminidase (NA), structurally parallel the two proteins that constitute a de novo designed nanocage. De novo protein design allows researchers to create proteins that are more stable than those produced in biological systems via evolution. The focus of this project is to use this designed nanocage to scaffold a more potent flu vaccine through a process called pseudo-symmetrization, which will allow the cage to display many different strains of HA and NA. To pseudo-symmetrize proteins, we used a modelling software to determine which protein components to modify to build a pseudo-symmetric protein. Then, we use molecular biology techniques to make modifications to regions of the protein that interact with other proteins when it self-assembles. As a result, the pseudo-symmetric protein is composed of building blocks that are all slightly different from one another, but can still assemble to create a nanocage with the same structure. So far, we have identified several variants of the modified cage component that can self-assemble, and we are continuing to test more interaction sites that make pseudo-symmetric proteins. These results, and continued experiments, will help us create new vaccines capable of displaying more flu virus antigens, thus protecting people from the flu using a single universal flu vaccine.

c-fos is an immediate early gene that is commonly used to mark cell activation and can be identified using immunohistochemistry techniques. In this project, we optimized immunohistochemistry conditions for a phosphorylated c-fos antibody in brain tissue obtained from the rat lateral habenula (LHb). Male, Long-Evans rats (n=4) were injected with cocaine (10 mg/kg) or saline. Ninety minutes following intraperitoneal injections, rats were perfused with 4% paraformaldehyde. Brains were dissected and 40-micron slices across the rostro-caudal axis of the LHb were collected. We tested two dilutions of the rabbit phospho c-fos antibody (1:500 and 1:800), three blocking agents (normal goat serum, donkey serum and bovine serum albumin) and two secondary Alexa Fluor antibodies (goat anti-rabbit Alexa Fluor 488, and donkey anti-rabbit Alexa Fluor 488) to determine which combination allowed for the best visualization of c-fos in the LHb. Fluorescence microscopy analysis of the tissue sections is underway, and imaging is expected to show differences in brightness and visibility of c-fos with respect to the varying conditions. These results will identify the optimal conditions for more accurate identification and quantification of cell activation in the lateral habenula.

Autism spectrum disorder (ASD) are a group of neurodevelopmental disorders characterized by impairments in social interaction and repetitive behaviors. One of the most common mutations leading to ASD is 15q11-q13 duplication, a CNV mutation where extra copies of a chromosomal region are expressed. The major gene within this region is ube3a. My lab has generated a mice model with increased copies of this gene and demonstrated a novel deficit in the mTOR/AKT signaling pathway. Other mice models of autism have exhibited deficits in this pathway and have rescued behavioral deficits with a drug, rapamycin. Rapamycin is an inhibitor of a key protein of the mTOR/AKT pathway suggesting a similar behavioral rescue could be observed with ube3a transgenic autism model mice. Rapamycin is administered to ube3a transgenic mice to normalize deficits in social interaction and repetitive behavior, two main behavioral hallmarks of autism. Mice are assessed using a three-chamber social interaction and repetitive self-grooming test. Successful behavioral rescue by rapamycin would be seen as increased social interaction and decreased repetitive grooming, similar to behaviors seen in wild type control mice. This would suggest that therapeutic treatment of the mTOR/AKT signaling pathway could be a viable target for patients with 15q11-q13 duplication.

 During development in most animals, tubes form as precursors to complex organs such as the neural tube, digestive system, and vasculature. To create a tube, cells within a sheet, or epithelium, must coordinate specific shape changes and movements. This coordination requires each cell to establish and maintain directional identity, thereby distinguishing the ‘top’ of the sheet from the ‘bottom’. While extensive research on a group of proteins, called ‘polarity proteins’, has elucidated how cells establish directional identity, little is known about how they maintain that orientation during the shape changes and rearrangements that occur during tube formation. To address this gap in our understanding, I am studying how these polarity proteins contribute to proper tube morphogenesis during the formation of specialized structures on Drosophila melanogaster eggshells called dorsal appendages (DAs). These appendages, which provide the developing embryo with oxygen, are formed from an epithelium that wraps into a tube, elongates, and then fills with eggshell protein. The epithelium sloughs off when the egg is laid, leaving the appendages as a visualization of the earlier tube formation. I used RNA interference (RNAi) to assess the role of 24 candidate proteins in DA formation. My initial results led me to hypothesize that one protein, Crumbs (crb), regulates the tube’s directional elongation. To explore this role, I am studying crb protein localization during tube elongation, assaying DA defects after knocking down expression using RNAi in subsets of cells, and analyzing the distribution of adhesion, motor, and other polarity proteins when crb is completely absent in null clones. These analyses will add to our understanding of the role of polarity proteins in the conserved development of epithelial sheets into tubes.

Lung transplantation is currently the only treatment solution for end-stage lung disease in a large cohort of patients afflicted with a range of pulmonary issues including cystic fibrosis (CF) and idiopathic pulmonary disease (IPF). Despite the successes, a major setback is the development of chronic lung allograft dysfunction (CLAD). This is characterized by chronic inflammatory host responses towards the transplanted lung resulting in injury of the donor tissue. Currently, we are investigating the role of cell-derived vesicles or exosomes, in a range of immune responses such as chronic inflammation. The composition of exosome membrane components and internal signaling within exosomes depend on the cell type from where it was secreted and the current immune state. A major benefit is that unlike cells, exosomes do not change phenotype after being secreted by the parent cell due to environmental stimuli, making them ideal targets for biomarkers associated with immune responses. In these studies, we assessed exosome surface markers and investigated exosome function from retrospective patient serum, which provides insight into potential novel mechanisms in the development of CLAD. We developed a novel ExoFlow that qualitatively and quantitatively assesses exosomes at 1, 3, 6, and 12 months posttransplant using CFSE, traditional fluorescent antibodies for T cell and macrophage markers, and ImageStream to generate immune profiles. We then correlated these results to clinical outcomes. In a preliminary study, it appears that macrophage exosome profiles show significance in differentiating patients who develop CLAD, while T cell specific exosomes seem to be unremarkable. This could be attributed to the heavy course of immunosuppression posttransplant. Functional studies show insight in potential polarization capabilities of exosomes as immune modulators and provides another novel area of investigation into the complex pathogenesis of CLAD.

Prior studies suggest that a dynamic shared care plan for children with medical complexity (CMC) may help meet the information needs of their diverse care teams. However, little is known about how these care plans should be implemented and kept updated; the primary objective of this study was to explore the optimal collaborative management model of a dynamic care plan which engages both parents and providers based on CMC parents’ perspectives. Following a user-centered design methodology, we conducted interviews with 21 CMC parents between 3/16-1/19 to elicit their preferences for engagement in the maintenance of a dynamic care plan. Between sessions, we conducted content analysis of interview transcripts to understand themes related to parents’ preferences for care plan (1) ownership, (2) reviewing, and (3) editing, as well as electronic alerts. CMC parents emphasized that the care plan owner should have the medical expertise necessary for caring for the child, a holistic understanding of the child’s health conditions, and frequent contact with the child. Since no one individual on the care team possessed all these qualities, we developed a “core team” model composed of the parent, as well as a parent-chosen physician representative and care coordinator, who would together oversee care plan content. Parents generally believed that healthcare providers had the necessary expertise to update medical information but desired the ability to review all sections of the care plan and edit family-related sections (e.g. care preferences, home nursing, social history). They also desired automatic alerts prompting them to obtain medication refills and review care plan updates made by other providers, as well as customizable alerts to help them keep track of appointments. Future usability studies will test parent engagement with a dynamic care plan through this model.

Transient receptor potential vanilloid-1 (TRPV1) ion channels are polymodal signal integrators of noxious stimuli including heat, vanilloids such as capsaicin, peptide toxins, acid, and inflammatory mediators. It is unknown whether activation of TRPV1 by different stimuli is achieved through the same structural mechanism or if different stimuli activate the channel through different structural mechanisms. Clinical trials using TRPV1 antagonists resulted in patients exhibiting hyperthermia, suggesting that TRPV1 plays a role in maintaining body temperature, and highlighting the need to ensure that therapeutics targeting the channel do not disrupt thermal homeostasis. Hence, knowledge of different structural mechanisms for channel activation would aid in the design of therapeutic agents targeting TRPV1. To address this, we have expressed a series of functional single-cysteine rat TRPV1 channels for spectroscopic analysis, with techniques such as electron paramagnetic resonance, double electron-electron resonance, and Förster resonance energy transfer spectroscopy. By probing several structural regions within TRPV1 we can determine which regions of the channels move during activation and whether those are the same for different noxious stimuli.

Both acute and chronic pain are universal, often debilitating sensations that lead to significant physiological, psychological and economic costs. Drug development and research have worked to counteract these adversities, but current therapies are often inadequate and have dangerous side effects. Targeted drug development, which relies on pre-selecting a target that is subjected to in-vitro testing, has been difficult, costly and ineffective in producing a drug that works to effectively relieve pain with minimal unfavorable consequences. An alternative approach would be to develop an untargeted screen in a system that employs complex pain behaviors. It would act as a means of modeling the nociceptive processes, which are the neural processes of encoding and processing noxious stimuli, in the organism. We utilize an unbiased, behavior-based, novel assay that uses zebrafish larvae to better understand pain sensation. We have screened thousands of small molecules on zebrafish larvae to identify ones that have analgesic properties. The potential analgesics should block sensitized temperature aversion which changes the larvae’s temperature zone inclination meaning we observe no preference between the two zones. Thus far, our untargeted screen identified three novel molecules with analgesic properties. We then performed a series of pharmacology and behavioral experiments to understand the impact of the compounds and to narrow down their effects in order to confirm if it is truly impacting nociception and/or temperature aversion with the intent to validate the compounds as prospective analgesics.

In neurons, microtubules are critical for cellular and developmental functions including neurite outgrowth and maintaining stable wiring of the nervous system. Tau, an intrinsically disordered protein, plays important roles in microtubule assembly and stabilization. However, tau can dissociate from microtubules and form amyloid fibrils in a set of neurodegenerative diseases termed tauopathies, which includes Alzheimer’s disease and frontotemporal dementia. Mutations in the tau sequence cause the disease FTDP-17T, providing evidence that alterations in tau alone can cause tauopathies. Most mutations occur in or near the microtubule-binding domain of tau, which suggests that mutant protein may be deficient in its capacity to stabilize neuronal microtubules, more prone to aggregate, or both. In particular, the disease-associated mutation P301L appears to enhance tau’s propensity to aggregate into fibers and alter its pathological activity. Molecular chaperones are responsible for maintaining protein solubility, promoting proper folding, and preventing atypical aggregation. The chaperone HspB1 delays wild-type tau fibril formation by weakly interacting with early species in the aggregation process. It is able to recognize aggregation-prone motifs within the microtubule binding repeat region of wild-type tau. Therefore, we used fluorescence spectroscopy to test the affinity and activity of HspB1 against wild-type and mutant models of the tau microtubule binding region, to see whether changes in the tau sequence alter interaction with the chaperone. In addition, tau oligomers are known to disturb lipid bilayers, a harmful interaction that could lead to potential cell death. Using a vesicle permeabilization assay, we tested the interactions between artificial lipid membrane with our tau constructs, and whether HspB1 can counteract these harmful interactions. This will extend our understanding of the characteristics and behaviors of disease-associated mutations of tau and the ways in which chaperone target aggregation-prone tau species.

The engineering of cells to be capable of detecting and responding to multiple input signals has major applications in the area of diagnostics, cancer therapy and biological computation. Current biological signal processing methods rely on single input single output computations. This proves ineffective when detecting and responding to multiple inputs such as determining a pathology based on combinations of different microscopic cues. The aim is to build non-linear computation in S.cerevisiae (baker yeast). A well-known example of nonlinear computation is neural networks, a complex framework of parallel computations talking together to come to a common output signal. The goal is to build several yeast strains that work together to perform these parallel computations. Each individual strain would process multiple inputs and release a singular output signal to another yeast strain. Combining many strains together would create a complex decision tree capable of higher-order computation. With this goal in mind, we decided to edit the native alpha-factor pathway within the yeast cells. Alpha-factor, a signaling molecule involved in yeast mating, is secreted by yeast and detected with surface protein STE2 (a protein in the GPCR family). We also inserted, a non-native version of STE2 from C.albicans yeast cells. This allowed our engineered strains to detect two different alpha-factor inputs (S. cerevisiae alpha-factor and C.albicans alpha-factor). When detected both alpha-factors trigger the pFUS1-yeGFP pathway causing the yeast cells to fluoresce. We investigated how different concentrations of STE2 and C.A.STE2 on the cell surface influenced the output fluorescents of the yeast strain. Mathematical models were used to interpret the data and predict signal response. These models can be used to design and predict more complex networks.

Fibrolamellar carcinoma (FLC) is a life-threatening variant of liver cancer affecting adolescents and young adults. FLC tumors do not respond to chemotherapy, thus, surgery remains the mainstay of therapy. FLC is characterized by a fusion event resulting in a novel chimeric protein that joins the N-terminal domain of DNAJ with the catalytic subunit of protein kinase A (PKAc). However, the underlying mechanism by which the FLC chimeric kinase drives FLC tumor growth remains unknown. Using CRISPR/Cas9 technology, the Scott Lab (UW) has re-created the human FLC fusion protein in a well-characterized and non-transformed hepatocyte cell line. The resulting cells retain hepatocytic differentiation while over-expressing DNAJ-PKAc compared with wild-type PKAc, akin to the human tumors. Using these model cell lines and a computational approach called, Kinome Regularization (KiR), I plan to uncover molecular mechanisms through which chimeric PKAc transforms hepatocytes in FLC. The concept of KiR is built around the fact that most kinase inhibitors are not specific but instead, affect a range of targets. By exploiting the property of polypharmacology in a set of well-characterized kinase inhibitors, we can make use of elastic net regression to compute and deconvolute the kinases that are responsible for a quantitative trait. To do so, we have identified a set of 30 broadly specific inhibitors that give us >85% coverage of the kinases studied. We can then test the effect of each of these 30 inhibitors on cell growth and use the results to identify the kinase important for cell growth. The KiR-based model will also be used to identify kinase inhibitors which can specifically reduce growth of FLC cells. Overall, these studies have a strong translational potential; a new therapeutic option for FLC could give unprecedented hope to the patients who currently face this disease with limited alternatives.

Age-associated diseases, like neurodegenerative disease, cancer, heart disease, and metabolic dysregulation limit healthy human lifespan. In recent years, biologists researching aging and longevity have turned their attention towards maximizing healthspan, the healthy portion of one’s life before the onset of age-related disease. By delaying age-associated diseases, we can fundamentally improve quality of life globally. Natural products and other pharmacological interventions hold particular promise as interventions to extend healthspan and lifespan. We seek to identify novel compounds that extend lifespan using the invertebrate model system Drosophila melanogaster (fruit fly). We have tested an extract made from Pterocarpus marsupium (PME), a tree native to India and Sri Lanka with uses in Ayurvedic medicine. PME extends cellular lifespan in budding yeast, another invertebrate model system. We also tested pterostilbene, a compound found in Pterocarpus marsupium extract. As a positive control for lifespan extension, we are treating other cohorts of flies with rapamycin. Rapamycin is a specific inhibitor of the nutrient sensing mechanistic Target Of Rapamycin (mTOR) pathway, a known longevity regulating cellular pathway. Using multiple fly genetic backgrounds, performed a dose response to identify concentrations of PME and pterostilbene that extend Drosophila lifespan. Through pharmacological methods, we seek to delay aging and minimize human vulnerability to age-induced diseases. Discovery of specific compounds that prolong lifespan is a first step in developing therapeutic methods to delay human aging and health decline.

Lift-off is a subtractive process used in wafer fabrication of Micro-Electrical-Mechanical-Systems (MEMS) devices to pattern deposited films, often metal, that are challenging to dry etch. To achieve a successful lift-off, the lift-off process begins by depositing and then patterning a photoresist masking material onto a silicon wafer using photolithographic techniques. An important characteristic of the photoresist is undercutting the resist sidewalls after developing, protecting them from being coated with metal during deposition. After deposition, the photoresist and metal layer is removed via solvent. The purpose of our research is to allow for a more varied range of fabrication using positive tone photoresist. Negative tone photoresist is typically used for this lift-off process because after development, this resist naturally forms the desired undercut. Alternately, positive tone photoresist is characterized by a tapered sidewall that is difficult to not coat with the metal film layer, making the removal step via solvent problematic. We hypothesized an advantageous undercut is possible with positive tone photoresist using a white light diffuser that would allow light to enter at angles through our mask during the exposure step. We evaluated resist undercut using the Nikon microscope and resist/metal sidewall characteristics using the Scanning Electron Microscope. We will assess the new resist exposure technique using both line-of-sight evaporation (physical vapor deposition in which a target material’s atoms precipitate into solid form, when heated up under high vacuum, coating everything in the vacuum chamber) and sputtering (metal deposition involving particle ejection of ions) to deposit a thin metal film. We anticipate evaporated metal to lift-off easily with the new technique, which indicates a crowning demonstration of success.

The receptor human protein kinase EphA2 has emerged as a therapeutic drug target for cancer and infectious diseases. However, the dearth of selective EphA2 inhibitors in the field is hampering EphA2-based research and therapies. Our lab recently developed a novel selective small molecule EphA2 inhibitor I268 using medicinal chemistry. Surprisingly, we observed the same inhibitory effects on cell proliferation paradoxically when inhibiting EphA2 with I268 or activating EphA2 with its agonist EphrinA1. We hypothesized that both I268 (inhibitor) and EphrinA1 (agonist) may lead to the same protein-protein interaction network of EphA2 that caused this paradoxical inhibition. We used a chemical proteomic method developed in our lab called “co-clickable precipitation (Co-CP)” to study the interaction network of EphA2 in colon cancer cell line HCT-116. We observed that the onset of inhibitory effect by I268 concurred with the association between EphA2 and a protein interactor called c-RAF, a key player in activating proliferation pathways in cancer cells containing Ras mutations such as HCT-116 cells. We are currently performing confirmatory experiments to characterize whether upon inhibition by I268 or activation by EphrinA1, EphA2 sequesters c-RAF in a form unable to transmit proliferative signals downstream. Our results may demonstrate a possible mechanism for inhibitory effects of EphA2 inhibition on cancer cell growth. These results validated EphA2 as a drug target and may provide novel ways for treating cancer cells by inhibiting cell proliferation pathways indirectly using EphA2 inhibitors.

Glycans are ubiquitous in biology and play a crucial role in cell to cell signaling, immunity, and disease. The A, B, and O blood groups are differentiated by different glycan structures and linkage patterns presented on either glycoproteins or glycolipids. To date, the exact glycan structures that differentiate blood types remain unknown. A clear understanding of these structures would increase the success of transfusion procedures and reduce the risk of complications. Our lab is currently examining glycans from glycoproteins isolated from red blood cell membranes, however we do not have the enzymes needed to remove glycans from glycolipids. ECGase I is an enzyme that was recently shown to effectively cleave glycans from a wide range of glycolipids. We have cloned the gene for ECGase I into a GST fusion vector for recombinant bacterial expression. With this we have been able to generate high yields of purified ECGase I. The EGCase I produced was more active than commercially available product. The ability to grow EGCase I enables large scale investigation of glycolipids involved in the ABO blood group system.

Synthetic CRISPR–Cas transcription factors enable the construction of complex gene expression programs, and chemically–inducible systems allow for precise, rheostatic-like control over the transcriptional dynamics. We have constructed a bio-orthogonal, chemically–inducible synthetic CRISPR regulatory system to activate and repress gene expression in yeast. By fusing chemically inducible transcriptional regulators to specific RNA binding proteins, we have expanded the tunability of this system of constructs. The RNA binding proteins are fused to half of one chemically-inducible system while the other half is fused to an effector. Upon addition of drug, the two halves come together to form the complete chemically-inducible system and either activate or repress the target gene. We use reporter gene assays to probe the dose-dependence, time-dependence, and reversibility of the systems. The use of multiple, orthogonal chemically-inducible systems and unique guide RNAs allows for more sophisticated, multi-gene programs that still maintain precise control of gene expression dynamics independently at different sites.

New techniques in genome mapping have revealed a high degree of 3D spatial organization in the nucleus. Long-range loops connect enhancers to their gene targets to regulate expression. In order to assess the mechanisms and functions behind 3D spatial organization of the nucleus we need a system that allows us to engineer DNA loops. We use programmable CRISPR-Cas DNA binding domains to target specific sites in the genome. The CRISPR-Cas complex is tethered to a targeting domain (TD) that can dimerize with another TD at a distant DNA locus. To promote interactions between TDs that are bound to DNA, we have designed an allosteric switch that assembles the TD only when the CRISPR-Cas complex has engaged its DNA target. To validate that our switch proteins can act as a DNA sensor we have developed a simple reporter assay; upon successful switch protein activation on DNA a transcription factor is recruited to drive expression of a fluorescent protein. Our results indicate that the protein switches activate when they are recruited to DNA, effectively acting as a sensor for DNA binding. Future steps include optimizing our reporter assay design and modifying the design for DNA looping.

In order for multipotent stem cells to properly differentiate into specialized cells, specific genes must be expressed at a specific time and amount during development. Many of the factors that regulate expression have been identified; however, it remains unclear how they work together to control the timing and amplitude of gene expression. Non-coding DNA elements, known as enhancers, can increase the the likelihood of transcription of a gene by integrating signals in the cell to provide regulatory logic for gene regulation. To understand how enhancers tune gene expression timing and amplitude during development, our lab has generated a transgenic mouse in which each of the two copies of the T-cell identity gene, Bcl11b, have been tagged with distinguishable fluorescent reporters, providing a sensitive readout for gene activity at the single locus level. Bcl11b turns on during T-cell development, and its activation executes a developmental switch from a hematopoietic stem cell to a T-cell committed progenitor. There is a non-coding region far downstream of Bcl11b which harbors a cluster of putative enhancers. To interrogate the function of individual candidate enhancers, we use CRISPR/Cas9 targeting to generate specific genomic deletions in T cell progenitors. From our preliminary experiments, we have shown that cutting off the entire enhancer region completely inhibits the expression of Bcl11b entirely compared to when we cut out only an individual enhancer peak which only partially inhibits it. This is promising because it shows that we have found an enhancer that controls the probability of activation while not being necessary for the activation of Bcl11b. This work will reveal the cis-regulatory logic that underlies the control of a master lineage-specifying gene. This better understanding will help us identify new strategies to control the expression of master regulatory genes for cellular reprogramming.

Genes encoding the RNA portion of the ribosome (rDNA) are present in essentially all eukaryotic genomes as tandem repeated arrays. In humans, rDNA copy number is highly variable and an undervalued potential source of genetic disease. Changes in rDNA copy number can occur through DNA breakage and repair as well as through errors in DNA replication. High transcriptional activity at the rDNA locus poses challenges for replication; all tested eukaryotes have evolved replication fork barriers (RFBs), ensuring that replication machinery does not collide with transcribing RNA polymerases. In yeast, the RFB is a specific sequence to which the protein Fob1 binds, blocking replication forks that converge with transcription. Mutants lacking Fob1 have greatly reduced variation in rDNA copy number. There are currently two models to explain how Fob1 binding to the RFB produces rDNA copy number instability. One model suggests that binding of Fob1 actively recruits DNA break and repair machinery which induces recombination between rDNA repeats. Another model proposes that the stalled replication fork at the RFB is inherently fragile, increasing the likelihood of breakage. To distinguish between these two models, I am generating yeast strains where Fob1 binds to the RFB but does not arrest forks. Using CRISPR/Cas9 gene editing technology, I am reversing the direction of the RFB in each of the 150 rDNA repeats in yeast to prevent replication fork stalling. By confirming the absence of replication fork stalling and determining whether rDNA instability has also been reduced, I can distinguish whether Fob1 binding to the RFB in the absence of fork blocking contributes to rDNA copy number changes. Clarifying involvement of the RFB in rDNA copy number changes will additionally provide insights into the connections between transcriptional activity, replication fork stalling and genome instability.
 

Different phenotypes of normal cells may influence genetic and epigenetic profiles and tumorigenicities of their transformed cells. The current study presents a comprehensive analysis of mutations in the whole mitochondrial genome for different immortalized (pre-neoplastic) cell types, which were derived from normal breast epithelial cells with two different phenotypes (stem vs non-stem). Duplex Sequencing, which shows the lowest error rates among currently available DNA sequencing methods, was applied to investigate rare and subclonal (low-frequency) mutations as well as homoplasmic (high-frequency) mutations. We found that the vast majority of mutations in the cells occur at low-frequency and are not detectable by conventional DNA sequencing methods. The most prevalent mutation types are C>T/G>A and A>G/T>C transitions. The overall frequencies of rare and subclonal mutations are significantly lower in stem cell-derived immortalized human breast epithelial cells (SV1) than in non-stem cell-derived immortalized human breast epithelial cells (SV22). The decreased mitochondrial mutagenesis of SV1 cells occurred mainly in the noncoding RNA region (rRNA and tRNA) of the mitochondrial genome. In addition, the predicted pathogenicity for rare and subclonal mutations in the tRNA region was significantly lower in SV1 cells than in SV22 cells. Our findings suggest that different phenotypes of normal cells lead to distinctive mitochondrial mutation profiles of immortalized cells. The identified mutation spectra and mutations specific to stem (versus non-stem)-derived immortalized cells have implications in characterizing heterogeneity of tumors and understanding the role of mitochondrial mutations in immortalization and transformation of human cells.

Precise spatiotemporal control over biochemical cue presentation is necessary to mimic the complex, heterogenous environments found in biological systems. Achieving this level of control within engineered microenvironments would allow for the manipulation of cell growth and differentiation, which could be utilized in tissue engineering and drug delivery. To this end, we developed a method that utilizes fusion proteins made from a novel PhotoCleavable protein linker (PhoCl) and a protein of interest (POI). This method allows for spatiotemporal control of POI release from hydrogels in response to cytocompatible violet light (λ = 405). This system is flexible, as PhoCl can be conjugated to many different POIs, including fluorescent proteins, enzymes, and growth factors, and was found to not affect protein function. Additionally, PhoCl undergoes a green-to-red transition after photocleavage, allowing for real-time tracking and quantification of POI release. As PhoCl cleaves in response to visible light, which is less damaging to cell function and has a greater tissue penetration depth than the traditionally used UV light, PhoCl fusion proteins hold promise for use in vivo. To demonstrate the feasibility of this system, PhoCl fusion proteins were formed with several fluorescent proteins (e.g., mRuby, sfGFP, mCerulean). Conjugating these fusion proteins into gels and exposing them to patterned light produced spatiotemporal localized release of proteins with micron scale resolution, which was demonstrated through fluorescent imaging of the photopatterned gels. To support the potential in vivo applications of this system, PhoCl was also used in mammalian cell studies with epidermal growth factor (EGF). These studies showed the expected increased cell growth in response to photomediated EGF release. This illustrates the potential versatility of the PhoCl system in biological applications, thus supporting the relevance of this novel system to tissue engineering and drug delivery methods.

Amyloid diseases are characterized by the aggregation and buildup of proteins in vital tissues and organs. Insoluble β-sheet amyloid fibrils were previously thought to be the major underlying cause of tissue degeneration and cell death. However, recent experimental evidence suggests that soluble oligomers, which form during protein aggregation and before polymerization into fibrils, are the principal cause of toxicity in mammalian cells. These toxic oligomeric protein assemblies are believed to share a common sequence-independent secondary protein backbone structure known as α-sheet. This project proposes the investigation of a synthetic peptide known as AP3 that is capable of forming toxic oligomers and β-sheet amyloid fibrils. This peptide was de novo designed with a completely randomized sequence which preserves the underlying chirality that produces α-sheet character leading to its exhibition of amyloidogenic properties under acidic conditions. Furthermore, AP3 aggregation was shown to be inhibited by three naturally occuring amyloid proteins implicated in their respective dieseases: Amyloid Beta (Alzheimer’s), IAPP (Type II Diabetes), and Transthyretin (Cardiac Amyloidosis). Analysis using dot-blot assays, soluble oligomer binding assays (SOBA), and BLITz assays will provide additional insight into the behavioral, binding, and kinetic properties of AP3. Upon further evaluation, we aim to demonstrate the ability of AP3 to serve as a synthetic model for naturally occuring amyloids and provide a better understanding of amyloidogenesis as well as the interactions between amyloidogenic species. This research will prove useful in the creation of more effective amyloid inhibitors and treatments for amyloid diseases.

The formation of dental biofilm (plaque) is promoted through the consumption of dietary sugars, which serves as a precursor to the metabolic process that bacteria must undergo. Organic acids are byproducts of this metabolism, and encourage the demineralization of the enamel which if left untreated leads to formation of dental caries (tooth decay). Evaluation of the acidity of plaque deposits on patient’s teeth can be used as a preventative measure to allow dentists to detect areas vulnerable to dental decay. A ratiometric fluorescence pH sensing device has been developed using an FDA approved dye, Fluorescein, and blue LED excitation. Fluorescent spectral profiles were collected using a spectrometer and analyzed with a spectral unmixing algorithm for calibration over the pH range of 4.5-7, the ideal range to measure the acidic environment of the mouth. A 420 nm LED housed with an electronic driver served as the fluorescein excitation source. Spectral profiles were collected within the 450-650 nm range. In an in vivo pilot study, we found that the dye solution which consists of fluorescein powder and deionized water causes retention issues on interproximal areas. In order to achieve accurate results from the spectrometer, the fluorescein dye must penetrate and diffuse through the dental plaque. To allow enough time for the fluorescein solution to adhere, glycerol was added to increase viscosity. Tests were performed to evaluate the effect of the glycerol addition on the spectral data. Solutions ranging from 0%-50% glycerol were measured. Glycerol addition increases the peak wavelength of the spectra by no more than 1%. The pH of the solution has no effect on this trend. An in vivo case study was performed for high caries risk patients to validate the device. The pH device has the potential to predict early caries, reducing oral restorative procedures and dental expenses.

Tuberculosis (TB) kills more than one million people annually. Bacteria of the Mycobacterium genus, including M. tuberculosis, build a complex cell wall containing mycolic acids. This cell wall is difficult to penetrate, so specialized antibiotics are needed. Even with newly developed drugs, bacteria adapt quickly and exhibit resistance at an alarmingly rapid pace. One adaptation that allows the community to survive is production of biofilms. Formation of biofilm is one of many quorum sensing behaviors known in pathogenic mycobacteria. This additional layer surrounds a microenvironment where bacteria can thrive with a very low concentration of antibiotic. Thus, one alternative method to treat TB is to control biofilm formation. In this research, a set of marine bacterial strains, including several bacteria that exhibited swarming behaviors and several from the same environmental samples that did not, were cultured, extracted, and analyzed by 1H NMR and LC-MS as well as in newly-developed biofilm and growth inhibition assays. Initial results showed one group of bacteria produced an organic compound that induced biofilm production in mycobacteria. This was an unexpected result. One representative strain producing a strong biofilm inducer was grown on large scale (10L) then extracted using progressively less polar eluents on a reversed-phase SPE column. The biofilm-inducing fraction was then separated using flask column chromatography. One major component was analyzed spectroscopically using 1D and 2D NMR techniques along with Mass Spectrometry. This compound could result in a strategy to interfere with biofilm formation in mycobacteria, thus making antibiotics more effective.

Due to antibacterial resistance and the high recurrence of urinary tract infections (UTIs), studies have shifted to focus on anti-adhesive therapies as alternative to antibiotics. Often treated with antibiotics, UTIs are caused by uropathogenic Escherichia coli (UPEC). The bacterial adhesin, FimH, found on the terminal end of fimbria, hair like structures expressed on the perimeter of UPEC, is the main etiological factor of UTI prevalence and recurrence. FimH increases the virulence factor of E. coli by mediating the initial binding of the bacteria to glycosylated cells in the urinary tract. FimH has two domains. The lectin domain (LD) recognizes and binds the terminal mannose on glycosylated cells lining the urinary tract, whereas the pilin domain acts as an anchor to the fimbria. Previous studies have shown that α-methyl-mannose (αMM) competitively inhibits glycoproteins, such as horseradish peroxidase (HRP), from the FimH active site. We hypothesize that αMM can non-competitively inhibit HRP through a novel mechanism of inhibition. To determine the mechanism of inhibition of HRP in the presence of αMM, we are using Enzyme Linked Immunosorbent Assays to measure the dissociation of HRP in the presence and absence of αMM, after the FimH-HRP complex has formed. We expect to see an increase in the dissociation of HRP in the presence of αMM. HRP in this case, will act as a model to the glycosylated cells lining the urinary tract. This study aims to assist in the design of innovative anti-adhesive therapies that inhibit binding of FimH once bound to glycosylated cells lining the urinary tract.

A person’s age is one of the greatest risk factors for many age-related diseases, such as Alzheimer’s disease, heart disease, and cancer. Developing interventions that target this common risk factor may allow us to slow down the progression of all age-associated diseases. One promising target for aging research is the mTOR (mechanistic Target of Rapamycin) signaling pathway, which is a master regulator of cell growth that allows the cell to fine tune its response to various environmental conditions. Inhibition of mTOR pathway has been shown to extend lifespan in yeasts, worms, fruit flies, and mice. In addition of being a target in aging research, the mTOR pathway is also a well-known effective target in some human cancers to prevent cancerous cells from dividing uncontrollably. We have previously developed a yeast-based system to identify mTOR inhibitors by comparing the growth of yeast strains that are differentially sensitive to mTOR inhibitors; a wild type strain, a tor1Δ mutant, and an fpr1Δ mutant. However, the system is not able to identify a few known ATP competitive inhibitors that have been shown to inhibit mTOR in human cells. This could be due to dissimilarities between yeast and human mTOR protein kinase domains. To address this issue, we are developing a humanized strain of yeast that should allow for identification of additional mTOR inhibitors that are effective in human cells. I am developing a novel hybrid gene containing the human mTOR kinase domain fused with the N-terminus of the yeast TOR2 gene. This new humanized strain will be utilized in our yeast-based assay with the goal of first detecting mTOR inhibitors known to be effective in human cells but unable to inhibit yeast mTOR. Successful completion of this goal will facilitate high-throughput screening of novel compounds that may have mTOR inhibitory and cancer fighting effects.

Dravet syndrome (DS) is a severe form of childhood epilepsy caused by a mutation in the SCN1A gene, which encodes the NaV1.1 voltage-gated Na+ channel. This channel is present in most GABAergic neurons, the main inhibitory neurons in the brain. Reduced activity of the channel in DS leads to loss of inhibitory activity in the brain; this, in turn, leads to seizures and developmental deficits. Through previous research using the mouse model of DS, the de la Iglesia lab has demonstrated that DS also affects circadian rhythms, which are the endogenous biological rhythms synchronized to the 24 hour day. These symptoms are likely caused by the loss of NaV1.1 in a sleep regulatory center called the suprachiasmatic nucleus (SCN), a set of cells which functions as the ‘master clock’ of the circadian system of mammals. However, the de la Iglesia lab found that selective deletion of the SCN1A gene from the SCN fails to replicate the abnormal circadian phenotype. We believe that these mutant mice are phenotypically normal either because there is a compensatory increase in the expression of another sodium channel, NaV1.3, or because the targeting strategy does not reach all cells within the SCN. To test the first hypothesis we employed in-situ hybridization to visualize the expression of the genes that code for NaV1.1 and NaV1.3 channels in either SCN-specific knock outs or their wild type littermates. My results will help explain the phenotype seen in the SCN-specific SCN1A mutants and determine whether developmental compensatory mechanisms are important in the SCN of DS mice.

Developmental and Epileptic Encephalopathies (DEEs) are a group of severe epilepsy disorders in children and infants characterized by prominent EEG (electroencephalography) abnormalities that disrupt brain function leading to cognitive decline. Identifying genetic causes of DEE is a key step to help researchers develop and personalize medical treatments for affected patients. Approximately 5% of DEE cases are caused by a copy number variation (CNV), where a region of DNA involving a disease gene has been duplicated or deleted. Historically, this type of mutation has been difficult to detect using sequence data. To address this, I have written a multi-step algorithm that analyzes smMIP (single molecule molecular inversion probe) targeted DNA resequencing data for known DEE genes to identify CNVs in patients’ DNA that are potentially disease causing. I have run this algorithm on the large collection of smMIP data for 1158 DEE patients available in the Mefford Lab and identified several potential CNVs. Among these, three CNVs ranging in size from 250,000-2,790,000 base pairs, each involving a DEE gene - GNB1, GRIA2, and UHRF1BP1L - were validated by a second method, array CGH, the current gold standard for CNV validation. To date, the validation rate of high-confidence CNV candidates is 50% or higher. Currently I am expanding the algorithm’s functionality to include the ability to selectively search for single-exon CNVs, which are as small as 500 base pairs, are more challenging to detect, have largely been missed by all CNV detection methods, but could still be pathogenic. To do this, I am leveraging the power of intersecting duplicate smMIP datasets to improve the sensitivity of single-exon CNV detection. As any disruption of a pathogenic DEE gene could be disease causing, inclusion of these smaller CNVs will increase our ability to solve DEE cases and improve patient care.

 Developmental and epileptic encephalopathies (DEEs) are an early-onset form of epilepsy characterized by intractable seizures and severe cognitive and developmental impairment. While most genetic variants that cause DEE reside in the coding regions of genes, splice-site variants can also be pathogenic. Splice-site variants are changes in the DNA close to, or on, the exon-intron boundary, which can cause aberrant splicing, resulting in exon exclusion or intron inclusion within spliced mRNA, generating a protein that is non-functional, partially functional, or aberrantly expressed. Aberrant splicing can have pathogenic consequences, but predicting which variants near splice-sites will have an effect on splicing is difficult. I am studying three potential splice-site variants in three genes associated with DEE: SYNGAP1, SCN1B, and WWOX. I used RNA extracted from fibroblasts from three DEE patients, each with one of these variants, to confirm whether the splice-site variants cause aberrant splicing and what the predicted consequences of this aberrant splicing is on the protein. I have been able to discover the effects of the variants on splicing in WWOX and SYNGAP1. In SYNGAP1, a synonymous variant at the exon-intron junction caused an exon 4 deletion, resulting in a severely truncated protein (SYNGAP1:p.Glu120Alafs*20). In WWOX, a duplication which included exon 5 resulted in a transcript with the inclusion of two copies of exon 5, leading to a frameshift mutation and predicted truncated protein (WWOX:p.His173Glyfs*13). We are still investigating the effects of the intronic variant in SCN1B, which appears to decrease expression of the gene. We are using nonsense-mediated decay inhibitors in order to better understand the mechanism through which this decreased expression occurs. This research could potentially improve the care of these patients by providing genetic evidence of the causes of DEE, which can be the basis for advancing better treatments.

Heterotopia are organized structures consisting of mixed cellular and neuronal elements arranged in a clear architectural pattern inappropriate to the considered tissue. Heterotopia are observed in the cerebral cortex and the cerebellum as a feature of many neurological disorders yet the we know little about the mechanisms driving their formation. We have analyzed a substantial number of Dandy Walker malformation (DWM) human fetal cerebella and found that a significant number of cases contain heterotopia. DWM is the most common structural birth defect of the human cerebellum and is characterized by an enlarged posterior fossa, enlarged fourth ventricle, and cerebellar vermis hypoplasia. A subset of cases are caused by loss of FOXC1, a transcription factor expressed in the mesenchyme during development. Our group has previously shown that loss of FOXC1 in mice causes loss of the mesenchyme-secreted factor SDF1alpha. Further, loss of SDF1alpha is sufficient to cause cerebellar heterotopia. This emphasizes the importance of mesenchymal signaling in the maintenance and development of the clear laminar architecture of the mature cerebellum. We hypothesized that granule neuronal progenitors (GCPs) are the primary cellular target of SDF1alpha mesenchymal signaling and therefore the main cell type causing heterotopia formation. To test our hypothesis, we excised the receptor for SDF1alpha from just GCPs in mice. Our findings show that loss of SDF1alpha in GCPs causes them to prematurely migrate into the developing cerebellar anlage and also caused other cerebellar cell types to form structured heterotopia. We observed defects in cerebellar foliation in the treatment group. Our data emphasizes the importance of SDF1-alpha dependent mesenchymal signaling in cerebellar development and identifies heterotopia as a new phenotype in DWM.

As of now, mRNA vaccines have been deemed as a potent replacement for current vaccine models against infectious diseases for their improvements in B-cell and T-cell immune responses. Usually, when soluble, subunit antigens are delivered, they are scattered and randomly bind to B-cell receptors, often loosely. However, with a nanoparticle carrier for antigens, there would be more effective crosslinking with B-cell surface immunoglobins as there is a higher density of structurally ordered antigen arrays presented by the nanoparticle. As a result, the B-cell creates a stronger immune response. Additionally, the multivalent particles also favors the creation of long-lasting immunity against a given virus. My team and I are currently developing a self-assembling protein platform using dn5A and dn5B protein components as a carrier for an mRNA vaccine against the flu. My project mainly focused on optimizing the co-secretion of the two particles by exploring different models and combinations of both. This is important as the translated cage not only has to be able to self-assemble but also be capable of doing so without producing excess protein in order achieve its purpose. To do so, I investigated 12 different constructs of dn5A and dn5B through transfections and analysis with western blots and electron microscopy. We used the data collected to improve the dn5A/dn5b protein platform utilized alongside flu mRNA vaccines, helping them better achieve potency. Overall, if effective, the new vaccination model can be utilized for other infectious diseases, including HIV and meningococcus.

Dandy Walker malformation (DWM) is the most common human cerebellar malformation, affecting 1 in every 3000 live births. DWM is an imaging diagnosis that is characterized by three features: cerebellar vermis hypoplasia, an enlarged posterior fossa, and an enlarged fourth ventricle. Although recent advances in neuroimaging have improved diagnosis of DWM, virtually nothing is known about the cellular and histological defects that lead to DWM. One major reason is that little human specific data is available describing the histology of normal and abnormal human fetal cerebellar development. Currently, there is limited published fetal pathology of DWM. There is no comparative analysis available and most studies are confounded by lack of molecular confirmations of diagnoses. We have carried out the first comprehensive histo-pathological analysis of human DWM. Such histo-pathological analysis, that I specifically was responsible for completing, included measuring the length and cell density of certain regions of the developing cerebellum in the 36 DWM cases, such as the external granule layer and the rhombic lip. Our results indicate a significant reduction in size and area of neuronal progenitor zones in the developing human cerebellum. We also observe aberrations in the developmental trajectories of specific cell types like Purkinje cells, and progenitor zones like the rhombic lip. Through our analysis of the human fetal DW cerebellum, we begin to directly address the developmental pathology of human DWM beyond that of the mouse models that share similar pathology. We believe our studies will fundamentally improve our view and understanding of the biology of the human cerebellar development and give us insights on the developmental pathogenesis of DWM.

Long non-coding RNAs (ncRNAs) play a significant role in transcriptional regulation; therefore, mutations in their sequences can lead to human disease. An example is provided by the promoter associated non-coding RNA (paRNA) that, when bound with Argonaut 1 (Ago1) and a miRNA, plays a key role in coordinating gene silencing of the tumor suppressor CDH1 in epithelial cells. The paRNA expression can be implicated with cancer. This is due to a single nucleotide polymorphism (SNP) at positions -160(C/A) relative to the CDH1 promoter. The -160(A) isoform favors a unique secondary structure of the paRNA, distinct from more common -160(C), which favors over-suppression of CDH1 and leads to increased cancer risk. To better understand how the -160(A) isoform mechanistically drives increased suppression of CDH1, our lab is applying NMR-based methods to determine the 3D structure of the paRNA. However, the T7 RNA polymerase used to synthesize RNA is prone to producing heterogeneous products in longer RNAs such as this one. Having exact lengths of the transcript is important for improving signal-to-noise and peak sharpness in NMR spectra, which is critical for 3D structure determination. To overcome this problem, I designed a pUC19 plasmid containing the paRNA -160(A) sequence with a downstream self-cleaving hepatitis delta virus (HDV) ribozyme. Following transcription, the HDV ribozyme undergoes self-cleavage, producing homogenous ends at the 3’ end of the paRNA. My results show that HDV incorporation produces a single species of RNA with no 3’ overhang and improves NMR spectra quality. This cloning tool is easily adaptable to other large RNAs, facilitating data collection for other large RNAs used in our lab. Ultimately, our ability to predict the 3D structure of this paRNA could someday lead to its use as a potential drug target.

Hearing loss is often due to the absence or degeneration of hair cells in the cochlea, thus it is important to understand the transcriptional control mechanisms specifying the prosensory cells that generate hair cells and support cells. Early B-Cell Factor1 (Ebf1) is a transcription factor that was brought to our attention from motif enrichment in replicated ATAC-seq peaks detected in prosensory cells and non-prosensory cells. We observed that in the open chromatin regions and dynamic regions during the development of prosensory cells showed greater enrichment of Ebf motifs. This suggested that Ebf1 could be acting as a pioneer factor. To determine whether Ebf1 influences cochlear development, we generated a Sox2CreER: Ebf1 fl/fl allele by crossing Sox2-CreER mice and Ebf1-floxed mice. Administration of tamoxifen to mice orally leads to Cre-mediating deletion of Ebf1 DNA binding domain and formation of the recombinant Ebf1-GFP fusion protein. To determine whether the Ebf1 knockout is successful we will compare Ebf1 expression between the Ebf1 knockout and wildtype through qRT-PCR. We will also compare the localization of Ebf1-GFP fusion protein in the Ebf1 knockout and wildtype. To determine whether Ebf1 affects cochlear differentiation, we will immunolabel for Sox2, Egfp, and Myo7a, this allows us to determine the presence of support cells (anti-Sox2), hair cells (anti-Myo7a) and the Ebf1 reporter (anti-Egfp) and compare support cell and hair cell counts between the Ebf1 knockout and wildtype. If the comparison shows a decrease or increase in support cell and hair cell counts in the Ebf1 knockout compared to the wildtype or the lack of support cells or hair cells in the Ebf1 knockout, then it suggests that Ebf1 is necessary for cochlear differentiation.

STUDIO is a peer mentoring program that focuses on connecting University of Washington (UW) undergraduate student mentors with low income, immigrant and refugee youth through Science, Technology, Engineering and Mathematics( STEM). The program is a partnership with the Neighborhood House community center in the High Point Neighborhood of West Seattle. Neighborhood House provides the facilities and is the gathering place for UW mentors and youth mentees on a weekly basis. As STUDIO is offered as an undergraduate course (EDUC 421), the mentors visit Neighborhood House once a week and then attend a weekly seminar class on campus as well. The program began as a research study that focuses on promoting youth interest in STEM. However, over the past few years it has made progress in undergraduate STEM learning research as well. As a lead mentor within the STUDIO program, I have an opportunity to participate in research aspect as well as cultivate mentorship with youth, and supporting other mentors. In this study, I explore how relationships between youth and mentors develop in STUDIO. I am doing this through analysis of documentation of STEM and non-STEM activities, written reflections of mentors and interviews. They are analyzed for types of relationships that mentors develop through this program as well as themes and patterns of development. The goal is to better understand how a mentorship program that focuses on bridging the gap of educational inequity for youth lends possibility for undergraduate learning through relationships building.

A protein's function is dependent on its structure, which is made up of amino acids. Proline, (an amino acid) is known to cause the kinks and turns in protein structures. However, little is known about the influence of pH on the isomeric preference of proline-attached dipeptides. For this experiment, the isomeric preference of methionine-proline was measured in 10% solutions for pH levels of 7 and 11, the pH of the human body falls between the range of 6 and 9. At pH 11, NMR-90 spectra showed that the cis- isometric form was preferred at a rate of 14% more than the trans- isometric form, measured by the alpha hydrogen. At neutral pH of 7 trans- isomers are preferred 36% more than the cis- form measured by the alpha hydrogen and 73% more measured by the delta hydrogen. In conclusion, this experiment supports the hypothesis that proline-attached dipeptides’ isomerization is pH dependent and is more likely to be in cis- form when in high pH in comparison to a neutral pH. The purpose of this experiment is to determine if pH levels can change the structure of a protien, with further research exploring if the change of structure changes the function. This is important to determine if medications containing amino acids can have an optimal or range of pH.

Polymorphisms in the gene, FKBP5, and its resulting protein, FKBP51, are associated with stress-related disorders. Although FKBP51 inhibitors may have antidepressant-like effects, the relevant brain regions mediating this effect are still unknown. We found that FKBP51 expression is elevated in serotonin neurons of the mouse dorsal raphe nucleus (DRN) after stress, so we tested whether FKBP51 inhibition in the DRN by the novel FKBP51 antagonist, SAFit2, has antidepressant-like effects. First, we implanted guide cannulas into the DRN of wildtype mice stereotaxically, then we habituated the mice to 2.5% sucrose-containing bottles in their home cages overnight. On the following two days, the mice were stressed through repeated forced swims after receiving either SAFit2 (n=8) or a vehicle (n=7) to the DRN via the cannulas, prior to each swim session. That evening, the mice underwent a sucrose preference test to assess motivation by quantifying sucrose versus water preference using lickometers. On the next day, the mice were tested in a three-chambered social interaction test, where one chamber contained a wired cup enclosing another mouse of the same sex and the other chamber had an empty wired cup. Our results show that the SAFit2 and vehicle mice had the same immobility time during the forced swim stress, signifying that SAFit2 did not interfere with our immediate stressor. The SAFit2 mice demonstrated an increased preference for sucrose after stress compared to the vehicle mice, indicating greater motivation to consume a pleasurable liquid. However, there was no significant difference in the time spent interacting with the same sex during the social interaction test. This suggests that SAFit2 may have blocked stress-induced anhedonia by inhibiting FKBP51 activity in serotonergic neurons, as measured by the sucrose preference test. Further studies of FKBP51 inhibition in the DRN can lead to potential therapeutic treatments of neuropsychiatric disorders.

With the discovery of radioactive materials, many institutions have conducted numerous radiation experiments in order to further understand the effects of radiation and radioactive contamination on human bodies and their environments. While doing so, there has been a great amount of unequal health disparities among the different communities involved. Institutions, like the University of Washington, while held in high regard, have participated in experiments that have affected the livelihood of many people. To this day, common knowledge of the University of Washington’s participation is still obscured. There are many institutions in the United States that are like UW and are known for their extensive and groundbreaking research; with the funding and power they hold, people often don’t question their research or their ethical approach to conducting it. From our research, we obtained a greater understanding of what occurred throughout the experiments - thus our goal is to bring attention to the actions of the University of Washington researchers’, C. Alvin Paulsen’s and Lauren R. Donaldson’s, unethical research practices in the Marshall Islands and UW Walla Walla Prison Radiation Experiments. Our methods include analyzation of interviews with Tom Carpenter - the Executive Director of Hanford Challenge, discourse analysis, reviews of relevant literature and interpretations of visual elements. Because the research of C. Alvin Paulsen and Lauren R. Donaldson is locked away in UW’s Special Collections Library, we were unable to analyze their research directly. We anticipate that this project will ensue more curiosity and lead to the general public learning more about the University of Washington’s participation in human radiation experiments.

Transcranial magnetic stimulation (TMS) of the visual cortex can induce phosphenes - highly transient, generally formless lighted areas at the periphery of the visual field - of stimulated test subjects while they look at a small visual target. Non-diagnostic ultrasound, applied to the visual cortex, can also induce phosphenes, studied while test subjects had their eyes closed during stimulation. Here, we sought to study potential visual alteration of a visual target during application of diagnostic ultrasound to the anatomical location where TMS induced phosphenes in the periphery of that visual target. First we used TMS, guided by anatomical landmarks, to stimulate the visual cortex and generate phosphenes in the periphery of a visual target. Next, we applied diagnostic ultrasound over 21 trials for 15 seconds per trial to a test subject while they looked at a white cross with a beginning baseline and a mix of random sham and treatment diagnostic ultrasound exposures. 10/11 test subjects observed TMS-induced phosphenes. Diagnostic ultrasound stimulation of the visual cortex induced visual effects in 7/10 test subjects, with no effects in 3/10 test subjects. The likelihood of the 7/10 test subjects to observe a visual effect increased as the experiment progressed, increasingly so as ultrasound exposure increased. Diagnostic ultrasound, delivered transcranially to healthy test subjects, altered the perception of a visual target and generated a lasting effect on their visual perception. This observation of a prolonged effect on visual cortex is consistent with recent work on stimulation by diagnostic ultrasound of the human motor cortex as well as earlier work on stimulation of the human amygdala. These results suggest diagnostic ultrasound may one-day find rapid application to humans for a variety of purposes, and also raises questions about the advisability of applying diagnostic ultrasound to the human brain without medical justification.

Understanding how adolescents explain gratitude in peer interactions uncovers how they view their identities and social worlds during a time when they are transitioning to the responsibilities of adulthood. Earlier studies define “gratitude” as a state of appreciation, and have shown that adolescent feelings of gratitude correlate with increased well-being, academic performance, and prosocial behavior. Furthermore, our previous findings suggest adolescents have differing rationale for why they feel grateful in situations when they either enact or receive calming, angering, avenging, and reconciling intervention from their peers. The goal of this study was to ask: 1) How do adolescents explain feelings of gratitude they feel following intervention in victimization events? 2) How do rationales for feelings of gratitude vary between actor and recipient of a peer intervention? 3) How does gratitude differ when adolescents calm, amplify, avenge, or reconcile a previously victimized peer? We qualitatively coded explanations provided by 265 adolescents for emotions in each condition. Our findings showed actors felt more gratitude when calming their peers, and recipients felt more gratitude after reconciliation. Peer gratitude was predicted to be more strongly linked to a desire for security when the subject was the recipient (e.g. “I was grateful she looked out for me”), and more strongly linked to a desire to display competence when the subject was the actor (e.g. “I was grateful she took my advice”). We carried out quantitative analyses using repeated measures analysis of variance and Epistemic Network Analysis. The findings from this study contribute to understanding peer relationships, which could help educators navigate student actions that de-escalate conflicts and promote well-being in student social environments.

Progeroid syndrome is a group of genetic disorders characterized by accelerated aging. Molecular mechanisms of progeroid syndrome include abnormalities in genomic stability, nuclear structure, lipid and carbohydrate metabolisms as well as mitochondrial functions. Genomic instability is a major category of progeroid syndromes in humans. Prototypical example is the Werner syndrome caused by the mutations in the WRN gene which encodes a DNA helicase. Cells derived from progeroid syndrome patients generally exhibit phenotypes of accelerated cellular senescence with a very limited replicative lifespan. International Registry of Werner Syndrome recruits progeroid syndrome patients from all over the world for the genetic study. Among the subset of those patients, a recurrent disease mutation has been identified in the POLD1 gene. The POLD1 gene encodes a catalytic subunit of polymerase delta, a major replication polymerase which is also involved in translesion DNA synthesis. The POLD1 mutation is known to cause MDPL (mandibular hypoplasia, deafness, progeroid features, and lipodystrophy) syndrome. In our study, cellular model of MDPL syndrome was generated by expressing mutant POLD1 cDNA in human fibroblasts via lentiviral system. Consequences of the POLD1 mutation are determined by various assays including cell growth, accumulation of DNA damage and sensitivity to genotoxic agents. The eventual goal is to develop therapeutic approaches that ameliorate cellular disease phenotypes. We plan to test candidate therapeutic approaches using established POLD1 cell lines and assay methods. More recently, a mutation in the SMAD4 gene, was identified in one progeroid patient. SMAD4 gene encodes a crucial member of the cellular transduction pathway. The wildtype and mutant SMAD4 are being expressed in human fibroblasts to determine whether cells expressing mutant SMAD4 exhibit phenotypes of accelerated senescence. This project would provide us with an understanding of recently identified progeroid loci and likely sheds a light on the previously unknown mechanisms of normal aging and age-related disorders.

The damascene process is an additive manufacturing technique commonly used in the nanofabrication industry to produce semiconductor devices. This process utilizes deposition and patterning of successive layers to produce interconnected copper patterns separated by interlayer dielectric. The goal of this project was to implement the damascene process to develop and refine methods at the Washington Nanofabrication Facility (WNF) for manufacturing multi-layer devices. For our purposes, we constructed an integrated passive device (IPD) that contains capacitors, resistors, and inductors. We utilized basic nanofabrication tools in our damascene process including deposition, photolithography, etching, electroplating, metrology, and polishing tools. In fabricating this method at the WNF, our main objective was to produce a highly repeatable device with structural integrity. The main challenges accompanied with this involved generating a successful etch on all three layers of the IPD and optimizing polishing conditions. Cross sections of the final product were analyzed in order to demonstrate that the layout expected from the process was achieved. The procedure we developed can be applied to future multi-layer damascene processes at the WNF. Multi-layer devices are significant in the semiconductor industry as they allow for high packing density and an increased variety of circuit configurations in a compact device.

Tin-doped indium oxide (ITO) is a n-type semiconducting material that have been widely used in solar cells, smart windows, and transistors. This material conducts electricity due to excess free charge carriers which charge-compensate the tin dopants. We are working to develop new chemical treatment methods to build optoelectronic devices from ITO nanocrystals. Colloidal nanocrystals are well-suited for solution-processing and low-temperature deposition techniques. These routes can be ideal for fabrication of thin-film electronics, especially on flexible substrates; however, increasing electronic communication between nanocrystals require post-deposition treatments. Few low-temperature methods to create conductive films from oxide nanocrystals have yet been reported. We seek to increase the conductivity of ITO nanocrystal films with ligand-exchange procedures to decrease the distance between each nanocrystal, then chemically crosslinking them. We synthesized ITO nanocrystals, then resuspended in solvents at high concentrations for film deposition. We first modify the surface of our nanocrystals by replacing the native long nonpolar ligands with short polar ligands. The modified crystals were deposited by spin-coating onto glass substrates and a variety of solvents/conditions were tested to optimize uniformity and thickness. Four-point probe measurements resulted in sheet resistance (R_s) ranging from 1 MΩ/square (ligand exchanged nanocrystals) to 2 TΩ/square (untreated nanocrystals). Profilometer measurements probed film thickness that can be used to calculate our films' bulk resistivity. Next, we used zinc and aluminum compounds as chemical solders to build bridges between nanocrystals to increase the material’s conductivity. R_s drops to ~0.15 MΩ/square with aluminum bridges and ~0.10 MΩ/square with zinc bridges. We plan on using other undoped metal oxides with this same scheme and apply them to thin-film field effect transistors (FET) to investigate how these treatments improve FET performance. We are also working on using similar techniques with SnO₂ nanocrystal films for application as electron-transport layers in perovskite solar cells.

Parkinson’s Disease (PD) is a neurodegenerative movement disorder characterized by muscular rigidity, slow movement of the limbs and resting tremor. The onset and progression of PD is attributed to the combined effect of environmental and genetic risk factors, with several specific disease-causing genes having been identified. PD onset is determined by the loss of more than 80% of dopamine-synthesizing neurons from the substantia nigra as well as formation of α-synuclein protein aggregates, called Lewy Bodies. Recent research has identified several potential pathogenic variants in the LRP10 gene on chromosome 14, suggesting that LRP10 may be a novel PD causative gene. It has been shown that the LRP10 protein is implicated in vesicular transport and that it may regulate α-synuclein aggregation, intracellular trafficking, and cell-to-cell transmission. The goal of my project was to analyze the association between LRP10 and PD by sequencing the regions on LRP10 that harbor these variants described in other families and check for co-segregation in the relatives of mutation carriers. DNA samples from 188 PD familial patients were sequenced. LRP10 regions of interest that were examined included exons 1-7 (protein coding regions) and intron 5 (a non-protein coding region). These LRP10 regions in patient DNA were amplified by polymerase chain reaction and sequenced subsequently using Sanger Sequencing. Relatives of LRP10 mutation carriers were then analyzed for co-segregation between the variant and PD. It was expected that certain LRP10 variants would be found to segregate with the disease in inheritance or increase the likelihood of its onset. The molecular function of both the normal and mutated LRP10 protein are still largely unknown and future research into their mechanisms will provide valuable insight into their specific role in Lewy Body formation and dopaminergic neuronal loss.

Alzheimer’s disease (AD) is characterized as an irreversible, progressive brain disorder that gradually destroys memory and thinking skills, and eventually the ability to perform simple everyday tasks. Despite many decades of research focus on AD, the cause and mechanistic understanding of the progression remains enigmatic. Identification of amyloid beta plaques and hyperphosphorylated Tau in post-mortem analysis of brain biopsy is viewed as definitive for diagnosis of AD. Dr. Su-in Lee’s lab has developed a machine learning method that integrates brain tissue pathology metrics with gene expression analysis from the same patient samples. Their analysis identifies genes and pathways that are statistically-associated with the concordance of pathological AD phenotypes. Working with researchers in the Kaeberlein lab, we are using the nematode C. elegans to directly test the impact that these genes have on the progression of human tau-induced neuronal dysfunction and death. The Kaeberlein lab previously identified a subset of genes in complex I of the electron transport chain (ETC) that are highly correlated with human amyloid beta-induced paralysis. We are now extending that work by examining the relationship of these ETC genes in the lifespan and healthspan of human Tau-model nematodes. Our preliminary results suggest that reduction of Complex I activity increases the lifespan of human tau-models nematodes. We are conducting further studies to determine the replicability of these findings as well as disrupting ETC function with RNAi from other genes. Through directly testing nematode orthologs of human genes that are statistically-associated with AD neuropathology we will generate a better understanding of the cellular mechanisms that influence normative aging and Alzheimer’s disease.

The application of quantum dots (QDs) is increasing in optical manufacture (solar cells), biological and chemical devices (cancer research). Exposure to QDs can potentially lead to a variety of adverse health effects especially for children and pregnant women. The goal of this study is to determine the toxicity of CdSe/ZnS QDs on brain development of both sexes using in vitro techniques. To determine sex-specific response, we used human neural progenitor cells (hNPC) lines derived from male (NSC-H14) and female (hNP1). An in-vitro model was established using an adverse outcome pathway (AOP) framework. We plated cells in proliferation or differentiation media for treatment to assess the morphology and biochemistry response in cells. Two QDs, differing in their surface chemical properties: carboxyl functional group – ITK and polyethylene glycol (PEG) without reactive functional group (Qtracker) were assessed. Cadmium chloride (Cd) was also tested to determine whether the toxicity of QDs is associated with ion release. The NSC-H14 and hNP1 cells were exposed to QDs of concentration from 0, 2.5, 5, 10, 20 and 40 nM on days in vitro (DIV) 0. After 24 hours, we evaluated the number of cells remaining (cell viability) with an LDH assay at each dose treatment. The results showed that exposures to ITK QDs demonstrated significant dose-dependent decreases in viability of both cell lines during the proliferative stage. Male (NSC-H14) cells were more responsive than female (hNP1) cells to the cytotoxicity of ITK. We did not observe significant dose response for Qtracker and Cd at the same dose range as ITK, either in proliferation nor differentiation stages of both male and female cell lines. Our results indicate that the coating on quantum dots may play a significant role in the exposure and toxicity of the cells, suggesting potential strategies to reduce the adverse health effects of quantum dots.

Exosomes are 40-50nm extracellular vesicles released by all cell types, and are involved in a variety of cellular mechanisms including modulation of immune response and cancer microenvironment. Exosomes embed proteins, lipids, and nucleic acids from the parent cells and circulate in abundance in human serum. Recently, exosomes have been shown to have potential application as cancer biomarkers with potentially increased sensitivity and specificity compared to traditional protein or nucleic acid markers. In cancer patients, exosomes are also shed in real time, allowing potential applications in directing therapy, monitoring treatment response, and surveying recurrent disease. Using a known tumor marker, carcinoembryonic antigen (CEA), we examined the role of CEA-specific exosomes as a colorectal cancer biomarker. To establish the methodology, we used CEA-secreting colorectal cancer cell lines LOVO and HT29. We compared exosome isolation methodologies and established an ultracentrifugation-based technique to maximize both purity and yield. Through this methodology, we isolated exosomes from cell-free media and demonstrated that CEA co-localizes with coxasackie-adenovirus receptor (CAR), a senescence-related marker of interest in our laboratory. This finding suggests that tumor-derived exosomes could be used to quantitatively define certain cellular events. We then used archival and fresh patient samples to examine whether the CEA-exosomes could be applied in disease monitoring in patients with metastatic colorectal cancers receiving chemotherapy. Efforts are ongoing to define whether CEA-specific exosomes are tumor specific, and whether exosome conjugate protein and nucleic acid can be further used as novel biomarkers in selective clinical situations.

 Adults use many forms of information when deciding how to act in any situation. We are investigating the types of information infants use when determining how to act in a novel reaching task. We are looking at costs (how far they must reach; Study 1) and the social value of the object they are acting toward (Study 2). In Study 1 (ongoing), we tested 6-month-olds (n=25) and 9-month-olds (n=14). Infants completed a warm-up task to measure their maximum reach. During test trials, they were presented toys at 3 different distances: one inch away (Easy condition), one inch past maximum reach (Hard condition), and 27 inches away (Impossible condition; Figure 1). Infants reached more in the easy (93.2%) than the hard (72.6%, β=-1.90, p<0.0001) and impossible conditions (19.3%; β=-5.45, p<0.0001; Figure 2A). Infants also reached more in the hard than the impossible condition (β=-3.5, p<0.0001; Figure 2A). A within-subjects one-way ANOVA revealed a main effect of condition on infants' latency to reach F(2,73)=127, p<0.0001. The average latency increased from easy (2.61+/-2.05seconds), through hard (4.93+/-3.21seconds), to impossible (9.29+/-1.13seconds; Figure 2B). This indicates that infants are less likely to reach for objects when costs are high. In Study 2 an actor will manipulate one toy to be exciting (“I love this toy!”) and one toy to be boring (“Eh”), 3 times, 10 seconds each. We hypothesize that if infants use social value in their decision-making process, they will persist more when reaching for the exciting toy, particularly in the hard condition when reaching is costly. Even though infants engage in less complex decisions than adults, we are able to see evidence of decision-making processes in infants’ actions, supporting the idea that humans’ ability to perform these processes may emerge during early infancy.

My research project involves the AP-3 (adaptor protein) complex, which plays a key role in membrane trafficking cells. Within all eukaryotic cells, there are membrane-bound sections of the cell that interact with each other in various ways to drive the cell's function. Vesicles mediate the transport of proteins and lipids among cellular organelles. These vesicles are created in various ways by proteins throughout the cell that form a "coat" around the vesicle as it travels to its destination. AP-3 is a protein complex that mediates vesicular transport from the Golgi apparatus to the lysosome. The current aim of my project is to analyze evolutionarily conserved features of the AP-3 complex by mutating subunits of the complex and observing resultant phenotypes in our model organism, Saccharomyces cerevisiae (baker’s yeast). In doing so, we utilize a gene reporter system called GNSI to analyze AP-3 function in different genetic strains. This reporter system is mainly analyzed through a colorimetric assay (chemical test to determine components), using qualitative observations of the intensity of colored halos around yeast colonies on a gel plate. We also use fluorescence microscopy by using signals from the Green Fluorescent Protein (GFP) to determine whether AP-3 was successful in trafficking to the lysosomal vacuole. So far, our results have shown that targeted truncations of proteins within the AP-3 subunit Apl6 yielded loss of function in trafficking. We are continuing our analyses by focusing on other proteins within AP-3. These basic studies will further our understanding of membrane trafficking and may provide insight into diseases linked to AP-3 function, including HIV-1 particle assembly and human genetic disorders.

In the US more than 100 million people are living with diabetes or pre-diabetes. The economic burden caused by these conditions, including medical costs, is approximately $327 billion annually. Conventionally, transgenic Escherichia coli has been the primary source of commercial insulin production, a process that requires extensive purification to ensure shelf stability and complete removal of contaminants. This study seeks to establish an alternative mode of insulin production using polyethelyne glycol (PEG) and agrobacterium to transform the oyster mushroom, Pleurotus ostreatus, with the human insulin gene. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins, rapid growth, and fully sequenced genome. P. ostreatus was transformed using PEG and agrobacterium with a plasmid containing the human insulin gene and a carboxin resistance gene. Transformed cells were selected using carboxin, extracted, and regenerated on plates composed of yeast extract, malt extract, and glucose (YMG). Integration of the human insulin gene in to the mushroom genome was confirmed through PCR analysis of the transformants. Successful transformation of P.ostreatus offers a new avenue for insulin production, potentially diversifying the market and treatment options for diabetics.

Ribonucleotides are essential components of the cell: they are the building blocks of RNA and DNA, key signaling molecules, and metabolic intermediates. Maintaining proper ribonucleotide balance is critical for cell survival. An essential, highly-conserved enzyme that regulates purine biosynthesis is IMP dehydrogenase (IMPDH), which catalyzes the first committed step in the production of GTP. In humans, there are two IMPDH isoforms: IMPDH1 is present in low quantities in most cells, and IMPDH2 is upregulated during proliferation. Both IMPDH isoforms form higher order structures in cells and assemble into filaments and bundles of filaments in vitro. Nine point mutations in IMPDH1 have been discovered that cause retinal degeneration and ultimately blindness in humans. Our current findings show that these point mutations in IMPDH differentially impact the protein’s ability to assemble into filaments in vitro. We hypothesize that the ability of IMPDH to polymerize will affect the protein’s activity because of the interplay between protein structure and substrate binding. We use negative stain electron microscopy to first characterize the polymerization behavior of IMPDH in the presence of different ligands: the substrates IMP & NAD+, as well as allosteric effectors ATP and GTP that promote polymerization. In addition, we employ kinetics assays that vary levels of substrate and GTP to quantitatively assess enzyme activity by measuring NADH output. Characterizing each mutant’s behavior will be the first step to understanding the molecular mechanisms that cause retinal disease in IMPDH. In a future study, we can use cryo electron microscopy to solve the structures of mutant protein.

Interferon-gamma Enzyme-Linked Immunosorbent Spot Assay (IFNγ ELISpot) is a laboratory technique that quantifies the number of cells producing interferon gamma (IFNγ) by utilizing antibodies that selectively bind to IFNγ molecules, resulting in spot formation corresponding to individual IFNγ-producing cells. Since cytotoxic T cells (CD8 T cells) and their helper Th1 cells (CD4 T cells) produce IFNγ to activate macrophages and inflammatory responses, quantifying IFNγ-producing cells allows for characterization of host immune responses. Our lab utilized a canine IFNγ ELISpot to test for immune responses against novel proteins expressed following gene therapy in dogs. Currently, we used a routinely given vaccine for canine flu, distemper, adenovirus, and parvovirus as a biological positive control. However, we tested our experimental proteins with peptides, making a whole-protein positive control, which required internal processing, flawed. Our goal was to optimize this ELISpot by identifying peptides from the vaccine that stimulates an IFNγ immune response in peripheral blood mononuclear cells (PBMCs) and splenocytes. Utilizing a commercially available canine IFNγ ELISpot, we stimulated PBMCs and splenocytes. These cells included lymphocytes (T cells) and macrophages, which acted as antigen presenting cells. We compared stimulation with the entire vaccine and various vaccine peptides in order to identify peptides that can be used as a biological positive control. These were compared to traditionally used mitogens that indiscriminately activated all lymphocytes. This optimization allows for greater confidence in the results obtained from our canine IFNγ ELISpot. The improved technique serves as a powerful tool to assist in preclinical trials of vaccine production and gene therapy. It is utilized in our lab to test for CD8 T cell-mediated immune responses against novel dystrophins following gene therapy in Duchenne muscular dystrophy dogs.

Barcoding of physical objects with molecular tags holds an advantage over traditional paper or electronic barcodes in that they are discreet, durable, and difficult to falsify. Here, we developed a DNA tagging system that labels objects to verify their authenticity and trace their origin. We chose DNA as our tagging medium due to its information storage capacity and chemical stability, allowing us to generate a wide variety of unique barcode sequences that can be read by Oxford Nanopore’s MinION sequencing device. The MinION contains an array of thousands of nanopore sensors that are capable of sequencing single strands of DNA. The nanopore sequencing process creates distinct disruptions in the ionic current through the sensors that are indicative of the DNA sequence. However, the DNA basecalling software that processes the raw ionic current is computationally expensive, making it impractical when our goal is to quickly “scan” and identify a tagged sample. Because of this, we designed our barcode sequences to generate unique current patterns that are identified using a simple classification algorithm as opposed to arduous basecalling. So far, we have synthesized and classified a set of 96 barcodes that can be indiscriminately combined to create multi-bit tags. In a given tag, each bit is defined by the presence or absence of a particular barcode, and in practice, we have assembled and read up to 16-bits. We have also explored increasing bit capacity by independently varying barcode lengths, which adds another dimension to the barcode space. We also tested the durability of our barcodes by drying them onto filter paper and sequencing them 24 hours later, proving that our barcodes could survive in a dehydrated state. Future experiments will aim to lengthen this duration and expose the barcodes to different environments, in order to better simulate intended tagging conditions.

In recent years, researchers have dedicated much effort to overcoming the ~250 nm spatial resolution limit of light in order to reveal biological details that have been obscured by diffraction. A new form of super-resolution microscopy called expansion microscopy (ExM) relies on physically expanding a fixed specimen in a swellable hydrogel polymer and offers a simple, inexpensive approach to achieving ~70 nm resolution with conventional confocal microscopy. A critical and understudied step in this process is the homogenization of the embedded sample by proteolytic enzymes, enabling artifact-free expansion. However, in large and complex samples like Drosophila, enzymatic digestion is time-consuming and sensitive to experimental parameters such as fixation, hydrogel composition, and tissue type. To overcome the limitations of enzymatic digestion, I have explored parameter space for rapid peptide cleavage using air-tight stainless-steel vessels to achieve high temperatures and pressures not typically accessible in the lab. Additionally, a small-molecule digestion agent, dimethoxyiodobenzene, was tested in order to provide site-specific peptide cleavage and enhance tissue homogenization. The modified digestion process was first validated using standard immunofluorescent microtubules in cell culture. Next, Drosophila tissue was treated using thermal digestion to confirm the applicability of this technique in robust, difficult-to-expand tissues. This improved ExM protocol holds the potential to increase sample throughput, reduce expansion-related sample distortions, and extend ExM to be applicable to a wide range of previously incompatible tissues types, enabling pathologists to better analyze and assess diseases in human tissues.

Neonatal mammalian heart tissues possess regenerative capabilities after injuries like myocardial infarctions that are mostly lost in adult mammalian tissues but conserved through adulthood in other vertebrates like zebrafish. Previous studies have shown that regeneration in ventricular cardiomyocytes (CM) occurs through de-differentiation and proliferation, but the underlying mechanisms that cause cardiomyocytes to enter the primed cell-cycle are unknown. Here we show that amino acid and metabolite levels in injured cardiomyocytes result in a primed state for regenerating cells. In chemically ablated zebrafish, it is shown that the amino acid profile activates the mTOR pathway to drive regeneration. Amino acid activation of mTOR is a result of high glutamine and leucine levels post-injury and in early heart regeneration in adult zebrafish, which is lost in adut mammals. Inhibition of the Wnt/β-catenin signaling pathway upstream of mTOR shows down regulation of mTORC1, showing that mTOR is necessary for CM proliferation in regenerating heart tissue. How Wnt signaling gets activated upon injury is unknown, and this study aims to understand the pathways upstream of Wnt signaling for activation. It is known that scarring needs to occur before regeneration occurs in heart tissue. This study also investigates why macrophages are essential for scar formation in ablated heart tissue and its underlying mechanisms. Further, single cell RNA sequencing one-week post injury is used to determine cell fates of the heart tissue. Cardiac cell types like CMs, endocardial and epicardial cells, and bulbus arteriosus (BA) cells were activated post-injury, with epicaridal cells promoting CM regeneration and BA cells activating signaling pathways during heart regeneration. This study demonstrates the signaling and metabolic pathways that activate cardiomyocyte regeneration in zebrafish hearts.

Proteins are essential to life, but many structural methods fail to capture the dynamic nature of proteins. This means researchers are left with an incomplete view of how protein function and structure relate. The Stoll lab uses electron paramagnetic resonance (EPR) to study the dynamic structure of proteins. The project I am working on is investigating the way in which the measurement conditions in EPR affect the determined protein structure. We use maltose binding protein (MBP) as a model system, since its structure has been previously well-characterized. My primary role has been to create new mutants for site-directed spin labeling, in which a radical spin label is attached to the protein. This radical is measured by placing the sample in a magnetic field and results in a probability distribution of distances. Common spin labels used in EPR, however, have many rotatable bonds, andd can cause uncertainty in the extracted distance distribution. Our project is investigating the contribution of spin labels to the EPR experiment. I use polymerase chain reaction to create new mutants of MBP by swapping a native residue with a cysteine residue in the mutants. Each mutant has one or two amino acids replaced with a cysteine. After mutating the DNA, I then transform the mutant DNA into wild type cells to grow the mutant protein and purify the protein. The spin label forms a disulfide bond with the cysteine residue in the mutant, providing an unpaired electron for measurement. By using various commonly used spin labels on many different site pairs in MBP, we hope to develop a model in which the contribution of spin labels to the probability distribution of distances is accounted for. This will ensure more accurate results in determining protein structure and will aid in structural characterization of many proteins.

Methods to differentiate stem cells into cardiomyocytes have been well established. However, a limitation for the successful application of these cells for research and medicine has been their fetal-like phenotype with respect to cell size, contractility, calcium handling, metabolism, and electrophysiology. We sought to increase the maturity of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) through metabolic pathway regulation. We hypothesized that switching the main metabolic substrates from glucose to fatty acids, mimicking the switch from placental to breast milk nutrient consumption that occurs during development, will increase hPSC-CM maturation. RUES2 embryonic stem cells were differentiated into cardiomyocytes and then treated with base media with varying glucose and calcium concentrations and fatty acid supplementation. Using quantitative PCR to measure gene expression, we measured an inverse relationship between glucose levels and markers of cardiomyocyte maturation: increased expression of cardiac troponin I relative to skeletal troponin I isoforms (TNNI3:TNNI1), increased expression of metabolic (CPT1B, PPARGC1A) and electrical maturity markers (KCNJ2, RYR2). These maturation markers were not influenced with fatty acid supplementation but were enhanced upon the addition of thyroid hormone and dexamethasone. We further quantified nucleation and sarcomere spacing to assess structural features of maturation using confocal microscopy. To understand the mechanisms by which media nutrients and signaling molecules cause phenotypic changes we investigated the relationship between maturation and global epigenetic state. Western blot revealed increases in global acetylation levels, measured by histone H3 acetylation, linked to maturation signaling. These findings depict a direct relationship between glucose metabolism and the development of a mature phenotype in hPSC-CMs, mediated by epigenetic mechanisms.

The goal of this project was to determine if mitochondrial biogenesis in zebrafish cone photoreceptors is circadian and/or regulated by light. Photoreceptors use more energy in darkness compared to light, and at night the number of mitochondria in zebrafish cones increases. We hypothesized that mitochondrial biogenesis would correlate with increased energy needs and increased mitochondrial number.  Zebrafish retinas from light/dark and dark-only cohorts were collected over a 24-hour cycle. Retinas were fixed and sectioned at 20 µm for immunohistochemistry. Sections were incubated with primary antibodies against mitochondrial biogenesis genes and cytochrome oxidase and imaged with confocal microscopy. Length measurements were taken from the center of single cone mitochondrial clusters. Retinas were also analyzed by Western blotting to assess cone mitochondrial protein content. 21 genes involved in mitochondrial biogenesis were analyzed via qPCR, in addition to ratios of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). Experiments were conducted in triplicate with three biological replicates and the ΔΔCt method was used to determine relative expression.  Immunohistochemistry revealed that cone mitochondrial clusters elongate 30% following dark onset. Western blot analysis is ongoing. mRNA transcripts for two genes involved with mitochondrial biogenesis, PolG1 and TFAM, increased roughly 6-fold and 3-fold respectively in the hours leading up to dark onset; transcripts for the mitochondrial fusion factor, Mfn2, increased 3-fold just prior to light onset. Cone mitochondrial clusters enlarge at night, and this is preceded by upregulation of mitochondrial biogenesis genes. Lastly, mitochondrial fusion prior to light onset may be followed by a decrease in mtDNA number. These effects occurred independently of light exposure. Overall, these results highlight a possible mechanism in photoreceptors to increase energy production in the dark while following the circadian clock.

This research explores 19th century Middle Eastern and European quarantine practices through the personal diaries of Joseph Mattias Svoboda, written in Iraq from 1862 to 1908. Joseph Mattias Svoboda’s diaries document a detailed account of daily life and information in the Basra and Baghdad regions of modern day Iraq. Joseph himself was from a prominent European family and worked for an English steamship company, traveling across Iraq during a time when cholera was prevalent and occasionally reached epidemic proportions. I used Joseph Svoboda’s notes as a basis to piece together common quarantine practices and procedures utilized in the 19th century by the Ottoman Empire. Using digital transcriptions of the diaries, I used data-mining techniques to extract references to cholera outbreaks, quarantine procedures, and numbers of people afflicted by disease before and after quarantines were put in place. Then, using contemporary European newspapers, medical journals, and historical records, I compared the Ottoman procedures to European quarantine practices. I examined the efficacy of quarantine procedures in both regions depending on the size of each given cholera outbreak. Ultimately, these early forms of public health governance may have influenced global trade patterns. Future projects stemming from this research could look into the influence of European public health practices on Ottoman medical thought, or the comparative role of physicians in the Ottoman Empire and in 19th century Europe. Regardless, hopefully this research encourages others to explore beyond the Eurocentric narrative of developing public health in the 19th and 20th centuries.

Building upon our rat testicular o-culture system developed to improve evaluation of chemicals and identify their potential adverse health effects on humans, we have designed in vitro mouse system to expedite such assessments. Models of in vitro cell cultures are, however, limited in their approach typically using a monoculture of single cell types. Using a 3 dimensional organotypic approach can provide a framework for evaluating systematic interactions between cells with a goal to reduce the need for in vivo assessments. We utilized a novel organotypic, in vitro model of testicular development that mirrors the development shown by in vivo studies. We used isolated testicular tissue harvested from C57BL/6 male mice on post-natal day 9 and digested into a single cell suspension. Testicular co-cultures were maintained for up to 16 days in 24 well plates. Data collections occurred at days in vitro (DIV) 3, 7, and 16. These timepoints represent “windows of potential susceptibility” for testicular development. Plates were stained with antibodies providing markers corresponding to morphological features of specific cell type; six different primary antibodies were used as markers to visualize the change in cell populations; markers for Sertoli cells corresponded to Vimentin, Leydig cells with 3b-HSD, Germ cells to DAZL, SCP3 and c-kit, and proliferation with PCNA. We used an immunofluorescence dual staining technique with Scanning Laser Image Cytometery (iCys) to quantify the proportion of cell types and to determine changes in phenotypic distribution of the cell population. Our observations in vitro demonstrated concordance of changes in Sertoli cells, Leydig cells and Germ cells throughout testicular development with similar trends in vivo. Our observation of similarities in testicular development in our 3 D in vitro experiment and in vivo studies is important and supports the current trends in minimizing the need for in vivo studies for chemical toxicological.

Giardia lamblia, a microscopic flagellated parasite that causes giardiasis, is a highly divergent eukaryote in which conventional Golgi, endosomes, lysosomes, and mitochondria are absent. Similar to other parasites of medical importance, Giardia lamblia has two life cycle stages - proliferative trophozoite form and water-resistant, nonmotile, infectious cyst form. During encystation when Giardia trophozoites transform into infectious cysts, they secrete cyst wall proteins (CWP1-3) that are trafficked and processed in Encystation Specific Vesicles (ESVs). These vesicles are thought to be stage-induced Golgi in Giardia. Previous work in the lab has shown that the signaling activities of G. lamblia’s single Rho family GTPase, GIRac play an important role in regulating this encystation process. The aim is to characterize proteins in Giardia lamblia that potentially interact with GIRac, currently focusing on homologs of known players in membrane trafficking by examining their order of arrival using morphology of the ESVs based on CWP1 staining. Since this is subjective, there is a need for stage-specific molecular markers. In other eukaryotes, Rab GTPases have been established as markers of membrane identity and directionality of trafficking. Only two out of nine Giardia’s Rab GTPases have been localized and reportedly found at ESVs and based on published images, they appear to be recruited at different stages of ESV maturation. By tagging the N-terminus of all 9 Giardia Rab GTPases with fluorescent tags, we can screen them for their localization to ESVs and perform multi-color imaging to determine the order of arrival of these markers. Ultimately, this finding of stage-specific molecular markers could be a powerful tool to further suggests its potential as a novel target for drug development to treat giardiasis.

Bacteria inhabit a world filled with threats, including antagonism from other species throughout different environmental conditions. One mechanism microbes employ as protection from these hazards is the type VI secretion system (T6SS) - a system bacteria utilize to inject toxic proteins into neighboring cells, leading to cell death. The H1-T6SS of Pseudomonas aeruginosa comprises 7 pairs of toxins and cognate immunity proteins (which prevent self-intoxication). I hypothesized that each of these toxins could be maximally effective against specific kinds of competing bacteria, however the function of many effector proteins is unknown. My research focuses on characterizing the protein Tse7, encoded by the gene PA0099, and elucidating its potential role in species-specific antagonism. To begin, I identified its key functional amino acids and the gene encoding its immunity protein. I designed and created mutant strains with several genes adjacent to PA0099 deleted and co-cultured these with wild-type Pseudomonas aeruginosa to identify any mutants with a loss in competitive fitness. This lead to the discovery that the gene PA0100 encodes the cognate immunity protein. To determine key functional residues, I identified potential candidates using conserved motifs in the toxin’s amino acid sequence. By creating mutants of these residues and analyzing their change in competitive fitness compared to wild type, I recognized histidine 230 as the residue vital for Tse7 function. Going forward, I will attempt to determine whether Tse7 improves fitness of P. aeruginosa against any specific families of bacteria, indicating the toxin targets that particular bacteria. Almost one-third of Gram-negative bacteria have T6SSs. These systems largely dictate the ability of bacterial species to compete with one another, dramatically affecting bacterial community structure and the landscape of human infections. Therefore, this deepened understanding of its function could further our knowledge of and ability to manipulate bacterial interactions’ impact on environmental and human health.

Increasing energy demand coupled with over-reliance on fossil fuels and other non-renewable energy sources has created a need for alternative renewable energy sources. The sun is one of the most promising sources and photovoltaic cells are one way to capture solar energy. Halide perovskite thin-films have recently emerged as ideal materials for solar cells due to low fabrication costs, bandgap tunability, high extinction coefficients, and high carrier mobility. Moreover, they have demonstrated rapid gains in power conversion efficiencies from 3.8% to 23.7% in nine years. Halide perovskites have the molecular formula ABX3, where A and B are cations while X is a halide. Past research has shown that methylammonium(MA) lead triiodide, a commonly used perovskite, can be changed into formamidinium(FA) lead triiodide by exchanging the A-site cation in a formamidinium iodide solution. This highlights high ion mobility and interchangeability in perovskites. However, in perovskites with mixed-ion composition, high ion diffusion adversely affects the device performance due to ion segregation, but little is known about inter-diffusion of different ions in perovskites. Here, we investigate the inter-diffusion of A-site and X-site ions in halide perovskite films by creating a lateral heterojunction of the ions. We confirm the creation of the gradient using UV-Vis and steady-state photoluminescence (PL) measurements. We further confirm that there is no change in the film morphology and crystallinity as evidenced by SEM and XRD, respectively. With PL line scans across the lateral gradient, we image the inter-diffusion of the ions as a function of position and time. Using Fick’s Diffusion equations to fit the PL line scans, we determine the ion inter-diffusion coefficient and extract the activation energy using temperature dependent measurements. This study demonstrates a facile quantitative method of probing the ion inter-diffusion in halide perovskites and furthers understanding of mixed-ion perovskite compositions.

Quantum confined nanomaterials have become an important field of study with many applications from color displays to low-energy alternative lighting sources. Discovered in the early 1980s, these semiconducting nanocrystals continue to draw attention; their unique properties differ from their bulk counterpart’s due to a quantum confinement effect rising from their small nanometer-scale size. Indium phosphide (InP), a group III-V semiconductor, is a promising nontoxic, environmentally innocuous material. The morphology of the synthesized InP nanocrystal is commonly a quantum dot quantum-confined in all three dimensions. However, the utility of the dot in biological imaging and display applications is hindered due to reabsorption resulting from overlap in its absorption and emission properties. This dims the light of an indicator and limits efficiency in catalysis. The subject of this investigation—InP quantum rods— offers a solution to this problem, as it is quantum confined in two dimensions and exhibits bulk semiconductor characteristics in the third, separating the absorptive and emissive features and improving the photoluminescent quantum yield. My procedure involves a hot-injection method. Currently, a magic sized cluster hot-injection synthesis in N-methyl pyrrolidone at 150 °C is known to afford InP rods (Dr. Friedfeld, UW Cossairt lab). I explore multiple experiment sets that manipulate the reaction length and temperature of the synthesis, cluster identity and concentration, and the identity of the aprotic polar solvent in which the reaction takes place. I characterize all material via UV-vis spectroscopy and imaged on a transmission electron microscope. The aim of the investigation is to learn why these rods form and what reaction conditions favor their formation. In understanding how to efficiently control the distribution of InP rods v. dots using chemical tools, the usefulness of nanocrystals in nanobiotechnology and clean energy science can be improved.

Polycystic kidney disease (PKD) affects nearly 600,000 people in the United States and it is caused by mutations in the genes that encode polycystin-1 (PC1) and polycystin-2 (PC2). These modifications are responsible for the growth of fluid-filled renal cysts that can impair kidney function over time, and lead to end stage renal disease. PC1 and PC2 have also been localized to the mitochondria, suggesting that they are responsible for regulating cellular metabolism and energy production, which can lead to mitochondrial dysfunction and increased PKD progression. Our lab uses human pluripotent stem cells (hPSCs) to generate kidney organoids that can be used to study the advancement of PKD, which contain the major proximal structures of the nephron including podocytes, proximal tubules, distal tubules, and endothelial cells. Orgnaoids that lack both PC1 and PC2 will develop cysts that can grow large enough to be seen by eye. Unfortunately, these organoids lack a collecting duct (CD) system, a crucial component of the kidney that expands greatly in PKD. To address this need, we developed a protocol to differentiate hPSCs into ureteric bud (UB) cells, the precursors of CD cells. First, we identified Dolichos biflorus agglutinin (DBA) as a key CD lineage marker in developing kidneys through immunofluorescence analysis on human kidney tissue. Then, we took undifferentiated hPSCs and investigated their ability to express DBA by treating them with a concentration gradient of small molecules known to induce kidney lineage cells. The resulting cells were stained with DBA and GATA 3, an additional, early CD marker, and we found that both markers were expressed in vitro. We will continue modifying this protocol to better study the increased metabolic rates seen in PKD, its effects in CD cells, and the mechanism behind the PC1 and PC2 pathway that leads to cyst formation.

 Antibiotic resistance is a growing threat to public health. This resistance, coupled with a dearth of new antibiotics, makes the study and development of antibiotics of critical importance. Many antibiotics derive from microbial pathways that synthesize complex natural products, and engineering these pathways to produce new antibiotics is an exciting prospect. I aim to use such a strategy to produce variants of the klebsidin lasso peptide, known for its distinct lariat knot structure and its antibiotic activity. Klebsidin is produced by three biosynthetic enzymes that modify and export a ribosomally-synthesized precursor peptide. We have developed an assay that relies on cellular growth to (1) investigate how mutation influences the antibiotic activity and biosynthesis of klebsidin, and to (2) identify variants of klebsidin that overcome a known resistance mutation. When expressed in E. coli, klebsidin inhibits growth of its host cell. I expressed a library of approximately 10,000 klebsidin variants, each within a single cell, and used next-generation DNA sequencing to count the frequency of each variant before and after a growth selection. Functional klebsidin variants should decrease in frequency after selection, whereas null variants should increase in frequency. Using these sequencing data, I generated functional scores for a majority of single amino acid mutations within klebsidin, identifying positions that tolerate mutation and positions for which mutation abolishes bioactivity. To better understand these trends, I am performing mass spectrometry experiments to study the biosynthesis of key variants. In addition, we are interested in how mutagenesis of klebsidin can combat known resistance to lasso peptides. By performing the same growth selection in an E. coli strain resistant to wild type klebsidin, we hope to identify variants that overcome this resistance. Together, these studies will provide a framework for understanding lasso peptide engineering and identifying novel peptide-based antibiotics.

The lysosome is a critical organelle affected by the aging process. Changes to lysosomal physiology during aging and their effects on cellular function are poorly understood. Here, we study the lysosome-like vacuole of the budding yeast during replicative aging. Using a microfluidic device, we trap hundreds of mother cells and visualize them over the course of their replicative lifespans. Using strains with fluorescent protein tags we investigate protein abundance, intracellular physiological conditions, and organelle morphology. We find that during aging, the vacuole becomes progressively less acidic. Using a vma mutant as genetic model of vacuolar pH dysfunction, we find that loss of vacuolar acidity triggers a loss of respiratory capacity, loss of iron-sulfur cluster protein activity, and iron starvation response. We find that iron supplementation rescues both the loss of respiratory capacity and shortened replicative lifespan of vma mutant cells. We see that during aging, a similar iron starvation response occurs, correlated on a single-cell level with the loss of vacuolar acidity. We propose that the loss of iron-sulfur cluster synthesis capacity due to loss of vacuolar acidity is a conserved process underlying multiple aging phenotypes.

Microphysiological systems (MPS), also known as “organs-on-chips,” provide a novel, ex vivo approach for evaluating the risk of drug induced toxicity and the impact of environmental toxins. Evaluating the chronic impact of exposure to environmental toxins in humans is difficult, due to ethical concerns, but the MPS provide an approach to examining the toxicological effects on human cells, without the need for human subjects. Aristolochic acid I (AA-I) is a potent, plant-derived nephrotoxin and carcinogen that has been implicated as the causative agent in both Balkan Endemic Nephropathy and Chinese Herb Nephropathy. The contribution of hepatic metabolism to the bioactivation of AA-I was previously demonstrated by microfluidically linking a hepatocyte containing MPS and a human renal proximal tubule eptileial cell (PTEC) containing MPS. PTECs are kidney cells from the nephron that lie between the glomerulus and the Loop of Henle. We studied the cytotoxic effects of AA-I on PTECs and human hepatocytes in MPS by exposing the MPS to concentrations of AA-I ranging from 0 µM to 10 µM for longer than seven days. AA-I was perfused into the MPS into either a linked hepatocyte-PTEC MPS or an unlinked PTEC only MPS. The chronic, low-dose effect of AA-I resembles Balkan Endemic Nephropathy, where individuals were exposed to low concentrations of Aristolochic acid from tainted wheat. Both PTEC only and linked hepatocyte-PTEC MPS provide experimental methods for further investigating how organ-organ interactions affect drug metabolism and xenobiotic toxicity.

Microbial toxins are a molecular weapon involved in pathogenesis, immune evasion, and bacterial competition. A prime example of such microbial toolkits are polymorphic toxin systems, which consist of multi-domain proteins and are widespread in all major bacterial lineages. A polymorphic toxin system called MuF has been newly identified and is the first to be discovered in temperate phages and their bacterial hosts. Though it is highly abundant in the human gut microbiome, its biological role has not been defined. To better understand the toxin system, our team is studying a model species Enterococcus faecalis, a commensal bacterium encoding a two-domain MuF toxin protein on one of its phages, consisting of an N-terminal MuF domain and a C-terminal toxin domain. The toxin domain is predicted to be an ADP-ribosyltransferase (ART), which post-translationally attaches ADP-ribose moieties to its target molecules and can profoundly impair cell processes, leading up to cell death. Using genetic approaches to generate phages with malfunctional ART activity, I have found that the mutations change phage infectivity and the morphology of the plaques formed (clear zones in a cell layer formed due to lysis by phage). Moreover, heterologous expression of the toxin domain in E. faecalis results in cell aggregation. From this, I hypothesize that the MuF toxin is delivered by phages to help infection and ensure phage DNA incorporation into host genomes. To further dissect the mechanism by which the MuF toxin system operates, our team is currently developing a fluorescent protein reporter system to investigate and track the detailed process of phage infection. In addition, by applying X-ray crystallography and electron microscopy, I aim to uncover structural information on the toxin, which may lend insight into the mechanism of MuF toxicity and its larger role in the human microbiome.

Space travel and exploration provides a new perspective of the universe and our place within it. Private companies are taking the leading role in driving the aerospace industry. Many of these companies are looking for new technologies that will lower the cost of spacecraft production and operation. This goal could be achieved through the development of a multi-use modular launch vehicle. This project created a scaled modular rocket utilizing an adjustable ring fin design. The adjustable ring fin allows the user to easily and quickly change the aerodynamics of the rocket to compensate for a variety of payloads. Four test models were created using Callisto rocketry kits. Three of the Callistos were modified with a variety of ring fin diameters, and one was kept as an unmodified control. The live test parameters were based on rail velocity, visual stability, and altitude. After the baseline performance of each ring fin was established, the lengths of the rockets were adjusted to simulate different payloads. This work demonstrates that adjusting the ring fin allows the same base rocket to fly a variety of payloads without needing to construct a new rocket. Successful flights of the test vehicles, with improved performance based on our alterations, provide a new avenue of research into incorporating small modifications to garner a wide array of uses without extensive and costly modification. Further research will involve scaling up to rockets with motors with an impulse up to 10,000 newton seconds, as well as testing other innovative concepts related to modularity and revisions to the ring fin design. The ultimate goal is the design of a single rocket with a changeable ring fin that can be used in a wide variety of applications, saving money on research and development of new launch systems.

Major Depressive Disorder (MDD) has the largest life time prevalence (17%) of mood and anxiety-related disease. The prevalence of MDD is also 1.7 times greater in women than men. Chronic stress and anhedonia are the primary symptoms of depression, and decisions are affected. Our laboratory has shown that corticotropin releasing factor (CRF), the stress hormone, potentiates dopamine release in the core of the nucleus accumbens (NAc), suggesting a relationship between stress and reward processing. While it is known that an individual’s decision making is altered in a depressed state, the precise neurological pathway between stress and reward processing in the brain is unclear. To characterize this “stress-reward” pathway, I used a novel decision-making framework where a cohort of male and female mice performed an operant concurrent-choice task choosing between 0.1M sucrose solution or water. Mice were injected intracranially with α-CRF, a non-selective CRF antagonist, or vehicle in the NAc prior to performing the task. I measured task performance. I hypothesize that mice injected with α-CRF demonstrate less appetitive and reward-seeking behavior compared to the vehicle group, implicating a lower sucrose nosepoke percentage, sucrose choice percentage, and higher sucrose latency during the decision-making task. I also hypothesize that females have a greater reward sensitivity system than males, resulting in an augmented decline in appetitive and reward-seeking behavior compared to males. If these results indicate a significance in further elucidating the “stress-reward pathway” through the decision-making task, this can pave way for potential new treatments targeting this pathway for depression.

When all goes according to plan, newly synthesized proteins within cells fold down an energetic funnel into a functional, minimal energy configuration. If a protein does not fold properly, it is both energetically unfavorable and nonfunctional, often with hydrophobic parts exposed to the aqueous environment. This creates the potential for misfolded proteins to form insoluble aggregates, which can become toxic to cells. These aggregates can crowd the cellular environment and impair cellular functions, which on a single cell scale can lead to cell death and on a larger organism scale, cause diseases like Alzheimer’s, Parkinson’s, and Huntington’s. To deal with this problem, cells have evolved protein quality control (PQC) systems that comprise two classes of action: chaperones that help proteins fold properly and ubiquitin-protein ligases that tag misfolded proteins with ubiquitin for destruction by the proteasome. Previous studies concluded that chaperones are required protein degradation. In our study, we find that Hsp70 chaperone dependence for protein degradation is variable along a spectrum of independent to dependent. My work specifically examined the function of yeast ubiquitin-protein ligase San1 by comparing degradation of various substrates between strains with or without San1 function, and with or without chaperone activity. By performing degradation whereby protein synthesis was halted and the stability of the synthesized pool of substrate was monitored by Western analyses, we were able to see the degree of substrate degradation by each strain over time. Degradation through San1 has been shown to require chaperones, but San1 also is known to recognize substrates independently without chaperones. From our work, San1 recognizes patches of hydrophobicity on misfolded proteins; a feature that is also recognized by chaperones. By studying the interactions of the folding and degradation enzymes, we are gaining a new understanding of how PQC pathways collaborate and coordinate to achieve optimal protection for the cell.
 

Auxin is a plant hormone that promotes root branching which allows improved anchoring in the soil and nutrient acquisition. At the molecular level, auxin promotes the activity of auxin responsive transcription factors called ARFs. I hypothesize that AGL42, a MADS box transcription factor works with ARFs during lateral root formation. I used CRISPR to remove the entire AGL42 gene from the genome and characterize lateral root phenotypes in the mutant. I characterized that AGL42 is regulated by auxin signaling. This was done by spraying Arabidopsis seedlings with auxin and measure AGL42 expression levels by RT-qPCR. I predict that increasing the levels of auxin will positively impact AGL42 levels. Alternatively, AGL42 may not be an auxin-responsive gene but may itself interact with the ARFs to affect downstream auxin signaling. To test this hypothesis, I performed a co-immunoprecipitation (Co-IP) assay to determine whether the AGL42 and ARFs interact with each other. By better understanding the lateral root specification, specifically the role of AGL42, we can learn how to improve the architecture of different crop plants, increasing their ability to thrive even in harsh environments.

The identification and characterization of natural and artificial protein-protein interactions are fundamental to science and engineering. Although many technologies have been established to expedite research in this area, all can hardly meet the need to analyze vastly diverse protein-interactome networks, dynamics, evolution, and druggability. To develop a high-throughput quantitative interaction profiling technology, multimeric protein interaction sequencing (mPI-seq), we performed a 20×20-plex binding assay in a single aqueous solution using a set of de novo designed heterodimeric pairs. First, we barcoded the proteins with DNAs by ribosome and mRNA display. Then, barcoded proteins were assayed en masse in aqueous solution and immobilized into an ultrathin polyacrylamide gel layer attached to glass surface for in situ sequencing. DNA barcodes were amplified into discrete DNA clusters (polymerase colonies or polonies) and then sequenced using sequencing by synthesis. Finally, the protein-protein interactions were measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Through this project, we aim to establish a robust quantitative method to measure protein binding affinity massively in parallel and to use the large-scale functional data set to guide computational protein design.

Previous research on JNK-mediated stress signals demonstrated that stem cells in the posterior midgut of Drosophila Melanogaster only undergo compensatory proliferation or apoptosis. However, our group discovered that the stem cells can also undergo the process of basal extrusion and dissemination, resulting in the cells being eliminated from the tissue into the hemocoel, the blood containing intertissue body cavity. The JNK signal promotes the cells to exit the epithelium of the gut, move through the muscle layer, and be released to the hemocoel, which resembles the process of metastasis in human cancer. In order to understand the mechanism of this extrusion process, we carried out a RNA Interference (RNAi) screen and sought to find genes that are necessary for the stem cell extrusion in the JNK activators, rasv12 and HepCA, expressed flies. We used the ESG-GAL4, UAS-GFP, TUB-GAL80TS(EGT) genetic system to study the knockdown effect from the RNAi of each gene. After 4 days of inducement, which is the sufficient time for the JNK signal to promote complete extrusion of the intestinal stem cells, the intestines were dissected, fixed, mounted, and examined with a fluorescent microscope for any presence of stem cells. Our previous research from last year focused on the first phase of screening of possible RNAi lines involved in cell extrusion. Our results from this year focuses on our second phase of further identifying characteristics of cell movement in rasv12 flies.

Loss of heterozygosity (LOH) is a form of genomic instability that is common in cancer and can result in loss of function of tumor suppressor genes. LOH can occur as a result of interhomolog recombination (IHR). We are studying the mechanism of IHR using a human cell line engineered to report on the frequency of IHR at the gene encoding the cell surface protein, CD44. In this line, one allele of CD44 carries an inactivating mutation in exon 1 and the other in exon 17, so no surface CD44 protein is produced and the cells are sCD44- by flow cytometry. Our laboratory has shown that double strand breaks (DSBs) targeted by CRISPR/Cas9 to sites between the two mutations will stimulate IHR and generate sCD44+ cells, which are readily quantified by flow cytometry. We are working to identify the factors that regulate IHR. This will enable us to control the frequency of IHR and may help to reduce the frequency of IHR that results in loss of tumor suppressor gene activity in tumors.

Chemically induced dimerization (CID), in which two proteins dimerize only in the presence of a small molecule, has been widely used to control cell signaling, regulatory, and metabolic pathways, and used as logic gates for biological computation in living mammalian cells. However, few naturally occuring CID systems and their derivatives are currently available. Creating a CID system with desired affinity and specificity for any given small molecule remains an unsolved problem for computational design and other protein engineering approaches. To address this challenge, we have used a novel strategy to select CID binders from a vastly diverse combinatorial nanobody library. We have created new CID systems that can sense cholecalciferol and artemisinin. We are validating CID biosensors by a yeast three-hybrid system and built structural models to understand the small molecule-induced dimerization. Our work is a proof-of-concept that can be generalized to create CID systems for many applications.

Youth sports participation can have many benefits, including positive psychosocial development and lifelong healthy activity levels. However, such benefits are often not realized due to attrition from sport. Parents play a key role in shaping youth sport experiences. High pressure sports parenting and disruptive sideline behavior contribute to attrition. Interventions aimed at reducing such parent behaviors exist; however, little is known about their effectiveness. The purpose of this narrative review was to synthesize the extant research on sports parenting interventions. A narrative literature review was conducted between December 2018 and February 2019. Studies were selected if they contained a critically evaluated sports parenting intervention (inclusive, but not limited to education and policies) for parents of children 18 or younger participating in organized sports. Additional articles were obtained via pearling of reference lists. Study quality was appraised using the Downs & Black checklist. Our preliminary search found 13 articles, which included 8 educational interventions and 6 policies. The majority of educational interventions were related to sportsmanship; few included any elements of sports psychology. Many educational interventions were implemented using online videos and non-interactive online modules. Evaluations were of poor methodological quality, consisting primarily of limited-scope qualitative evaluations. No randomized controlled trials have as-yet been published on this topic. High quality research into the effectiveness of sports parenting education and sideline behavior policies is needed to better understand whether existing approaches are effectively changing this important parenting behavior. Future research should seek to use randomized designs and measure behavioral outcomes. Further, studies should move beyond efficacy measures and collect implementation data to understand how policies and educational programs are being used in a range of youth sport settings.

Eighty-four percent of genes associated with human disease have a zebrafish counterpart. This is one reason that zebrafish are staples for developmental and genetic studies. Recently, there has been an interest in studying genetic control of skeletal phenotype, particularly in the craniofacial skeleton. Experiments typically use micro-computed tomography (micro-CT) to capture skeletal phenotype in 3D, and rely on manually annotated anatomical landmarks for statistical shape analysis of individual bone structures. However, manual segmentation has some fundamental problems. Subtle differences in how anatomical landmarks are recorded create differences between data sets that can influence the fidelity of the work. Varying lab practices can make exchanging data between researchers and reproducing findings difficult. Additionally, the significant time it takes to manually annotate an image makes large data sets an obstacle. Here, we propose the use of a fully segmented synthetic template of the zebrafish skeleton, created from common inbred strains, to segment and isolate cranial structures in a nearly automated procedure. Instead of segmenting each image individually, manual segmentation only needs to occur once on the template. Then, large quantities of images may be segmented by matching their skeletal landmarks to those of the template. This would not only standardize these micro-CT studies but also reduce the time burden of image analysis. We demonstrate an application of nearly automated shape analysis in proof-of-concept studies that compare the template-segmented volumes of 200 zebrafish otoliths with the volumes measured with manual annotation. Further proof-of-concept studies will compare measurements of other skeletal structures, specifically cranial bone width and parasphenoid bone length. Future work will include developing a micro-CT scanning protocol for the zebrafish Weberian apparatus and applying the template segmentation method to studying the relationship between swim bladder defects and the Weberian apparatus by examining the skeletal phenotypes of a number of zebrafish mutants.

Understanding how genetic variation shapes phenotypic variation for complex quantitative traits is fundamental to developing more accurate disease prognoses and therapeutic interventions. Genes that are important in early development contribute to adult quantitative traits, such as height, vision, and health. The fruit fly Drosophila melanogaster is a model organism for studying complex traits, such as aging. Drosophila possesses many well-developed genetic tools and shares evolutionarily conserved age-regulating pathways with our species. One such conserved pathway is the mechanistic Target Of Rapamycin (mTOR) nutrient signaling pathway. Rapamycin is a specific allosteric inhibitor of mTOR signaling that extends lifespan in adult Drosophila melanogaster and delays development in larvae. However, the functional explanation for these effects is incomplete and a genetic association between development and lifespan is unknown. We used the Drosophila Genetic Reference Panel (DGRP), a highly inbred fruit fly population representing natural genetic variation, to measure rapamycin-mediated developmental delay. We used these data to carry out a Genome Wide Association Study (GWAS), and combined our data with data from a screen for the effects of rapamycin on lifespan in the DGRP, also carried out in our lab. GWAS analysis will help us to identify genetic variants associated with rapamycin efficacy and to discover novel variants associated with developmental timing. By connecting lifespan and development genetically, we identified shared candidate genes that modify these two very different molecular genetic programs. Accomplishing this is the first step towards identifying early life biomarkers that are predictive of rapamycin success as a longevity intervention in later years. These pharmacogenomic analyses advance a precision medicine approach where interventions are tailored towards genetic background to maximize human health.

Studying zebrafish embryos allows us to understand features of vertebrate embryonic development. Neuro-mesodermal progenitor cells at the very posterior end, or tailbud, of an embryo are bipotential. This is because the presence or absence of Wnt signaling commits them to either neural or mesodermal fate. Directed by environmental cues, mesodermal cells exit the tailbud, migrate anteriorward, and become somites, structural segments from which muscles differentiate. The Kimelman lab has found that Tbx16/Spadetail, a major driver of mesodermal morphogenesis, downregulates Arhgap29 and Arhgap35, members of Rho family GTPase activating proteins. This suggests Arhgap29 and Arhgap35 may be involved in mesodermal cell movement. My work in the lab is focused on finding out what roles these two genes play. I used heat shock promoter hsp70 to overexpress Arhgap29 and Arhgap35 in transgenic fish lines. Previously, our lab showed that sustained Arhgap35 affected somite morphology, and that sustained Arhgap29 also decreased the number of somites. In my experiments, I carried out in-situ hybridization in wild-type, Arhgap29- and Arhgap35-expressing embryos to examine genes regulating specification/differentiation of muscle cells and genes involved in transmembrane cell adhesion. I will present data on cell tracking and cell protrusions collected from Arhgap29- and Arhgap35-expressing embryos. These results will help me compare cell migration between Arhgap-expressing and wild-type embryos. The purpose of these analyses is to understand how these two proteins control cell movement in the embryo. In the future, I will continue to investigate cellular mechanisms underlying vertebrate posterior elongation.

Crops are constantly exposed to pathogens and pests, but being sessile, they cannot physically move away from danger. In order to defend themselves, plants have mechanisms to recognize and respond to threats. One way that plants do this is through the Jasmonic acid pathway, which is specifically activated in response to the wounding of plant tissue. Surprisingly, there are many functionally similar Jasmonate-ZIM domain (JAZ) proteins involved in this response, and the reason for this redundancy is unknown. Studying this pathway in depth presents a challenge when using low-throughput methods. In order to better understand this complex system, we built a workflow, which will be integrated into an automated system using the Aquarium Laboratory Operating System, designed by the Klavins Lab. This will help us track data from high throughput experiments. By developing automated methods to track maintenance of cell cultures, production of protoplasts, and delivery of dCAS9 transcription regulators, we hope to produce sufficient data to better understand the JA-signaling pathway. We anticipate that these experiments will illustrate a relationship between the manipulated activity of JAZ gene regulators and subsequent immune response activity, and have potential implications in the agricultural and food industries, where pests and stress conditions are a significant concern.

Lecture demonstrations have a long history of use in science learning spaces. While extant research in STEM education show that demonstrations can be used to rouse student interest, some studies suggest that demonstrations have little to no effect on improving students’ understanding of the concepts taught in class. In chemistry, demonstrations are commonly used in lectures with the belief that student understanding of chemical concepts improves from observations of chemical phenomena. The purpose of this mixed methods study was to investigate students’ perceptions of lecture demonstrations and their usefulness in preparing for exams. To measure students’ perception of lecture demonstrations, a survey was administered to first year students (n <1500) in nine introductory chemistry classes over two quarters. Initial findings reveal that while students do in fact remember and enjoy lecture demonstrations, they found it difficult to apply demonstration concepts to problems on exams. Results also suggest a strong link between student engagement and exam performance. Overall this study highlights the importance of soliciting student feedback for improving and designing course materials and tools. Various strategies and implications for practice are also addressed.

The plant hormone Jasmonic acid (JA) interacts with JAZ proteins, proteins that play a crucial role in the regulation of signal cascades triggered by jasmonates, and controls the activity of MYCs, a family of genes that code for transcription factors. In the absence of JA, JAZ proteins bind to downstream MYCs and limit their activity. JA is produced in plant cells in response to tissue damage, and is crucial to plant fitness. The genome of model plant Arabidopsis thaliana encodes 13 JAZ and 5 MYC isoforms, but as of now, it is unclear why this apparent redundancy exists. Such redundancy is a common feature of plant genomes. In the case of JA-signaling, it may be that interaction strengths differ between JAZ and MYC isoforms and that this is important for system behavior. In A. thaliana, there are 65 pairwise combinations alone, which provides many opportunities for engineering incredibly fine-tuned and controlled gene expression. Our past work has been to clone and create a library of JAZ and MYC proteins. Our goal now is to create yeast mini-circuits, which are yeast cells that contain plasmids that carry certain selected JAZ and MYC proteins. After constructing these yeast mini-circuits, we will study the JA-response dynamics of these circuits to better understand the role of each of these proteins and study differences in strengths of interaction between the different proteins. We suspect that the absence of certain JAZ and MYC proteins will lead to different phenotypes in defense mechanisms. Additionally, by investigating this relatively familiar pathway through the construction of yeast mini-circuits, we are attempting to develop an efficient technique which can be used to characterize other unexplored plant systems.

Obstructive sleep apnea (OSA) is the condition of repetitive stops of breathing during sleep due to complete or partial occlusion of the upper airway. OSA affects around 34% in men aged 30–70 years and is associated with significant cardiovascular mobility. Studies have indicated that retro-palatal region is the most common site of upper airway collapse leading to OSA. However, few studies have addressed the respiratory dynamics of soft palate, tongue base, and epiglottis during sleep. The purpose of this study is to analyze the respiratory motions of soft palate and its relation to oropharyngeal structures during drug-induced sleep in verified obese/OSA and non-obese/non-OSA minipigs. The hypotheses is that obese/OSA minipigs would have larger variance of soft palate motions and altered motion pattern as compared to non-obese/non-OSA controls, which may contribute to OSA. Four obese and two non-obese minipigs were first revived live sleep monitoring using wireless BioRadio system to verify OSA, and all 4 obese showed different degrees of OSA by apnea/hyponea index (AHI) while 2 non-obese did not. Sleep videofluoroscopy was performed under sedation when subjects were places in the prone and lateral positions. The ImageJ was used to trace the movements of soft palate tip in inhaling and exhaling phases. The static reference was defined to be the intersection of mesial surface and occlusal plane of upper second molar. The purpose of reference is to compare relative location of soft palate tip and its relation to other oropharyngeal structures. Dynamic movement of soft palatal tip presented significantly larger variance in obese/OSA than non-obese minipigs. Going forward, I will further map the dynamic shape changes of the soft palate and to define how spatial relationships between soft palate, tongue base and epiglottis lead to oropharyngeal airway patent or collapse during respiration.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ inflammation and damage, including skin and kidney. Ultraviolet B light (UVBL) is the only environmental factor known to precipitate both skin and kidney disease. How UVBL-triggered sterile inflammation in the skin influences kidney injury remains a pressing question for SLE patients, about 70% of whom suffer from sensitivity to UVBL. We previously demonstrated that neutrophils are the first immune cells to infiltrate the inflamed skin tissue after exposure to UVBL. To investigate the neutrophils’ role in UVBL-induced kidney injury, C57BL/6J mice were irradiated with a single dose of UVBL (500mJ/cm2). Cells in the bone marrow (BM), skin, blood, and kidney were characterized using flow cytometry. Gene expression of inflammatory mediators and adhesion molecules was evaluated using qPCR. Following acute exposure to UVBL, we observed a 10-fold increase in skin neutrophils, associated with a decline in neutrophils from the BM and a 5-fold increase in circulating neutrophils relative to baseline (no UVL). Relevant to SLE, neutrophils increased up to 10-fold in the kidney after skin UVBL injury (vs. no increase in monocytes/macrophages). Local skin response was characterized by rapid induction in inflammatory cytokines (IL1b, TNFa, IL6, IL33) and neutrophil chemoattractants (G-CSF, CXCL1, LIX) (day 1-2) that returned to baseline by day 6 after UV. Neutrophil infiltration into the kidney was accompanied by endothelial activation and inflammation: increased VCAM1, E-Selectin, IL1b, Ngal, and s1008/9 gene expression, markers of kidney injury in SLE, as well as by transient proteinuria. Neutrophils recruited to the kidney demonstrated two phenotypes: early activated CXCR2hi (day 1-2) and late aged CXCR4hi (day 2-6), which followed expression of CXCR4 ligand CXCL12, another marker of kidney injury. Together, our findings propose a novel neutrophil-dominated skin-kidney axis of pathogenesis and provide a model for UVBL-triggered disease UW flares in SLE.

Fatty Acid Binding Proteins (FABPs) are intracellular proteins that facilitate the transport of fatty acids and other lipophilic substances into the cell for various biological functions. For example, FABP5 is important for binding endocannabinoid anandamide (AEA) which helps regulate cognition, learning, and memory. Compounds such as tetrahydrocannabinol (THC), the psychoactive component of cannabis, competitively inhibit FABP5 and prevent catabolism of AEA in the cytosol by fatty acid amide hydrolase (FAAH). Because many drugs (like ibuprofen and progesterone) are also lipophilic and poorly water-soluble, we predict that FABP5 has a role in their metabolism. The goal of this investigation is to better understand how FABP binding may affect drug metabolism by determining the binding affinities of different drugs to FABP5 using a fluorescence displacement assay. We first validated an assay for screening drugs and determining drug-FABP5 binding. Both ANS and NBD-Stearate, molecular probes that generate a fluorescence signal upon binding, were evaluated. FABP5 was titrated with increasing concentrations of each probe and the resulting changes in fluorescence plotted a hyperbolic curve used to calculate Kd (dissociation constant). In these trials, NBD-Stearate was found to have high background fluorescence and low sensitivity to changes in fluorescence upon alternative ligand binding, so ANS was selected for further method development. We found that factors such as mixing and time to equilibrate affected the Kd, possibly from non-specific binding. The binding affinities for ANS were 1.177 ± 1.005 μM (Morrison equation fit average), and 4.744 ± 1.387 μM (One Site Specific average). The preliminary data showed that fluorescence decreased by 48.28% with the addition of arachidonic acid (positive control) which confirmed ANS as a suitable assay. By adding increasing concentrations of various drugs to competitively inhibit the binding of ANS, a decrease in fluorescence will be measured and used to calculate Ki (inhibitor binding affinity).

The Hille Zebrafish Lab studies embryonic development and is particularly interested in cell motility and adhesion. We use zebrafish as a model organism due to its small size and rapid development, as well as for having clear embryos that allow researchers to observe somites form. One way that cells can crawl is through extending filopodia with actin microfilaments. The cell is anchored to other cells with adherens junctions and can push off against these anchor points, then form new adherens junctions with cadherin and other proteins. This method of walking is used for cells to move between other cells and is highly visible in premesoderm cell walking and somite formation. Cells need to regulate both elongation and adhesion, and a protein called p120 catenin activates both cadherins (which are needed for adhesions junctions) and the proteins RAC1 and CDC42 that stimulate motility through actin microfilament elongation and filopodia formation. Our hypothesis is that p120 catenins with phosphorylated tyrosines bind to Cadherins. In our mutant p120 RNA sequences, we test the activity of phosphorylatable residues by mimicking post-translational modifications with residues that can not be phosphorylated. For example, aspartic acid can mimic phosphorylated serine. Our lab has made and injected mutated mRNA sequences for p120 catenin to test if the mutant protein is able to rescue morpholino knocked-down embryos. We predict that if the tested amino acid is necessary for binding to a motility protein, then we will see rescue in phosphorylation mimics and no rescue in dephosphorylation mimics. My project was to make a p120 mutant that is missing most of the predicted regulatory amino acids. The truncated p120 sequence is called 4A and is a splice variant of wild type p120 that starts at the fourth methionine, omitting the regulatory region containing many phosphorylatabe sites.

Utilizing the Type III Secretion System (T3SS), Shigella spp. uses a cascade of proteins to manipulate, penetrate, and colonize host eukaryotic cells. Inducing epithelial necrosis, Shigella spp. infection is responsible for moderate to severe diarrhea in millions of children and immunocompromised individuals — the majority from low and middle income countries. Previous translational research for Shigella spp. has been limited due to a lack of robust in vitro and in vivo models to screen therapeutic efficacy. Understanding infectivity of Shigella spp. heavily relies on imprecise estimations of intracellular Shigella spp., which ultimately impacts vaccine and antibiotic efforts. This project aims to address this problem by developing a novel red intracellular reporter to quantify successful invasion of Shigella flexneri. By using a series of polymerase chain reaction (PCR) and Gibson assemblies, we have constructed a plasmid with a RFP reporter to be expressed during successful invasion of Shigella spp. The IpaH9.8 MxiE promoter, which has been shown to be expressed upon cell entry, is integrated within the pUltra RFP plasmid and electroporated into a streptomycin-resistant S. flexneri strain. We will test the recombinant strain in vitro by activating ipaH9.8 in broth culture and inoculating a mammalian cell line (HCT-8). RFP expression will be monitored with a BioTek FLX-800 plate reader and visually imaged using a FX-EVOS microscope. The expected outcomes of this project will provide an accurate and efficient method of quantifying invasive S. flexneri. in vitro and in vivo, as well as quantifying pharmacodynamics and pharmacokinetics of new treatment therapies. The implications of this project are crucial to the advancement of shigellosis research and in furthering the efforts of the international community to abate the steadily increasing cases of drug resistant shigellosis cases observed worldwide.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is well known for its role in sensing numerous “noxious” stimuli. It responds to noxious heat, acid, capsaicin (the active compound that makes chili peppers “spicy”), and inflammatory signals, which can be perceived by the brain to be painful. When the TRPV1 channel senses a signal such as capsaicin, it goes from a closed, non-conducting state, to an open state that allows the passage of ions into the cell, generating an electrical signal. Since TRPV1 is activated by a variety of different stimuli one of our aims is to determine if the structural mechanisms that open the channel is the same or different for each stimulus, which could have important clinical implications. For example, failed clinical trials of some TRPV1 antagonists resulted in elevated body temperatures in patients, indicating that the body’s ability to regulate body temperature is impaired when TRPV1 is completely inhibited. Hence, if TRPV1 can be inhibited in most cases, but still respond to temperature, it will be a better target. We aim to combine cysteine-scanning mutagenesis with spectroscopic techniques to map the structural changes of TRPV1 activation in response to different stimuli. Since introduction of mutations can result in impaired channel function, each mutant needs to be functionally verified. My research will be to generate a broad library of single-cysteine TRPV1 mutants and verify their function using calcium imaging. Mutants that preserve TRPV1 function can then be used for further spectroscopic studies.

Social determinants of health (SDOH) are non-medical factors that contribute to a person’s health and well-being. The Centers for Medicare/Medicaid Services recently released a standardized 26-question survey designed to capture SDOH in the following 13 domains: living situation, food insecurity, transportation, utility help, personal safety, financial strain, employment, family and community support, education, physical activity, substance use, mental health, and disabilities. However, there is limited study using this survey tool in a safety-net institution. Harborview Medical Center (HMC) is the public hospital serving King County, WA and provides care to a wide variety of patients. This project was a prospective, observational study which aimed to investigate implementation of this survey tool and to measure relevant SDOH and other medical factors in this setting. My role included administering the survey to patients admitted to the HMC Hospital Medicine service from October to December, 2018. I collected data related to the duration of the survey and any challenges patients encountered responding to the survey, as well as review of patient charts for demographic information, medical system utilization, and medical history. In total, only 24 of 90 patients (27%) completed the survey. Major challenges included patient lack of interest, language barrier, ability to consent, and competing time demands. These results have implications for the generalizability of this tool and whether it comprehensively identifies SDOH needs.

Skin plays an important function as a highly innervated sensory organ. This sensory function is critical to organism survival, but the skin, and its axons, are easily damaged and need to be repaired to maintain homeostasis. Zebrafish are an excellent model to study tissue repair because they regenerate tissue efficiently and share conserved skin architecture with other vertebrates. A cell type shown in previous lab experiments to contribute to skin repair is a macrophage like cell, the Langerhans Cell. I hypothesize that if Langerhans Cells are not present within zebrafish epidermis then tissue repair will be incomplete or delayed. I investigated these cells using two different methods. First, I analyzed mutations that cause a loss of function in genes required for Langerhans Cell development. Second, I used the transgenic ablation technique to inducibly kill Langerhans Cells in the skin by addition of the antibiotic metronidazole. I imaged fluorescent Langerhans Cells in the zebrafish epidermis in both of these genetic scenarios to quantify the reduction in Langerhans Cells. Following the completion of these experiments, we expect to see that a lack of Langerhans Cells will induce slower or halted recovery from damage. In the loss of function method, I expect that various mutants will not have as many Langerhans Cells and that following induced ablation, Langerhans Cells will die out when the drug is introduced and slowly recover following metronidazole removal. The key implication of this is finding more about our own biology that can lead to faster healing. In theory, if Langerhans Cells have a major effect on this process then knowing more about our own skin can help us translate this understanding to our own healing process.

Research has shown that wildfire smoke exposure is associated with various respiratory health effects. To help protect the lungs, respirator masks that are labeled as N95 can be worn to filter out fine particulate matter produced from wildfire smoke. Previous studies show that video-based education is effective in increasing knowledge and intervention practices among viewers, and it was hypothesized that an N95 mask instructional video could help educate the lay-public. A video-content needs assessment was conducted using semi-structured key informant interviews. Eight experts knowledgeable in air quality, message communication, N95 respirators, and respiratory and cardiovascular health were interviewed for important content needs. Response themes were identified and used to inform the content and style of the video entitled, Smoke from Fires: N-95 Respirator Masks. A Knowledge, Attitude, and Practice (KAP) survey, using a 5-point likert scale, was created to assess the video’s knowledge transference, and effect on attitudes and commitment to practice use. The KAP survey was given to undergraduate students before and after viewing the video. The results from our key informant interviews included instruction on proper use, mask limitations, incorrect alternatives, and appropriate use of language, images, and tone. The KAP survey results indicate the video enhanced the participants’ knowledge of N95 masks with regards to proper fit, incorrect alternatives, and limitations. Additionally, attitudes of increased ease of use and mask effectiveness were demonstrated. Since this study investigated a video-based intervention, possible future research includes testing the effectiveness of different education methods on N95 mask fit, in addition to knowledge, attitudes, and commitment to practice.

For many globular proteins, the sequence and native structure are known. However, less is understood about how a string of amino acids folds into a functional protein. Experimental study of folding presents challenges due to the transience and variability of folding/unfolding transition states and intermediates. Alternatively, computational study of unfolding can provide significant insight into folding. Here, molecular dynamics simulations have been used to study the unfolding pathways of the SH3 domain structural family and to investigate the factors that determine the path and outcome. To separate folding determinants from amino acid sequence, 17 SH3 proteins were chosen with an average sequence identity of only 27%. Six unfolding simulations were performed for each protein, and the unfolding transition state ensemble was identified by locating the large, rapid conformational changes that signal the start of unfolding. Contact analysis was used to characterize the structure of the transition states ensembles. Two general pathways at the transition state were identified, distinguished based on the specific β-sheet structure lost at the transition state. In the first, more populated pathway contacts in the β-sheet containing the N- and C- terminal β-strands were lost while the second pathway was defined by structure loss in the other β-sheet. Though many of the investigated proteins went through both pathways in different simulations, most showed a clear bias towards one pathway. This work demonstrates that similar protein structures can fold through different pathways. The bias of many SH3 proteins towards one folding pathway also suggests the presence of some elements of primary structure that direct folding. Further investigation of the SH3 domain may yield ‘rules’ that determine the structure and folding pathway of the domain, and these rules may inform the study of other, similar proteins.

Metal-organic frameworks (MOFs) represent an important new class of adsorbent materials, catalysts, and catalytic supports. As such, it is important to fundamentally understand its adsorption capacity and selectivity of simple gases. NU-1000 is a prototypic zirconium-based MOF which has shown to be thermally stable up to 250 C and has a number of interesting catalytic and adsorbent properties. It is composed of zirconium oxide nodes connected by pyrene linkers with COO- end groups. We determined the isosteric heats of adsorption (Qst) versus coverage of four gases (N2, CO, Ar, and CH4) on NU-1000 by measuring volume-pressure equilibrium isotherms at very low coverages (under 0.1 monolayer) and above 90K. We then compared our experimental measurements to density functional theory (DFT) calculations of adsorption enthalpies at 77 K for the zero-coverage adsorption of the same gases at seven different types of sites of the MOF lattice. These comparisons showed remarkable agreement between the measured and theoretical isosteric heats in trend as well as reasonable agreement in magnitude, indicating that the sites predicted by DFT calculations are populated sequentially in order of decreasing absolute enthalpyt. This study further increased our understanding of adsorption on this prototype MOF at very low coverages and reaffirmed the accuracy of theoretical calculations.

Hydra vulgaris are some of the simplest animals with neurons and have only two thin, near transparent layers of tissue: myo-endodermal and myo-ectodermal layers. Each cell in the animal can be examined simultaneously due to their small size, simple body pattern, and stereotypical (regular and defined) behaviors. This makes Hydra great animals for examining simple signal transmission pathways from which the complex pathways in vertebrates derive. Cells in the ectoderm and endoderm use calcium signaling to coordinate contractions and cause the animal to move, but how this mechanism of cell-to-cell calcium signaling functions is not well understood. Invertebrate gap junctions, intercellular proteins that cells use to send signals to adjacent cells, are coded from the innexin gene family. It has been found that the genome of Hydra magnipapillata has fourteen predicted innexin genes. Recent data suggests some of these innexins are expressed in the ectoderm, specifically innexins 1,4,5, and 13. I hypothesize these proteins are necessary for the animal to perform coordinated contractions. To determine the role of these proteins, I used shRNA techniques to knockdown innexin expression. After examining wild type Hydra, I examined an existing line of transgenic Hydra which express GCaMP in ectodermal cells to identify when cells use calcium to signal other cells. In these animals, signals can be viewed as a wave of fluorescence passing across the ectoderm. I knocked down the genes by electroporating shRNA molecules into adult animals who express GCaMP, and will image these animals’ behavior. There should be quantifiable differences in the fluorescent waves, as I postulate that some cells will be excluded from these waves if an innexin is knocked down, and analyzing these differences should clarify the role of innexins in gap junction signaling.

Endothelial cells within the pancreatic islet produce key factors for survival and functionality of insulin-secreting β cells. Laminin is one such factor, thought to be responsible for enhancing insulin release. Laminin is a heterotrimeric protein composed of three isoforms: α-chain, β-chain, and γ-chain. Islet endothelial cells from diabetic (db/db) mice show a significant decrease in the expression of laminin α4, β1, and γ1, and show a reduced ability to secrete insulin. To test the correlation between these in vivo observations, we used an immortalized islet endothelial cell-line (MS-1 cells), to decrease genes of interest; and generate conditioned media (CM) in which to culture isolated islets to test their insulin secretion. Islets cultured in CM from MS-1 cells with decreased laminin α4 expression did not have blunted insulin release. However, we found that that decreased laminin α4 increased expression of laminin α1, perhaps as a compensatory mechanism. We inhibited expression of laminin α4 and α1 in MS-1 cells, and cultured islets exposed to this CM also did not have decreased insulin secretion. Therefore, we hypothesize that reducing all three isoforms of laminin in MS-1 cells is necessary to cause a decrease in insulin secretion in islets exposed to this CM. To determine if the knockdown of laminin-411 is sufficient to impair islet insulin release, CM will be collected from MS-1 cells exposed to oligonucleotides designed to inhibit the expression of laminin-411 or a nonspecific control oligonucleotide for 24hrs. After the media is collected, glucose concentrations of the two types of CM are matched. Isolated islets are exposed to these CMs for 48hrs and insulin secretion will be determined for basal and glucose stimulated conditions. We anticipate that CM generated by cells with reduced laminin 411 to decrease insulin secretion in isolated islets, replicating the results observed from db/db mice.

Autism Spectrum Disorder (ASD) is neurodevelopmental disorder that impairs an individual’s social, communicational, and behavioral skills. Boys are 4.5 times more likely to be diagnosed with ASD compared to girls. A potential cause for differences in diagnostic rates may be that girls tend to be more socially focused, allowing them to better camouflage their autism symptoms. Puberty is a period in an adolescent’s life where teens experience significant changes in social experiences and expectations. While puberty often increases social motivation, it’s unclear if males and females improve in the same ways or at the same rates. The goal of this study is to investigate sex differences in the relation between autism behaviors and stages of pubertal development. Participants included 140 children (70 males, 70 females) between the ages of 8 and 17 with a diagnosis of autism. Participants or their parents completed the Pubertal Developmental Scale, which provides a score of the child’s physical progression through puberty. Parents of the children also completed the Social Responsiveness Scale (SRS-2) which scores their child’s social and autism-related impairments. Our analyses (1) Compare SRS scores between youth with ASD based on their pubertal groups (pre/early/mid/late/post). We expect an increase in social awareness, motivation, and cognition for children going through puberty or in post puberty relative to those who have not yet started puberty. (2) Examine sex differences in SRS subdomain scores within each pubertal group and in the pattern of differences between pubertal groups. We expect to find no sex differences before puberty, but lower SRS scores in girls during and after puberty. Investigating the relationship between puberty and social behaviors could help us develop better supports for youth with ASD that incorporate biological and physical changes that come with development and consider how that might impact the youth’s social environment.

 Alcohol use disorder (AUD) is associated with both acute and chronic alcohol-related harm. Marginalized populations, such as those experiencing homelessness, are both less inclined to successfully achieve abstinence and at higher risk of experiencing alcohol-related harm. Identifying strategies for safer use are thus particularly important for this population. To this end, we analyzed interview data collected from 86 adults experiencing homelessness and AUD who received harm reduction treatment (HaRT-A) during the parent randomized controlled trial. Participants were administered the demographic and alcohol measures at baseline (week 0) and were provided with HaRT-A, at which point interventionists introduced a list of safer-drinking strategies. Participants’ endorsement of safer-drinking strategies was assessed at HaRT-A appointments at weeks 0, 1, 2, and 6. For the current analysis, we conducted a mixed methods approach to characterize and evaluate endorsement and implementation of safer-drinking strategies at week 0 and week 6. Findings indicated use of strategies to buffer the effects of alcohol (e.g., eating while drinking), change the manner in which one drinks (e.g., counting drinks), and reduce alcohol use. Quantitative analysis indicated all participants were able to successfully endorse and implement safer-drinking strategies over the course of the study. These findings support that people experiencing homelessness and AUD adopt strategies that minimize alcohol-related harm, including ways that change the amount and manner of drinking and buffering the effects of alcohol. This extends the findings from our parent study among chronically homeless individuals to people experiencing transitional or episodic homelessness.

Harmful radiation, such as UV photons from the sun, can damage DNA in a manner that results in cation-radical formation which leads to nucleobase loss and strand breakage. In the Frantisek Tureček research group, DNA cation-radicals were generated in the gas phase, isolated and subjected to incremental wavelengths between 210-700 nm with intent to observe their absorption action spectra that can be used to assign structures and intrinsic properties. The difficulty with these experiments is the ability to effectively communicate the experimental set-up of mass spectrometers, tunable lasers, and various other pieces of equipment in a supporting manner that delivers concise information to be understood regardless of prior knowledge in this specific field. I had the unique opportunity to take two-dimensional, over-simplified schematics and transform them into three-dimensional, professional and photorealistic images of our experimental arrangements using Siemens NX CAD modeling software.

Thermoregulation, the maintenance of core body temperature in a constantly changing enviroment, is a critical aspect of homeostasis. Despite its importance, the neural mechanism by which thermoregulatory processes occur is not very well understood at the circuit level. Afferent skin temperature information travels through the spinal cord to the parabrachial nucleus (PBN), where it passes on to the preoptic area of the hypothalamus (POA). A subset of prodynorphin (Pdyn)-expressing neurons in the PBN (PdynPBN neurons) are activated when mice are exposed to warm environments, and 80% of these neurons project to the POA. The exact role of PdynPBN neurons has not been characterized, however, and their full projection profile is not established. Using genetic and viral techniques, we inserted a Cre-dependent designer receptor exclusively activated by designer drugs (DREADD) into mouse PdynPBN neurons and labeled their synaptic projections with GFP-bound synaptophysin, an abundant synaptic vesicle protein used for neurotransmitter trafficking. The use of Cre-dependent DREADD and synaptophysin-GFP allowed us to specifically label and activate PdynPBN neurons. We found that activation of these cells increases tail-skin temperature with a concurrent drop in core-body temperature. These data suggest that PdynPBN neurons may convey environmental temperature information that is sufficient to activate heat-defense responses. Establishing the genetic identity of neurons in a circuit that helps to maintain constant core body temperature will allow for the elucidation of downstream nodes in this circuit.

Enteric diseases, or diseases of the Gastrointestinal (GI) tract, remain one of the most prevalent killers of children in sub-Saharan Africa. The most practical way to prevent such diseases is through vaccination, but antigens for enteric diseases need to be delivered directly to the GI tract to be most efficient, making vaccination difficult. Recent studies by the von Adrian group at Harvard University have found that both T and B cells are reprogrammed to home to the GI tract when they encounter retinoic acid, a metabolite of vitamin A. The King Lab at the University of Washington is working to develop a novel vaccine candidate using recently developed self-assembling protein nanoparticles, that can simultaneously package all-trans retinoic acid (ATRA) and multivalently display enteric antigens. Previous work has suggested that two cystine mutations to Cellular Retinoic Acid Binding Protein I (CRABP-I) create a disulfide bond as a result of the conformational change that CRABP-I undergoes when it binds ATRA. This disulfide bond would essentially lock ATRA into CRABP-I, reducing its dissociation constant in vivo and maintaining the gut-homing properties of the nanoparticle post-injection. In order to assess the efficacy of these cysteine mutations, I expressed two versions of CRABP-I, the wildtype protein with no cysteine residues, and a version with no cystine residues except for the two that create the disulfide bond. After establishing that these new CRABP-I mutants folded into the approximate shape of wildtype CRABP-I via circular dichroism, I designed and tested new assays that measured free thiol concentrations of each protein after binding ATRA, as well as free ATRA concentration overtime. This data will help us determine whether these two cystine mutations make a significant difference in the ATRA binding quality of CRABP-I, which could improve the immune response generated by our vaccine candidate.

Cellular growth and aging requires constant replication of the genome. The processes of genome replication and segregation must be carefully regulated to prevent DNA damage which can cause cell death or oncogenic transformation. Damage can arise from errors in cellular processes, as well as environmental factors such as UV radiation or chemical agents. In mammals, the protein Retinoblastoma (Rb) prevents cells from beginning the process of DNA replication for cell division if internal conditions are not suitable for replication. This function is disrupted in virtually all cancers, and mutations in Rb have been widely studied as a result. Whi5 is the functional analog to Rb in S. cerevisiae. Whi5 activity is modulated by phosphorylation at 4 different sites, triggering Whi5 export from the nucleus and allowing transcription of DNA replication genes. Previous research suggested Whi5 export initiates irreversible progression through cell division. However, using single-cell observation technology, we show that Whi5 can re-enter the nucleus prior to cellular division, arresting the cell cycle. Cells that undergo Whi5 re-entry events exhibit a longer average replicative life span, counted in number of divisions. Additionally, cells without a functional copy of Whi5 have a reduced replicative lifespan. Our results suggest Whi5 may play a role in arresting the cell cycle in response to DNA damage to prevent the cell from making fatal errors during replication and ensure healthy aging. To investigate the role of Whi5 in response to DNA damage, I am using CRISPR gene editing technology to modify the phosphorylation sites on the Whi5 gene, modulating Whi5 activity. The ability of modified Whi5 strains to recover from UV induced DNA damage and their relative replicative life spans will clarify the role of Whi5 in DNA damage response. Our results may help us better understand the interactions governing Rb dysfunction in cancer cells.

Aging is driven by the time-dependent disruption of cellular processes.  Collectively these changes lower physiological resilience and increase the probabilitity of death.  As the population of elderly humans gets larger, studying pathways that mitigate age-dependent changes has taken on increasing importance.  The round worm Caenorhabditis elegans has proven to be a powerful model organism for studying the basic mechanisms of aging.  Here we have investigated a novel life-extending pathway acting in these animals that is triggered in response to mitochondrial electron transport chain disruption, a phenomemon that occurs naturally in humans with age.  The pathway is comprised of mitogen-activated protein kinase (MAPK) signaling cascade comprised of DLK-1, SEK-3, and PMK-3 and the downstream reporter gene tbb-6.  Our primary objective in this study has been to identify signaling factors triggered distal to PMK-3 activation that mediate life extension.  We have utilized several independant approaches including both targeted and unbiases genetic screens, as well as co-immunoprecipitation.  Among a family of 33 candidate bZIP transcription factors, we have identified six that are required for PMK-3 mediated life extension.  Using an ENU mutagenesis screen we have identified ten genetic mutations that trigger constitutive tbb-6 activation.  Sequencing tests suggest that these lines contain specifc allelic mutations that form on the X chromosome.   Finally we have generated transgenic strains containing tagged versions of PMK-3 and SEK-3.  We have succeeded in immunoprecipitating both proteins from whole worm lysates, in preparation for future mass spectral analyses.  Updated results from our studies will be presented.

Dementia is a neurological condition that impairs memory, cognitive functions, and the performance of daily activities. It is estimated that 5.5 million Americans suffered from dementia in 2018. Currently, the predominant method used to treat persons with dementia is through pharmacological means. However, through reviewing current research, we have found evidence to suggest that cognitive behavior therapies (CBT) may be more beneficial to patients, with the potential to reverse some symptoms of dementia. Our study aims to improve CBT through virtual environments. Virtual Reality (VR) is not merely a visual platform but an immersive environment that stimulates the brain in the same way real environments do. However, in VR we can track certain markers like head and eye movement, metrics that can be valuable in tracking the progression of the disease as well. We have developed a virtual experience that can calm and stimulate seniors while being able to track a person’s ability to perform free recall and spatial recognition through a series of memory games. This experience has the potential to have both therapeutic and diagnostic capabilities, as well as provide insight into how VR can affect memory. Over the past 6 months, our research team has worked with seniors at the Greenwood Senior Center to test different virtual reality platforms such as the Oculus Go and HTC Vive, as well as various virtual environments. Using our experience from the center our team is creating a platform tailored to fit seniors who are affected by dementia. In the near future, our team will be volunteering at a local assisted living facility to work with these seniors. This project will offer understanding of how implementing VR therapy can improve patients’ morale and cognitive capabilities, as well as exploring the use of VR as a diagnostic tool.

The mammalian brain is composed of a wide variety of neurons, all of which signal through structurally and biochemically heterogeneous synapses. Understanding the proteomic makeup of these synapses is crucial for understanding brain function. However, biochemical isolation and characterization has proven difficult due to the morphological complexity of neurons. MS-based proteomics could be a useful tool for studying such diversity, but spatial information is lost because the analysis is performed post-lysis. In this study we proposed to overcome this limitation by using proximity biotin labeling of genetically specified neuronal subpopulations. I thus constructed APEX2-GFP fusion constructs with post-synaptic proteins which could be expressed in vivo using an adeno-associated virus (AAV). Four post-synaptic proteins were chosen: LRRTM1 and LRRTM2 to localize excitatory synapses, and NGLN2 and SLITRK3 to localize inhibitory synapses. A pAAV-CAG (AAV plasmid with CAG promoter) backbone was digested using NotI and AscI, and an APEX2-GFP fragment with NotI and AscI ends was generated via Phusion PCR. Following NotI/AscI double digests, the pAAV-CAG backbone and APEX2-GFP fragment were ligated together in order to make a pAAV-CAG-APEX2-GFP vector. The pAAV-CAG-APEX2-GFP vector was opened with a NotI digest, and gene fragments of the four post-synaptic proteins with Gibson overlaps were then generated via Phusion PCR. Gibson assembly was then used to assemble the pAAV-CAG-APEX2-GFP vector with the gene fragments, and pAAV-CAG-gene-APEX2-GFP vectors were generated for LRRTM1 and LRRTM2. The vectors were then expressed in HEK cells and monitored for GFP expression and biotinylation activity. The pAAV-CAG-APEX2-GFP vector showed bioluminescence and biotinylation activity, but the vectors with gene inserts did not. Although we successfully cloned two of the target constructs, current investigations are still under way in order to optimize them.

Prion diseases occur from the misfolding of the Prion Protein cellular form (PrP^C) under low pH conditions to the infectious scrapie species (PrP^Sc), which can aggregate further into insoluble fibrils. Previous studies have demonstrated that along with other amyloid oligomers, the prion scrapie oligomers cause neurotoxicity by disrupting the membrane, increasing its permeability and affecting calcium ion influx; however, the molecular mechanism for this effect is unknown. Molecular Dynamics simulations were performed to gain insight into the molecular mechanism of PrP^Sc-induced misfolding of PrP^C and oligomer toxicity in a membrane environment. The system was composed of the hexameric bovine PrP^Sc spiral model oligomer and the di-glycosylated human PrP^C attached to a POPC membrane via a glycophosphatidylinositol (GPI) anchor. Prior unpublished membrane simulations of this system have suggested that PrP^Sc induced PrP^C conformational changes as well as significant membrane disruption from oligomer-binding. Here we confirm and build upon these earlier studies demonstrating the reproducibility and robustness of oligomer binding affinity by varying the proximity of the oligomer to the membrane, providing key insight into infectious scrapie propagation and PrP^Sc cellular toxicity.

Have you ever had to motivate the team? What was the result? The English language Wikipedia is notable for its large number of articles. However, 288 other active language editions of Wikipedia have also developed through the intricate interactions of contributing editors. While the editor interactions in the English Wikipedia have been researched extensively, these other language editions remain understudied. To understand how editors currently come to consensus in article building in the French language, a team of researchers has leveraged an existing English framework that depicts how power and policies play a role in mass collaboration. Using this English language framework, we are using qualitative coding methods to build a unique model of the editor interactions on the French language Wikipedia. The results of this study will help contribute to a deeper understanding of how a framework in a different language edition of Wikipedia differs from the English. Our preliminary results show that policy plays a large role in justifying editor decisions for the edits they make on various articles. Furthermore, our research findings have expanded our knowledge of the issues surrounding replication of an English framework in a different language platform.

The influenza virus is known for its rapid evolution, or the ability to fix many mutations over a short period of time. Some of these mutations lead to amino acid substitutions in regions of the virus targeted by the immune system. Such changes are often selected for because they confer a fitness advantage by allowing the virus to "escape" immune response. This pattern of repeated immune escape is a detriment to public health because it necessitates an annual update to the influenza vaccine. Therefore, identifying sites on the influenza virus which are targeted by the immune system could help predict which influenza strain will circulate in the future, inform vaccine design, and help understand basic evolutionary questions. Using molecular phylogenetic techniques, we can identify sites potentially targeted by the immune system by looking for "positive selection", a phenomenon which manifests as a higher than expected rate of evolution. To identify sites evolving faster than expected, we defined a null expectation of the evolutionary rate of influenza in the absence of immune pressure. This null model is defined using empirical measurements from a high-throughput functional assay known as deep mutational scanning. This null model differs from traditional phylogenetic models in that it describes the constraints on influenza on a site-specific basis and, as a result, has been shown to be a more accurate and powerful null model. I have implemented an empirical Bayes approach to identify sites which deviate from the null model by an unexpectedly high evolutionary rate, suggesting positive selection. Preliminary results show that my method outperforms other methods for identifying sites under positive selection. Next, I will apply these methods to the influenza virus surface protein, hemagglutinin, which is a major target of the immune system.

Natural proteins often assemble into various complex geometric structures based on their interactions with each other. These structures can hold and transport "cargo" as well as display antigens, making them extremely useful in vaccine design. The King Lab at the University of Washington uses the way these proteins assemble to develop computational models that help them design novel self-assembling protein cages, or nanoparticles. These nanoparticles are then used to develop vaccines or treatments for diseases. Components of the designed protein cage can be modified and expressed individually before being assembled together into the nanoparticle. I am working on stabilizing one of these protein cages known as T33DN2, so it can be used towards creating a vaccine. T33DN2 is a tetrahedral cage comprised of two trimeric proteins known as T33DN2A and T33DN2B. When expressed individually through E.coli, DN2A is produced in a soluble form while DN2B is produced in a mostly insoluble form. T33DN2 is currently an unstable cage, as only the A component is expressed solubly. Soluble proteins are generally more stable and thus easier to work with than their insoluble counterparts. To increase the solubility of DN2B, I have been making mutations to specific amino acids in the DNA that produces this protein, as well as expressing and purifying this component to determine its stability.

Hydrogen is a non-polluting, sustainable energy alternative to fossil fuels. One method of generating hydrogen is by splitting water with sunlight and a semiconductor photocatalyst. In this study, determining the most viable semiconductor in terms of efficiency and safety for the environment is critical for making hydrogen energy available for commercial usage. When the catalyst absorbs UV light, water molecules in contact with the activated surface are split. Hydrogen is produced more rapidly when the photocatalytic crystals have a high surface area to volume ratio. Therefore, greater efficiency can be attained by reducing the size of the photocatalyst crystals. Nanometer-sized crystals can be synthesized within nanopores, thereby avoiding loss of surface area by agglomeration. Particular attention has been focused on placing the most efficient and cost-effective semiconductor inside a matrix of cellulose fibers and introducing doping, quantum dots, and other engineering strategies to improve the suitability of the photocatalyst. Selecting the most fitting photocatalyst candidate for large scale mass production of hydrogen is imperative for providing a viable source of renewable energy.

 In vivo animal models are an advanced tool for testing complex biological phenomena and functions in humans, yet are difficult to adapt due to high cost, biological and anatomical differences, and ethical concerns. As an alternative to in vivo models, in vitro organotypic models have been created that can accurately and efficiently reproduce biological processes, allowing researchers to probe specific factors without the difficulties associated with in vivo models. We present further development of a novel microfluidic platform, called Stacks, which uses open and suspended microfluidic principles to enable spatial and temporal manipulation of 3D cultures in suspended collagen plugs. We are developing the Stacks platform to accurately reflect morphology exhibited in the air-liquid interface (ALI) of the epithelial layer of the lung and allow analysis of lung pathophysiology, cell signaling, and immune responses of the lung in both monoculture and coculture. Furthermore, using the flat faces of the device that allow for stacking, we have connected a layer of human lung endothelial cells, representing a model vascular system, with a second layer of immune cells and mycobacterium, representing a model infection system, to study soluble factor signaling in granulomatous infections. Through the modification of the Stacks device, we can achieve granuloma formation and utilize soluble factor analyses (e.g., immunoassay) to observe and characterize infection-dependent changes in vasculature resulting from the exchange of chemical signals generated by the infection model. Future development of the platform will build on these newly developed lung models to combine them for studies involving signaling and disease mechanisms between the lung ALI and the underlying tissue layers.