The delivery of cell and drug-based chemotherapeutics to tumors have presented major challenges in effective cancer treatment. Opportunities to improve current small molecule drug delivery systems exist by increasing overall delivery specificity and decreasing harmful off-target effects. Towards this, we have recently developed a chemical framework for creating user-programmable hydrogels that undergo programmed degradation in response to multiple environmental cues following Boolean logic. Exploiting this methodology, user-specified combinations of environmental inputs (e.g., tumor-presented enzymes, reducing conditions) yield material breakdown and accompanying therapeutic release. To translate these materials for chemotherapeutic delivery in vivo, we established strategies to formulate these stimuli-sensitive materials into nanogels that circulate in the bloodstream before acting on the desired target site. We developed techniques to formulate gels on the 50-250 nanometers size scale, one which should enable circulation in the blood and uptake within tumors based on the enhanced permeability and retention effect. Different ultrasonication conditions allowed us to tune nanogel, size and dispersity. This system is scalable, translational, and simple to recreate. In the future, these materials can effectively hone and selectively deploy small molecule chemotherapeutics to tumors in patients.

Despite being individually uncommon, rare diseases are a heterogeneous set of nearly 7,000 diseases estimated to affect 6-8% of the global population, making them collectively common. Rare diseases are infrequent and their determinants are usually unknown which negatively impact clinicians’ ability to make an accurate and timely diagnosis. With the inception of consortia such as the Undiagnosed Diseases Network (UDN) and ontologies such as the Human Phenotype Ontology (HPO) there is now a wealth of information on rare diseases. This rich information has enabled researchers to infer gene-disease relationships and clinicians to assign genetic diagnoses. However, rare diseases that are caused by unique or uncommon gene combinations remain unsolved. In this study, we address this problem by assuming that a disease can be described by a combination of clinical features or phenotypes and, thus, a combination of genes. By developing a pipeline to incorporate known gene-phenotype relationships from publicly available resources such as HPO and using various combinatorial search algorithms, we can prioritize the most likely sets of genes to result in a given set of phenotypes. Integration of our pipeline with individual patient data is expected to further reduce the search space and produce higher quality predictions. We anticipate the results from this study to advance the quality and accuracy of gene-disease relationship predictions for rare and undiagnosed diseases associated with one or more genes. Our pipeline could be used as a tool for the diagnosis and discovery of likely oligogenic, rare and undiagnosed diseases that are normally turned away from consortia like the UDN.

The release of chloride in response to the binding of the neurotransmitter GABA to the ligand-gated ion channel GABAAR mediates neurotransmission that plays critical roles during early development. Disturbed chloride homeostasis in early neuronal circuits results in imbalances in neuronal differentiation, cell growth, and synapse formation that are thought to trigger irreversible paths into neuropathological brain states associated with autism, epilepsy, and schizophrenia. However, the pathogenesis and fundamental physiological mechanisms underlying these disorders are unknown. To dissect these diseases, researchers must understand, in neurodevelopment, the specific role that chloride-mediated neurotransmission plays in triggering the imbalances of inhibitory and excitatory neuronal pathways observed in neuropathological states in adulthood. Zebrafish serve as a powerful model, as the fundamental mechanism of neurotransmission and the microarchitecture of neuronal circuits are preserved among vertebrate. Furthermore, the zebrafish central nervous system is accessible for experimental manipulation from early development. I aim to precisely control chloride-mediated neurotransmission during early development through the creation of versatile transgenic zebrafish lines in which GABA-producing (GABAergic) and glutamate-producing (glutamatergic) neuronal subtypes express the light-activated chloride channel iC++. We are currently designing DNA constructs encoding iC++ and UAS, a promoter element that binds to the yeast transcriptional factor Gal4, to be injected into embryonic wild-type zebrafish. Upon successful integration of these transgenes, Gal4 driver lines that express the transcription factor in specific neuronal subtypes such as GABAergic (Gal4:gad1b) and glutamatergic (Gal4:vglut2a) cells will be crossed with our UAS response line to achieve our desired expression. Ultimately, I will non-invasively activate iC++ in live animals through light stimulations to drive chloride signals with millisecond precision at any time throughout the embryonic and larval stages. This approach will serve as a high-throughput system, allowing us to collectively study a large number of conditions that trigger permanent neuropathological impairments.

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.

Blood transfusion is a cornerstone of modern medicine, with a transfusion performed every 2 seconds in the United States. It is critical to accurately determine both patient and donor blood type prior to transfusion, as mixing non-complementary blood types can trigger life threatening reactions. While the ABO antigen system was first described over a century ago by Nobel Laureate Karl Landsteiner, to this day safe transfusion remains burdened by the nuance of blood type. Many of the current blood typing tests over simplify classification and often disregard ABO subgroups, despite widespread recognition of their significance. Moreover, our understanding of the ABO blood types (A, B, AB, and O) is still incomplete, as the full structure and microheterogeneity of these ubiquitous blood group antigens is not yet fully described. This project characterizes the biochemistry of ABO blood types through an interdisciplinary collaboration between UW Bioengineering, Medicinal Chemistry, and Bloodworks Northwest (the regional blood center). Our study employs exhaustive isolation of red blood cell (RBC) membranes from genotyped donors for comprehensive biochemical and biophysical analysis. The RBC membranes are treated with cocktails of enzymes - namely PNGase F, EGCase and Neuraminidase - to cleave glycan structures at specific locations. Reactivity to different antibodies and lectins provides insight into the structure of the glycan antigen. Results have shown that the clinical anti-A antibody binds disproportionally to N-linked associated antigens. These findings inform ongoing mass spectrometric and biosensing work to further elaborate ABO structure and bioactivity, with implications for transfusion and transplant medicine.

Current methods of drug delivery include the use of antibody drug conjugates (ADCs), lipid and polymeric nanoparticles, and top-down modified viruses. While successful in certain applications, these delivery platforms involve laborious production methods and have limited engineering opportunities. Synthetically designed proteins are viable drug delivery candidates that can be precisely modified to overcome the engineering challenges associated with other delivery platforms. Specifically, synthetic nucleocapsids (synNCs) are computationally designed icosahedral protein assemblies evolved to have virus-like genome packaging. The linkage between the encapsulated mRNA (genotype) and exterior proteins (phenotype) allows for directed evolution to be achieved through library selection. The synNC was evolved to increase the in vivo circulation half-life from less than 5 minutes to over 4.5 hours. The next step is to optimize the targeting ability of synNCs toward specific organs. We performed site-directed mutagenesis to introduce several mutations on the exterior of the synNC, performed two rounds of in vivo library selection, and identified several mutations that favored specific organs. Our current work involves producing five promising synNC mutants, biochemically characterizing the mutants, and examining their biodistribution in mice. The ability to target specific organs will be a successful step towards utilizing nucleocapsids in drug delivery and other biomedical applications.

With reduced operating hours and additional pressures, there is a rising importance in the development of an accurate surgical simulation. In order to improve the effectiveness of surgical simulations, there must be an accurate model of the human body that can be used for practice. Although cadaver and animal models are imperative to the training of future and current surgeons, there is a rising ethical interest paving the way towards alternative solutions. My undergraduate research at the Center for Research in Education and Simulation Technologies (CREST) aims to develop an accurate model for adipose tissue in the greater and lesser omentum. We have developed four prototype recipes for simulated adipose tissue using chemically-modified polydimethylsiloxane (PDMS) composites. In order to evaluate how the prototypes compare to in-vivo and in-situ adipose tissues, we have collected quantitative data through uniaxial tensile, coefficient of friction, and puncture testing in addition to qualitative data collected through conducting surveys of physicians. By utilizing this collection of both quantitative and qualitative data, we have developed an accurate synthetic adipose tissue model.

Self-assembling synthetic nucleocapsids have been at the center of increasing development as a medium for targeted drug delivery. By identifying certain cell markers of tumor cells, researchers at the Baker Lab have been evolving desired characteristics to this protein assembly by attaching different functional domains to specifically target cells. This screening of functional domains is possible because of the nucleocapsid’s ability to encapsulate its mRNA sequence, thereby linking genotype to phenotype. Moreover, this enables us to evolve desirable features in these functional domains through library selection. However, subsequent attachment of functional units to its exterior surface proved to decrease its mRNA packaging efficiency, thus limiting applications like mRNA delivery and evolution of functional units. This project aims to enhance the amount of encapsulated mRNA by mutating certain interior amino acids. Methods include cloning our I53-50-v4 protein assembly gene into Escherichia coli (E.coli) strains and over-expressing the proteins through IPTG induction. After the cells were harvested and lysed, proteins were purified through Immobilized Metal Affinity Chromatography (IMAC). RNA encapsulation levels were measured through Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) to compare between the original and mutated nucleocapsid. Promising results would demonstrate an increase in RNA concentration per nucleocapsid when compared to the original assembly across different constructs. The success of this project would allow for attaching multiple domains for evolution without compromising the nucleocapsid’s capacity to retain its biomolecular cargo. This research would greatly expand the utility of these nucleocapsids for evolving displayed proteins for desirable characteristics as well as enhance their efficacy for drug delivery applications.

Chronic pain is debilitating for millions of people worldwide and our understanding of how the central nervous system processes painful information is limited. If we are to create more effective therapeutics to treat chronic pain, it will be necessary to develop a better understanding of the circuitry and functionality of the somatosensory system. Our lab is interested in the calcitonin receptor-like (CALCRL) receptor, which is expressed throughout the central nervous system and is known to respond to pain-inducing neuropeptides. We aim to profile CALCRL receptor-expressing neurons within the dorsal horn to elucidate their functional roles in nociception and to determine how they process painful information. This was done by 1) utilizing an adeno-associated viral vector (AAV) to preferentially infect and fluorescently tag CALCRL-positive neurons in the dorsal horn, 2) utilizing a retrograde, monosynaptic rabies viral vector to infect neurons that are synaptically connected to CALCRL-positive neurons, and 3) immunohistochemical staining and analysis to detect a variety of neuronal markers of inhibitory and excitatory neurons, laminar markers, and several other known dorsal horn neuron subtypes. Thus far, CALCRL-positive neurons were successfully infected via direct injection using both AAV and rabies vectors into the dorsal horn. In addition, we have also been successful in costaining for multiple neuronal markers. We have begun to profile and classify these neurons according to known neuronal subtypes. Our next goal is to modulate the activity of this specific neuronal population followed by behavioral testing to better understand its functional role in the processing of painful stimulus. If we are able to create a clear profile of these neurons, then we may be able to identify future targets for specific therapeutics.

The average number of coronary artery bypass surgeries performed annually in the United States is roughly half a million. Recently, extensive research has been conducted on the use of acellular tissue engineered vascular grafts which could be implanted into the body to replace the blood vessels that fail due to cardiovascular disease. The Ratner lab is working on creating a novel vascular graft based on a pro-healing porous material which is best suited to guide native blood vessels to heal into the material so that the vascular graft can transform into a living blood vessel. However, a paradox in this design is that right after the graft replaces the blood vessel, before the healing happens, the pores in the graft could give rise to bleeding if not monitored properly. This project addresses that problem by creating a hydrogel that seals the pores, preventing initial bleeding, while degrading at a rate in sync with the rate of healing and is ultimately replaced by vascular tissue. Initially, a series of hydrogels with varying crosslinker levels were made. Subsequentially, an in-vitro degradation assay was used to test each hydrogel in a cell culture medium. This assay showed that the higher the concentration of crosslinker, the slower the hydrogel degrades. In addition, the hydrogel was implanted under the skin of a mouse and the observed degradation of the hydrogel in vivo closely matched the in vitro data but was slightly slower. In the future, the hydrogel with the optimized crosslinking will be applied to a vascular graft for large animal experiments in sheep and pigs and the healing and degradation rates will be observed to measure the effectiveness of the hydrogel as a sealant.

Reactive oxygen species (ROS) such as the superoxide anion (O₂-) and hydrogen peroxide (H₂O₂) are produced by enzymes such as NADPH oxidases (NOX), which have been identified in most cell types and tissues. Additionally, ROS is identified as a second messenger which regulates the activity of G-protein-coupled receptors. For example, ROS decreases the sensitivity of the kappa-opioid receptor (KOR), a signaling pathway in the brain involved in drug addiction. Thus, a fluorescent ROS sensor protein would allow us to monitor KOR signaling in animal models of drug addiction. However, the best performing ROS sensor called HyperRed is not suitable for in vivo studies due to the weak amplitude of fluorescent signal output. Therefore, the overall goal of the project is to increase the magnitude of fluorescent change of HyperRed, ease the detection of low H₂O₂ concentration, and decrease the time of fluorescent response after the addition of H₂O₂. First, using the structure-based engineering approach with the help of Dr Berndt, I construct a library of thousands of semi-randomized HyperRed variants, where mutations are only introduced to the amino-acid linkers between the main domains. Then I test the fluorescent output of all the variants in the library at high throughput utilizing a novel protein engineering platform. This approach allows me to rapidly identify the best performing ROS sensors and optimize their signal amplitude in several rounds of repeated mutagenesis mimicking an accelerated evolutionary process. The expected result is to detect one variant with the signal amplitude having at least a 100% change of fluorescence over baseline values which has been determined to be the minimum for applications in animal models. The improved sensor enables direct measurements of ROS signaling during drug-seeking behaviors and could potentially facilitate the development of novel therapeutic approaches.

Since 1982, with the introduction of insulin as the first recombinant protein therapeutic, peptide and protein drugs have grown to encompass 10% of the pharmaceutical market and are the fastest expanding class of drugs. Advantages of using peptides over small-molecule drugs include high potency, selectivity, and capability to be engineered for a diverse range of targets, most commonly to disrupt or facilitate key protein-protein interactions (PPIs) in the human body for a therapeutic effect. To search for strong binders for a therapeutic target, combinatorial peptide libraries of up to billions of different sequences are synthesized and screened against promising targets. Due to the enormous library size, screening for high affinity binders often requires multiple rounds of enrichment in order to isolate the most potent molecules, a laborious and potentially bottlenecking step in developing protein drugs. Current approaches that have strategies for enrichment, such as phage display and yeast surface display, are limited to screening a library of binders against one target instead of multiple targets (library-on-library). This proposal describes the development of a peptide binder screen utilizing a simple workflow of repeated mating and sporulation of genetically engineered S. cerevisiae, or baker’s yeast. This technology improves the throughput of established screening methods through a library-on-library format that efficiently isolates high-affinity peptide binding interactions.

While there is an abundance of computational tools for protein design, the vast majority of them focus on static structures. Data for the most probable rotamers of an amino acid are often obtained from mining its appearances across crystal structures, which do not take into account the native dynamics or conditions of a protein. Additionally, conformational secondary structure propensities are often derived from the frequency that an amino acid appears within a certain range of dihedral backbone angles in static structures. However, these data do not emulate realistic conditions, because proteins are dynamic and surrounded by solvent; thus, incorporation of dynamic data should improve the design process. Wrangler, our in-house software for peptide design, integrates dynamic data for both rotamers and secondary structure propensities. The data are obtained from molecular dynamics (MD) simulations in explicit water, taking into account interactions with surrounding solvent to better sample the most probable conformations. Several amyloid peptide aggregation inhibitors were designed using Wrangler’s scoring metrics to validate the software. These inhibitors were shown via MD simulations to better retain secondary structure than our current standards. Future work involves synthesizing the peptides in the wet lab to determine whether they experimentally perform better than our current inhibitors.

Approximately 25% of inpatients in a hospital setting receive a catheter during their stay. This statistic corresponds to a diverse group of practitioners inserting catheters which, if done improperly, can lead to urethral trauma and catheter associated urinary tract infections. My undergraduate research at the Center for Research in Education and Simulation Technologies is aiming to address the gap between practitioners in the hospital by developing a high-fidelity urethral catheter simulator, CREST Sim. Modified elastomeric materials were developed to simulate the male genitourinary anatomy and mechanical behaviors. Quantitative studies between the CREST Sim and LifeForm Male Urethral Simulator were conducted using a uniaxial load cell to measure the force of insertion of a lubricated Bardex catheter. In collaboration with the UWMC OR nursing staff, the integration of CREST Sim in the onboarding training of nurses provided feedback in the form of a survey. Similar insertion forces were observed between CREST Sim and LifeForm Male’s simulator. Preliminary results from the survey have reported positive training responses regarding the state of the CREST Sim. Through our collaboration with UW Willed Body, we will characterize the biomechanical properties from genitourinary tissues of deceased males in-situ. This data, along with more robust survey data, will be integrated into the next generation of CREST Sim.

Membrane trafficking in the eukaryotic cell is a highly controlled and significant process that is related to various inherited disorders and cancers. Among numerous regulatory proteins, Uso1 protein – an essential, long, coiled-coil protein – plays a key role in the tethering process, capturing and pulling the vesicle toward the target membrane. Despite many years of work, the tethering mechanism has not been fully understood. Although several tethering models have been proposed, none of them were tested with Uso1 protein. Hence, to test these models, we engineered a mechanically deficient version of Uso1 protein, which lacks the critical tethering region for functioning. After the protein characterization, in cell survival tests and in test tube tests in chemically defined fusion system have been performed. The failure of the trafficking event, which would support our hypothesis, along with other functional test data, would provide a promising demonstration for the tethering mechanism of the long coiled-coil tether and the role of Uso1 protein.

Neurological diseases like stroke, paralysis and spinal cord injuries are some of the leading causes of disability and death across the world. Current medical treatments are not effective, and there is a world-wide effort to investigate new ways to restore function in the central nervous system. A treatment option that is gaining momentum is the use of brain-computer interfaces (BCIs), which has the potential to treat neurological diseases through reading and analyzing signals from the brain and sending electrical impulses to disease-affected areas. A significant obstacle that BCI implementation faces is biocompatibility, the ability for invasive devices to coexist with living tissues. Current BCIs are metal-based interfaces; their conductive properties allow them to efficiently record and send electrical brain signals. However, the human body elicits a foreign body reaction (FBR)- an immune reaction- in response to the “foreign” metal material. As a result, a capsule of scar tissue forms around the site of implantation, which mitigates the efficiency and longevity of BCIs. Hydrogels are an exciting organic material that have the potential to reduce FBRs and create biocompatible BCIs because of their elasticity, pliable material properties, and complex network structures. My project focuses on using poly(hydroxyethyl)methacrylate (pHEMA) as the base material of BCIs due to its ability to be accepted by the brain tissue after implantation. While pHEMA is biocompatible, it can not be used as a BCI in its current form because it is not conductive (not able to send and receive electrical signals in the brain). As a result, the gel is copolymerized with the conductive monomer 3,4-ethylenedioxythiophene (EDOT). This research project balances the conductivity and biocompatibility of the pHEMA-EDOT matrix to produce a new breed of long-lasting, efficient BCIs.

Depression is a neuropsychiatric disorder with a high rate of recurrence and increased mortality. One in six people experience depression at some point in their life, and the disease burden was over $200 billion in 2013. While little is known about the underlying neurobiological processes, the presence of inflammation in the central nervous system has been observed in those suffering from depression. This inflammation, mediated by microglial activation, astrocytosis, and pro-inflammatory cytokine production is involved in the progression of this disease. Past research has focused on the response of microglia in adults, but there are limited studies on the developmental features. Therefore, using a Sprague-Dawley rat model of lipopolysaccharide (LPS)-induced inflammation, this study aimed to understand the progression of the inflammatory response in the development of depressive-like behavior. We determined a suitable dosing scheme to induce inflammation while minimizing mortality to be an intraperitoneal injection (i.p.) of 0.05 mg/kg LPS on postnatal day (P) 7 and 0.1 mg/kg LPS on P9, P11, and P13. Control animals were injected with saline. The presence of inflammatory response was evaluated at P14, P21, and P35. This study showed that rats exposed perinatally to LPS had an increased number of microglia in the hippocampus as well as a hyper-ramified microglial phenotype in comparison to control animals. Similarly, rats treated with LPS showed decreased expression of anti-inflammatory TGF-β and increased expression of pro-inflammatory cytokines TNF-α and IL-6. LPS treated rats also tended to display more anxiety-related behavior including avoiding the open arms of an elevated-plus maze. These results support that LPS exposure in rats can be used as a model to study the effects of inflammation in the developing brain of rats displaying depressive behavior.

Non-coding regions account for approximately 98% of the genome and play a significant role in regulating and maintaining levels of gene expression via transcriptional and translational control mechanisms. Our lab focuses on the functional characterization of non-coding regulatory regions in the human retina and identifying mutations within these elements that contribute to inherited retinal diseases. In pursuit of this long-term goal, my aim is to establish an in vitro model to dissect non-coding regulatory elements in the human retina. Retinal organoid cultures are a promising new system for studying the development of the human eye, however it is currently unknown if organoids recapitulate all of the features of the adult human retina. Based on similarities of cell types and gene expression, we hypothesized that retinal organoids recapitulate some non-coding regulatory features of the normal human retina. To identify both shared and specific non-coding regulatory elements, I profiled the epigenetic landscape of developing human retinal organoids using a genome-wide sequencing approach (ATAC-Seq) to quantify chromatin accessibility - a feature of non-coding regulatory elements. I then used bioinformatics techniques such as genome track assembly, peak calling, and gene ontology to compare my organoid dataset to adult human retina data we previously generated and to a published dataset on the developing human retina. We found distinct sets of non-coding regulatory elements that were shared between retinal organoids and the human retina, as well as elements that were unique to each system. These comparative data have yielded additional insight on which non-coding elements may influence retinal disease phenotypes. Furthermore, these data will potentially give us a clearer understanding of the epigenetic landscape of the developing and adult retina in a context that will drive progress towards regenerating damaged photoreceptor cells in the future.

Myocardial infarction (MI) is the leading cause of death globally. Methods to regenerate cardiac tissue after MI has focused on inducing proliferation in adult cardiomyocytes near infarcted tissue or injecting stem cell-derived cardiomyocytes with proliferative capacity into the infarcted tissue. However, optimal regeneration has not been achieved with these methods, as the mechanism behind adult cardiomyocyte proliferation is not well understood and proliferative stem cell-derived cardiomyocytes are phenotypically and functionally immature. Exploration of the mechanism of cardiomyocyte proliferation is therefore necessary to enable optimal regeneration of cardiac tissue and function and MI. We hypothesize that the sarcomere structure, the basic muscle unit of the cardiomyocyte, is the limiting factor in proliferation of cardiomyocytes. To investigate this hypothesis, we have performed a thorough characterization and comparison of stem cell-derived wild type cardiomyocytes (WTC-CMs) and troponin I double knock out cardiomyocytes (TNNIDKO-CMs) which have an incomplete sarcomere structure due to the lack of troponin I. After confirming TNNIDKO-CMs and WT-CMs only vary in their sarcomere structure, we developed a coculture platform to demonstrate the mechanical weakness of TNNIDKO-CMs sarcomere structure. We then performed proliferation assays utilizing multiple proliferation markers to observe if proliferation was higher in the TNNIDKO-CMs with the incomplete sarcomere structures. Preliminary results have shown that TNNIDKO-CMs are more proliferative than WTC-CMs, thus implicating that sarcomere structure plays a role in controlling cardiomyocyte proliferation. Successful characterization of TNNDKIO-CMs and their increased proliferative capacity will elucidate the sarcomere structure’s role in proliferation as well as develop a more comprehensive understanding of the underlying mechanism behind proliferation to help progress therapies for regeneration of cardiac tissue after MI.

Prostate cancer, the second most common cancer in American men, has a relatively low fatality rate – only around 2% of men will actually die of the disease. Prostate cancer has many treatment options. Each of these treatments comes with varying and oftentimes severe symptoms, so choosing a treatment plan is often based on patient preference. Since treatment is based heavily on patient’s values, it is important that patients are well informed about various treatments and related side-effects. This study aims to reveal how a patient’s socioeconomic background influences their decision making processes, with particular focus on how they receive information related to prostate cancer treatments. We review articles and studies from the past 10 years to determine how they address ethics in prostate cancer decision-making. The three framework topics used are: 1) how healthcare providers influence the decision-making process with their ethics and values, 2) how the socioeconomic and cultural factors of a patient informs their decision-making in prostate cancer treatment, and 3) how patients receive information and whether that information is reliable and accurate. In this literature review we find that patients are presented treatment options differently based on their education levels, race, and socioeconomic background, which has potential ethical implications. As this review progresses, we expect to organize these studies to inform how to best provide information to patients of varying cultural backgrounds. We aim to clarify future areas of study towards arriving at ethical consensus in decision-making in prostate cancer. Since decision-making in prostate cancer is a cost-benefit analysis, usually a trade-off between quantity and quality of life, studying how to best involve patients of varying backgrounds in the decision-making process is crucial to treating the disease.

Many illnesses such as influenza and the common cold present similar symptoms, which renders them difficult to diagnose without a formal molecular diagnostic test. Designing a diagnostic test that could be run in patients’ homes would address limitations of existing assays, but with the risk of patients attempting to self-treat without consulting healthcare professionals. As a result, this project has developed  a lateral flow assay (LFA), the same type of assay as an at-home pregnancy test, that is adapted to detect an infectious disease while blinding the end user from the result. Traditional LFAs indicate the presence or absence of a biomarker by capturing the biomarker with antibodies and labeling the capture event with colored nanoparticles; two lines indicate the presence of the biomarker and one line indicates its absence. We've proposed two methods of blinding the end user from the results. One is a system where two lines always appear in an LFA even in the absence of the biomarker, preventing the end user from interpreting the result. The color intensity of the two lines will be proportional to the amount of biomarker present; this relies on humans’ inability to reliably detect absolute color intensities to blind the assay results. Preliminary testing of an influenza LFA suggests that the proposed scheme successfully blinds immunoassays to the end user, but the limit of detection (LOD) is 20 times worse than traditional LFAs. The second method uses a randomized array of antibodies spotted on an LFA as a way to obfuscate the result but through imaging can later be interpreted. We've optimized the proposed schemes to improve the LOD, demonstrate that the assay can be quantified with a cell-phone, and enable quantitative detection of infectious disease biomarkers in the home with a blinded immunoassay.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that results in progressive degeneration of upper and lower motor neurons, leading to muscle weakness, paralysis and death. Currently, ALS affects 15,000 Americans in the U.S., who have an average life expectancy of three to five years at the time of diagnosis. There are only two FDA-approved medications for ALS treatment, which minimally slow but do not reverse the progression of the disease. Understanding the genetic causes of ALS can help us to identify more effective treatments, therefore we seek to identify pathogenic variants and investigate how they contribute to the death of motor neurons. We previously showed that a tandem repeat in an intron of the gene WD-repeat-containing protein 7 (WDR7) may be involved in ALS. To better characterize this repeat, we PCR-amplified the repeat region and quantified repeat size in 500 ALS samples, including those from sporadic cases and those with known pathogenic variants. These repeat sizes were compared to approximately 500 Parkinson disease samples, 100 Primary Lateral Sclerosis samples, and 500 control samples from the Coriell Cell Repository, to verify whether expansion of this repeat was specific to ALS. Furthermore, to determine if repeat blocks were enhanced in certain subsets of patients, the lengths of the intronic repeats in genomes of ALS patients were profiled and compared with de-identified phenotypic information. To resolve the exact sequence of repeat in ALS samples, a subset of patient DNA samples and controls were sequenced using single molecule real-time (SMRT) sequencing. Together, these findings help us gain insight into the disease and guide us to develop a better treatment.

My research has worked to develop an automated system that utilizes techniques that will modernize pathology and reduce diagnosis times from days to hours. The current method of diagnosing cancer (histopathology) requires a tissue biopsy that is fixed with formalin for 6-72 hours, dehydrated and embedded with wax, then manually sectioned, mounted on slides, and stained for imaging. This manual process is extremely time and labor-intensive and is the reason diagnoses can take up to ten days. The preferred, and least invasive, way to obtain tissue from a patient is the core needle biopsy (CNB), which is taken by inserting a needle into a region where cancer is suspected to exist, and removing a cylindrical section of tissue. Although CNBs are rapidly becoming the standard-of-care, their small diameters make it difficult to prepare and section the tissue for use in conventional histopathology. My research project over the past year has been to develop CoreView, a device designed to handle CNBs, automatically staining and imaging their entire surface for more complete images used to diagnose cancer. We use deep ultraviolet light to image the outer 2-3 cell layers of fluorescently stained tissue in a lab-built microscope to produce sharp images that can be equivalent to standard histology images from thin sections. I used LabVIEW to develop the rotational and imaging control to image the tissue at different focal points so the biopsy surface stays in focus during rotation. Along with designing necessary components of our system, I have worked on improving processing techniques for staining fixed porcine liver to produce clearer images with high contrast between the nuclei and the cytoplasm/cell boundaries. Our collaborating pathologist has confirmed that these images are at diagnostic quality, proving that CoreView and my improved techniques for processing tissue have the opportunity to revolutionize current histopathology.

Current methods of modeling tissues rely on two-dimensional (2D) cell cultures which fail to incorporate the three-dimensional (3D) aspect of native tissues in the body and therefore cannot mimic tissue-specific conditions in vivo. The inability to accurately mimic the native properties of tissues in vitro makes characterizing physiological cell behavior challenging and limits the applications of these models. As such, the lack of sufficient in vitro tissue models necessitates the need for a more advanced engineered tissue models that accurately recapitulates the native morphology and function of cells in vivo. Previously developed in vitro platforms that attempt to model human vasculature in vivo have been limited in their ability to imitate the circumferential architecture of smooth muscle cells which surround the veins and arteries. Through the production of a more advanced vascular tissue engineered platform that accurately recapitulates biomimetic conditions of native tissue, we could better study cardiovascular biology, disease modeling, and drug-response in a dish. We propose the fabrication of an architecturally-controlled multi-layered 3D smooth muscle cardiovascular model that mimics the tubular structure of blood vessels in vivo to study structure-function relationships. Utilizing a thermoresponsive nanopatterned film, we are able to direct cell alignment and layer sheets of cells to create a circumferentially aligned 3D smooth muscle tissue model that mimics the physiology of the vascular tunica media. Furthermore, we have designed a fibrin gel casting method to produce tubes with a hollow intraluminal space that has mechanical properties that are physiologically relevant to human tissue. We aim to determine how anisotropic alignment of smooth muscle affects vascular compliance. This model is highly versatile in nature and can be functionalized with a wide variety of cell types to accommodate different tubular tissue structure throughout the human body.

Dynamic-contrast enhanced (DCE) MRI has a very high sensitivity for breast cancer detection. However, the high costs, long scan-times and safety issues associated with injecting gadolinium-based contrast agents prompt the need to explore non-invasive, non-contrast-based diffusion-weighted imaging (DWI) as a possible alternative. DWI reflects the microscopic cellular environment and at high sensitizations (b-values), DWI can highlight malignant breast tissues without the aid of gadolinium. Acquiring images at high b-values increases image distortions and lengthens scan times. By simulating these high b-value images, lesion conspicuity can be increased while minimizing scan time and maintaining image quality. The purpose of this study was to compare lesion conspicuity across b-values and between acquired (aDWI) and computed (cDWI) DWI. Twenty women with invasive breast cancer were enrolled to undergo a research DWI scan. aDWI was acquired at multiple b-values of b=0/100/800/1500/2500 s/mm². Apparent diffusion coefficient (ADC) maps were generated and cDWI images were then computed for b-values ranging from b=200-2500s/mm² using: S_b = S₁₀₀ e^-Δb*ADC. Lesion contrast-to-noise ratio (CNR) was calculated for both aDWI and cDWI at each b-value. CNR measures across b-values from cDWI and aDWI were compared by Wilcoxon signed-rank test. Lesion conspicuity, as measured by CNR, increased with increasing b-value, with no significant difference between aDWI and cDWI. Our findings show the maxium lesion conspicuity on DWI is achieved at b=1100-1500s/mm², which is higher than typical diagnostic breast DWI protocols. However, lesion conspicuity likely varies with breast density and other patient and tumor characteristics. Potential advantages of cDWI include shorter scan times and flexibility to retrospectively generate images at any b-value for optimal interpretation, warranting further exploration of the value of this technique for breast imaging.

The use of targeted microbubbles to image the molecular expression of vascular factors is an active area of ultrasound research. Combining the imaging advantages of ultrasound (e.g. cost, ease of use, availability) with the potential of molecular imaging makes targeted microbubbles especially attractive for studying the expression of vascular factors. During imaging, signals from molecularly attached microbubbles need to be separated from signals of non-attached free-flowing microbubbles in the vasculature. Thus far, different indirect approaches have been used to isolate stationary microbubbles. In this work, we present a direct approach to classify bound microbubbles in the presence of free-flowing microbubbles by processing nonlinear Doppler acquisitions. Nonlinear Doppler sequences are programmed on a research platform where Doppler processing separates low frequency stationary microbubbles signals from high frequency flowing microbubbles signals. In-vitro experiments are conducted by imaging stationary microbubbles surrounded by free-flowing microbubbles in a dialysis tube. In-vivo experiments are conducted by applying this approach to image the extent of inflammation associated with spinal cord injury (SCI), which plays a critical role in progressive tissue loss after injury. Both targeted and non-targeted microbubbles have been imaged in a rat SCI model. Targeted microbubbles were made for the inflammation marker p-selectin. Our in vivo results show successful separation of a limited number of non-targeted microbubbles adhering around spinal cord contusions. We believe this may be due to interactions between microbubbles and activated leukocytes. We expect to observe increases in bubble adherence and differences in the spatial distribution in using targeted bubbles, hopefully elucidating the extent of inflammation due to SCI. This work demonstrates the potential to separate bound targeted microbubbles from of free-flowing microbubbles to image a vascular factor for inflammation, which demonstrates practical pre-clinical ultrasound molecular imaging and opportunities for broader applications.

Osteoporosis — a condition characterized by abnormally low bone density — primarily afflicts women over the age of 65 and is estimated to cause almost 9 million annual fractures worldwide. The current gold standard for clinical osteoporosis screening is dual-energy x-ray absorpiometry (DEXA), which can be used in combination with demographic metrics to estimate an individual’s likelihood of fracturing a bone. Early detection of osteoporosis enables preventative dietary, lifestyle, and pharmaceutical interventions to improve patient outcomes. However, DEXA requires access to expensive equipment and specialized facilities. This motivates the need for an inexpensive and ubiquitous osteoporosis screening technology, bringing access to osteoporosis screening to individuals in low resource settings. In this work, I designed, implemented, and evaluated a smartphone application called OsteoApp that attempts to infer bone density indirectly by measuring the resonant properties of bone. Using my smartphone application prototype in parallel with a custom hardware setup, I collected data from retirement community members with known DEXA scan results  as well as from a control group of University of Washington students. I analyzed these data to evaluate the feasibility of a smartphone-based osteoporosis screening solution.

Microfluidics is a field that consists of the manipulation of fluids in microchannels and chambers for a variety of biomedical applications from drug screening to the study of cell biology. Currently, the majority of microfluidic devices are made from drug-absorbent materials and manufactured using processes that are expensive, labor-intensive and time-consuming. These constraints have limited the commercialization and dissemination of microfluidic technology into healthcare markets. Digital manufacturing is a computer-based manufacturing method which integrates digital designs, automated fabrication, and device testing in order to increase fabrication efficiency. Through recent advancements in digital manufacturing technologies like 3D printing and laser cutting, and the development of non-drug absorbent resins for 3D printing, the Folch lab has been inexpensively prototyping complex 3D microfluidic platforms capable of testing the effectiveness of personalized cancer therapies that utilize multiple drug exposures. Current methods for modeling cancer lack the ability to replicate the human microenvironment in which a tumor develops, and prevent high throughput analysis of the effects of therapeutics. This need to efficiently recapitulate physiologically relevant effects of disease progression have been addressed by patient-derived tumor organoids. Composed of patient-derived cancer cells cultured in vitro, tumor organoids are a promising method for accurately modeling cancer because they serve as a representative snapshot of the types of cancer cells seen within the patient. By performing tests on tumor organoids to observe the effect of different combinatorial therapies, personalized cancer treatment regimens can be developed to most effectively treat individual patients. Combining the advantages of digital manufacturing with tumor organoids, I have been able to 3D print a microfluidic device capable of trapping and treating organoids with different combinations of drugs through the use of biocompatible, non-drug absorbent resins. This device’s functionality and manufacturability demonstrates that digital manufacturing is vital for the implementation of microfluidics into healthcare industries.

One method of comparing strain fitness is to compete strains head to head; over time, the competitor with a fitness advantage will increase in frequency in the population. To track the frequency of each strain, they must be differentially marked such that their frequencies can be measured by plating the culture and counting colonies of each type - a time and labor-intensive process. One common marker used by our lab and others is using pigment production to produce colonies of different colors. My project is to create a continuous culture monitoring device named a chromostat that uses a color sensor to measure the relative abundance of different colored strains in solution, removing the need for plating and increasing the automation of competition experiments. By comparing the color of the individual yeast strains to the color of them mixed together, the chromostat can calculate, in real time, the relative abundance of each strain in solution and determine which strain is more fit and by how much. I built the chromostat on a raspberry pi minicomputer using an open source Java library, pi4j, to control the attached color sensor. This sensor converts light waves to red, green, and blue color values, which are then converted to frequency values for each strain and displayed to the user. The chromostat is controlled through a text-based interface that operates on the command line and has a variety of functions to modify data acquisition to increase overall accuracy and allow for data analysis. It will be used as part of a high school teaching laboratory in which students conduct evolution experiments and later compete different yeast strains against each other to generate fitness data.

Polycystic kidney disease (PKD), commonly caused by defects in polycystin-1 or polycystin-2, results in the formation of fluid-filled cysts and progressive loss of kidney function. Our laboratory has established a cellular model for ADPKD using kidney organoids, multicellular tissue that functionally and structurally resembles the organ of interest, derived from gene-edited human pluripotent stem cells (hPSC). To address the need surrounding a cure for PKD, we have also discovered a basis for a form of treatment involving myosin. Non-muscle myosin is a protein that controls cell protrusion and adhesion; we have established that lack of adherence increases cystogenesis dramatically. We discovered blebbistatin, a non-muscle myosin (NMMII) inhibitor, is a robust inducer of cystogenesis. Our previous finding begged the question if activators of NMMII have the opposite effect and reduce cystogenesis in PKD organoids. To better understand the role of myosin, we differentiated kidney organoids from human PKD hPSCs in a 24-well plate coated with Matrigel. We proceeded with microdissecting the organoids at 3 weeks before they formed any cysts and transferred the organoids to suspension culture where they were treated with either DMSO as a control or a pharmacological myosin activator for one week. The organoids were then imaged and number of cystic organoids were quantified. The treatment using the myosin activator compound resulted in decreased cystogenesis, in terms of the size and number of cysts. Although we have yet to clarify myosin’s effect in the PKD pathway, we have identified a myosin activator as a potential inhibitor of cystogenesis. Further experimentation is to be done with other compounds such as actin activators, and proceeding with experimentation on ex vivo kidneys of mice. Our current findings suggest that the polycystin proteins positively regulate actomyosin’s contractility, therefore myosin may be an important factor for keeping kidney tubule integrity and preventing cystogenesis.

Cancer metastasis, or spreading to another site, is the leading cause of cancer-related deaths. Invasive cancer cells migrate through the extracellular matrix (ECM), a meshwork of protein fibers that gives structure to tissue. These fibers normally have a random arrangement, but as a cancer cell migrates it rearranges them, leaving a trail of aligned fibers that can induce other tumor cells to follow. Tumor-specific aligned matrix topography is critically lacking in current cancer models and drug screening platforms. Here we explored the different mechanical factors that can be used to induce type I collagen, an important ECM protein, to form aligned fibers as they polymerize. Our preliminary experiments showed the effects of fluid flow induced by mechanical motion on gel formation. Collagen gels were incubated in polydimethylsiloxane molds of varying dimensions on a rocking platform to guide the flow direction and collagen fiber alignment. We then imaged the gels with a second harmonic generation microscope and analyzed the images using the MATLAB-based program CT-FIRE. The deviation between the angle of fibers and the alignment were used to determine the parameters for optimizing the method. The results suggested that the dimensions of the chamber and amount of time on a rocker affect the alignment of the collagen fibers. Further experiments will be conducted to quantify the effects of fluid flow, shear force, and surface tension on collagen fiber alignment. We plan to determine and combine two parameters which are most effective to align collagen fibers to establish an easy and reproducible method. We will further optimize the protocol to achieve uniformly aligned collagen fiber in a high-throughput manner. The tumor matrix topography specific 3D model we develop in our study will be valuable as a platform to study the behavior of migratory cancer cells and to identify novel anti-metastatic therapeutics.

 Alzheimer’s disease (AD) is a severe neurodegenerative disease and the most common cause of senile dementia in the United States. AD affects approximately 5.7 million Americans. Presently, there is no treatment for the disease and the mechanisms underlying the development and progression of the disease are not well understood. Human induced pluripotent stem cells (hiPSCs) are a promising technology for the study of Alzheimer’s disease pathogenesis, but current stem cell maturation methods, used to produce functionally mature neurons for accurate disease models, are too time-consuming to allow for the efficient study of disease mechanisms. Therefore, there is a need for a more rapid maturation method to produce cultures of functional neurons without the extended time commitment that is standard for common differentiation protocols. To achieve accelerated neuron maturation, optogenetic stimulation will be used to periodically stimulate neural stem cells according to an optimized stimulation protocol. To accomplish this, we have created a lentivirus using a construct that expresses the channelrhodopsin, Chronos, under an EF1α promoter, designed RT PCR primers to measure Chronos expression, and collected RT PCR data on transduced cells. This virus will then be used to transiently transduce hiPSC derived neurons that have previously been gene edited to express a NeuN (marker of maturity) RFP (Red Fluorescent Protein) tagged fluorescent reporter that will function as a cell maturity readout. This stem cell line will be used to develop an optimized protocol for the light driven maturation of stem cells in vitro. The maturity of the neurons will be measured using electrophysiology to evaluate cell function and RT PCR to monitor gene expression. The success of this project will allow for more efficient study of Alzheimer’s disease in stem cell based disease models, which could lead to the discovery of disease mechanisms, revolutionizing the understanding of this burdensome disease.

Biological mineralization is the formation of minerals in hard tissues guided by proteins. Unique aspects of these minerals include the molecular control of hierarchical structure, intricate architectures, and multifunctional properties for inspiration in bionanotechnology and nanomedicine applications. Numerous biomineralization strategies have been developed in hard tissue regeneration therapies. However, there is currently no in-depth understanding of how proteins regulate the synthesis of these inorganics or the physiological formation of the minerals. The ability to control mineral formation for biomedical applications, therefore, is still limited to the use of a few mineral-directing proteins extracted from tissues. Biomineralization can also be controlled using short peptide domains derived from natural proteins known to have a regulatory role in mineralization. Our laboratory has designed peptides derived from amelogenin (ADPs), the key protein in tooth formation, using combinatorial selection and computational design, whose utility in rebuilding hydroxyapatite (HAp) mineral on tooth has been demonstrated in numerous case studies. The goal here is to understand the fundamental mechanisms of biomineralization guided by ADP5 and develop a methodology to form HAp with exclusive control of its growth kinetics and mineral crystallography. We designed mutants of ADP5 to investigate changes in mineralization kinetics, nucleation, and morphology. In the current study, we are establishing the conditions for ion-peptide interactions on the onset pH for mineral nucleation using calcium/phosphate and mutant ADPs. The goal is to gain insights into the correlation between sequence domains and biomineralization outcomes eventually facilitating greater control over the reaction and further optimize remineralization approach. The developed method has a high potential to develop non-invasive oral health care materials and methods by restoring mineral loss, the root cause of dental ailments and, eventually, help bring clinical and over-the-counter dental products into the market with preventive, restorative, therapeutic, and cosmetic characteristics. Sponsored by SoD Spencer Funds.

Recently, microfluidic technologies have been developed to allow higher throughput collection of yeast replicative lifespan data. Adoption of these devices has been limited, in part, due to the high cost of the motorized microscopy instrumentation from mainline manufacturers. Inspired by recent development of open source microscopy hardware and software, we developed minimal-cost hardware attachments to provide long-term focus stabilization for lower-cost microscopes and open-source software to manage concurrent time-lapse image acquisition from multiple microscopes. This functional 3D printed scaffold along with attached electronics has shown to capture accurate yeast lifespans without costing as much as other third party autofocusing system. We hope that this can help spur the wider adoption of microfluidic technologies for the study of yeast replicative aging.

All around the world, humans depend on cultivating plants for medicine, food, landscaping and building materials. Understanding how plants grow helps people make more informed decisions about what to grow and how best to grow it. The plant hormone auxin plays a key role in determining almost all aspects of plant growth and development. The Nemhauser Lab has previously determined that two versions of the receptors for auxin, TIR1 and AFB2, function at different speeds in the model plant Arabidopsis thaliana. Currently, we do not know how this speed disparity affects specific plant functions. My hypothesis is that there are measurable differences in organism functions caused by this difference in molecular speed. In the past, full knockout mutants for TIR1 and AFB2 did not exist, making it difficult to answer this question. To complete my research, I developed these mutant plant lines, determined functions that can be quantitatively measured, and collected and analyzed the data. Because the auxin-signaling network affects so many plant functions, any additional information we can gain about how this network functions in the model organism helps us move towards a more complete understanding of plant growth. This could positively improve the efficacy of agriculture and other plant-based industries, as well as moving us towards other new discoveries in the field of plant growth.

Spinal cord and traumatic brain injuries (SCI and TBI) result in secondary injury causing further damage to neural cells, due to mechanisms of inflammation and cytotoxicity. Though some anti-inflammatory drugs have shown promising results in reducing secondary injury in animal models, their use is controversial in clinical practice due to systematic side effects at the required high dosage with questionable efficacy. An electrically controlled drug release system will allow for spatiotemporal control of drug delivery and might solve some of the issues with systemic dosing. We designed and fabricated a polypyrrole (PPy) microneedle array for control release of dexamethasone for application to the nervous system. Using micro-scale 3D printing, microneedles design were 3D printed in photoresist and sputter coated in gold to form a conductive surface. PPy was electrodeposited onto the microneedle arrays from a monomer solution of pyrrole and dexamethasone. Dexamethasone can then be released from the PPy layers by applying a negative potential. Dexamethasone release was quantified using absorbance spectrometry, after purification through high pressure liquid chromatography. Dexamethasone PPy arrays were then tested in an in vitro model of neuroinflammation. One hour after inflammation activation, cells received treatment. After 48 hours of incubation, cells were assayed for nitric oxide and cytokine production. Experimental results show sufficient release using electronically controlled dexamethasone release from PPy microneedles to decrease nitric oxide. There was a trend towards decreasing inflammatory cytokine release, but future experiments with higher sample sizes will be needed to prove significance.

For women to have protection from unintended pregnancy and human immunodeficiency (HIV), current lead prevention options use oral antiretroviral drugs (ARV) for pre-exposure prophylaxis (oral PrEP) along with a form of contraception. Failure to adhere to these drug therapies will increase the risk of contracting HIV or pregnancy. We have proposed to integrate drug-eluting materials onto a copper-intrauterine device (IUD) that could provide both HIV prevention and contraception. We will evaluate two methods to formulate a matrix release drug delivery system. Injection molding is a method to inject material into a mold that can be used for constructing drug-eluting medical devices with low drug degradation. For our purpose, we injected a polymer and drug combination into a mold to construct a solid slab. Whereas, electrospinning is a method that uses electric force to formulate stable and high surface-to-volume ratio nanofibers with high drug encapsulation and porosity compared to the molded slab. Both delivery systems will be used to administer ARV drugs to the female genital tract for a year. We optimized the molded slab and electrospun nanofibers technique for maximum polymer-loading, and used 3-D printing and nanofiber wrapping technique as a process for slab integration and fiber integration onto the IUD respectively. The polymer and drug combinations for both electrospun nanofibers and molded slabs were chosen to have the maximum drug-loading and stable mechanical properties. Drug release was measured in vitro to predict daily release rates out to three years. The ideal matrix release drug delivery system method for the dual HIV prevention and conception IUD is determined based on the mechanical properties and drug release rate of the polymer and system combination. We also investigated the drug delivery systems for cytotoxicity to verify dosage safety.

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.

Implanted biomedical devices are becoming increasingly common for the treatment of organ or tissue loss. The efficacy of these implants, however, is continuously hindered by attempts of the host immune system to eliminate foreign biomaterials, a process termed the foreign body reaction. The long-term outcome is the collagenous encapsulation of the implant that prevents it from integrating into the body. While there are many approaches towards guiding or suppressing the host immune response, we are particularly interested in modulating the macrophage phenotype at the biomaterial-host interface to dampen foreign body reaction and restore tissue homeostasis. Specifically, vascularization, the formation of blood vessels and capillaries, in and around a porous biomaterial is shown to be partially driven by the expression of different macrophage phenotypes which in turn are modulated by the biomaterial pore diameters. Thus, we hypothesize that activating the pro-inflammatory M1 macrophage phenotype in the biomaterial microenvironment promotes vascularization and dampens collagen deposition. To test our hypothesis, I am currently optimizing the seeding and activation of primary inducible engineered pro-inflammatory M1 macrophages (i-M1macs) on biomaterial with different pore diameters in vitro. The focus following the optimization of their M1 phenotype expression will be investigating their effect on the foreign body reaction to biomaterial with different pore diameters in vivo. I will implant the i-M1macs seeded biomaterials in laboratory mice and harvest them at selected time points. Using histology and image analysis, we will determine whether the primary i-M1macs increase vascularization in and around the biomaterial and whether i-M1macs-dependent vascularization is affected by the pore diameters. Obtaining positive results will confirm the potential of our cell therapeutic approach to improve the integration and biocompatibility of various biomedical devices.

Conditioned pain modulation (CPM) is the alteration of pain sensation upon the application of a separate, conditioning stimulus. In healthy patients CPM is experienced as a pain-inhibits-pain phenomena, but patients living with fibromyalgia (FM) and other chronic pain conditions have been shown to lack this pain inhibitory CPM response. While the deficiency of CPM in chronic pain patients has been widely documented in meta-analysis, the impact of other risk factors on CPM has largely yet to be understood. A limited collection of previously conducted studies has examined the impact of daily activity, depression, and anxiety on CPM in non-FM patients but the low number of participants and studies available warrants further investigation. To do this, we analyzed baseline data from a clinical trial in FM patients underwent thermal stimulus testing to determine CPM, wore an actigraphy watch for seven days to provide objective measurements of daily activity, and completed several questionnaires and psychological assessments. A series of analyses were completed using Stata SE, including bivariate correlations, linear regression, and logistic regression. Results indicate that there were no significant correlations between daily activity level, depression, anxiety, pain catastrophizing, pain intensity, and physical functioning with CPM. However, a significant correlation was observed between CPM and PTSD score, with individuals more likely to be suffering from PTSD experiencing greater hyperalgesia CPM effects. Indeed, the odds of hyperalgesia were 3.1 times higher among those with PTSD than in those without PTSD (95% CI: 1.4, 6.6). This suggests that PTSD contributes to neurological deficits in pain processing. If this result is confirmed in other samples of patients, then future research should evaluate the effectiveness of PTSD-targeted treatments for improving pain outcomes in FM patients with PTSD.

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.

Cortical plasticity is the substrate for learning and memory. It is the basis of an organism’s ability to adapt in response to a changing environment and is central to functional recovery after an injury involving the nervous system. Vagus nerve stimulation (VNS) has already been shown to be effective in altering neuroplasticity. Most VNS research has been conducted on epileptic animals and thus offers little information on how VNS may affect a healthy human brain. The final goal of this project is to establish a minimally invasive VNS protocol aiming to augment neuroplasticity and ultimately affect behavioral performance in a cognitive task. Because this study is conducted using non-human primates, and because of the limited invasiveness of the procedure, the outcomes can be directly translated to applications for human subjects. Non-human primates are implanted with cortical electrodes allowing both recording and stimulation of the cortex at select locations. Closed-loop cortical stimulation triggered by the troughs of beta (15-25Hz) oscillations leads to phase-dependent changes in excitability which outlast the oscillatory episodes for a couple of seconds. By delivering current through a clip electrode attached to the right or left cymba concha, we paired the cortical stimulation with auricular branch VNS (abVNS). This stimulation protocol is used to investigate the effects of cycle-triggered cortical stimulation with abVNS. Preliminary results show a suppression of excitability when the abVNS is delivered in phase with cortical stimulation which lasted for minutes after the cycle-triggered stimulation has ended. However, more control experiments need to be run before conclusions can be drawn. These findings of lasting change to cortical plasticity are novel and suggest that vagus nerve stimulation can affect medium-term neural plasticity.

Cysteine is an infrequently occurring but important amino acid critical to tertiary and quaternary structure. While peptide chains typically undergo post-translational modifications, cysteine is susceptible to further modification due to its reactive thiol group. The effects of these modifications are unclear, as they could either represent normal cell-cell communication or potentially harmful by-products. Common agents that affect the thiol group of cysteine are by-products from other biological processes, for example metabolic processes (which creates H₂O₂), natural toxicant defense (p450 or glutathione), and inflammation (HOCl). This experiment sought to discern the products that form when cysteine is exposed to biologically relevant concentrations of these oxidants. Liquid chromatography-mass spectrometry (LC-MS) was used to perform the analysis of the oxidation products. Cysteine was mixed with 5 mM bleach in vitro and pH buffered and the resulting solution was analyzed. Methods were developed by optimizing instrumental conditions—including molarity of solutions, voltage spray and flow rate, and acidity of the samples—in order to use LC-MS. Pending results will allow for the identification and quantity of the cysteine oxidation products.

The tumor microenvironment (TME) dictates the outcome of many immuno-oncology therapies for solid tumors. To better understand the dynamic milieu of the TME, new multiplexed, spatially-resolved histologic techniques are being developed. A key limitation to evolving these techniques is identifying specific and selective antibodies that perform well in an immunohistochemistry (IHC) platform. The over-arching goal of this project is to develop a flexible, high-throughput platform to empirically test the IHC staining characteristics of antibodies in formalin-fixed paraffin-embedded (FFPE) tissues with improved throughput and lower test volumes. Essential elements in our platform design include: 1) flexibility to test a variety of biologic materials, which is dependent on the test antigen, 2) compatibility with manual and semi-automatic tissue microarray (TMA) builder, 3) easy use for pathologic assessment, and 4) future compatibility with fully-automated tissue staining instrumentation. Using computer-aided design software, and a stereolithography printer, we prototyped a guide template to build recipient TMA FFPE blocks. To screen for a variety of antigens in TMA "cores", we prepared cell lines, and acquired mouse tumor xenografts and human tissues. Standard IHC techniques were used to screen hybridoma supernatants generated from mouse immunizations. We have designed and tested six different multi-well slide-based IHC screening platforms. The current format consists of a six by four array of 2 mm cores. We have screened antibodies from two different mouse hybridoma campaigns. Thus far, we have identified candidate IHC antibodies for exogenous epitopes to identify chimeric antigen receptor T cells used to treat solid tumors, and a fusion protein hypothesized to be an oncogene in pediatric liver cancer. This project developed a single-slide antibody screening prototype for IHC. The device offers the flexibility to test multitude of tissues, and is built with design considerations for future automated tissue staining compatibility.

Malaria is a disease associated with a significant global burden of illness, including hundreds of thousands of deaths every year. Severe malaria is most often due to the Plasmodium Falciparum species of parasite, the pathogenesis of which involves avoidance of host detection through endothelial sequestration. This process allows infected erythrocytes (red blood cells) to adhere to the vessel wall and avoid filtration via the spleen. Severe cases of malaria are often also associated with endothelial activation and release of the adhesive glycoprotein, von Willebrand Factor (VWF). Preliminary data from our laboratory suggests that erythrocytes infected with P. Falciparum may be able to bind to VWF fibers formed under in vitro conditions, and we believe that VWF binding may prove to be a novel mechanism contributing to endothelial sequestration. This study was conducted in order to understand the interactions between plasmodium infected erythrocytes and VWF fibers formed following endothelial activation. Because of the high demand for technical replication for such a project, and the labor and time expenses associated with the lab’s commonly used microvessel system, modifications were made to the geometry and housing to characterize it for the high-throughput platform for the study of VWF’s role in the binding of infected erythrocytes to activated endothelium. By utilizing nanofabrication techniques and soft lithogaphy with Polydimethylsiloxane (PDMS), we have successfully completed design and manufacturing of the novel devices that have allowed an increase in speed and efficiency up to three times that of previous assemblies. In these newly fabricated devices, we saw consistent formation of robust VWF fibers upon activation and aim to continue their use to observe and quantify binding of plasmodium infected erythrocytes in VWF-rich regions within vessels.