The majority of the population living with HIV resides in African countries where the cost-effectiveness and ease of implementation of the technology is imperative. Our goal is to develop an assay that eliminates the need for expensive nucleic acid sequencing technology, to test and diagnose drug resistance by detecting variation in nucleic acid sequence of the virus. One of the missing pieces that will allow us to reach this overarching goal is to design a method for isolating the viral particles in a manner appropriate for point-of-care application. Our approach, which will contribute to the assay being developed at the same time, is to use protein G magnetic beads, which are affinity molecules, to isolate the HIV virus from plasma sample. The protein G on the magnetic beads interacts with the coat antibodies on the viral particles and facilitate their removal from plasma. We show that the protein G magnetic beads have comparable efficiency at isolating the viral particles compared to the gold standard, Qiagen extraction, without the use of strong chemical reagents and complicated instruments. Yet, further work is needed to demonstrate that the viral RNA can be extracted starting from clinical plasma specimens with a protocol applicable in point-of-care settings defined by limited use of complicated insturments and cost effectiveness of the solution. 

Vascular injury causes platelets to form hemostatic plugs. Platelets are activated by high shear and collagen, and adhere and contract to protect a clot from mechanical forces. The role of collagen and the anticoagulants heparin and ASA (Acetylsalicylic Acid), which inhibit thrombin and TxA2 (Thromboxane A2) respectively, are not fully understood. Our lab has designed a microfluidic device to measure clot forces and area. Microfluidic channels with block-post sensors generate shear gradients that induce platelet adhesion. Clots grow towards the post and contract, pulling the post closer. Fluorescence microscopy allows post deflection to be analyzed with MATLAB code using Hooke's Law, and phase images monitor area. First, channels were coated with and without collagen. Then, channels were collagen-coated and blood inhibited with heparin or ASA. Blood was incubated with 15 USP (U.S. Pharmacopeia standard) heparin lithium in H2O or 0.3 mM ASA in DMSO for 20 min. Clots in uncoated channels had lower forces than in collagen-coated channels. Blood with ASA in DMSO had less platelet adhesion and contraction than in blood incubated with DMSO. Blood with heparin in H2O had lower force but higher clot area than without heparin. Reduced force of clots formed in uncoated channels relative to collagen-coated channels suggests that collagen is important in force. Inability of platelets to adhere or produce normal force with ASA indicates that TxA2 promotes platelet aggregation and force. Heparin reduced force but not platelet aggregation, thus thrombin is involved in force generation only. This clot-on-a-chip technology can shed light on the clotting process and diagnose patient clotting issues.

Cancer is a leading cause of mortality worldwide, and the number of new cases is projected to reach 22 million in the next two decades. Surgical resection of tissues is a standard of care for treatment of a variety of cancer patients. For the prostate, surgically removed tissues usually undergo a process of bread-loafing (cutting into 0.5-1.0 cm thickness), chemical fixation, embedding in paraffin (wax), sectioning (5-10 μm thick), staining, slide mounting and finally microscopic examination to determine whether all tumor growths have been excised. The procedure is labor-intensive, time-consuming and expensive. In addition, the majority of the prostate specimens are normal tissues that do not require detailed microscopic examination. These factors reveal the need to significantly accelerate the screening process and increase the efficient use of healthcare resources. Therefore, to reduce unnecessary processing time and costs, we proposed a fast and inexpensive imaging system to screen the surface of thick tissues for abnormality, before full histological processing. This system was designed to enable a wide imaging area and integrate deconvolution algorithms, which achieve high contrast and resolution. More specifically, the project was carried out in three phases: 1) design, construction, and optimization of a fluorescence microscope; 2) system calibration and optimization of deconvolution algorithms; and 3) system validation using fresh human tissues. The system is capable of generating a 1 x 1 cm² image in a few minutes with 4 nm resolution. Success of such a system will facilitate clinical translation and increase efficiency of histopathological procedures, which are beneficial to both patients and healthcare providers.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a propensity for early dissemination. As little is known about this process, we sought to identify the preferential sites of initial distant metastatic MCC in order to guide imaging evaluation and clinical follow up. In this retrospective study, we analyzed the clinical and imaging records of 442 initial distant metastases from 305 MCC patients enrolled in our data and tissue repository. Initial distant metastases were defined as the first lesions detected beyond the regional lymph nodes of the primary tumor. Among 305 patients, 69% had 1 initial metastasis, 19% had 2, 9% had 3, and 3% had 4 or more. Of the 305 patients, 50% were evaluated only by radiologic and/or clinical criteria, while 50% also underwent biopsy-confirmation of MCC metastases. Of the 442 initial distant metastases, the distribution of sites was: 26% to distant lymph nodes (typically supraclavicular, retroperitoneal, or iliac), 15% to liver, 13% to skin, 13% to bone, 13% to other visceral sites, 6% to lung, 5% to pancreas, 3% to brain, 2% to kidney/adrenal gland, 2% to gonads, 1% to chest wall, and 1% to stomach. Although there were similarities in initial metastatic sites between MCC and melanoma, the brain (3%) and lung (6%) were markedly less common in MCC than in melanoma (20% and 36%, respectively), while the pancreas was more common in MCC (5%) than melanoma (<1%). Given the aggressive nature of MCC, with ~40% of patients eventually developing distant metastatic disease, these findings may facilitate improved detection and management.

Post-traumatic stress disorder (PTSD) is a widely known and generally understood condition, however, in pain medicine, it is often overlooked and not considered in treatments. PTSD has been shown to greatly affect mood and quality of life in the general population but has not been studied extensively in general pain medicine. Data collected from the University of Washington Center for Pain Relief’s web-based questionnaire that patients fill out prior to clinic visits was used to analyze how PTSD correlates with common components of pain such as intensity, interference with common tasks, quality of life, and substance use. PainTracker^TM is a web-based patient reported outcome tool that assesses pain treatment over time. PainTracker incorporates a variety of questions and instruments to effectively understand the various components of pain. This research illuminates the importance of PTSD in chronic pain patients and how it affects the population suffering from chronic pain. Current analysis has indicated that as PTSD severity increases, so does pain intensity, interference in daily life, mood, anxiety, and quality of life. This illustrates the importance of understanding PTSD’s role in pain and may potentially lead to more effective pain treatment by understanding the roots of pain in post-traumatic stress disorder.

Due to the rise of pathogen genetic knowledge and implementation methods of diagnoses pathways for patients, an increase in access to speedy and efficacious therapies is needed. The development of inexpensive, high-performance point-of-care (POC) tests does not only improve healthcare in low resource settings, but also moves the diagnoses out of hospitals and into homes and primary care offices. Paper-based microfluidic devices help both identify and screen for pathogens by allowing for more rapid care and treatment. The multiplexed, autonomous, disposable nucleic acid amplification test devices fabricated in the Yager Laboratory performs sequential functions - sample preparation, nucleic acid amplification, and lateral flow detection – for target identification. My project aims to bridge the last two segments into a single unit by introducing a method of inhibitory competition that permits for measurable, fluorescent-based analysis of isothermal DNA amplification from dry reagents in porous media. To prove this concept, a simplified experimental model of a multi-region, paper-based nucleic acid amplification test was designed, and new interference amplification mixtures developed. The isothermal strand displacement amplification (iSDA) technique utilized in these devices incorporates a fluorescent hybridization probe that allows for the detection of the increase in amplicons using an optical detection method. By incorporating a dilution of comparable, secondary strand of DNA as an internal control, a competition for reagents is established which creates different chemical sensitivities in the amplification zones. As a result, target DNA from the pathogen is amplified against a competitive threshold in a range. In the long run, fluorescence imaging of all regions can yield a quantitative measure of the concentration of pathogens in a sample. Overall, this project tests the fundamental groundwork for a new family of optical-based, quantitative devices by testing the use of fluorescent detection for POC tests, and investigating the principle of inhibitory competition.

While, according to the World Health Organization, the successful application of antiretroviral therapy reduced global deaths from HIV/AIDS by 24% between 2005 and 2011, a corresponding rise in viral resistance to these drugs has been measured. This rise is of particular concern in low-resource settings that are collectively burdened with over two-thirds of worldwide HIV infections while lacking access to robust diagnostic assays. In response to this need, the Lutz Lab, in collaboration with the Frenkel Lab at Seattle Children’s Research Institute and the Lai Lab at UW BIOE, aims to engineer a low-cost paper-based oligonucleotide ligation assay (OLA) for drug-resistant HIV. In order to lower the chance of user errors and reduce diagnostic turnaround time, it is desirable to be able to detect a set of six point mutations indicative of drug-resistance to first-line antiretroviral treatment in a single device. However, with current techniques we are limited to being able to test for one point mutation per device. To address this challenge, we have investigated a DNA hybridization method of OLA probe capture. Using this method, each point mutation is assigned a specific DNA “barcode” that could be used to generate spatially distinct capture lines in a diagnostic device. We have designed a set of six engineered barcodes that display highly specific and sensitive binding to their engineered complements. Furthermore, the performance of this DNA barcode system has been compared to barcodes constructed from non-natural nucleic acid analogs in order to develop a method that optimizes sensitivity and specificity of probe capture. The completion of this project brings the OLA one step closer to being able to make a previously expensive, technology intensive, and operationally complicated test, accessible to those in low-resource settings.

This project aims to develop a better model for testing gene therapy vectors in vitro. Gene therapy uses a modified virus to insert a functional copy of a gene into a patient’s cells, but testing new viral vectors on 2D cell culture does not accurately predict success in vivo. To address this need, I have designed a 3D-printed microfluidic device for optimized viral vector testing. Cells are cultured in 3D in one channel, while media and viral vector load can be easily adjusted in a connected channel through a gravity perfusion system. The device is currently undergoing tests for cell viability and demonstrating its utility for gene therapy applications. The primary advantage of this device is that cells cultured in 3D mature more similarly to true skeletal muscle and thus are better models for predicting the success of a viral vector in a patient. The small size and fine tune control over the cell’s environment also save time and money during testing, allowing the maximum number of options to be explored. A new, physiologically relevant model for testing viral vectors would expedite gene therapy research to help bring treatments faster to patients in need of gene replacement therapy.

Approximately half a million mortalities occur in the United States due to cardiac arrest alone; only a few survive from the initial shock. To improve these chances, first responders must resuscitate patients before their arrival to the hospital for proper medical treatment. Unfortunately, none of the devices on the market can be deployed to guide resuscitation by generating useful hemodynamic information to the responder. Animal studies further simulate that obtaining hemodynamic data during medical procedures such as CPR provides optimum patient outcomes. We have created a non-invasive ultrasound-controlled system that measures flow patterns of oxygenated blood within the ascending carotid artery, as a means to guide resuscitation during cardiac arrest. We designed and tested our ultrasound transducers on a water-submerged string phantom that mimics the flow properties of blood. The transducer propagates a pressure wave towards the moving string, and then records the reflected and frequency shifted wave resulting from the Doppler effect. Once the data has been collected in MATLAB, we produce a graphical user interface (GUI) containing a real-time pulsed Doppler spectrogram displaying the blood flow speeds, which are proportional to the Doppler-shifted frequencies. Our team collected data from multiple swine in-vivo to capture the flow rate in the carotid artery. We analyzed and compared the baseline, trauma, and resuscitation measurements to significant parameters such as heart rate and mean arterial pressure. Successful analysis of those pig studies will motivate retrospective human trials that, in turn, should provide sufficient results to motivate commercialization of this technology.

Breast cancer is the most common cancer in women. Even with advancing treatment techniques, breast cancer is still the cause of tens of thousands of death in the U.S. alone. Combined treatments including surgery, radio and chemotherapy are commonly implemented for breast cancer. However, the survival rate of metastatic breast cancer remains low, so new targeted therapy strategies are needed. In recent years, nanotechnology has opened a new route for targeted therapy of breast cancer. Nonetheless, some challenges still remain for nanoparticles (NPs) used in breast cancer treatments. After loaded with anticancer drugs, fluorophores and targeting ligands, NP drug delivery systems suffer from issues of size limit, stability, target cell internalization and biodegradability. Anti-human epidermal growth factor receptor 2 (HER2/neu) monoclonal antibody is a good targeting ligand for some breast cancer cells for two main reasons. Firstly, about 1/4 of breast cancer patients overexpress HER2/neu. Secondly, monoclonal antibodies have a better targeting effect than small molecule, protein and aptamer-based targeting ligands. However, the bulky size of the antibodies can change the desired physiochemical properties of NPs. Therefore, an anti-HER2/neu peptide, which is a small exocyclic peptide derived from the antibody, is utilized for targeting purposes. Moreover, paclitaxel (PTX) is a strong anticancer drug that can restrain mitotic spindle assembly and chromosome segregation, and in turn prevent breast cancer growth. Here we report anti-HER2/neu peptide-conjugated and PTX loaded iron oxide NPs (IONP-PTX-AHNP) that are small size, highly stable and biocompatible in biological medium for breast cancer treatment. Significantly, the IONP-PTX-AHNP showed targeted cell uptake into HER2/neu overexpressed breast cancer cells and PTX-mediated cancer cell death.

Macrophages are cells of the immune system that play key roles in surveillance, destruction of pathogens, and tissue repair. In the context of cancer, macrophages can infiltrate solid tumors and adopt an anti-inflammatory profile resembling that of the “M2” macrophage subclass. These M2-like tumor-associated macrophages (TAMs) promote tumor growth and metastasis by suppressing the immune response. Therefore, selective destruction of TAMs using a biomolecule that targets and binds to TAMs may have therapeutic benefit in cancer. We have previously identified a novel peptide, M2pep, that selectively binds to M2 macrophages over other immune system cells. In other experiments, a specific pattern of amino acids, named the M2pep motif, was observed to be critical for peptide binding to TAMs. This project seeks to improve M2pep binding and selectivity to TAMs by constructing and screening a library of peptides containing the M2pep motif for binding to M2 macrophages. The library was constructed by genetically engineering bacteriophages to display all possible peptides containing the M2pep motif. This was achieved by: (1) designing DNA sequences encoding for peptides with certain amino acid positions “fixed” with the M2pep motif, and the other positions randomized; (2) inserting these sequences into bacteriophage DNA; and (3) incorporating the engineered DNA into bacteria to generate bacteriophages that display the peptide library on their outer surface. Then, the peptide library was screened for binding to M2 macrophages, and the peptides that bound were sequenced. Several candidates were evaluated for M2 macrophage-binding and compared with M2pep to identify sequences with improved binding and selectivity. This method of peptide library construction and screening using a bacteriophage platform can be a powerful tool for future drug development, and enable further refinement of binding molecules with established binding motifs.

Macrophages are broadly classified as pro-inflammatory M1 and anti-inflammatory M2 macrophages. The majority of tumor-associated macrophages (TAMs) in tumors are known to express M2 phenotypes (M2-TAMs) which secrete cytokines and growth factors to promote cancer cell proliferation, tumor angiogenesis, and metastasis. Hence, depletion of the M2-TAMs is a promising adjuvant therapy to the current cancer treatments. Our lab has previously developed M2 macrophage-binding peptide (M2pep) which can bind to both M2 macrophages and M2-TAMs. However, in vivo targeted delivery of cytotoxic drugs to M2-TAMs using M2pep as a targeting ligand was limited by poor serum stability of M2pep. Previously, we engineered and tested different linear variants of M2pep; however, none of these was able to improve serum stability while retaining M2 macrophage-binding activity. In this study, we investigated cyclization of M2pep as a strategy to improve its serum stability since cyclic peptides are usually more serum resistant than their linear counterparts due to their higher structural rigidity. To synthesize a cyclic M2pep variant, the original M2pep was synthesized with two flanking cysteines on each terminus. These cysteines were then oxidized to form a disulfide bond yielding cyclic M2pep. The serum stability of cyclic M2pep was evaluated by incubating the peptide in serum and withdrawing aliquots of the serum at various time points for analysis by mass spectrometry. Compared to linear M2pep which was rapidly degraded in serum within 4 hours, the cyclic variant was detected even after incubation in serum for more than 24 hours. In conclusion, we have developed an optimized cyclic M2pep which exhibits enhanced serum stability while retaining M2-TAM binding activity. Our peptide can be utilized as a more effective ligand for targeted delivery of cytotoxic drugs to M2-TAMs. Future development of M2pep-based TAM-depleting therapeutics would serve as an effective adjuvant platform to potentiate current cancer therapies.

In diagnostics, the ability to detect small quantities of molecules with high sensitivity and specificity is crucial for a quick and accurate diagnosis of the presence of target antigens. The ELISA assay, which uses fluorescence to detect the presence of target molecules, is a standard diagnostic and research method for antibody-antigen detection. However, current ELISA methods do not have fast binding kinetics at low target antigen concentrations, requiring several hours for complete results. Therefore, there is a significant need for development of a simple and inexpensive method to improve the molecular binding kinetics in ELISA assays. My hypothesis was that rotational stimulation during enzyme incubation would greatly increase the sensitivity of the assay in a much shorter amount of time. To test this hypothesis, I compared the measured fluorescence of two different types of incubations – the traditional static incubation and a rotational incubation method. I measured the fluorescence generated by each set of assays at varying enzyme concentrations to determine the limit of detection, the minimum concentration that can be detected in a sample, for each method. I also analyzed the binding kinetics of each method using time-based data of each method. It is expected that the rotational method increases the sensitivity of the assay using half the concentration of target molecules compared to that of the static method. It also significantly decreases the time required to reach a binding equilibrium state by several hours. Promising results would suggest that rotational stimulation could significantly improve the molecular binding kinetics in diagnostic assays and could be implemented in future protocols to produce a more efficient diagnostic assay procedure.

Pancreatic cancer is ranked fourth amongst the most lethal cancers in US and first in mortality due to its low five-year survival rate. Accurate, definitive diagnoses are determined by pathologists who visually inspect and evaluate tissue slices from patient-procured biopsies traditionally processed into 2D slides. 3D microscopy makes 3D pathology, consisting of the imaging, visualization, and evaluation of unsectioned tissue biopsies in 3D, become a reality. Similar to the advantages of CT over traditional X-ray, 3D pathology may enhance pancreatic cancer detection and further streamline the diagnostic process in pathology. However, similarities with traditional pathology in optical information needs to first be achieved in 3D pathology in order to pair with current skilled pathologists. Enough color contrast and resolution of intracellular and extracellular structures can provide desirable features of biopsies to pathologists. Hence 3D pathology project in Human Photonic Laboratory aims to accomplish this goal by performing staining, imaging, reconstruction, and 3D visualization for tissue biopsies. Preliminary result illustrates that the reconstruction algorithm with appropriate parameters based on frequency spectrum of data determines the resolution of reconstructed images. Specific 3D manipulation software Amira helps 3D visualization of reconstructed images and preserve color contrast using its colormapping feature. The investigation of proper colormapping protocol starts from colormapping in-silico image of processed 2D microscope slides. An imaging systems with designed image stitching program creates grayscale image of microscope slides. Spectrometer is used to take spectral reflectance measurement. A developing algorithm will take the data and calculate coordinate values of the specimen in color space of human vision. Those values will be used in Amira in colormapping for in-silico image of microscope slides to pair with full color image of slides. This colormapping technique has potentials to extend into unsectioned sample and hence combine with reconstruction algorithm to achieve colormapping for 3D visualization.

Cancer cells can grow into life-threatening tumors and metastases partly because of their ability to evade the immune system. Immunotherapies combat cancer by boosting the natural immune response towards cancer cells. Immunotherapies are often more successful than standard treatments like chemotherapy because they are more targeted and have longer lasting effects with milder side-effects. A key component of immunotherapy is the identification of ligands that selectively target immune cells for drug delivery or immunomodulation. Peptide ligands are of special interest because they are inexpensive to synthesize and can be identified by a library screening technique called phage display. The purpose of this project is to identify peptide ligands to address two needs in cancer immunotherapy. The first need is to enable selective depletion of M2 tumor-associated macrophages, which promote tumor growth and suppress the adaptive immune response. We successfully identified 6 peptide ligands that preferentially bind murine M2 macrophages over M1 macrophages by using phage display and Illumina next-generation sequencing. The second need is to develop technologies for T-cell based immunotherapies. T-cells are an effective target for immunotherapies because they kill pathogens and regulate short-term and long-term immune responses. The identification of inexpensive peptide ligands to specific surface receptors on T-cells will be useful in increasing accessibility and efficacy of these treatments. We applied the successful next-generation sequencing methods from M2 macrophage ligand identification to target the CD28 and CD3e surface receptors on T-cells.

Highly controllable adhesives refers to adhesives that can be activated to attach strongly when needed but deactivated for easy and non-damaging detachment. There currently is a need to develop highly controllable adhesives that work optimally in wet environments, like those found inside the human body. Single fimH proteins, found at the tip of E. coli type 1 fimbria, form bonds with mannose that can be activated to bind strongly when pulled at high speeds, but are not activated under low speeds and as a result bind weakly. We hypothesize that thin-films composed of E. coli type 1 fimbriae can act as a nanoscale locking seatbelt, where they lock in at high speeds but not at low speeds, and that we can use mechanical force to control the adhesive behavior. Using atomic force microscopy (AFM) we tested the adhesive behavior between fimbriae thin-films and mannose covered surfaces. We observed that the force required to break the surfaces apart, as well as the number of activated bonds formed, increased the faster the surfaces were pulled away from each other. Also, by using the AFM to preload the bonds with high force, we observed that we could increase the number of activated bonds by a near 4 fold, as opposed to pulls with no preload. Inactivation after preload was also observed by allowing enough time for bonds to inactivate under no force, which decreased the bonds activated to only a 2 fold increase. These results show that these adhesive thin-films are capable of acting like a nanoscale locking seatbelt and can be controlled through mechanical force. An adhesive that can display these properties can have the potential to be incorporated into devices like pill endoscopy, giving an increased ability of controlled precise movement in fields like medical micro-robotics.

A cell’s outer membrane displays a broad range of proteins and other biomolecules that play an essential role in mediating interactions between the cell and its environment. Some cell surface molecules, known as antigens, can be used by clinicians and researchers to identify specific cells for use in diagnostics and targeted therapies. Cell identification based on surface antigens is known as phenotyping, which is typically performed in the clinical laboratory using a flow cytometer. In flow cytometry, cells are labeled with fluorescently-labeled antibodies that bind to a specific antigen on the cell surface in order to identify the cell’s phenotype. Conventional platforms for flow cytometry are costly, and fluorescence-based phenotyping involves tedious labeling steps and advanced detection capabilities. For laboratory facilities without the resources or need for a flow cytometry platform, a method for simple, specific, and direct cell characterization is desirable. The Ratner Lab uses silicon photonic technology in biosensing applications to detect when mass binds to a sensing surface. In this project, the sensing region of the silicon photonic chip was coated with antibodies to target a specific antigen displayed by a cell. A silicon photonic sensor was functionalized to phenotype prostate carcinoma cell lines for prostate-specific membrane antigen (PSMA). The cells were fragmented to yield membrane microparticles that could be flowed across the antibody-coated photonic sensor in order to characterize the cells for the presence or absence of the cancer marker. This study employed model cell lines as a proof-of-concept for simple, label-free cell phenotyping, departing from traditional fluorescence-based methods for cellular analysis.

Epigenetic analysis is a rapidly growing field of interest within the research community, due to its potential to advance our knowledge of the origins of a variety of diseases. It involves analyzing modifications of DNA as well as modifications of proteins associated with DNA, which affect overall chromatin structure and lead to potentially detrimental effects on gene expression. Aberrant gene expression has been shown to be involved in the development of diseases such as cancer, diabetes, and kidney disease. The most commonly used method for epigenetic analysis is chromatin immunoprecipitation (ChIP). However, current ChIP assays involve time-consuming and labor-intensive pre-analytical sample preparation and have low throughput, resulting in concerns about accuracy and sample loss. Thus, the current assays are limited in their clinical utility. The purpose of this project is to design an optimized protocol for DNA-protein crosslinking and chromatin shearing using novel high-throughput ultrasound technology for increased efficiency and reduced sample loss in ChIP. Various conditions were tested with different stages of ChIP, including DNA-protein cross-linking, and chromatin shearing. Effects of these conditions on the ChIP assay were analyzed utilizing a microplate-based matrix ChIP assay and quantitative polymerase chain reaction (qPCR). Efficacy of chromatin shearing was analyzed using gel electrophoresis. My findings will contribute to the development of a fully automated platform for chromatin shearing and ChIP, and lead to advances in epigenetic biomarker research of diseases that burden our society. Its application to human tissues will allow for quick and accurate evaluation of chromatin alterations and gene-bound enzymes, factors, and receptors in a wide variety of human biospecimens. It will also allow for discovery of innovative diagnostic and therapeutic biomarkers based on epigenetic pathways, and determination of genomic pathways that are associated with drug resistance development.

Current chemotherapies exploit the larger pore size of tumor vasculature to reach cancer cells. However, this rudimentary form of targeting offers low selectivity and carries the risk of severe systemic toxicity. In addition, chemotherapy distribution to tumors is limited by the large distance between blood vessels in tumors, increased interstitial fluid pressure, and physiological resistance by the extracellular matrix (ECM). With the goal of addressing these limitations, researchers have developed drug-bearing polymers of various sizes and architectures that selectively target tumor cells. The Pun lab previously produced a novel drug delivery polymer, the sunflower polymer, that can be utilized for anticancer drug-delivery. The sunflower polymer is a macrocyclic brush polymer that can be functionalized with a folic acid targeting ligand and an anticancer drug, doxorubicin, that is released in the acidic tumor microenvironment. In order to maximize the potential of anti-cancer drug-delivery polymers such as this, the impact of polymer size and architecture on tumor penetration must be better understood. This is because the efficacy of any anticancer drug, regardless of its mechanism of action, is limited by its ability to navigate the tumor microenvironment and reach all viable cells in a tumor. This project seeks to discover how polymer size and architecture impacts tumor penetration by using a 3D in vitro perfusion chip model of the tumor microenvironment. The model consists of microfluidic channels seeded with KB carcinoma cells and ECM, and has been optimized using model polymers of polyethylene glycol (PEG) and dextran. Tumor penetration is studied by perfusing fluorescently labelled polymers of various morphologies through the seeded microfluidic channels. The information obtained from this project could potentially be used to optimize the size and architecture of drug-delivery polymers for tumor penetration. In turn, these optimizations could present a significant contribution to the development of anticancer drug-delivery polymers.

The ability to recapitulate the dynamic presentation of signals in a stem cell’s microenvironment remains a major hurdle in tissue engineering. Controlling cell growth and differentiation in 4 dimensions (i.e., time and 3D space) would allow for heterogeneous synthetic tissues to be produced that match the complexity of their native counterparts. Combining strategies in both light-programmable hydrogels and recombinant protein engineering, we control the cellular microenvironment using proteins site-specifically modified with a bioorthogonal handle and able to covalently bind to a photocaged reactive group in the hydrogel. Previously we’ve demonstrated the ability to photopattern gels with fluorescent proteins that excite at different wavelengths, and have shown that multiple proteins can be patterned independently within the same material with 4D control. These techniques have many potential applications, including improving joint replacement. Current joint replacement therapies often involve use of metals, plastics, and ceramics, and commonly require future revision surgeries. We propose that through our techniques, we can generate a patterned bone/cartilage interface to improve joint replacement, creating a longer-term option for joint replacement. Towards this, we have generated two photopatternable recombinant growth factors, BMP-2 (bone morphogenic protein 2) and TGF-β (transforming growth factor β), known to direct human mesenchymal stem cell (hMSC) osteogenesis and chondrogenesis. I will encapsulate hMSCs in a hydrogel and direct cell differentiation and growth in 4D using a combination of photopatterned BMP-2 and TGF-β proteins. This research will have significant impact in tissue engineering, as it will enable recreation of complex physiological structures, grown outside the body with the patient’s own cells, that can be used for personalized medicine. Our approach is unique in that it allows for unprecedented control over microscale tissue structures, ultimately matching the complexity of native tissue.

Gene delivery has successfully been accomplished with the use of polycations, both in vivo and in vitro. However, a major problem in this category is the inherent cytotoxicity associated with a high density of positive charges. Polymers that can be degraded within the cells are typically better tolerated compared to their non-degradable counterparts. This research studies the use of disulfide bonds to reversibly connect cationic polymer moieties to a stable polymer backbone. Reducible and non-reducible, statistical co-polymers of 2-aminoethyl methacrylate (AEMA) and oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) were synthesized and evaluated for a comb-like, backbone chain. These monomers have been chosen due to their low cytotoxicity as well as their high degree of polymerization control in reversible addition-fragmentation chain transfer (RAFT) polymerization. Backbones containing 50 and 100 monomers per polymer have been successfully synthesized. The current phase of the project is to functionalize AEMA with a unique biomolecule called dibromomaelimide that contains two points for positively charged moieties. This final polymer will be used to transfect cells with genetic material to test its feasibility for gene delivery. Success of this project will create a well-defined polymer containing positively charged cations that are easily dispersed upon internalization by cells.

Optical coherence tomography (OCT) is an non-invasive imaging technique that is able to produce micrometer resolution images of human tissue. Building upon this imaging technique, the Wang lab has developed novel OCT based techniques to image the microvasculature of the human eye. With these high resolution OCT images, it would be possible to provide quantitiatve measurements of ocular features that can assess eye diseases non-invasively. These measurements would include blood flow rate, vessel density, vessel tortuosity, fractal dimensions, etc. The goal in this research is to determine which of these physical parameters in the eye can provide objective measures that correlate well with the severity of diabetic retinopathy (DR), the leading cause of blindness worldwide. Specifically, this research focused on five quantifiable metrics - vessel diameter, vessel area density, vessel skeleton density, vessel perimeter index, and vessel complexity index - and their correlations with the progression of the disease. The main hypothesis was that at least one of these parameters would show significant correlation with DR progression.Each of these metrics were quantified from OCT images of patients with DR using semi-automated computer software. Then, statistical analysis was done to calculate the correlations of each metrics with disease progression. Finally, an evaluation was performed to see which of these objective parameters can serve as sensitive biomarkers, possessing the potential to be used in diagnosing or monitoring the therapeutic treatments of diabetic retinopathy. 

Protein homooligomers, multibodied assemblies built from identical polypeptide chains, comprise a large fraction of known cellular proteins. Homooligomers prove to be particularly amenable for many biological applications; they hold the potential as oligomerization domains, often have enzymatic functionality as a byproduct of their oligomeric configuration, and can serve as structural scaffolds for bionanomaterials. While there exists a multitude of protein homooligomers in the Protein Data Bank, the finite number of existing homooligomers limits the potential for custom applications. Our current work involves designing novel cyclic protein homooligomers from a set of de novo designed repeat proteins that share no sequence or structural homology to any known protein structures. We chose these proteins because of their modular properties, large variety of topologies, and unprecedented thermostability. Using the Rosetta software suite, we generated a set of de novo homooligomer models by designing the oligomeric interface to direct self-assembly into a target configuration with two to six identical chains. After a round of refinement, we will express the designs in Escherichia coli and purify them by immobilized metal affinity chromatography. Their oligomerization state will be validated by measuring the molecular weight in solution by size exclusion chromatography paired with multi-angled light scattering and comparing it to the predicted molecular weight of the design. Designs that exhibit the desired molecular weight will be submitted to collaborators for small angle X-ray scattering data and X-ray crystallography. The exclusive use of de novo proteins in homooligomer design grants a greater control over the shape and stability by nature of the repeats, thus making one successful interface design useful for a multitude of shapes and sizes. This variability opens up a wide scope of scaffolds for bionanomaterial engineering.  

A barrier to quality healthcare in low-resource, rural areas is the lack of surgical care, and current anesthetic delivery devices either are expensive or require resources such as electricity. The Bioengineers Without Borders have proposed a low-cost, point-of-care anesthetic vaporizer, and seek a tool to assist in designing the device. To this end, a finite element model was constructed to predict the performance of a device in different operating conditions. We have imported the prototype’s geometry into finite element multiphysics software and added a domain representing the liquid anesthetic. Conservation equations and boundary conditions for mass, momentum, and heat transfer were applied to the domains of the device and numerically solved over clinically relevant time periods. Physical properties of the liquid halothane, stainless steel, and air were approximated using literature values of similar compounds. The model is currently being validated by comparing the vaporizer’s benchtop measurements of concentration and temperature to the computational model’s prediction. Computational results indicate that a vaporizer with no external heat transfer will experience a significant temperature and concentration drop, which in turn lowers the amount of anesthetic vaporized. When validated, the model will prove useful to engineers as proposed modification may be tested computationally before production, allowing for design refinement and less chance of device failure.

While there are currently drugs available to treat patients with human immunodeficiency virus (HIV), there is no known cure for this infection. As the HIV virus integrates into the host’s DNA, reservoirs of latent HIV are established. Because antiretroviral drugs only attack replicating HIV, the presence of these latent reservoirs prevents the eradication of the virus from the body. Once the use of anti-retroviral drugs is discontinued, the active virus reemerges in patients. The purpose of this project is to identify transcriptional and epigenetic pathways to reverse HIV latency, knowledge that could pave the way for new drug design to kill all existing virus in the body. To accomplish this goal, we are using a microplate-based matrix chromatin immunoprecipitation (ChIP) assay and quantitative polymerase chain reaction (qPCR) to measure more than one hundred different transcription and epigenetic events along the latent HIV genome upon its transcriptional activation. We applied cluster analysis to our data and have found that Protein Kinase A (PKA), a class of enzymes which phosphorylate proteins, and its target, the transcriptional factor CREB, are recruited to HIV genome. This finding, which has not previously been reported, suggests that PKA plays a role in HIV genome transcription, and if so, could be a possible path for developing pharmacological reversal of HIV latency.

Heart failure is one of the leading causes of morbidity and mortality worldwide. Heart failure (HF) is the inability of the heart to keep up with its workload, and at least half of HF patients suffer from decreased ventricular contractility or systolic dysfunction. Improving cardiomyocyte contraction is a potential therapy for diminished heart systolic function, but there is no current effective therapy that directly improves cardiomyocyte contraction. Previous studies from the Regnier Lab have shown that heart muscle exhibits a significant increase in contractility and force when naturally occurring 2-deoxyadenosine triphosphate (dATP) is used in place of adenosine triphosphate (ATP) as the substrate for contraction. Intracellular dATP levels can be elevated by the overexpression of the enzyme ribonucleotide reductase (RNR). In order to assess the efficacy of this alternative myosin binding substrate as a potential therapy, it is necessary to design a reliable and accurate method to quantify the dATP levels in cardiac tissue and/or cultured cells. The goal of this work was to design a protocol for tissue sample preparation to run using High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) for quantitative assessment of nucleotides. A standard curve was produced to allow for absolute quantification of the nucleotides. Finally, these methods were implemented to study the cardiac levels of nucleotides in a transgenic mouse that overexpressed the enzyme attributed for de novo dATP synthesis on dATP levels. Correlation of dATP levels and cardiac function can provide validation of the influence of elevated dATP to improve cardiac muscle contraction for use in studies of animal models of heart failure.

 Light Chain Amyloidosis (AL) is a disease defined by the deposition of aggregated immunoglobulin light chains in the form of fibrils in several organs. While these fibrils are insoluble, the soluble oligomers that precede them in the pathway involving unfolding and then aggregation of the protein are toxic to the cell. A novel secondary structure, alpha-sheet, was hypothesized to be the cause of toxicity. This structure is rarely seen in normal proteins, but it is populated in all peptides and proteins involved in amyloid diseases studied so far in our lab. In addition, the correlation between thermal stability and fibril formation has led to the study of the impact of amino acid mutations on the unfolding pathway. As is the case for most amyloid diseases, Light Chain Amyloidosis is incurable and fatal . The determination of specific patterns and mutations that destabilize the protein and lead to increased toxicity is important to better understand how to intervene. To this end, we are comparing two patient-derived light chains, one non-amyloidogenic and the other amyloidogenic. By analyzing their differences, we can determine regions of the protein that contribute to aggregation. We performed molecular dynamics simulations of each protein and analyzed their unfolding pathways and characterized the structure of the amyloidogenic interemediate associated with toxicity. The docking of pre-existing amyloid inhibitors developed in the lab to the resulting structures was done to determine a lead compound. Through rational and evolutionary design, several iterations were done on the lead compound and an inhibitor with an overall better binding score was developed.

Thrombotic microangiopathy is characterized by endothelial activation and microvascular thrombosis, and is associated with a group of life-threatening disorders, such arteriothrombosis, sepsis, and other small vascular diseases. With systemic inflammation, the arteriolar and capillary vessels are often found to be obstructed by platelet rich thrombi, leading to tissue ischemia and organ failure. Understanding the mechanism behind endothelial activation and induced thrombi formation during inflammation and vessel injury has important clinical implications that can lead to therapeutic development. Here, I developed a novel in vitro microvascular platform that mimics the in vivo arteriole architecture and function. This platform is generated based on a biocompatible polymer, polydimethylsiloxane, using injection molding. The device is lined with an extracellular matrix (ECM) using type I collagen for structural support. Human coronary artery smooth muscle cells (hCASMCs) are seeded within the bulk collagen to allow spontaneous reorganization and migration. A channel is created within the ECM using needle-based subtraction molding and is then seeded with human umbilical vein endothelial cells (HUVECs) to form an endothelium. Optimization was performed to provide consistent cell seeding of HUVECs within the channel, to allow cellular reorganization and alignment, and to form a functional small-diameter arteriole. We observed reorganization and alignment of both HUVECs and hCASMCs after 3 days of culture under continual flow. In addition, luminal patency was maintained with proper hCASMC contractile function. On-going studies include development of functional assays to recapitulate the inflammatory response and thrombi formation in arterioles. The study is expected to not only further elucidate the fundamental mechanism of arteriothrombosis, but to also provide a base platform to develop preventative or therapeutic medicine for those affected by these pathological phenomena.

Spinal cord injury (SCI) can severely impair quality of life. While there are a variety of accommodations for individuals with SCI, there are no clinically available treatments which restore pre-injury levels of function to these patients. The optimization of emerging treatments for SCI will require a large amount of detailed experimental trial data in animal models. Assessments of trained motor behavior in animal models of SCI recovery typically fail to capture information about fine gradations in the course of recovery, which interferes with the development of new restorative treatments. To better characterize these gradations during the study of an emerging method for cervical SCI rehabilitation, I monitored forelimb movements of a cohort of injured rats during a motor behavior task using a high-precision haptic interface device - a machine which simulates physical interactions with virtual objects or events by applying forces to its user. I used the motion information gathered by this device to trigger electrical stimulation of the spinal cord to enhance attempted movements and encourage Hebbian plasticity within the spinal cord. Functional recovery of strength and range of motion was assessed relative to pre-injury baseline, demonstrating the utility of 3D haptic interfaces for the assessment of promising SCI treatments. Our goal is to help individuals with disabilities due to spinal cord injury regain function and independence by quantifying, optimizing, and the accelerating the translation of potential new treatments for SCI using these precise and clinically relevant recovery measures.

Over a quarter of a million individuals live with Spinal Cord Injury (SCI) in the United States and experience debilitating chronic conditions, reduction of quality of life, and an accumulation of high healthcare costs. Recent clinical studies have shown that epidural spinal stimulation can lead to improvements in motor function, weight bearing, and autonomic dysreflexia among patients with SCI. It is thought that epidural stimulation creates an excitatory environment in the spinal cord and allows descending signals that were previously too weak to effect a motor output to reach a supra-threshold level. While the results are promising, both the mechanism of action and optimal parameters for stimulation and rehabilitation remain unknown. Therefore, in order to further develop this potential therapy, it is necessary to create a clinically relevant research model for the study of the treatment in question. This project addresses this through the development of an automated rehabilitative device for animal subjects following spinal cord injury, designed to mimic the extensive rehabilitation received by human patients with SCI and measure functional recovery following injury and during epidural stimulation treatment. Initial results show that subjects interact with the automated device, indicating a potential for more widespread implementation. Specifically, this high-throughput and automated system will be initially utilized for the study of the effects of epidural spinal stimulation in the rat cervical cord following contusion injury in order to quantify both transient and lasting motor improvements.

The primary function of the kidneys is to filter waste from blood, and the kidney glomeruli are the major site for filtration. The glomerulus is a blood filtration barrier with low resistance to small molecules, but is relatively impermeable to macromolecules. Each glomerulus consists of a blood capillaries network with endothelial cells on a basement membrane, the other side of which is lined with specialized epithelial cells known as podocytes. The integrity and function of the glomerular filtration barrier is critical for kidney function, and disruption of this barrier correlates with kidney disease progression, and eventually kidney failure. Current research models for kidney disease include animal models, 2D, and 3D kidney epithelial cell cultures. However, animal models suffer from high cost and are too complex for studying detailed cellular interactions, while existing in vitro models lack the architecture and components to fully reproduce in vivo conditions. We propose a 3D microphysiological system that recapitulates the endothelial-epithelial interface found in the kidney glomerulus. We have developed a method to form independent microscale channels within a collagen I substrate, separated by a very thin layer of collagen. Collagen I is a native matrix protein that supports cell growth, invasion, and exchange of cellular signals. Primary endothelial and epithelial cells has been seeded into the channels to form closed lumina, whose overlaps form the endothelial-epithelial interface. However, culture conditions remain to be optimized to support both types of cells. Structure, morphology, and function of the interface are monitored by confocal microscopy, biochemical assays, and electron microscopy. When fully developed and validated, this culture system can be used to study the transport properties and injury responses of glomerular filtration. It can also provide a platform for drug screening to identify compounds toxic to the kidney, or therapeutic compounds for kidney diseases.

Bacterial endocarditis is an infection on the inner lining of the heart. When studying bacterial adhesion, researchers often ignore that blood is pumped through the heart in a pulsatile manner. Because of this, all studies have been done with constant flow. We hypothesize that pulsatile flow conditions play a critical role in this disease. We studied how Streptococcus gordonii, a strain of bacteria that is known to cause bacterial endocarditis, bound in constant and pulsatile flow. The rate of adhesion in both constant and pulsatile flow was measured at different shear stresses. When suspended in buffer, the bacteria bound at the same rate in both flow conditions. The overall trend showed the rate of bacterial adhesion was highest at 10 dyne/cm². At the shear stresses predicted to be present within the heart, 20 – 80 dyne/cm², only about 10% of bacteria bound as compared to the peak value. In a solution with 50% buffer and 50% red blood cells (RBCs), the percentage commonly found in the blood, there was a dramatic change in the trend of bacterial adhesion in constant flow. Instead of having a peak value, the rate of adhesion increased with the level of shear stress, allowing for a much higher rate of binding in the 20 – 80 dyne/cm² range. We were unable to repeat this experiment using pulsatile flow due to limitations in our flow device but this could mean a dramatic change in the trend observed in pulsatile flow as well. We have created a new device to further investigate our hypothesis and better simulate the conditions found in the heart.

Antibodies play a critical role in our health system, adding specificity to diagnostics and therapeutics with their strong binding capabilities. Unfortunately, the binding cannot be controlled over time, introducing contamination of the target and preventing further testing with the bound sample. We propose an alternative to antibodies: a switchable binding protein. This protein has affinity for its binding target which can be modulated by environmental change without directly altering the binding site, otherwise known as allosteric regulation. This presents the potential to later change the binding target to a medically relevant antigen. A binding protein FimH, found on the end of bacteria pili, was used as the scaffold for the design because its binding depends on a significant structural change. I predict that if this protein is locked in place with a cross-linker or covalent bond, which experiences a large structural change under a simple external stimulus, FimH binding could be precisely triggered. Inserting a disulfide bond or a photoswitchable linker are two methods I am investigating with their binding triggers being dithiothreitol (DTT) and ultra-violet (UV) light, respectively. The photoswitchable linker design is ideal because it would induce reversible binding, allowing for reuse of our protein. Using past structural knowledge, multiple mutation designs are being created and their binding tested under external stimulus. The design requires mutation placed far from the binding site in order to create this allosteric switch. Initial results indicate the design with a disulfide bond between residues 112 and 189 does switch binding affinity, triggered to unbind, when DTT is added. This research also has the potential to contribute a novel approach to regulating large proteins and add specificity in diagnostics by concentrating the target.

The University of Washington graduates 74% of its students: meaning that 26% of students do not complete their studies. Students who churn (or “drop out”) from the University of Washington end up spending a significant amount of time and money and ultimately receive no benefit. State governments and the federal government together spend ~$2.9 billion per year in grants and loans to first year students who will not return to school, representing a significant waste of taxpayer dollars. The first step in improving student retention at the University of Washington is identifying the students who are at the greatest risk to churn from the school. This information can be passed to counselors or department specific administration staff who can then target retention efforts at the students who need it most. The work is twofold: first, machine learning algorithms were trained on University of Washington transcript data from the period of 1970 to 2013. The feature set was loosely classified into four categories: classes taken, class performance, demographic data, and high school performance. These algorithms provided an 87% prediction accuracy of student dropouts. A feature engineering approach is now underway to further improve performance. This involves expanding the feature set by creating composite features and modified features. Composite features will allow the machine learning algorithms to weight relationships between features in addition to the features themselves (e.g. an engineering student with a poor math GPA may be more likely to churn). Modified features include some mathematical function of the original feature (log, square, multiplied, etc.). This allows for different (and potentially much greater) feature weights without suffering the penalties imposed by algorithm regularization. These two approaches to feature engineering will allow for an improvement over the previously observed 87% accuracy.

The Joint United Nations Program on HIV/AIDS (UNAIDS) reported in 2014 that approximately 36.9 million people in the world are currently living with HIV/AIDS. Many of these individuals reside in low and middle income countries where access to STD prevention methods is limited and risk of infection is high. Electrospun nanofibers are a promising drug delivery platform in development, with the potential to provide either pericoital or sustained protection at an affordable cost. Here, we fabricate and characterize nanofibers composed of polyvinyl alcohol (PVA) and agarose (AGR), a naturally occurring polysaccharide that is both mechanically strong and biocompatible. Previous studies in our group have reported challenges in electrospinning polymer blends from AGR and PVA, likely due to the high gelling properties of agarose. Subsequently, a non-uniform distribution of fiber diameter resulted in variable mechanical properties with an undesirable burst release of hydrophilic small molecule drugs (tenofovir, TFV). In this study, the electrospinning process was improved to achieve higher AGR loading in the nanofibers. We used a melt-electrospinning technique where the polymer solution was heated at a constant temperature of approximately 60°C, which is below the glass transition temperature of PVA. Constant heating of the polymer solution reduced AGR gelation and improved fiber throughput. The melt-electrospinning process enabled fabrication of nanofibers with up to 30% AGR content. Mechanical testing on the nanofibers revealed that the Young’s Modulus was approximately 100 – 200 MPa. Drug release studies revealed that AGR loading in the nanofibers did not significantly affect the burst release of TFV from the fibers. Fiber properties indicate that the PVA-AGR system must be optimized to further validate experimental results. In general, the melt-electrospinning process was key to enable the fabrication of PVA-AGR nanofibers with increased AGR content, which allows for enhanced characterization of these fibers as an antiretroviral drug delivery platform.

Many biological, socioeconomic, and cultural factors increase women’s risk of acquiring HIV, especially in low-resource settings. Currently, there aren’t any options that allow women to discreetly protect themselves from HIV acquisition. To address this, we developed a topical nanofiber mesh to be inserted into the vagina to deliver antiretrovirals for HIV prevention. Benefits of this dosage form include high drug loading, tunable drug release, and imperceptibility to users while protecting them from HIV over the course of a week. We designed a “fiber-in-fiber” system: the “burst” release nanofiber delivers an initial bolus of drug, while the “sustained” release nanofiber (polyester) prolongs drug release over a week. Nanofibers are synthesized via electrospinning, which involves exposing a polymer and drug solution to an electric field to create a nanofiber mesh. After polyester fibers are electrospun, they are micronized using a food processor and electrospun directly into the burst release fibers. The final product is a burst release nanofiber mesh that contains small polyester fiber particles. Initially, the polyester fiber particle sizes were large and variable, ranging from 8 to 2000 micrometers. After optimizing the micronization process, the median particle size was reduced to 179 micrometers. We compared this to the median particle size of post-electrospinning, which was 26 micrometers. The data indicate that smaller nanofibers are more likely to be electrospun into the fiber-in-fiber mesh. Thus, minimizing particle size is essential to achieve the desired drug loading. Furthermore, loading the micronized fibers with a fluorescent dye revealed that they are evenly distributed throughout the burst release fiber mesh. Recent experiments assessed the drug release profiles of this fiber-in-fiber system, which showed that it is able to sustain drug release. Evaluating the drug release benefits of this fiber-in-fiber system will allow this system to be applied to other topic drug delivery systems.

Francisella tularensis and Burkholderia pseudomallei, the agents of tularemia and melioidosis, respectively, have been identified by the Centers for Disease Control and Prevention (CDC) as bioterrorism agents that could lead to mass casualties and severe threat to public health. These pathogens are highly infectious and aerosolizable intracellular alveolar pathogens that can cause fatal respiratory tract infections. The intracellular compartmentalization of these pathogenic organisms within alveolar macrophages is a significant barrier to bacterial clearance and contributes to their associated morbidity and mortality. Currently, there is no effective treatment to eradicate melioidosis and tularemia other than prolonged antibiotic therapy and even with several months of intensive antibiotic treatment, complete clearance of these pathogens is not guaranteed. Therefore, there is an urgent clinical need to develop new therapeutic drug nanocarriers that can deliver antibiotics intracellularly to alveolar macrophages to increase treatment efficacy of tularemia and melioidosis. The goal of this project is to design biodegradable and nontoxic polymeric nanoparticles with various drug release rates and drug loading densities to overcome pathogens’ drug resistance mechanisms. The nanoparticle scaffolds will be synthesized using Polysorbate 80 via Thiol-ene and Thiol-Michael “click” reactions to maintain biocompatibility while incorporating functional groups that are amenable to chemical modification for enhanced drug conjugation. Once the nanoparticle scaffolds are characterized, they will be conjugated to antibiotics such as ciprofloxacin via hydrolytically degradable ester bonds. Drug release profiles will be characterized in serum containing media via reverse phase HPLC to evaluate their efficacies. Once ciprofloxacin is conjugated to the nanoparticle scaffolds, in vitro studies will be conducted to evaluate the polymeric nanoparticles’ therapeutic efficacy and toxicity in co-culture macrophage models of bacterial infection. Development of these polymeric nanoparticles will lead to rapid clearance of tularemia and melioidosis with shorter antibiotic administration while reducing the chances of relapse and antimicrobial resistance.

Point of care diagnostics devices are aimed at addressing health disparities in low resource settings. They must be cheap, easy to use and offer quick results. Nucleic acids (DNA, RNA) can be used to identify infectious pathogens, and employ molecular diagnostic tools such as PCR to do so. PCR is a process which utilizes DNA Denaturation, where the hydrogen bonds that form between DNA and complementary strands break under various temperatures, forming to single strands of DNA. The melt temperature, the point where half of a given solution is single stranded DNA, and half of the solution is double stranded DNA, can be calculated by using fundamental thermodynamic equations. Our goal is to create a lab teaching module in which students can perform experiments measuring the melt temperature of various pieces of DNA with its complementary strand. Learning Objectives were produced, and a lab was designed in order to fulfill each objective. The module consists of a prelab portion, in which students review the basic concepts behind thermodynamics, read and analyze scientific papers, and calculate melt temperatures (by using thermodynamic values as well as industry standard software). The actual lab portion consists of an experiment which tests the effect of DNA length on melt temperatures. Students will then interpret and analyze the melt curves generated in lab. Lastly, during the post lab portion students will brainstorm future experiments and solidify their understanding of how thermodynamic tools can be used to measure various nucleic acid processes, namely denaturation.

Hydrostatic pressure is important to many vascular processes, including endothelial activation (inflammation), vasodilation, vasoconstriction and angiogenesis (branching of new blood vessels from existing ones). The problem of pressure regulation in in vitro studies is usually solved by syringe pump based systems. Harvard Apparatus provides a catalogue of “Constant Pressure Syringe Pumps,” the cheapest of which costs $4500. This is prohibitively expensive for many applications. Thus, there is a need to create an affordable device that can reliably regulate hydrostatic pressure. I present a syringe pump based system that maintains liquid pressure within a flow chamber at a fraction of the cost of commercially available devices. My pump was designed from open-source and off the shelf components, including a pressure transducer and Arduino Microcontroller, at a total cost of ~$350. I have tested the pump’s ability to maintain constant liquid pressure across a high-impedance microfluidic system and have shown that it maintains pressure at a user-defined set point between 0 and 70 cmH2O of gauge pressure. This device illustrates the power of open-source software and hardware, 3D-printing and rapid prototyping to allow labs to design customized hardware to meet specific research needs at a reasonable price. Once completed, I will use it to study the pressure dependence of angiogenesis and vasculogenesis (formation of new blood vessels by endothelial cells embedded in interstitium). We will vary hydrostatic pressure to optimize and document blood vessel network formation. This is relevant to studying embryonic development and tumor vascularization and to engineering grafts to repair the damage from myocardial infarction (heart attack). Other possible applications include eliminating the confounding variable of pressure variation in Doppler echocardiography or in studies of endothelial cell response to variation in microvessel geometries.

Extracellular matrices are more than structural scaffolds used merely to provide mechanical support to cell and tissue organization—rather, they provide physical and biochemical signals to change the cell structure and function. Here, my project is to investigate on the diffusion properties of human kidney extracellular matrix (kECM) and their impact on proximal tubular epithelial cells (PTECs) in an engineered 3D physiological system. Outcomes of the project include: 1) diffusion coefficients of dextran of different sizes in the matrices made from pure type I collagen, pure kECM, and mixtures of type I collagen and kECM (3:1, 1:1, and 1:3). 2) A standard protocol and an assistive Matlab program for diffusion measurement and vessel perfusion experiments. 3) characterization of structure, morphology and transporters (Immunostaining) of proximal tubular epithelial cells inside the flow-derived microphysiological system. I expect the study to provide supportive understanding of cell-matrix interactions and facilitate the further development of the bioengineered human kidney microphysiological system to study kidney function and response to drugs and toxins.

Cardiovascular disease is the leading cause of death worldwide due largely to the inability of the heart to respond effectively to injury through tissue regeneration. Coronary artery narrowing initiates cessation of arterial blood supply to the heart generating an ischemic injury, characterized by low oxygen and nutrient supply and the accumulation of metabolic waste. Currently, research approaches to study ischemia rely on high-cost and low-throughput in vivo models. As such, there is a need for an in vitro platform for mimicking ischemia/reperfusion injury in a three dimensional cellular environment. The research and design pursued here addresses this deficit in modeling capabilities by developing an in vitro ischemic injury and reperfusion model utilizing engineered cardiac micro tissues. Cardiomyocyte microtissue particles (MTPs) are formed through centrifugation of a single cell suspension of stem cell derived cardiomyocytes without scaffold materials into polydimethylsiloxane micro-wells. Ischemic injury is induced in MTPs by exposing tissue cultures to mineral oil layers on top of small volumes of spent culture media to restrict oxygen supply and metabolic waste exchange. This simulated injury results in an observable reduction in live cells as evidenced by a decreased live cell fluorescence signal associated with duration of injury from a calcein and EthD-1 live/dead stain. Optimization of this staining protocol for characterizing the extent of the induced injury in an adapted plate reader format is currently underway. Thus far, percent of live cells has been shown to positively correlate with normalized calcein fluorescence intensity and current efforts are directed towards increased efficacy of EthD-1 dead cell marking and sufficient tissue infiltration of the stain. Implementation of this protocol will create a baseline metric for monitoring the state of the engineered cardiac tissue during injury and reperfusion and provide a foundation for development of an accurate and cost effective cardiac ischemia injury model.

In developed regions, laboratory-based molecular assays are routinely performed to enable early diagnosis and improve patient outcomes. However, several key technology gaps prevent the widespread use of molecular assays in low-resource areas. For instance, the lack of trained laboratory personnel in resource-poor settings can limit their capacity to perform labor-intensive tests. Additionally, many commonly used enzymes (e.g. polymerase and ligase) require costly cold chain storage. To simplify molecular assays for use in sub-optimal laboratories, we are developing a dried reagent platform for drying, storing, and dispensing test reagents. Our current work is focused on optimizing the platform for PCR and ligation, which are key steps in the Oligonucleotide Ligation Assay (OLA)-based HIV drug-resistance test being developed by the Frenkel lab (Seattle Children’s Research Institute) and the Lai and Lutz labs (UW Bioengineering). Multiple formulas containing preservatives up to 10% w/v were evaluated based on their assay compatibility and enzyme-stabilizing effect during freeze-drying. We found that the presence of trehalose and poly(ethylene glycol) in the dried reagent mixtures best maintained assay performance for both PCR and ligation. Preliminary data also showed that ligation performance of the new formula with the dried reagent platform was comparable to the performance of freshly prepared mixtures and that the dried reagents remained stable after 28-days of ambient storage (<15% relative humidity, 20-25°C). Long-term storage studies with the preserved PCR mixture are still in progress but we have demonstrated that lyophilized mixtures stored overnight produce amplified products in the same range as those from freshly prepared PCR mix. Future work will aim to achieve dried reagent stability up to 3-months and incorporate the platform into the first prototype of our drug-resistance test. Ultimately, the platform will reduce user manipulation and assay preparation time for the OLA and could be expanded to simplify other complex molecular assays.

In the United States, kidney cancer has consistently been in the top 10 most prevalent cancers with 62,700 new cases and 14,240 fatalities each year. Renal cell carcinoma (RCC) derives from the epithelium of the kidney’s proximal convoluted tubule, and comprises the overwhelming majority of all kidney cancers. A critical step in the formation and spread of all cancers, including RCC, is angiogenesis: the formation of new blood vessels. Cancerous cells secrete numerous signals to recruit endothelial cells to form new blood vessels; these vessels provide primary tumors with oxygen, nutrients, and a path to metastasize throughout the body. While cell and animal models have been utilized to study angiogenesis and test anti-angiogenesis drugs in RCC, successes fail to translate in clinical human studies. Because of the added complexity of the human body, a better model to represent human RCC is required to study angiogenesis in vitro. In order to address this need, we are developing a tissue engineered vascularized RCC microphysiological 3D system. Microfluidic devices will be utilized to recapitulate the angiogenic interaction between blood vessels and RCC cells. Sprouting from the endothelial-lined channel, simulating kidney microvasculature, toward collagen suspended RCC cells will be analyzed to determine the efficacy of our device. In addition, we have unprecedented access to normal and cancerous tissue sample pairs from the same kidney cancer patient at the UW Medical Center. Cultures from the tissue sample pairs will be utilized to validate the device and display differences in angiogenesis between RCC and normal kidney tissue. Tissue pairs were also utilized in qRT-PCR to determine up regulated pro-angiogenesis genes in RCC. Commercial drugs along with those currently in development or clinical trials will be administered to our device to determine their efficacy in preventing angiogenesis in RCC.

The plasma protein von Willebrand Factor (VWF) is secreted by vascular endothelial cells. VWF’s primary function is to recruit platelets to the site of blood vessel injury. The interaction between VWF and platelets happens through binding between the VWF A1 domain and the platelet surface receptor, glycoprotein Ibα (GpIbα). VWF requires shear to be activated. Recently, it has been shown that oxidizing agents increase the platelet-binding activity of VWF, although the exact molecular mechanism is not understood. To explore whether neighboring domains of A1 play a role in the oxidant-induced activation of VWF, we used a flow chamber microscopy assay. Two experimental constructs of VWF, one containing the A1 domain and the neighboring A2 and A3 domains, the second containing A1 and the neighboring D`D3 domain were expressed and bound to separate plates. One control construct containing only the A1 domain was also expressed and bound to plates. Then GpIbα coated beads were washed at different shears over plates that had either been pre-oxidized or not. The movement of beads was recorded. Subsequently, rolling velocities and total number of beads were measured and analyzed using MATLAB. The results showed that, at high shear, both the A1A2A3 and the D`D3A1 constructs were more active in binding to beads when oxidized compared to the unoxidized controls. The control construct did not show a difference in binding to beads when oxidized compared to unoxidized controls. These findings suggest that the A2, A3 and D`D3 domains could have an inhibitory function on the A1 domain and that oxidation removes this inhibition resulting in an increased binding activity of VWF to platelets. Understanding how oxidation alters the function of VWF domains is necessary to explain the increased risk of thrombosis in patients suffering of chronic or acute inflammation.

Vessels of different organs have heterogeneous permeability to serve distinct physiological functions. Differences in basal vascular permeability may result from heterogeneity in the endothelial cells that line the vessel walls, which are influenced by many factors—including differences in the complexity of cell-cell junctions and the expression of various diaphragms that span the cell body. In addition, many diseased states are often associated with elevated levels of permeability, including viral hemorrhagic fever and tumor-induced angiogenesis. To understand such fundamental heterogeneity of the blood vessels and further investigate the change of vascular structure and function in disease scenarios, we characterized the permeability of endothelial cells harvested from different areas of the body. Human primary cells—including endothelial cells from various organs and epithelial cells from the proximal tubules of the kidney (PTECs)—were seeded on gelatin-coated transwells. We measured transendothelial electrical resistance (TEER) using a Millicell ERS Voltohmmeter, and determined solute permeability by tracking the perfusion of fluorescent dextran across the cell monolayer over time. Immunostaining was performed to observe monolayer formation, while transmission electron microscopy (TEM) was utilized to observe ultrastructure. Preliminary TEM images demonstrate distinct morphological differences between endothelial cell types, specifically in the complexity of cell-cell junctions. Investigations into the impact of these morphological differences on vascular permeability are ongoing, and a definitive conclusion will be made upon the study’s completion.

Point-of-care diagnostic tests allow clinicians or care providers to make rapid, informed treatment decisions in settings where patient follow-up is unlikely or not an option (i.e. rural regions in developing countries). The Anisotropic Ratchet Conveyors (ARC) platform developed in our laboratory can transport small, individual droplets with a passive surface pattern and orthogonal vibration. In this work, a novel ARC system is characterized for future use in automated point-of-care diagnostic devices. Effects of rung radius on droplet transport were characterized with respect to the acceleration amplitude of the vibration required to initiate transport. The acceleration amplitude was significantly lower on the 1000 µm radius and 1500 µm radius rungs compared to the 500 µm radius rungs and slightly lower on the 1000 µm radius rungs compared to the 1500 µm radius rungs. Interestingly, the radius of the rungs was also observed to influence the natural frequency of the transported droplets. The results of this work will be applied to optimize droplet transport across functionalized detection regions on chip. Thus, ongoing work aims to functionalize the silicon dioxide rungs for immunosensing. In order to demonstrate proof-of-concept immunosensing in droplets, antibodies (e.g. antibodies targeting human immunoglobulin gamma - anti-IgG) will be bound to the silicon dioxide rungs using a linking molecule (e.g. silane-PEG-NHS or APTES) via micro contact printing, a common technique used to transfer proteins to a surface with elastomeric stamps. This process will be optimized to control the density of bound antibodies. Fluorescent microscopy will be used to verify and characterize the capture of fluorescently tagged target molecules (e.g. FITC-IgG) suspended in droplets transported on ARC tracks. Experiments will also be performed to characterize how antibody capture affects the velocity droplets transported on rungs of each radius group. The ability to capture target molecules suspended in droplets will present the potential of using ARC devices as a platform for rapid, efficient healthcare diagnostics.

The prefrontal cortex has the potential to play an important role in the development of future brain-computer interface technologies, which have the potential to greatly improve mobility and quality of life for people who suffer from central nervous system injuries or diseases . Most brain-computer interface systems collect neural signals from the motor cortex to control the effector; while this has long been the standard in the field, this approach is not viable if the motor cortex is damaged (e.g. by traumatic brain injury or stroke). The prefrontal cortex plays a role in executive function; it has signals that modulate with motor planning, is susceptible to operant conditioning, and its activity could possibly control brain-computer interfaces. This project aims to explore the efficacy of volitional control of local field potential signals in the prefrontal cortex for potential brain-computer interface applications. Two macaques were bilaterally implanted over the prefrontal, premotor, and motor cortices with dual surface-and-depth electrodes to collect differential local field potential signals. Through repeated training sessions with operant conditioning, the macaques learned to control the cursor on a computer screen, performing center-out tasks by modulating the frequency of local field potentials in the prefrontal cortex. These initial results show that volitional control of local field potentials in the prefrontal cortex is possible. My role in this project is to both collect and analyze data. Current and future work includes data collection from another macaque and examination of interactions between cortical sites during task performance.

M. tuberculosis (TB) infects one third of the global population and causes 1.5 million deaths annually. Most of the disease burden occurs in low-income countries. Although TB is curable, the lack of proper diagnostic tools in developing areas restricts treatment and propagates the spread of this highly contagious airborne disease. Currently, the gold standard for pulmonary TB detection is a sputum culture, which is not only time intensive, invasive, and requires a clinical setting with laboratory analysis, but also increases risk of transmission and is difficult to obtain. In collaboration with Dr. James Lai of UW Bioengineering and Dr. David Horne of UW Global Health, we are developing a noninvasive and highly sensitive TB test for point-of-care settings. We propose a sequence specific capture modality of TB transrenal DNA (trDNA) in easily collected urine samples. Recent literature has shown the ability to detect TB trDNA in patient urine samples, but the current methods target long trDNA fragments which are unlikely to pass through the renal system and lack both sensitivity and specificity. As a result from specific DNA capture and targeting short DNA fragments, the proposed protocol allows detection of TB trDNA fragments from urine at a thousand-fold lower concentration.

Mussels are bivalve mollusks that construct a byssus – an assemblage of protein threads with a strong adhesive plaque that attaches to underwater substrates. A mussel’s survival relies on a strong byssal attachment. Research on this adhesive can help explain trends in mussel attachment and inform how to emulate this glue or design surfaces that mussels cannot adhere to. Our work seeks to understand how abiotic factors affect the properties of mussel attachment, particularly at the thread-substrate interface (plaque). Specifically, we wanted to understand how increased temperature, decreased _pCO₂, and maturity (age) of the plaque affect its mechanical properties. This study involved three local species of mussel, as well as hybrid mussels between two species. The degree of hybridization was determined through PCR analysis. Byssal threads were cut from the animal and allowed to soak in a variety of treatments, and then stress-strain curves were generated for individual plaques. Initial findings indicate no difference in plaque strength after treatment in high temperature seawater. The results of this work could help explain trends in seasonal dislodgement of mussels, which are relevant ecologically and for aquaculture businesses. It can also give us insight into how changing environmental factors associated with ocean acidification will affect mussel attachment, independently of how they affect the organism itself.

Studies have shown that Electrical Stimulation (ES), in the form of electric or electromagnetic field can enhance wound healing by promoting cellular proliferation and expression of growth factors. We hypothesized that ES mediated by negative electric charge is predominantly effective in inducing cellular rehydration of dehydrated mammalian cells, resulting in increased viability and survival. This was tested by analyzing the effects of ES induced in a cellular model through negative electric charge build-up in a parallel plate capacitor system. The system consisted of two identical gold-plated surfaces separated by two polytetrafluoroethylene pillars. Electric charge built up on the plate was manipulated by altering the voltage of the power supply connected in turn to the gold plated surface. Experiments were conducted on the commonly used standard cellular model of mouse embryonic fibroblast NIH/3T3 cells under sterile conditions to reduce extraneous variables. The strength of ES was predetermined through a quantitative and qualitative analysis of temperature, pH, and bubble formation in different standard solutions. Cells challenged by partial dehydration with an established range of sodium chloride concentrations will be subjected to the predetermined ES and transferred to different cell culture media- Phosphate Buffered Saline (PBS) or Dulbecco's Modified Eagle Medium (DMEM). Cellular viability will be analyzed by Trypan Blue dye-exclusion method. In this way, we will determine whether negative charge promotes viability.

Factors such as temperature, pressure, presence of a catalyst, and reactant concentration are all known to affect the rate of chemical reactions and are well exploited in industry to drive chemical synthesis to favor certain mechanistic pathways. Here we consider the effect of using magnetism to control the rate of reaction for the Briggs Ruascher reaction. This reaction was chosen as a model for magnetic manipulation because it is mediated by both radical and non-radical pathways that compete with oscillatory behavior to produce the same product. Dramatic color changes serve as a visual cue to indicate which path is dominating. Here we put forth magnetic radical arrest hypothesis for the Brigg’s Ruascher reaction. It is known that electrons act like tiny magnets that pair with opposite spins. Because radical species carry a lone unpaired electron, presumably their magnetic fields are not concealed. We proposed that due to the exposed nature of the unpaired radical electrons in the Briggs Ruascher reaction, the progress of the reaction would be affected by the application of a magnetic field. Upon exposure to strong magnets, the radical intermediates of the Briggs Ruascher reaction were expected to aligned their spins with the applied magnetic field affecting their reactivity. Several types of neodymium magnets with various strengths were applied to the reaction in order to determine whether magnetism could prevent or greatly reduce the velocity of the color phase changes for the radical mediated portion of the reaction. We also built an electromagnet to expose the reaction to in order to achieve even greater field strength. The Brigg’s Rouscher reaction was found to be significantly robust and there was insufficient evidence to suggest that the reaction was affected by neodymium fields below a range of 5.8 mT. Results of exposure to the electromagnet are still in progress. The color changing cycles of the Briggs Ruascher reaction were found to follow a decreasing sigmoid mathematical model before going to completion after 9-10 oscillations.

Current tissue-engineered vascular grafts (TEVG) lack physiologically relevant internal structural organization which limits their mechanical properties and contractility. It has previously been shown that through the utilization of anisotropically nanopatterned scaffolds, vascular smooth muscle cells (vSMC) can be manipulated to express enhanced contractile phenotypes. We hypothesize that by using vSMCs cultured on nanopatterned surfaces we can create a TEVG, which exhibits native internal structure, that has increased mechanical properties and contains vSMCs with increased expression of contractile markers compared to a graft which lacks internal organization. In this study, we developed a flexible thermoresponsive nanofabricated substratum (fTNFS) which allows for the fabrication of multi-layered cell tubes with well defined internal structural configurations and diameters. Using the fTNFS, individual cell sheets can be stacked into a multi-layered tissue construct which can then be wrapped into a cylindrical shape. The cell tube structure can then be stabilized using a hydrogel. Here, we fabricated multi-layered vSMC tubes with structural characteristics similar to native blood vessels. The TEVGs were then immaged using immunofluorescence microscopy for expression of contractile proteins. The  vSMC tubes produced in this project maintauned internal structures analogous to native blood vessels and expressed contractile proteins.

Duchenne muscular dystrophy (DMD) is an incurable disease affecting approximately 1 in every 3600 males in the United States that is caused by a genetic mutation leading to the loss of dystrophin expression in muscles. Dystrophin is a protein that links the cytoskeleton of muscle cells to the extracellular matrix (ECM), and a lack of dystrophin leads to extensive damage to muscle fibers and subsequent loss of muscle function. Proposed methods of treatment, such as direct stem cell injections, have not been effective due to poor cell viability and difficulties associated with the culture of muscle stem cells in vitro. However, the development of implantable, vascularized and functionally mature skeletal muscle tissue patches may be able to address some of these issues. These tissues are made by growing primary muscle cells on biodegradable nanopatterned scaffolds that have been functionalized with sphingosine 1-phosphate (S1P), a potent angiogenic and myogenic biomolecule. The nanopatterning of these scaffolds is designed to mimic the nanotopography of native muscle ECM such that the anisotropic alignment and maturation of cultured muscle cells is induced. With this combination of topographical and biochemical cues, there was an observed increase in the formation of mature, contracting myotubes and the presence of differentiated endothelial cells. Furthermore, increased expression of myogenic and angiogenic genetic markers were found in cells cultured on substrates functionalized with S1P. Current work is focused on the validation of these results with cells featuring a knockout of the S1P receptor 1 gene. Lack of this receptor should lead to S1P-functionalized substrates having little to no effect on cell differentiation and tissue maturation, thereby illustrating the importance of S1P on the observed results. With further research and development, these tissue constructs may be used in the future to treat chronic muscle diseases such as DMD.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by mutations in the gene that encodes the protein dystrophin. This disease is incurable and causes severe muscle weakness, respiratory failure, cardiomyopathy, and early death. Understanding the causes of DMD and developing new treatment strategies relies on in vitro models. However, current animal and cell based disease models of the cardiomyopathy experienced by patients with DMD do not accurately recapitulate the human presentation of the disease. Stem cell derived cardiomyocytes provide a platform for improved and personalized disease modeling, however the cells are functionally immature, exist in two dimensional cultures, and do not experience the mechanical stresses of the human heart. There is a need, therefore, for a way to generate three-dimensional constructs of cardiomyocytes and subject them to physiologically relevant mechanical loads for the purpose of promoting maturation and disease phenotype testing. Induced pluripotent stem cells (iPSC) derived from the urine of healthy and diseased patients were used to generate cardiomyocytes in vitro (iPSC-cardiomyocytes). Differentiations of stem cells were enriched using metabolic methods, allowing us to generate cardiomyocyte cultures with greater than 90% purity. Three-dimensional cardiomyocyte constructs were generated in polydimethylsiloxane (PDMS) molds using a cardiac-stromal co-culture method with a combination of collagen and geltrex. In order to further replicate the human heart, a device was designed and fabricated that was capable of applying controllable, uniaxial mechanical loads to the three-dimensional cardiomyocytes and was used to measure the force generation of DMD or healthy iPSC-cardiomyocytes. Future studies involving the device will increase our understanding of the pathology of DMD and allow for novel, personalized treatments to be developed.

As a consequence of unhealthy lifestyles, the prevalence of hypertensive heart disease is rising both in the United States and globally. Hypertension often results from the chronic activation of adrenergic receptors found in the walls of blood vessels. These receptors are normally activated in times of extreme stress by the sympathetic nervous system. However, chronic activation leads to elevated blood pressure which puts extra stress on the heart and can lead to complications that kill millions of people annually. Drugs such as beta-blockers treat this disease by blocking the endogenous ligand, epinephrine, from binding to the receptor, which relaxes the blood vessels and lowers blood pressure. However, this class of drugs ignores another important receptor found in vessel walls, the alpha-1D adrenergic receptor (ADRA1D). ADRA1D is known to contribute to hypertension and benign prostatic hypertrophy in humans, but its mechanism of action is poorly studied. The Hague Lab conducts pharmacological studies on ADRA1D to elucidate how this receptor confers disease. My project focuses on understanding how the receptor localizes to the surface of the cell where it can be activated. Recently, our lab conducted proteomic screenings to identify intracellular binding partners. The results showed that ADRA1D binds to two unique proteins, a signaling protein, alpha-syntrophin (SNTA1), and a cellular polarization protein, Scrbble (SCRIB). This interaction was later attributed to a conserved binding region known as a PDZ domain that interacts with the C-terminus of ADRA1D. SNTA1 has one of these domains while SCRIB has four. My project attempts to quantify the strength of these interactions to give a better understanding of ADRA1D’s intracellular landscape. By better understanding how this receptor locates to the surface and how it signals, drugs can be created to disrupt these processes and better treat patients with hypertensive heart disease.

Shear-enhanced adhesion is a mechanism in which binding is enhanced at high flow rates. Some species of bacteria within the genus Streptococcus demonstrate shear-enhanced adhesion under high flow conditions, which are present throughout the human body. The purpose of our research is to understand and compare the effect of constant and pulsatile flow in Streptococcus gordonii, a strain of bacteria that causes infections by binding to human platelets. Our research focuses primarily on cases of infective endocarditis, a life-threatening infection of heart valves caused by various strains of bacteria. Rates of incidence in a general population has been estimated between 2 and 6 cases per 100,000 person-years. In our model system, we utilized a flow chamber to mimic pulsatile flow conditions in heart valves, which oscillate between a low and high flow, with a maximum shear stress of 3 Pa. We took videos of S. gordonii, which uses a serine rich-repeat adhesin to bind to platelets, and determined the number of bacteria that bonded to the surface under constant low, peak, average, high, and pulsatile shear stresses. We analyzed the videos to determine the conditions that enhance adhesion and compared our results under pulsatile flow to constant flow for the same range of shear stresses. Our results indicated that pulsatile conditions promote low amounts of adhesion above 1-2 Pa, demonstrating that pulsatile flow does not explain how Streptococci bind to heart valves at 2-8 Pa. These results disproved our hypothesis that pulsatile flow enhances adhesion at 2-8 Pa. Further research may look to testing the mechanism behind which Streptococci bind to heart valves in bacterial lesions exposed up to 8 Pa in the circulatory system. By understanding the adhesive properties of S. gordonii, our research may contribute to the development of anti-adhesive therapies to treat bacterial endocarditis.