Providing quality surgical care and anesthesia in low-resource communities has proven to be quite challenging, as many anesthetic devices are either too expensive or require electricity. Anesthetic vaporizers work by evaporating a liquid anesthetic into an air flow, which is then delivered to the patient. As the liquid anesthetic is evaporated, the temperature decreases, leading to a decrease in delivered anesthetic, which is problematic if not addressed. Current devices address this issue by encompassing the vaporizer in a large steel heat sink, but this makes the device unsuitable for transport long distances. A low-cost anesthetic vaporizer has been proposed and developed by Bioengineers Without Borders that will utilize phase change material to hold the vaporizer temperature constant, and they are seeking a predictive tool to guide design choices. A computational model of this vaporizer has been previously built utilizing laminar flow dynamics. However, this model did not sufficiently predict the dynamics of the vaporizer under various operating conditions. The model has now been converted to utilize turbulent flow dynamics, and is undergoing validation steps through comparisons with the experimental measurements of concentration output and halothane surface temperature. Additional adjustments have also been made to the laminar flow model, resulting in a more accurate model. Currently, both the turbulent and laminar models predict a decreasing temperature over time, resulting in decreasing amounts of anesthetic being delivered. Both models perform very well at high flow rates, but constantly overestimate deliveries at low flow rates. Upon validation, this model will give Bioengineers Without Borders the ability to computationally test possible design modifications to the vaporizer prior to production, saving time and resources. 

The study of gene regulatory networks, both synthetic and natural, is at the center of modern medical and agricultural science. Gene regulatory networks -- gene circuits -- are the foundation of life and have been widely applied towards molding biology to better serve human interests. This potential, however, cannot be fully leveraged because of the complexity of genetic systems and the amount of time it often takes to get an accurate characterization of them. They are often complex, nonlinear, and responsive to a confounding number of environmental factors ranging from temperature, to light, to media composition. We aim to exploit data obtained from multiple kinds of experiments to enhance the accuracy of estimations of system parameter values of gene circuit mathematical models. For this aim, we first demonstrate a computational analysis method for automatic parameter estimation through use of Sequential Monte Carlo (SMC), a learning algorithm that uses randomness to estimate the parameters that govern a system. Using species input from real cell culture and a simulation model of the genetic circuit we are measuring, the algorithm can estimate parameter values over the course of many iterations. Though this computational method is not new, we will combine it with a transformation function that can compare estimated parameter distributions from different growth conditions in order to understand how the circuit in question has been affected by the change in environment. Often in research only one type of growth environment is used to demonstrate how cells or other biological functions work, but cells produce distinct results in different conditions. The transformation function will use the correlation and mean of predicted parameters for the same circuit in different growth conditions to understand how different factors influence the system and give researchers the ability to compare various kinds of experimental conditions.

Antibodies play a crucial role in both therapeutics and diagnostics due to their high target specificity. However, a characteristic that accompanies this high specificity is the reluctance to release the bound target. This presents challenges when either the protein or target needs to purified and analyzed or the protein needs to be removed from a site of interest. Current methods to reverse binding are often toxic and require large environmental changes that are not always feasible. These environmental changes can negatively affect the ligand structure, thus hampering analysis to characterize the target ligand. Therefore, a need exists for a protein whose binding can be externally controlled in a nontoxic manner. The Thomas Lab proposes using a light-activated azobenzene linker to create a crosslinked protein whose binding affinity can be controlled by light. The protein of interest is the bacterial adhesion protein, FimH, which has two known conformations that are regulated by force. The goal is to replicate the two conformations in the absence of force by using the linker. FimH has been mutated to have two free cysteines across the two domains for crosslinking. An assay has been developed to differentiate between crosslinked and non-crosslinked proteins to determine whether the linker has bound to the free cysteines. The azobenzene linker has been crosslinked to the mutated FimH and the change in binding to the target under UV light has been characterized. If a change in binding is detected, the design of a crosslinked protein whose binding can be externally controlled by light will be successful.

Influenza is a viral respiratory tract infection responsible for 15-60 million infections and more than 200,000 flu related hospitalizations in the United States annually. Diagnostics can help stop outbreaks and treat infections, however the current flu detection methods lack high sensitivity. The Yager Lab at the University of Washington is developing a simple diagnostic test to identify subtypes of the influenza virus. The assay targets the viral surface protein hemagglutinin (HA) and can specifically detect two strains of the influenza virus, the Solomon Islands strain of H1N1 and the Switzerland strain of H3N2. We have developed an automated device for this assay, supplying the reagents sequentially using a pinched valve and water soluble paper. I have tested and modified this device in order to enable hands free operation once the sample (a nasal swab) is inserted in the system. The paper based assay is fast, specific, and easy to use, allowing rapid diagnosis of influenza in any setting.

The overwhelmingly complex nature of cellular networks found in biological cells makes it challenging to build predictive models. It takes substantial amounts of time and energy to experimentally model a new metabolic or signaling pathway. My research focuses on evolving biochemical networks with specific behaviors using computational evolution. The desired behavior could be described by a set of experimental data and the aim would be to evolve networks that are consistent with those experiments. Modeling a network begins with generating random networks. These networks are then simulated using a reaction network simulator. Networks are selected according to their fitness which is computed based on the desired behavior. The fittest networks are mutated and re-populate the next generation of networks. Repeating this step with many generations will eventually produce networks that express the behavior we desire. The initial part of the research focused on evolving networks with a specific steady state value given an input concentration of a node species. The computational program was successful in producing networks with a specific steady state value capable of performing simple mathematical operations such as square roots and multiplications. My current research focuses on evolving networks with a desired transient behavior of a given species concentration. Being able to develop a computational program capable of doing that will enable us to model networks given a constraint from wet-lab experiments. Biomedical research applications, such as drug delivery, will be much easier with computational programs reducing the challenge to discover signaling and metabolic pathways.

 The biliary system is a network of tubular ducts that both transport and process liver-produced bile into the upper intestine for digestion of fats. These bile ducts are lined with cholangiocytes, which share a closely related lineage with hepatocytes, the parenchymal i.e. main functioning cell type of the liver. Hepatocytes are known for their plasticity to become cholangiocyte-like upon certain conditions, such as being co-cultured with fibroblasts in 3D multicellular spheroids known as aggregates. What is unknown, however, is how other nonparenchymal liver cell types influence hepatocytes to shift their phenotypes to become cholangiocyte-like. Here, we hypothesize that inclusion of nonparenchymal cell types into hepatocyte aggregate culture induce a more widespread, stronger hepatocyte to cholangiocyte-like phenotype shift. To test this, we created aggregates with varying cell type combinations and ratios from primary rat liver. During culture, we performed assays to test the aggregates’ ability to transport and process bile. Then the aggregates were fixed, sectioned, and immunostained for various cholangiocyte markers such as CK19, Sox9, and EpCAM. Aggregates created with a wider variety of cell types improve cholangiocyte function in the aggregates and had a greater expression of these markers. Information on how nonparenchymal cell types influence hepatocytes’ phenotypes is vital in developing novel methods to create liver tissue engineered constructs that include a biliary system, since most do not currently. Without a biliary system, functional engineered liver tissue becomes toxic over time from cholestasis, thus inclusion of an artificial biliary system will better recapitulate native liver tissue. These new developments could potentially lead to applications in ‘organ-on-a-chip’ purposes for disease modeling and regenerative medicine.

 Human immunodeficiency virus (HIV) is a serious global health issue with 36.7 million people infected worldwide and over 70% of these people living in low- to middle-income countries. Despite increased access to antiretroviral therapy (ART) to manage HIV and recent declines in HIV-related deaths throughout these countries, the emergence of drug-resistant (DR) strains of HIV threatens the continued efficacy of available ART. Drug resistance testing can be used to inform proper ART regimens, improve patient outcomes, and prevent further transmission. However, the gold-standard method (Sanger sequencing) is costly, requires sophisticated laboratory resources, and is unable to detect low amounts of DR HIV that can lead to treatment failure. To address this technology gap, the Frenkel Lab at Seattle Children’s Research Institute developed a sensitive, inexpensive, laboratory-based Oligonucleotide Ligation Assay (OLA). The OLA identifies single nucleotide polymorphisms (SNPs) known to confer resistance to ART via highly specific ligation of DNA probes targeting a SNP of interest. In order to expand application of the OLA to lesser-equipped settings, the Frenkel Lab is collaborating with the Lai and Lutz Labs (UW Bioengineering) to re-engineer the OLA into an integrated, paper-based device. As part of this effort, the present project aims to simplify the critical ligation process into a solid-phase enzymatic reaction system that can be integrated with a previously developed paper-based detection device. Compared to the laboratory assay, the solid-phase format provides a less intensive user protocol, protection from contaminants, and seamless transfer of analyte to downstream detection. Several affinity-based and bioconjugation-based enzyme immobilization methods will be investigated to create the solid-phase system. Quantitative analysis of enzymatic activity and immobilization efficiency will then be used to identify the best-performing method(s). Ultimately, simplification of the ligation process is expected to move the OLA closer to implementation in resource-limited areas impacted by HIV.

Bacterial endocarditis is a life-threatening disease that occurs when bacteria bind to the inner surface of cardiovascular tissue. One group of bacteria in particular, the viridans group Streptococci, cause a high percentage of cases of endocarditis. It is known that adhesion is mediated by specialized proteins on the surface of bacteria capable of binding to specific glycans expressed outside of host cells, and that binding is also dependent on shear stress from flow through allosteric regulation of the binding site. However, the exact mechanisms and resulting rates of binding are not sufficiently understood. If adhesion of the viridans group Streptococci can be experimentally tested and characterized, it could lead to the development of anti-adhesive therapies to treat or prevent diseases such as endocarditis. Current methods of testing bacterial adhesion utilize a parallel plate flow chamber to test the binding of one species of bacteria to a single glycan under one flow rate. Significant progress could be made through the construction of a microfluidic flow chamber compatible with a glycan array capable of testing bacterial adhesion to various glycans under a range of shear stresses in a single run. I have designed such a device using CAD and COMSOL fluid modeling software, and will construct it through the process of 3D stereolithography and laser-cutting. Additionally, I have conducted experiments to identify conditions that minimize non-specific adhesion, maximizing specificity for the device. Bacteria were fluorescently tagged, run through the flow chamber and across the glycan array, and then adhesion was analyzed using a microarray scanner. Data gathered from this device will help collaborators develop treatments to treat and prevent this type bacterial endocarditis. The flow chamber is also now accessible to others in the field to be used to study other clinically relevant types of bacterial adhesion.

Kidney cancer is the 7th and 10th most common cancer in men and women in the United States respectively. Renal cell carcinoma (RCC), a cancer of the proximal convoluted tubule, accounts for >90% of these kidney cancer incidences. Most cancers, including RCC, stimulate angiogenesis – the sprouting of existing blood vessels. By upregulating the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), tumors promote vascular sprouting within their microenvironment. This increased vasculature allows for increased oxygen transport as well as an opportunity to metastasize. This tumor-induced angiogenesis is of particular interest to researchers hoping to continue to develop alternative cancer therapeutics to inhibit angiogenesis, starving the tumor of oxygen, or normalize the vasculature, making drug delivery more effective. Common cancer models like 2D cell cultures and animal xenografts have been shown to be poor predictors of drug efficacy in patients. A lack of cellular complexity in 2D cultures along with genetic differences in animal xenografts make them insufficient models of the human pathophysiology seen in vivo. To address this problem, we are developing a 3D, vascularized, flow directed, microphysiological device deemed a “kidney-tumor-on-a-chip”. To engineer a perfusable microvessel, a 120μm collagen channel was lined with human umbilical vein endothelial cells (HUVECs). Primary RCC spheroids that mimic the 3D structure of solid tumors were then introduced into the surrounding collagen, recapitulating the tumor-vessel microenvironment. Initial results have shown no endothelial sprouting in non-RCC control devices, compared to significant endothelial sprouting in devices with RCC spheroids. These results suggest RCC cells may contribute to angiogenic sprouting in our microphysiological device, simulating what is observed in vivo. The ability of this kidney-tumor-on-a-chip to mimic the tumor-vessel microenvironment demonstrates its potential to be a more effective cancer model for drug testing.

Biological recognition proteins are widely used for a variety of biomedical applications, including in vitro diagnostics (IVD). IVD tests that use antibodies as recognition proteins, such as rapid antigen tests, are one of the fastest methods for providing point-of-care diagnosis of infectious diseases. However, many current applications of antibody-based molecular diagnostics are restricted by poor sensitivity in a sample with low target antigen concentration. One method which has shown promise for improving the sensitivity of rapid antigen tests is to pre-concentrate a target sample. We are using selection from a random library of Escherichia coli bacteria expressing the adhesion protein, FimH, to develop an activatable recognition protein for capture-and-release pre-concentration of a target antigen. FimH is a two-domain bacterial adhesion protein that exhibits conformation-dependent binding to mannose. We predict that mutations in the CDR2 and CDR3 loops of the FimH binding pocket can be used to design a protein with binding specificity for a diagnostic target while maintaining conformation-dependent affinity. In previous work, random mutations of the amino acid residues in the FimH binding pocket produced a library of FimH variants with variable binding specificity. Our results suggest that using a biomagnetic selection assay to screen this library of FimH mutants against the small molecule targets digoxigenin and cortisol enriches FimH variants with target binding specificity. Currently, we are designing and optimizing an assay, iterating on the wash conditions, conjugation strategy, and other parameters, to screen the library against protein targets such as the viral antigens influenza hemagglutinin and dengue NS1. In future work, we seek to demonstrate that a variant with binding specificity for a diagnostic target has distinct conformation-dependent affinity which can be actively regulated to capture and release a target antigen.

The World Health Organization reports that seasonal influenza (flu) epidemics result in 3 to 5 million cases of severe illness with upwards of 500,000 deaths. In the United States, 200,000 hospitalizations and 36,000 deaths occur annually with an economic burden of $87 billion. Hence, there is an urgent need to develop highly-sensitive, low-cost diagnostics for use at the point-of-care (POC) to facilitate immediate treatment at the early stages of infection. POC diagnostics can be implemented in paper-based, lateral flow assays to capture influenza nucleoproteins (NPs) with antibodies. Despite their utility, these assays suffer from moderately poor sensitivity. The highest sensitivity measured in a study evaluating six common colloidal gold colorimetric flu tests was 71%. To improve assay sensitivity, fluorescence detection has been demonstrated to detect analyte concentrations 1000-fold lower than with colloidal gold in lateral flow assays. My project aims to develop a fluorescence-based immunoassay for influenza A NPs to achieve improved sensitivities over those of colorimetric tests. The assay is carried out on lateral flow strips, making it readily translatable to point-of-care use. Upon capture of NP by immobilized antibodies, a biotinylated immunoglobulin G (IgG) binds to the NP at a different epitope. Subsequently, streptavidin-bound horseradish peroxidase (HRP) binds to the IgG. HRP oxidizes dihydrorhodamine 6G, a non-fluorescent substrate, into the fluorophore rhodamine 6G (R6G). Hence, an observable fluorescent signal is generated in the presence of NP. In preliminary experiments, R6G adsorbed to the membrane; R6G accumulates near HRP, increasing the signal-to-background ratio. An immunoassay for NP shows observable fluorescence signals at the capture and control regions for their respective positive and negative sample conditions. The remainder of the project aims to optimize reagent concentrations to further improve the assay’s sensitivity. Ultimately, the fluorescence-based assay developed would serve as a translatable platform for highly-sensitive detection of other viral analytes.

Parenchymal disease of the liver, known as diffuse liver disease, led to 1.4% of the total deaths in the United States in 2013. It causes impaired liver functioning and may lead to portal hypertension, encephalopathy, or hepatocellular carcinoma. Current methods of diagnosing diffuse liver disease are often invasive, subject to heterogeneous variation, and fail to distinguish between intermediate disease stages. Ultrasound imaging has long been used to monitor changes in tissue structure, and it is known that sound attenuation changes with disease progression. Tissue is acoustically nonlinear, meaning that propagating sound waves are distorted due to tissue properties and generate higher order harmonics. Since fatty deposits and fibrosis change with disease progression, we hypothesize that associated attenuation changes may be detected by changes in the nonlinear signal distortion and can be quantified as a marker of disease. We have designed a methodology based in Tissue Harmonic Imaging to use a quantified marker of attenuation to specifically detect diffuse liver disease. This project comprises of (1) a theoretical and experimental investigation of the influence of sound attenuation on harmonic content in tissue, (2) clinical patient data collection and development of MATLAB analysis tools utilizing the disease marker developed in Phase I, and (3) development of new imaging sequences capable of grading diffuse liver disease when coupled with the analysis tools. The noninvasive, easy to use, sensitive, and clinically usable methodology developed in this project has the potential for aiding in diagnosis of diffuse liver disease and enhancing quality of patient care.

Point-of-care diagnostics are needed to improve the detection of pathogens like the Zika virus. The Zika virus can be transmitted by mosquitoes, or by the exchange of blood or other bodily fluids. The implications of the virus in pregnant women and their children are still largely unknown. So monitoring, and if possible, preventing the spread of the disease is critical. Paper-based diagnostic assays enable complex assays in settings without laboratory assets. Today, sample preparation for molecular testing requires training and expensive machinery. The Yager lab recently created a fully automated multi-material paper-based system that can extract DNA from urine, but had not yet developed an automated system for blood targets. My initial work centered on adapting the paper-based system to enable diluted blood processing. Our updated design successfully flowed ~1.6 mL diluted blood sample through the paper in ~20 minutes. By passing large volumes through the system, low-concentration DNA can be extracted from the sample, removing assay inhibitors at the same time. This work continues the study of fundamentals of fluid movement through porous networks in the Yager lab, enabling a foundation for DNA sample preparation necessary for blood diagnostics. These advances will help detect the Zika virus in blood samples outside the laboratory, contributing to the monitoring and prevention of the disease.

Heart disease and other cardiac degenerative diseases account for 1 in 4 deaths in the United States. Current in vitro tissue models for cardiac disease modeling and potential drug testing lack dimensionality, scale, biomimetic behavior, and longevity. Manipulation of biophysical cues via biomaterial scaffolds in 3D bioprinting methods can alleviate these limitations as have been shown in 2D models. By mechanically inducing alignment in 3D tissue models, cells will respond to extracellular cues and form more organized structures similar to in vivo. This project focuses on implementing cardiac muscle cells into a mechanically aligned structure using a decellularized extracellular matrix (dECM) “bioink”. A BioBots extrusion based bioprinting system is utilized to manufacture cardiac cells laden in porcine left ventricle cardiac muscle dECM. We hypothesize that a polycaprolactone (PCL) post system design will induce cell alignment in a band-like tissue structure suspended in a Pluronic F-127 polymer bed along the axis of tension during tissue manufacturing. Induced bioink tension will enhance cardiac structure and function compared to current bioprinted models. This bioink composition and biomaterial process will provide cardiac cells with a better niche for development into a mature state that accurately mimics in vivo conditions. Bioprinted tissues will exhibit increased mechanical properties and structural organization with expression of contractile markers via uniaxial cell alignment upon bioink gelation in culture. This process is high throughput and provides a streamlined standard method for drug screening that more accurately tests possible side effects inside native heart tissue.

The beta-amyloid (Aβ) peptide, implicated in Alzheimer’s Disease, forms toxic aggregates known as oligomers that cause brain degeneration. This aggregation process can be inhibited by designed peptides that have a novel structure, known as the alpha-sheet. This structure is similar to a beta-sheet except that the carbonyls all point in one direction and the amine groups in the other, generating a molecular dipole moment. The Daggett group hypothesizes that at some point, Aβ undergoes a conformational change that gives it this alpha-sheet character and the resulting molecular dipole causes Aβ monomers to be attracted to each other and form oligomers. There is also evidence to suggest from simulation that this structure occurs in many other amyloid-related diseases. As such, designed peptide inhibitors from the Daggett group all target this structure. One of the control designs, AP3, aggregates much more rapidly than Aβ does under acidic conditions. My project involves using AP3 to probe the reasons for Aβ toxicity as well as its aggregation mechanism – both of which are not understood. The residues involved in the turn sequence of AP3, which is hairpin peptide, are of particular interest due to their propensity to display β-sheet character. After synthesizing these “turn sequence” peptides, I found that the presence of these turn sequence peptides can increase the speed of AP3 aggregation, which is an analogue to the seeding of amyloids. I am characterizing these turn sequence peptides using circular dichroism to observe their secondary structure. In addition, I along with other undergraduates in the lab have optimized ELISA to quantify the interactions between our designed peptides and another peptide of interest. The behaviors of these turn sequence peptides as shown by characterization and various assays prove enlightening in understanding the pathology of amyloidogenic diseases.

The leading cause of mortality in the developing world is infectious disease. Because such diseases are often treatable, the expanded use of accurate and rapid diagnostic devices plays an important role in reducing the burden of these illnesses. Point-of-care (POC) diagnostic devices can be inexpensive, which expands use in low resource settings, and rapid, which expands options for immediate patient care and treatment. The Yager lab designs low-cost, rapid and easy-to-use POC diagnostic devices based on paper microfluidics. These devices perform sample preparation, nucleic acid amplification and visual result detection in sequential, automated steps. As with lateral flow tests (LFTs), these devices have generally employed colorimetric detection, but this method increases test duration compared to fluorescence detection, which can give results in real time. Additionally, we hypothesize that fluorescence detection could provide a semi-quantitative evaluation of pathogen load via competitive thresholding. When a second DNA species is introduced, competition for reagents can generate different chemical sensitivities by varying the concentration of the secondary species. The competitive threshold, which we aim to calibrate by this method, is the point at which a known concentration of competitive targets amplifies in preference to the pathogenic target. We first performed amplification reactions in aqueous solutions with the number of copies of the pathogenic target and the competitor varied across five orders of magnitude. We visualized this calibration matrix as a surface plot, which we used to establish an accurate, order-of-magnitude competitive threshold that did not hinder pathogenic target amplification. Implementation of this method in paper-based amplification reactions will lead to shorter test times and, possibly, semi-quantitative detection in a new family of optics-enabled POC diagnostic devices.

The use of graphene in nanoscale systems serves many applications not only in solar cells but also in biomaterials because of the combined optical, electrical, and structural properties. Conventional methods including chemical vapor deposition and electrospraying to produce a layer of graphene on nanostructures is costly. Reduction of graphene oxide using chemical reagents presents an easier and cheaper alternative while maintaining the unique properties of graphene. But these methods are typically solution-based and further steps must be taken to coat reduced graphene oxide onto nanostructured surfaces. Dopamine chemistry was applied in a simple simultaneous approach of self-polymerization to reduce graphene oxide while creating an adhesive to incorporate a heterogeneous coating of polydopamine and reduced graphene oxide on a nanostructure. Use of various concentrations of graphene oxide demonstrated control over the sample's surface conductivity profile. Surface analysis characterizations confirmed the presence of the coating through X-ray photoelectron spectroscopy (XPS) while the electrical properties were determined using conductive atomic force microscopy (cAFM). The overall topographical morphology will be confirmed with atomic force microscopy (AFM). A potential application of the proposed device serves to provide functionally and structurally mature human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) for use in research and clinical drug trials.

Preclinical screening and toxicity studies are crucial steps in the drug development processes to ensure safety of new compounds when exposed to patients during clinical tests. Cellular responses in blood vessels play an important role in drug screening in terms of permeability and control of blood flow. The structure of the smooth muscle cell layer is crucial to the function of vessels in terms of contractility, mechanical properties and extracellular matrix production. Through recapitulating native tissue properties, tissue-engineered vessel models provide insights to study cellular responses when exposed to different environments, such as drugs. Although much research has been done in the development of tissue-engineered blood vessels, limitations in current in vitro models are yet to be addressed. Existing models such as synthetic and autologous vessel grafts are limited by thrombosis, non-native cellular resonses and poor mechanical properties. Bioreactors or microfluidics device are limited in terms of the complexity of the model and the scale to mimic larger vessels. The project addresses the need for an in vitro tissue-engineered arterial vessel model that replicates in vivo functionalities of the vessel by developing a fabrication method to form smooth muscle cell tubes that is reproducible and functional. The project is divided into three phases: (1) Formation of three-dimensional smooth muscle cell tubes using a cell sheet rolling method, (2) Optimization of the rolling method and (3) Validation of model by contractility analysis. Ultimately, the success of this project allows formation of vessel graft with functionalities in terms of mechanical properties, compliance and contractility. Vascular grafts can be created by human or patient derived sources and serve as drug screening or disease modeling purposes.

Vaccines save approximately 2.5 million lives every year, and vaccine development is an ongoing area of research in immunology. A vaccine is formulated with antigen and adjuvant, the latter of which enhances the host immune response to an antigen via stimulation of antigen presenting cells (APCs) like dendritic cells (DCs). Only a few adjuvants are currently approved for clinical use, posing a significant obstacle for new vaccine development. Multiple studies have investigated a diverse array of biomaterials and highlighted chemical analogues of pathogen associated molecular patterns recognized by immune cells. However, the immunogenic effects of physical properties like stiffness and porosity, which play an important role in cell function, are not fully understood. The goal of this project is to investigate how the stiffness and porosity of electrospun nanofibers modulate DC activation states. Recent studies showed that macrophages exhibit higher activation on stiffer substrates. Both macrophages and DCs are APCs derived from myeloid progenitors, thus motivating the hypothesis that stiffer nanofiber meshes could also induce higher DC activation. We investigated poly (vinyl alcohol) (PVA) and chitosan (CS) nanofibers that were cross-linked to modulate stiffness and porosity while also improving stability in water for cell studies. PVA and CS nanofibers were both thermally cross-linked at 150°C. PVA nanofibers were additionally treated with methanol for 8 hours. Thermal treatment did not cause a significant change (p>0.05) in the stiffness of the PVA nanofibers whereas combined methanol and thermal treatment for 20 minutes produced PVA fibers that were twice as stiff. Cross-linked nanofibers will be incubated with murine bone marrow-derived DCs for cell viability and DC activation studies. DC activation state will be measured by cytokine secretion along with CD86 and CD80 surface marker expression. The results from this study have the potential to guide engineering of immune-modulating biomaterials for novel adjuvant development.

Current approaches to deliver prophylactic drugs against HIV are ineffective for many reasons, including drug leakage, low drug loading, and lack of drug release tunability. To protect against HIV infection, a versatile prophylactic drug delivery platform that offers flexible dosing schedules would be an attractive and empowering option for many women. In the Woodrow Research Group, we have developed a fiber-in-fiber (FIF) drug delivery platform that addresses these requirements. The primary benefit of FIF is that it provides both immediate and prolonged drug release to protect against HIV acquisition. To fabricate the FIF platform, two nanofibers are used: a rapidly dissolving burst release fiber with encapsulated, slowly-degrading sustained release nanofibers. To preserve the release properties of the sustained release nanofibers and to ensure that they are of an imperceptible size to the user, we micronize the sustained release nanofiber in a blender. While this process is effective for water-insoluble drugs, water-soluble drugs like tenofovir leak out during micronization. Thus, improving the encapsulation of water-soluble drugs in the sustained release nanofiber component is needed to expand the functionality of FIF. I have developed a new strategy using dry micronization to improve the encapsulation of water-soluble drugs, allowing the FIF system to deliver diverse classes of drugs to protect against HIV acquisition. Retention of water-soluble drug drastically increased from 0% using previous strategies to 90% with dry micronization. Current experiments are assessing the drug release profiles of the dry micronized fibers compared to those intact nanofibers. SEM images of dry micronized fibers show that nanofiber architecture still exists, meaning that the benefits of nanofiber technology apply to dry micronized fibers. Therefore, dry micronized fibers have the potential to be their own drug delivery platform that can be delivered orally, intravenously, as well as intravaginally as a part of the FIF system.

With the rising prevalence of drug resistant bacteria, there is a pressing need to develop novel approaches which do not rely upon antibiotics. Anti-adhesive therapies target the ability of bacteria to adhere to tissue via adhesin receptor interactions. Yet, commonly used methods for characterizing these interactions involve parallel plate flow chambers (PPFCs), and are consistently done without consideration of mass transport effects. Here, we demonstrate that measurements of adhered bacteria taken within a large adhesive spot can vary significantly based upon the location measured. A computational model of a PPFC experiment is developed and validated in order to use partial differential equations to determine how various parameters influence spatial variation in adhesion. Use of a computational model allows investigation into a wide range of parameter values which would be impractical to experimentally test. Based upon these results, analytic models are established to accurately predict how transport conditions affect adhesion measurements. Interestingly, the measurement which best demonstrates a spot’s affinity for a bacterial ligand is the difference between the concentration of bound bacteria at the start of the spot and the consistent concentration reached well into the spot, according to these models. This is validated through PPFC experiments. Additionally, we demonstrate that within a multispot array, the adhesion in the spot of interest is highly affected by the preceding spot. It is vital that experimenters recognize this issue while interpreting their data. Guidelines to help experimenters set up and interpret their data in a manner that accounts for mass transport effects are described. Ultimately these guidelines could help reduce or negate the systematic error which mass transport introduces to adhesion experiments, and lead to better anti-adhesive therapies.

Water-absorbent polymer networks known as hydrogels are attractive materials from which functional tissue substitutes could be developed using patient-derived stem cells. While hydrogels demonstrate biocompatibility conducive to harboring cells, they lack mechanical and biochemical aspects of the native extracellular matrix (ECM), the intricate microenvironment about which cells function. Conventional photopolymerization-based techniques provide an avenue for incorporating these stimuli within hydrogels, but at the expense of introducing propagating free radicals that are prone to non-specific reactions with biological systems. In light of these limitations, we have developed a strategy for hydrogel formation and modification absent of propagating free radicals, proceeding through oxime ligation moderated by a photocaged alkoxyamine. Upon mild UV light exposure, the photocage is cleaved, liberating the alkoxyamine and permitting localized condensation with an aldehyde to form an oxime-based linkage. After synthesizing multi-arm crosslinkers, functionalized with either benzaldehydes or photocaged alkoxyamines, I demonstrated successful formation of oxime-based hydrogels within minutes of light exposure in the presence of live cells. Through a series of rheological studies, I found polymerization rates and final mechanical properties of these gels could be systematically tuned by varying crosslinker concentrations, light intensity, aniline catalyst concentration, and pH. Harnessing the light-driven aspect of this chemistry, I controlled hydrogel geometry and final mechanical properties by dictating the location and extent of UV exposure, respectively. I then translated photomediated oxime ligation toward the biochemical modification of hydrogels, where full-length proteins containing photocaged alkoxyamines were immobilized in user-defined regions exposed to UV light. The programmability afforded by photomediated oxime ligation can recapitulate mechanical and biochemical stimuli found throughout the ECM, which are dynamic and heterogeneous in their presentation. Consequently, photopolymerized oxime-based hydrogels will enable an enhanced understanding of cell-matrix interactions by serving as improved 3D cell culture platforms, thereby leading to advancements in tissue engineering and regenerative medicine.

As antibiotic resistance becomes a growing problem, there is a need to research alternative bacterial infection treatments without relying on antibiotics. Anti-adhesive therapy, which prevents bacteria from adhering to tissue, is an alternative solution to the antibiotic resistance problem. Flow rate (or shear stress) is one of the factors that affect bacterial adhesion in solution. Bacteria adhere to tissue in vivo where shear stress can go up to 8 Pascal, but they are unable to adhere in vitro in shear stresses above 1 Pascal. Hydrodynamic lift is the force known to push microparticles away in high flow and prevent the particles from getting close to a surface. However, there is no tool that describes how lift affects bacterial adhesion. This project aims to determine whether lift pushes the bacteria away from the wall and prevents bacteria from initiating binding in continuous flow in vitro as opposed to pulsatile flow in vivo. To do this, we developed a computational model which uses discrete finite element method to determine how lift affects initial adhesion. The model showed bacterial concentration depletes by the wall but concentrates where lift and gravity are balanced. The flow rate or wall shear stress affected where the bacteria accumulates thus influenced their adhesion. Moreover, this model has improved the mass loss problem, which the previous model faced. We validated the model by existing parallel plate flow chamber experiments with different bacteria. The validation experiments also gave some understanding regarding how lift affects the binding of different bacterial species under multiple continuous and pulsatile flow conditions. The results of this project will help researchers make informative decisions to design bacterial binding experiments by providing a tool to understand how lift and gravity affect initial bacterial adhesion.

Characterizing fundamental mechanical properties of individual molecules is essential to understanding and treating disease, because proteins have diverse responses to stimulus. The ability to predict a response enables targeted treatments. Although atomic force microscopy (AFM) and magnetic tweezers are able to measure the response of single molecules to mechanical force, it is challenging to ensure single molecules are being measured. In this project, structural DNA nanotechnology (DNA origami) is used to create a template which spaces molecules for single molecule force measurements. DNA is an excellent nanoscale building material due to its self-assembly properties, nanoscale structural precision, and the capability for precise control over placement of functionalization. By attaching functionalized DNA at specific sites and polymerizing the structure, a repeated precise interval is created for molecules of interest. This nanostructure was designed using caDNAno and canDo. Nanostructure folding time, temperature, and salt conditions were optimized using agarose gel electrophoresis. Nanostructures were imaged with transmission electron microscopy to confirm correct formation of the structure. Polymerization will be imaged using AFM, and force characteristics of the structure will be measured via AFM, to establish a base point for measuring the molecules of interest.

Myocardial infarctions (MI) are ischemic events which cause heart tissue to die and scar tissue (fibrosis) forms to maintain integrity of the walls of the organ. Fibrosis is crucial in the initial healing process but continued formation can compromise the elasticity of the tissue and lead to heart failure. Fibrosis is formed by myofibroblasts. These cells differentiate from fibroblasts after MI. Currently, the differentiation process is not well understood. The RNA binding protein muscleblind like (MBNL1) is known to be critical in this process, however. This project aims to use fluorescent imaging to track the localization of mRNA bound by MBNL1, particularly serum response factor (SRF) which has also been shown to play a role in myofibroblast differentiation. We aim to use regularly interspaced short palindromic repeats (CRISPR) and nuclease-null, RNA-targeting CRISPR associated protein 9 (RCas9) to specifically bind to the desired sequence of SRF mRNA. Enhanced green fluorescent protein (EGFP) is bound to the RCas9 in order to be able to visualize the RNA. Two target sequences on SRF mRNA were identified and protospacer adjacent motif presenting oligonucleotides (PAMmer) and single-guide RNA (sgRNA) were designed to bind to these sequences. PAMmer and sgRNA allow Cas9 to bind to the target sequence. This system was transfected into mouse embryonic fibroblast (NIH 3T3) cells and differentiation induced using transforming growth factor beta. During differentiation, live cell imaging was used to visualize RNA dynamics and localization. Results are expected to show colocalization with MBNL1 in the cytoplasm. This data will allow for the correlation of RNA trafficking and cell differentiation. It will also allow for a better understanding of myofibroblast differentiation and fibrosis formation after a myocardial infarction.

In Plasmodium falciparum malaria, infected erythrocytes (IEs) adhere to vascular endothelium, causing occlusion, coagulopathy, organ dysfunction and inflammation. While IE cytoadhesion occurs in many organs, cerebral malaria in particular is associated with high IE cytoadhesion and life-threatening complications. However, IE cytoadhesion in cerebral malaria remains incompletely understood. Research into human cerebral malaria pathogenesis has been limited by the inaccessibility of the human brain, and existing experimental platforms fail to recapitulate human vascular conditions and disease pathophysiology. In vitro microfluidic models lack appropriate geometry and materials to mimic in vivo flow conditions and matrix, while animal models do not adequately replicate human disease pathology due to use of non-human malaria parasites and non-human tissues. To address these limitations, we propose a 3D in vitro brain microvascular model to study P. falciparum cerebral malaria pathogenesis. Microvessels are created by molding collagen I with micropatterned stamps and incorporating brain endothelium and supporting cells into the collagen channel. Micropatterned stamps allow specification of complex physiological vessel network structure, and when these networks are designed using computational flow simulation, they allow specification of physiological flow properties in the fabricated microvessel. Collagen comprises the vascular extracellular matrix in vivo and regulates cell morphology, lumen formation and vessel stability, so its use as a biomaterial scaffold increases physiological relevance of the in vitro model. By constructing a physiologically relevant brain microvascular bed, we will characterize the molecular and biophysical mechanisms of cytoadhesion and the resulting changes in local endothelial function. The completion of this project will improve understanding of IE cytoadhesion, thereby improving understanding of P. falciparum pathophysiology to guide the development of treatments targeting disease mechanisms.

Endothelial cells (ECs) are cells that line our blood vessel walls. They differ throughout the circulatory system. For example, high pressure arteries typically have tighter endothelial junctions/gaps than the leaky, low pressure veins. ECs are known to sense fluid shear stress and hydrostatic pressure. These cues play a large part in embryonic development of the vasculature and induce short term responses in adults, such as dilation and constriction of arterioles, but it is unknown if adult ECs adapt to them in the long term. This matters for many medical procedures, such as venous graft bypass surgery, where the saphenous vein is implanted to bypass a blocked artery. These grafts are often lifesaving successes, but they sometimes re-occlude. Venous and arterial endothelium are very distinct, and it is important to know if the venous ECs in the graft can adapt to their new arterial environment. If not, that could explain the pathological re-occlusion. Here, we measure adaptation by changes in gene expression from a venous to an arterial phenotype. We designed a novel in vitro pump system to expose a flow chamber with endothelial cells to controlled flow and pressure. HUVECs (human umbilical vein endothelial cells) were then cultured and seeded into the chamber. Finally, a quantitative real time polymerase chain reaction (qRT-PCR) will measure gene expression at various time points in the venous (20 cmH₂O, low flow) and arterial (60 cmH₂O, high flow) cases. We analyze the markers EphrinB4 (EphB4) (higher in veins), EphB2 (arteries), Cx40 (veins) and endomucin (arteries). We present preliminary gene expression data from the 24-hour time point. We hypothesized that HUVECs will respond to arterial conditions with increased arterial gene expression, while retaining their venous state in venous conditions. Supporting or falsifying this will illuminate endothelial cell response to changing conditions in the body.

Vascular remodeling, the modification of preexisting vasculature, often occurs during events such as cardiovascular disease. Little is known about the perivascular factors controlling vascular remodeling. A small-diameter, single-channel arteriole model was developed to study vascular remodeling under various perivascular conditions, such as those of disease models or under the addition of drugs. The housing device consists of polydimethylsiloxane (PDMS), a non-toxic silicone-based polymer, held by a plexiglass casing. Type I collagen gel embedded with human coronary artery smooth muscle cells (HCASMCs) is cast within the housing, and the lumen is formed using a needle-based subtractive molding method with a 180um acupuncture needle. The original diameter of the lumen is retained while human umbilical vein endothelial cells (HUVECs) are seeded into the lumen by hand-perfusion. Migration and realignment of both the HCASMCs and the HUVECs is observed after applying physiological flow rates of growth media for 3 days in co-culture. Additionally, endothelial barrier function and smooth muscle contractile function are maintained. The functionality of the model is confirmed through observed calcium wave propagation and epinephrine treatments, where dilation of the arteriole occurs upon perfusion of the hormone. Current studies include observing the effects of combined pressure and flow on perivascular remodeling and function with a focus on modeling atherosclerosis and arterial thrombosis. This study will demonstrate the role of pressure in controlling the vascular remodeling process, specifically the migration and alignment of HCASMCs and HUVECs. It will also provide insight into vascular remodeling during coronary artery disease.

Many cases of chronic kidney disease are caused by loss of podocytes, kidney cells that are part of the filtration barrier between the blood and urine, resulting in declining kidney function. Current therapies do not prevent podocyte loss and rely on systemic small-molecule drug delivery, which is less effective and can lead to significant complicating side effects. Therefore, targeted drug delivery to podocytes could more effectively protect against injury, prevent further loss, and halt disease progression to kidney failure. To effectively deliver drugs to these cells, ligands that bind with high affinity to proteins specific to podocytes must be developed. Aptamers, DNA- or RNA-based ligands, are advantageous because they can be generated using standard techniques and are easily reproduced. Here, systemic evolution of ligands by exponential enrichment (SELEX) was used to identify aptamers that bind to His-tagged nephrin and NEPH-1, two membrane proteins highly specific to podocytes. First, a large library (on the order of 6×1014 sequences) of randomized single-stranded DNA (ssDNA) was incubated with nephrin or NEPH-1, and protein-aptamer complexes were isolated using His-tag capture beads. Beads were washed to remove any unbound ssDNA, and bound ssDNA were then isolated. This pool will be narrowed further by applying additional selection stringencies over the course of several rounds, ultimately resulting in a small final pool of aptamers with high specificity and affinity to these desired targets. Libraries from selected rounds will be tested for binding to target proteins to monitor selection. Through SELEX, we anticipate finding a small pool of aptamers capable of binding to recombinant nephrin and NEPH-1, and will next validate individual aptamer sequences for their ability to bind to these proteins in vivo. These identified aptamers can be further used to develop a targeted drug delivery platform to podocytes.

The most common form of cancer treatment is the use of small-molecule chemotherapeutic agents. However, due to non-specific distribution and uptake, these drugs have a host of complications that limit their efficacy and usage. Polymer nanocarriers have been demonstrated to improve drug pharmacokinetics, enhancing therapeutic benefit of the drugs. The feasibility of cyclic micelle-like polymer nanoparticles for use as a delivery vehicle for doxorubicin (abbreviated as DOX), a potent chemotherapeutic drug, is discussed here. It is hypothesized that cyclic polymers will afford a more efficacious pharmacokinetic release profile than their linear counterparts. A linear polymer is functionalized with “click”able moieties at each end, which are then joined to create a single cyclized polymer. The polymer is decorated with poly(ethylene glycol) (PEG) chains to reduce immunogenicity, and carboxylic acid groups to electrostatically complex with the positively-charged DOX molecule. Upon addition of DOX, the polymer self-assembles into unimolecular micelle-like structures. When the construct accumulates in a tumor microenvironment due to the enhanced permeability and retention (EPR) effect, the low pH of the tumoral interstitium protonates the carboxylic acid groups, releasing DOX locally. Using a unimolecular micelle-like approach, less polymer can be used to deliver the same therapeutic payload, relative to other similar approaches that utilize pluralities of potentially toxic polymers to form micelle structures. The polymer’s average molecular weight is around 11530 Da and the polydispersity of the reaction is 1.067. Gel Permeation Chromatography, Nuclear Magnetic Resonance, Fourier-Transform Infrared Spectroscopy, Ultraviolet-Visible Spectroscopy and Dynamic Light Scattering are used to confirm chemical identity, drug loading efficiency, particle size, and polydispersity. Cytotoxicity studies conducted with MDA-MB-231 invasive ductal carcinoma cells in vitro demonstrate the feasibility of this construct.

Recent research has been dedicated to investigating ways to promote angiogenesis using microphysiological systems that more accurately represent native organs. The Zheng lab has recently created a kidney microvascular model on a chip that utilizes primary isolated endothelial cells. However, this model requires the implementation of a biologically representative matrix and the incorporation of perivascular and parenchyma cells to better mimic the native microenvironment. We hypothesize that the incorporation of a biomimetic matrix will confer quiescence in the endothelium of the microvascular model and that the integration of perivascular cells within the matrix will promote improved vessel maturation and the establishment of a basal lamina. We have synthesized a biomimetic material from decellularized human kidney tissue that has a complex modulus of 930 Pa, making it stiff enough to use in semi-3D cell culture. Furthermore, use of this material has led to decreased endothelial migration and fewer alpha smooth muscle actin positive perivascular cells, indicating representative in vivo behavior of both cell types. In the microvascular model, we expect the integration of perivascular cells to the matrix will lead to increased endothelial quiescence. Results in collagen I matrices have shown irregular perivascular homing to and coverage of the endothelium, denoting increased but not complete vascular maturation. We expect the utilization of this technique in the biomimetic matrix to show increased perivascular coverage of the endothelium and the formation of a more complete basal lamina. If proven to be biologically accurate, implications of an in vitro kidney microphysiological model include use in therapeutic tissue regeneration studies as well as the ability to replace less accurate animal models during drug testing.

Development of antibody-based diagnostics, biomolecular sensors, and biomaterial with reactive surface requires the ability to control the adsorption of bioactive protein at a surface. All medical devices exposed in an in vivo biological environment are immediately coated with a layer of adsorbed protein that plays an important role in the body’s behaviors towards the devices and the effectiveness of the devices themselves. In this study, we use a multi-technique approach to characterize the effect of protein G B1 (6 kDa) orientation on multi-layer antibody binding. By conjugating a cysteine group at different positions along the protein’s surface, we can immobilize five variants of the protein (V21C, D35C, E42C, T11C, and WT) onto maleimide and bare gold surfaces. We have confirmed through X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry experiments that the proteins adsorb onto these surfaces. In addition, we introduced primary IgG and secondary F(ab’)2 antibodies to investigate if the protein’s orientation will affect the binding of the antibodies. The adsorbed mass are measured using quartz crystal microbalance with dissipation monitoring. We have demonstrated that the cysteine mutants orient better on maleimide surfaces and encourage more IgG binding. We will also study the effect of temperature on adsorption mass and extend the experiments to amine and carboxyl functionalized surfaces. Fully developed techniques to accurately characterize the orientation and quantify the amount of protein and secondary adsorption will significantly contribute to understanding the interactions of biomaterials with the biological environment.

Type II diabetes mellitus (T2DM) is a prevalent disease that relates to pancreatic islet beta-cell failure. With no cure and numerous secondary complications - blindness, kidney failure, heart attack, and stroke - T2DM places an enormous burden on our society and healthcare system today; the disease is projected to be the seventh leading cause of death by 2030. Within approximately 90% of T2DM cases, the majority of the amyloid deposits is made up of amylin, also known as islet amyloid polypeptide (IAPP). Unlike other organisms, human IAPP is capable of self-aggregating and forming amyloid fibers or beta-sheet plaques through its unique chemical properties. Our group’s previous findings suggest that T2DM and other amyloid diseases contain misfolded protein aggregates and form toxic oligomers through a non-standard alpha-sheet secondary structure. We propose a peptide-based therapeutic to combat T2DM via utilizing synthetic alpha-sheet compounds complementary to the amyloid-associated alpha-sheet structure to target the toxic oligomer form of IAPP. In this study, the efficacy of this peptide-based therapeutic is assessed through inhibitory and toxicity reduction effects of designed peptides on IAPP aggregation and oligomer cytotoxicity. The designed peptides are co-incubated with IAPP and their aggregation is monitored via a Thioflavin-T assay. The preliminary results on IAPP suggest that our peptides are effective in reducing IAPP aggregation and allow us to conduct cytotoxicity experiments. Effects of our compounds on cell viability are currently being conducted on a human-derived neuroblastoma cell line (SH-SY5Y). The cytotoxicity of oligomer determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay. MTT is a yellow compound that will be reduced to purple formazan only in living cells. The cells are plated in a 96-well plate with 15x10⁴ cells/mL.The IAPP is co-incubated with designed peptides and applied to cells. The results of the MTT assay provide the relationship between inhibition of oligomer formation and reduction of oligomer toxicity. The alpha-sheet platform provides a novel potential therapeutic for treating T2DM and other amyloid diseases.

The Oligonucleotide Ligation Assay (OLA) specifically detects point mutations in the HIV polymerase gene conferring drug resistance. Screening patients for drug-resistant HIV is a necessary step in determining the proper treatment. However, the traditional OLA is complex and lengthy, making it non-ideal for low-resource settings. We are exploiting a novel approach to adapt the OLA into a point-of-care, paper-based format. To enable downstream isothermal ligation and paper-based detection steps, the amplification step of the OLA must be designed to preferably generate many copies of single-stranded HIV DNA (ssDNA). Typically, this can be achieved using a Linear-After-The-Exponential Polymerase Chain Reaction (LATE-PCR), which uses an uneven ratio of forward and reverse primers. Though the LATE-PCR is a useful tool in producing large amounts of ssDNA, it involves a plethora of parameters whose contributions to the reaction output are not well understood. Thus, the LATE-PCR is often optimized via trial and error and may not yield the most desirable outcome. In order to minimize wasted resources from unnecessary trials and achieve a high-efficiency amplification, we aim to understand the behavior, predict the outcome, and develop a platform for LATE-PCR optimization. To accomplish this, we have observed the behavior of LATE-PCR reactions by measuring ssDNA output when altering key reaction parameters such as the number of cycles run, primer concentration ratios, enzyme concentrations, enzyme rates, salt concentrations, and primer sequences. Analysis of these results will allow us to understand the effects of these parameters on the thermodynamics and kinetics of the LATE-PCR. This will form the basis of a computational model which will accurately predict reaction behavior, and provide specific LATE-PCR reactions given user-defined reaction parameters and desired ssDNA output. Future research will focus on expanding the LATE-PCR model to other instrument-free isothermal amplification approaches for a simplified point-of-care OLA.

In the US, someone needs blood every two seconds, making blood transfusion a frequently performed procedure in hospitals. Transfusion of incompatible blood due to clerical error in the clinical blood typing procedure can induce a multitude of life-threatening immune responses in the recipient’s body. The most prevalent typing procedures are based on the overly simplified characterization of surface antigens on an individual’s red blood cells (RBCs) into the commonly known ABO blood type system. However, studies have shown that the antigens embedded in the RBC membrane, comprising of glycolipids and glycoproteins, are structural determinants of variations in the blood type outside of the ABO system, elucidating the limitations of current typing methods. This project follows the hypothesis that gas-phase hydrogen/deuterium exchange (HDX) MS techniques can be employed to characterize and further elaborate the structure of these RBC antigen constituents. To test this hypothesis, genotypically and phenotypically defined donor blood samples are processed to obtain RBC ghosts, containing only the membrane components with the hemoglobin removed through an osmotic lysis procedure. Glycoprotein and lipid fractionation, followed by digestion are employed to isolate carbohydrate components in preparation for HDX-MS analysis, which provides insight into structural composition of the carbohydrate antigens. Through this study, we expect to develop a deeper understanding of the variations in the carbohydrate RBC antigens which dictates the blood type of individuals. This knowledge will be crucial in developing glycobiological sensors that can be functionalized onto silicon chips used by Ratner Lab, facilitating novel blood typing techniques.

Nucleic acid (NA) extraction is a critical step in molecular diagnostic assays, since it can impact the sensitivity and specificity of the subsequent amplification step such as the Polymerase Chain Reaction (PCR). Most commercially-available NA extraction approaches capture both disease target NA and other non-target NAs. However, these non-target NAs compete with the target NA for resources and as a result can reduce the efficiency of PCR to detect a disease target of interest. Here, we present a strategy to specifically capture NA sequence of HIV from lysed infected human CD4 cells. After cell lysis, restriction enzymes are used to excise the HIV pol sequence from proviral DNA integrated in human genome. After a heat-denaturing step, biotinylated DNA probes hybridize to the HIV pol sequence. This solution containing the duplexes of pol gene/biotinylated probes is transferred to a microcentrifuge filter tube containing streptavidin agarose bead resin. Subsequent centrifugation retains the duplexes of biotinylated probes and HIV pol while removing other undesirable non-target NAs and possible contaminants in cell lysate. Finally, the target sequence is eluted from the biotinylated probes and with sodium hydroxide and subsequently neutralized prior to addition to PCR. For initial optimization of this capture system, synthetic HIV pol at a concentration ranged from of 50 – 100,000 copies were used as mock specimens. The method presented achieved 60% ~ 100% HIV DNA target recovery. In the next stage of this work, the effectiveness of capture will be tested with the upstream sample preparation procedures. We are currently investigating capture efficiency and restriction enzyme activity in different sample solute conditions in an effort to streamline the two steps. The successful development of this sequence-specific capture will potentially improve the sensitivity of the PCR assay and allow for early HIV diagnostics.

Over the past few years’ silicon photonics has proven to be a viable bio-sensing platform for various diagnostic applications. The small size, label free detection and ability to be multiplexed with various assays make it a very attractive technology and potential replacement for many clinical tests. Silicon photonics has proven to be a viable technology for ABO blood typing. This technology has the potential to achieve a higher level of automation than the current clinical method along with a lower run time and lower costs. Two large limitations in clinical translation of this technology are reducing the time it takes to run the assays and the amount of processing that must be done before running them. Indirect blood typing using this technology currently requires centrifuging whole blood to isolate plasma or serum to use in the assay. This sample processing can take anywhere from 20-40 minutes and is not ideal for a clinical setting. This project seeks to increase silicon photonics clinical viability by designing an inline filtration system to isolate the plasma or serum from whole blood as it is running through the assay. This would mean that whole blood would not have to be centrifuged for indirect blood typing. Instead, the blood will be filtered in the assay and then travel through a paper network for reagent delivery recently developed in the Ratner Lab. Additionally, this assay will be developed for the commercial Genalyte silicon photonic test setup. This requires the development of a custom mount for the system along with determining positioning for the paper network. This research may help to replace the agglutination diagnostic test that has been in use for over 100 years and help save medical and monetary resources as well as patient lives.

Prolonged activation of cardiac fibroblasts after acute wound healing post myocardial infarction results in excessive extracellular matrix deposition in the myocardium and formation of fibrotic scar at the site of injury. This adverse fibrotic remodeling disrupts physiological function in the heart and leads to increased risk for heart failure which is detrimental for patient outcomes. The reasons why activated fibroblasts, known as myofibroblasts, persist after wound stabilization are poorly understood due to the lack of a myofibroblast reporter system capable of direct longitudinal study. Current reporting modalities are invasive, resource and time intensive, wasteful, and indirect. The present project aims to design and develop an in vivo, repeatable, and real time reporter system for myofibroblast cell fate as a surrogate for cardiac fibrosis. To achieve this, we have designed a chimeric promoter to drive firefly luciferase vector expression specifically in myofibroblasts. We have built 9x tandem repeats of serum response factor (SRF) and nucleated factor of activated T-cells (NFAT) binding sequence since they are key mediators in myofibroblast activation. These repeats were ligated onto smooth muscle alpha actin (ACTA2) and collagen type 1 alpha 2 (COL1A2) minimal promoters, and these constructs were cloned into pGL3 luciferase reporter vectors. The specificity of the promoter was evaluated and optimized through in vitro luciferase assays with NIH 3T3 primary mouse fibroblasts.  With the successful creation of this reporter, wasteful use of animal models will be mitigated and direct mechanisms in the pathobiology of chronic cardiac fibrosis can be elucidated. Furthermore, efficacy of novel therapies can be evaluated in vivo.

Anti-retroviral therapies (ART) are an effective strategy to treat HIV patients by suppressing viral replication and reducing transmission. However, error-prone HIV reverse transcriptase (RT) can cause single nucleotide polymorphisms (SNPs) associated with drug-resistant HIV. To ensure effective treatment, HIV patients must be tested for drug-resistant SNPs prior to initiating ART. The Oligonucleotide Ligation Assay (OLA) is a lab-based sensitive method for detecting drug-resistant SNPs in HIV, developed and validated by the Frenkel lab at Seattle Children’s Research Institute (SCRI)- however complex workflow has limited implementation in low-resource labs. To address the need for an HIV drug resistance test in these regions, the UW Bioengineering Lutz lab, in collaboration with the Frenkel lab and the UW Bioengineering Lai lab, are attempting to re-engineer the lab-based traditional OLA into an equipment-free, point-of-care format. We are currently exploiting innovative chemistry and autonomous wicking properties of paper-based microfluidics to integrate the OLA into a credit-card-sized cartridge device. Multichannel capillary-based sequential delivery platform is used to supply the reagents required in all main steps of the OLA: nucleic acid extraction, amplification, ligation, and detection, to the different regions of the paper-based device. To enable incubation during amplification and ligation a pull-tab technology is used to physically disconnect the circuit. After these reactions are completed, the tab is pulled to re-activate the next sequential delivery steps. Currently, a proof-of-concept integrated device has been designed. To initially test the microfluidic physics, five fluid sources were colored to permit visualization of the flow. A successful controlled sequential delivery to mimic all steps in the OLA has been demonstrated. Next, the chemistry of each step will be incorporated into this cartridge device. The success of this work will enable non-instrumented, sensitive detection of HIV drug resistance and viral load that requires only two user steps: addition of a rehydration buffer and activation of pull-tabs.

Hepatocellular Carcinoma is the most common form of liver cancer accounting for a quarter of a million deaths annually across the globe. Microbubbles have been shown to be an effective vector for both gene and drug delivery. In combination with ultrasound, the microbubbles can be locally burst to deliver a drug payload via sonoporation – the temporary disturbance of the cell membrane. Ultrasound and microbubbles drug delivery is a strong candidate for the treatment of HCC because the liver that is easily accessible with ultrasound and the organ itself is highly perfused allowing for easy microbubble access. In microbubble gene and drug delivery, ultrasound parameters that induce cell death when used in combination with microbubbles are not well defined. While the literature agrees on general trends that greater exposure times and greater pressure lead to higher cell death, the specific combination of parameters that cause cell death are still disputed. We will design an enclosure such that cells can be cultured and exposed to ultrasound with microbubbles. We hypothesize that at greater exposure times, higher duty cycle, and greater pressures the cell viability will decrease. We hypothesize when multiple parameters are increased in combination, there will be a compounded decrease on cell viability. The rationale behind these hypotheses is that with greater power and force, greater stress and strain will be placed on the cell causing lowered cell viability. The goal of the project is to conclude specific values at which cell viability is compromised. From the study, it was found that while stronger acoustic parameters caused higher cell death, the variable that made the most difference was bubble concentration.

Currently, only metals are known to be affected by nearby magnets. Ferromagnetic materials, such as iron and nickel, exhibit a strong attraction when exposed to an external magnetic field. Paramagnetic materials, such as magnesium and lithium, also experience an attraction to magnetic fields, but the attraction is weaker than that experienced by ferromagnetic objects. This experiment shows that non-metallic materials can be attracted to magnetic fields when floating in water. When a small piece of paper was buoyed in a closed petri-dish filled with water and exposed to an external magnetic field, the piece of paper was observed to float towards the magnet. Additionally, the speed of the paper continuously increased as the paper got closer to the magnet. This effect was also observed with small pieces of Nafion and Teflon tubes. This means that items that we would consider to be non-magnetic may in fact be magnetic in a very small scale. Future work will be needed to find out what allows these materials to experience positive susceptibility to magnetic fields.

36.7 million people are living with HIV worldwide, with most in low- to middle-income countries. Among many clinical interventions for controlling HIV, Antiretroviral Therapy (ART) has shown success in reducing HIV transmission. However, the effectiveness of these ART is undermined by rising drug-resistant HIV. The lab-based Oligonucleotide Ligation Assay (OLA) can specifically detect HIV drug resistance as an alternative to expensive complete-genome sequencing, but its reliance on moderately expensive lab equipment prevents its universal access. Our lab, in collaboration with the Frenkel Lab (Seattle Children’s Research Institute) and the Lai Lab (UW Bioengineering), is developing a simplified OLA-based HIV drug resistance test. In the OLA, PCR amplification of the HIV Pol region is necessary before subsequent detection of Single Nucleotide Polymorphisms (SNPs) conferring HIV drug resistance. Current amplification relies on a thermal cycler, a >$2.5k instrument not widely available in low resource settings. Isothermal Helicase Dependent Amplification (HDA) eliminates need for a thermal cycler. HDA has been used to amplify the conserved Gag and Env regions of HIV, however it has never been used for amplification of the Pol region, a non-conserved region containing mutations that interfere with DNA annealing in isothermal amplification. We have designed and validated three sets of primers that successfully amplified three regions in the HIV Pol gene covering six common SNPs that confer HIV drug resistance against 1st-line ART. These primer sets each could detect down to 10 copies of HIV Pol DNA per reaction.  We are currently working on a multiplexed HDA assay that simultaneously amplifies three regions in a single tube to reduce cost and consolidate workflow. Implementation of HDA will allow OLA to be further simplified for use in low resource settings by allowing accurate diagnoses of HIV-infected individuals, controlling transmission rates, and reducing the evolution of drug-resistant HIV within entire populations.