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. 

 Oncolytic virotherapy is a cancer therapy that uses a modified virus to attack and kill cancer cells. These viruses can naturally only replicate in cells that lack a functional p53 gene, a gene that normally regulates the cell cycle and prevents cancer. To replicate, many of these viruses disable the p53 gene themselves, so that they can reproduce in healthy cells. However, by interrupting the processes used to disrupt the gene, virologists have engineered viruses that can only reproduce in, and kill cancer cells. One limitation of this process is that the patient’s innate immune system can prevent virus replication in cancer cells that naturally produce defensins - a class of small antimicrobial proteins. My lab has previously shown that one of these, synthetic Human Defensin 5 (HD5), blocks replication of Adenovirus 3, which is one virus used for oncolytic virotherapy. It would be important to understand in which types of cancers HD5 is produced, so that virologists can better design oncolytic viruses to bypass HD5. My research was the first demonstration that HD5 is secreted and upregulated in its active form by epithelial ovarian and lung cancer cells in situ. This makes the cancer cells especially hard for the virus to kill. To demonstrate this, I stained sections biopsies of a wide array of human tumors using a mouse anti-HD5 antibody. There was a clear difference in the staining of ovarian and lung cancer samples and their respective controls with no anti-HD5 antibody. This is especially significant because so little is known about what factors contribute to the effectiveness of oncolytic virotherapy.

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.

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.

Understanding the relationship between the parts of the brain that initiate behavior and those that execute movements is a fundamental goal for basic neuroscience and important for effective clinical treatment of many behavioral disorders. Electrically evoked potentials (EEPs) are a measure of the strength of neural connections between cortical sites and have been used to document connectivity both in animals and in humans. Past research has shown that there are spontaneous state-dependent changes in basal-cortical EEPs and cortico-cortical EEPs in non-human primates; the goal of this project is to examine state-dependent changes in connectivity between the prefrontal and motor cortices. We hypothesize that prefrontal cortex-motor cortex EEPs will show robust state-dependent changes, as the prefrontal cortex is heavily involved in motor planning. For this work, we collect neural data and stimulate the brain to produce EEPs using the Neurochip-3, a portable, head-mounted neurophysiology system, while the monkey is freely behaving in its home cage. Behavioral state is defined using signals from a head-mounted accelerometer and power spectral density analysis of the neural signals directly prior to stimulation events. We hypothesize that EEPs in the motor cortex evoked from the prefrontal cortex will have higher amplitude during waking movement than during rest or sleep.

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.

 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.

Anterior cervical discectomy and fusion (ACDF) is a well-established surgical technique used for the treatment of various disorders of the cervical spine. The two most common ACDF procedure methods are: standalone systems and traditional plating techniques. Dysphagia, the inability to properly swallow, is an extensively described complication; it has been proposed that mechanical irritation, additional dissection, or displacement of the esophagus by plate placement may contribute to a greater incidence of postoperative dysphagia. The aim of this retrospective cohort study is to assess differences in pain, clinical outcomes, and dysphagia, including complication and readmission rates, following ACDF using either a standalone cage system or a cage with anterior plating (traditional plating). Between 2014 and 2015 we identified 377 consecutive patients meeting study criteria (standalone, n= 211; plate and cage, n= 166). Pre-operatively scores were collected for patient specific characteristics, surgical characteristics, and Numeric Pain Rating Scale (NRS). In addition, complication and readmission rates, the Dysphagia Disability Index (DDI), and NRS scores were collected at one year and two years post-operatively. There was significantly greater improvement in neck pain scores for the plate and cage group after one and two years post-surgery ( 2.3 and 3.1 versus 1.6 and 1.5, respectively; p <0.01). This could presumably result from biomechanical advantages when compared to stand-alone cage systems. There was no statistical difference between DDI scores either postoperatively or at two years post-surgery. These results indicate that mechanical irritation may not differ between procedure types. Further research is needed in order to determine factors that may lead to a higher rate of nonunion for patients undergoing standalone cage placement. 

Spirometry is among the most common tests for characterizing pulmonary function and plays a critical role in detecting and improving outcomes related to chronic lung disease. During a spriometry maneuver, a patient rapidly exhales into a machine which records the total volume and flow of air over time. However, patient error in performing the spirometry maneuver, such as by coughing or taking multiple breaths, can lead to clinically misleading results. As a result, spirometry must take place under the supervision of a trained specialist who can detect and correct patient errors. To reduce the need of specialists to coach patients during spirometry, we demonstrate the ability to automatically detect four common patient errors. Applying machine learning techniques on features derived from spirometry data, we were able to automatically classify errors from spirometry data with an F-score between 0.784 and 0.907. Our work provides the first step toward reducing the need for trained individuals to administer spirometry tests by demonstrating the ability to automatically detect specific errors and provide appropriate patient feedback. This will increase the availability of spirometry, especially in low resource and telemedicine contexts.

Cerebrovascular reserve (CVR) is the dilatory response of blood vessels to match the perfusion needs of the brain. CVR is necessary for regulating oxygen and nutrient transport to maintain normal brain function. Individuals with Alzheimer’s disease (AD) often exhibit vascular pathology due to reduced perfusion. We believe that in patients with early AD, CVR can serve as a biomarker for early vascular pathology. Using breath-hold BOLD fMRI, CVR can be measured quantitatively and spatially. Older adult subjects (n = 48, age >65 years) were asked to perform normal paced breathing for 20s followed by a 15s breath-hold. This process was repeated 6 times. MRI data was processed using Python and FSL. Python was used to extract breath-hold signals from the MRI scanner. With FSL, BOLD fMRI data was corrected for motion and baseline drift. The breathing time course was used as a stimulus regressor in FSL FEAT to obtain voxel-wise maps of CVR. The CVR maps were registered to the anatomical T1 image and subsequently to the standard MNI template to obtain region-specific values of CVR. As data processing is ongoing, we have not obtained conclusive results. However, we expect to see in patients with onset of AD, CVR ability is lowered throughout regions of the brain, most notably in the frontal and parietal brain regions. Our preliminary studies in 30 older adults indicate that overall gray matter CVR in healthy older adults was 0.42±0.13%, but was 0.38±0.16% in individuals with cognitive impairment and at risk for AD. The results of this study will demonstrate the usefulness of cerebrovascular imaging to understand vascular pathology in patients with AD at an early stage, thereby allowing for treatment to begin sooner, and slowing down disease progression.

Managing cultures is a vital task in synthetic biology, but constantly measuring and adjusting culture conditions is both tedious and labor intensive. Modern assays that accomplish this, such as High Precision Liquid Chromatography, can also be prohibitively expensive. Our project aims to reduce the amount of time and effort needed to maintain cultures through the creation of an affordable image analysis system that autonomously reads visual data to measure the current state of a culture and then determines whether to release inducer chemicals based on user input. Our project utilizes the violacein pathway to simulate other metabolic pathways by producing four colored signals dependant on a two gene switch. These outputs are then measured by an open-sourced Raspberry Pi (a small, standalone computer) setup, which captures visual data via camera, measures the culture’s RGB value, and then directs the gradual release of inducer chemicals to maintain or change the culture’s color over time. Currently, yeast strains capable of coexpressing both a violacein and a non-visible pathway are being cloned. In addition to this, a prototype containing 3D-printed syringe pumps, a chemostat, and image analysis software in development. By combining both biological and hardware systems, we expect our unique design to be able to generate previously unavailable visual data regarding certain biosynthesis processes, such as those involved in antibiotic production and fermentation.

 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.

Current trends in the healthcare industry are focused on providing precision medicine for more individualized patient care. Many researchers are actively designing specialized devices specific to the imaging and care of breasts. However, we are unaware of any comprehensive large-scale studies that analyze population breast sizes that provide design guidelines for such precision medicine machines. In order to fill this knowledge gap, we collected nearly 15,000 mammograms from the Seattle Cancer Care Alliance and built a database of the images and DICOM (Digital Imaging and Communications in Medicine standard) header information. Images which were selected to be analyzed were run through a MATLAB algorithm to extract length and width measurements of each individual breast in the General Electric mammography unit. Around 2,400 images with Cranio-Caudal view, without magnification, and without foreign objects, were run through the MATLAB algorithm. With the measurement results from this algorithm, we studied the statistical distribution of the breast size and shape from this population. The extent of the breasts from the chest wall into the scanner had a mean of 96.19 +/- 29.04 mm , while the width of the breast had a mean of 184.69 +/- 29.00 mm. With the database in place, the next phase of our research was dedicated to analyzing breast density among the population. Women with dense breasts are at higher risk of false negatives in cancer screening and many states require that clinicians notify a woman if she has dense breasts. Nevertheless, there exists the problem of intra-and interobserver variability for judgement of breast density. To address this, some researchers have proposed automated algorithms to quantify breast density. We used our existing database to analyze the variability within these automated algorithms.

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.

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.

Glioblastoma multiforme (GBM) is the most common malignant adult primary brain tumor, accounting for 70% of malignant brain tumor diagnosis. However, regardless of therapy – surgery, radiation, and anticancer drug therapy – virtually all GBM patients eventually succumb to the progressive disease within 12 – 15 months of diagnosis. This is indicative of the proliferation and differentiation of neural stem cells into various cell types in the tumor which is thought to be a main contributing factor of malignant tumor resistance to standard treatments. This project aims to address this therapeutic challenge by identifying and testing potential new therapeutic regiments that leverage the underlying genetics of glioblastoma to improve treated response. We propose that certain genetic modifications (i.e. modifications in IDH1 or MGMT) can be made in glioma stem cells such that when a gene loses or gains a function, it will increase sensitivity of cells to a combination of therapies that is greater in magnitude than a single drug by itself. Towards this, we have generated whole exome sequencing data that will guide us in building a genetically-defined glioblastoma cell line and allow us to identify potential new therapeutic combinations predicted to have high specificity for GBM cells. Implementation of these predicted synthetic lethal combinations in a co-culture system will determine if the predictions are tumor, genotype, and treatment-specific. Results of this project will help us better understand therapeutic vulnerability and its determinants, and test specific combination therapeutic regiments that could be applied to improve the treated outcome of patients with this deadly 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.

Core needle biopsy (CNB) is used as a minimally invasive method to diagnose prostate and other cancers. However, this method still has limitations of getting non-diagnostic and inadequate CNBs due to sampling error. Thus extra CNBs are often taken and other time-consuming tests looking at surface cellularity such as histological evaluation on tissue smears are done to reduce probability of this error, which leads to needless pain and cost. Therefore, we propose to design a rapid-on-site evaluation system by imaging the outer surface of needle biopsy samples. This system generates a whole surface image of CNBs that contain cellularity information as a quicker and more informative adequacy testing of CNBs. This proposed project aims to develop cost-efficient imaging for obtaining sub-cellular and structural information from CNBs at the point-of-care. There are three phases: Design of System, Development Image Stitching Algorithm, and Integration, testing, and generating surface image. Phase I aims to determine the optimal optical imaging system and developing a staining protocol for CNBs. Phase II is developing an image stitching algorithm that takes in images from Phase I. Phase III will integrate I and II and generate a whole surface image of CNBs to provide morphological features for clinicians so that they can determine the adequacy of biopsy and needs for additional biopsies. The main criterion of this design is to perform evaluation within 20 minutes while still maintain intactness of specimen. If the design is successful a commercial device will speed the process by 10x (<2 minutes), while maximizing use of CNBs and reducing the number of biopsies taken to minimize suffering of patients. It may also accelerate diagnosis speed for prostate cancer. Importantly, this design can be applied to other types of cancer diagnosis, such as remote radiology clinics where a pathologist is typically not available.

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.

It has long been known that certain species possess the capacity to regenerate bony appendages following amputation through a process called epimorphic regeneration. For instance, zebrafish possess the ability to regenerate their tail fin bones when amputated. Early regeneration involves the formation of a blastema, similar to the blastema that mediates limb regeneration in salamanders. After the blastema forms, the bone regeneration process resembles the bone developmental process in humans. Thus, a better understanding of epimorphic regeneration holds promise to enhance our understanding of regenerative biology, allow for medical advances in bone tissue engineering, and increase understanding of the skeletal developmental processes. A challenge in understanding the regeneration process is the inability to immobilize fish to generate time lapsed images of various stages of regeneration. Typical methods for anesthesia in zebrafish only enable 10-20 minutes of sedation. Several studies (including those by our lab) have developed specialized methods to do long-term imaging with the use of tricaine methanesulfonate and benzocaine. However, both anesthetics are sodium channel inhibitors, which inhibit the regeneration process itself. In order to circumvent this problem, we designed a chamber for anesthesia-free imaging with a restraint system, and a removable glass-bottom window to facilitate imaging. The chamber is coupled to peristaltic pumps so water can flow in and out. The fish is secured and unanaesthetized, thereby bypassing the issue of lack of regeneration due to the anesthesia. In pilot studies we have found that the zebrafish quickly acclimate to the chamber, suggesting that the zebrafish can remain in the chamber for several hours. Our studies indicate that this anesthesia-free, long-term imaging chamber may allow us to see details of dynamic processes that unfold over a period of 24 hours, which currently can only be seen through snapshots of short-term imaging.

Many in vitro and in vivo approaches (e.g. overexpression or depletion) have established that proteins’ expression levels can affect the cellular response to stresses and stimulus. Here, I describe an approach to examine the lowest protein amount necessary for a physiologic cellular response. My studies are focusing on Fanconi Anemia (FA), a heritable human genetic disease characterized by cellular and developmental abnormalities, including congenital abnormality, bone marrow failure, and an increased risk of malignancy. The FA pathway responds to inhibition of DNA replication due to DNA damage, particularly interstrand cross-links such as mitomycin-C (MMC). Mutations in the FANCA gene, however, can disrupt this pathway and cause cellular defects. The specific goal of this research is to investigate how much functional FANCA (wildtype FANCA) is required to suppress these cellular defects. To modulate FANCA protein amount, we are currently developing vectors with variable strength promoters that allow for graded expression (from 0-100%) of wildtype FANCA gene. These vectors will be integrated in a unique chromosomal location in corresponding FANCA-deficient isogenic cell line to generate a cellular library. The integrated transgene consists of two regions: a variable strength promoter and a common EGFP linked via a T2A self-cleaving sequence to the FANCA coding sequence. FANCA protein expression will, hence, be quantified using Western blot and using the co-translationally expressed fluorescent protein (EGFP). Lastly, the cellular library will be subjected to differing doses of MMC and promoter sequencing will be used to quantify promoter-specific survival (aka FANCA protein expression-specific survival) as a function of MMC dose. The findings from this study will enable FA-specific drug/therapeutic research by establishing the quantitative requirements for FANCA protein function in human cells.

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.

Protoporphyrin IX (PpIX) is a red fluorescent metabolic by-product of oral bacteria and serves as an indicator of early and late stage caries. PpIX has a strong absorption band centered at 405 nm and exhibits an emission range from 615 to 710 nm. Spectroscopic and imaging devices targeted at PpIX require a fluorescent standard for calibrating their detection sensitivity. However, PpIX lacks photostability and an alternate molecule is needed to represent the excitation and emission fluorescence characteristics of PpIX. Evaluation of instrument performance over time is especially important for long range clinical studies examining changes in fluorescence in response to disease therapies. The scanning fiber endoscope (SFE) is an optical imaging system currently used in clinical dental research to quantitatively monitor the fluorescence of PpIX. Water based solutions of PpIX are unstable and led us to develop a dye-in-polymer standard that offers photostability, portability, and uniform fluorescence. Organic europium complexes model the fluorescence spectrum of PpIX closely, with a similar red emission range from 615 to 710 nm. Preliminary photobleaching experiments using a 405 nm laser illuminating a europium dye embedded in a polyurethane polymer demonstrated that it offers significantly better photostability than PpIX, exhibiting a 3-4% drop in europium fluorescence after one hour of continuous laser excitation. Under similar laser illumination conditions another PpIX phantom molecule, octaethylporphyrin (OEP), suffered a >20% drop in fluorescence after only 3 minutes of excitation. The photodegradation curve over time for europium was evaluated using the same 405 nm laser and LED excitation sources used for measuring the pH dependence of PpIX fluorescence in oral biofilms. Dye-in-polymer europium materials were also exposed to elevated temperatures in addition to laser illumination to further assess their suitability as fluorescence standards.

The goal of this research is the generation of induced pluripotent stem (iPS) cells and derived kidney organoids from urinary cells. iPS cells are somatic cells reprogrammed to an undifferentiated state, making them an exciting advancement in regenerative medicine as they could model an in-vitro progression of many disorders and show great promise for replacing or repairing tissue damaged due to disease or injury. To generate new kidney tissues, adult somatic cells were isolated from a patient’s urine sample. This method of sample collection is advantageous over the traditional skin biopsy because it does not require a physician and is easier for the patient to supply. This protocol was further optimized to accommodate remotely collected urine samples, showing the ability to isolate somatic cells from patients located across the globe. We successfully isolated somatic cells with various morphologies, ranging from epithelial to mesenchymal-like cells, from 25 different patients. By introducing into these cells RNA encoding four specific transcription factors, a safer alternative to introducing DNA as it eliminates the risk of inserting a mutation into the patient’s genome, we successfully reprogrammed urinary cells of an international patient with autosomal recessive polycystic kidney disease into iPS cells. These iPS cells were further differentiated into kidney organoids, which have the capacity to become a powerful tool in improving current therapies for kidney disease as an in-vitro model. Thus, for the first time, we have generated patient-specific kidney tissue from a urine sample. This will increase our understanding of the disease and improve current methods for the screening of investigational therapeutics. The ability to create new kidney tissues in the lab also raises the possibility of transplanting these tissues back into their original patients, where they could potentially function without the need for immunosuppression.

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.

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.

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.

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.

Neural injuries (either peripheral or central nervous systems) affect a large number of the human population world-wide and in general are considered irreparable due to the lack of regenerative capacity of the neural tissues. Various neural tissue engineering approaches have been employed to differentiate human neural stem cells (hNSCs) into neurons for the purpose of using them in regenerating the damaged neural tissues. Recently, electrical stimulation was found effective to enhance neuronal differentiation of hNSCs. Light-triggered generation of free electrons on the surface of semiconducting materials is an exciting option to achieve electrical stimulation on hNSCs. However, the band gap of most light-harvesting semiconductors falls in the UV range which is harmful to cells. The main hypothesis of this study is that hNSCs will preferentially differentiate into neurons when cultured on dopamine-modified titanium oxide (TiO2) substrates followed by irradiation by visible light (~ 420nm). We have discovered that TiO2 grown on polydopamine (PDA) coated substrates exhibits an absorption peak in the visible light range. The surface chemistry was confirmed by various surface characterization methods including UV-Vis, SEM, EDS, and XPS. Using this substrate, we investigated the effect of laminin on hNSC adhesion, surface free electron generation on TiO2 by visible light irradiation, and preferential differentiation of hNSCs into neurons. The results from this research open up a new possibility for implantable neural tissue engineering scaffolds that can be activated by light irradiation.

Zinc pollution is a large problem in underdeveloped countries with mining industries. Leaching of brass and galvanized iron can add substantial amounts of zinc into water resources. Furthermore, high zinc concentration is usually indicative of lead and cadmium, which, when once accumulated in the human body, can negatively affect the immune system. Currently, there are no rapid, inexpensive methods to determine the concentration of zinc in water. The sample is usually sent to a lab and analyzed using sophisticated and prohibitively expensive instruments. Our goal is to create an affordable and easy-to-use diagnostic tool using Saccharomyces cerevisiae—a yeast commonly used in industry. By designing a gene circuit that connects a zinc-sensitive promoter for Green Fluorescent Protein (GFP), we hope to engineer a strain of yeast that will give a green fluorescent output of different intensities when grown in different concentrations of zinc. After building and testing different strains of yeasts-- each using a different zinc sensitive promoter, we found that there was no significant variation in GFP output between different concentrations of zinc and between different yeast strains. In addition, after comparing the results with a wild-type control, we also found that our recombinant strains are not much better at detecting zinc than the wild type strain. For the next steps of this project, we will continue to build gene circuits involving other zinc sensitive genes. Once a few genes are identified as being suitable for our purposes, we will focus on amplifying and fine-tuning GFP expression as well as replacing the GFP with that a color output that can be detected by the naked eye.

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.

Variation in gene expression can be used as a way to test for or track the progression of large scale diseases. Although existing methods to evaluate expression such as RNA sequencing or microarrays are widely used in medical research, clinical application is limited because of time and cost. By monitoring gene expression levels clinicians could perform earlier diagnosis, evaluate therapeutic efficacy and predict recurrence of disease. Gene profiling by a linear DNA-based molecular circuit to classify samples with varying gene expression patterns would provide a novel approach resulting in a rapid, cost-efficient and reliable alternative to existing gene expression diagnostics. Previous work done on the project shows that multi-stage strand displacement cascades can be engineered to perform catalytic signal amplification with high sensitivity, amplification and specificity. In the Seelig lab, already a linear DNA molecular classifier has been engineered to distinguish between bacterial and viral infections and could correctly label 94.0% and 80.5% samples respectively. Although the previous experimental data supports the mythology of the classifier, it is not an ideal representation of clinical use. For that reason, I am working to scale up the molecular circuit by using RNA derived directly from human cancer cell lines to see if the classifier can robustly identify the originating cell line. I will take amplified RNA from each cell and add it to the classifier. The accuracy of initial binding will be analyzed by a fluorescent readout measured across both channels, corresponding to successful strand displacement. Upon demonstration of rapid, cost-effective and accurate classification of human cancer cell lines I then hope to translate this research into classification of clinical samples with the goal to adapt the current methodology into a point of care diagnostics device.

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.

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.

  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 bind the Fc region of antibodies. 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 three 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 using these homooligomers for near atomic scale structural characterization by cryo-EM.

Metatarsal stress fractures are among the most common injuries sustained in both recreational and competitive long-distance runners. However, current research lacks a proper understanding of how elevated plantar pressures may pose as a risk indication for stress fractures. Therefore we are proposing a research experiment investigating whether or not elevated plantar pressures exist in women with confirmed stress fractures. We hypothesize that elevated plantar pressures will retrospectively correlate to an increased likelihood of metatarsal stress fracture. In this study, each participant will have a personalized pressure mask of their feet designed that is specifically measured and scaled using their x-ray images. After the successful creation of the personalized mask, all data will be analyzed to investigate absolute, relative and cross-foot ratio comparison between the injured and non-injured contralateral foot. It is anticipated that patients with confirmed metatarsal stress fracture will have relatively larger plantar pressure under the respective fractumetatarsal. The information generated by this experiment may provide valuable insight into understanding metatarsal stress fracture pathogenesis. Further implications could even warrant a secondary study investigating the provision of personalized footwear that lower plantar pressures under the at-risk metatarsal.

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.

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.

Nickel sulfate hexahydrate (NiSO4.6H2O) is an important industrial compound most commonly used in the nickel-plating industry. Approximately 40,000 tons are produced and consumed each year by manufacturers requiring corrosion-resistant components ranging from consumer electronics to medical devices and aerospace engineering. Freitas et. al. found that zinc sulfate heptahydrate crystal growth increased by 38% under a magnetic field of 0.3-0.7T compared to crystallization under earth’s magnetic field alone, whereas Lundager et. al. have presented evidence that paramagnetic materials such as CuSO4.5H2O do not exhibit such magnetically-induced increases in crystal growth rate. We present here our progress on confirmatory experiments aimed at fitting Nyvlt theory of cumulative crystal weight fraction M(L) and average growth rate G in batch synthesis of diamagnetic ZnSO4.7H2O and paramagnetic NiSO4.6H2O under external, uniform, homogeneous magnetic fields 0.0T<B<1.0T. Our study will examine the differences in the growth rate of paramagnetic and diamagnetic sulfate crystals under the influence of a manipulated magnetic field. Recrystallization is a method of purification during the synthesis process of many compounds. If recrystallization can be improved to increase percent yield by influencing the magnetic field surrounding the compound, it may be a profitable method for drug manufacturers to consider.

Merkel cell carcinoma (MCC) is an aggressive, neuroendocrine skin cancer and one-third of patients develop metastatic disease. Specific sites and timing of distant metastases are largely un-described and, if characterized, could inform staging and surveillance protocols as well as optimize clinical  management. A cohort of 292 patients with metastatic MCC were prospectively followed and then grouped by the site(s) and timing of their initial distant metastases. Initial distant metastases were defined as the first distant metastatic lesions detected clinically and/or radiologically. Presentation of multiple lesions in a single organ site only counted as one initial distant metastasis for that organ site. MCC-specific survival was analyzed using a competing-risks regression, where 200 patients died of MCC. 292 patients had 420 distant metastatic sites, most commonly non-regional lymph nodes (40% of 292 patients), skin / body wall (26%), liver (22%), bone (20%), lung (9%), pancreas (8%), and brain (4%). Patients with only liver lesions (HR 2.1; p=0.02) or patients with lesions in multiple sites (HR 2.7; p<0.01) were over twice as likely to die of MCC than patients with distant metastases limited to the skin / body wall. Additionally, patients whose first distant metastasis occurred >3 years after initial diagnosis were twice as likely to survive MCC than patients whose MCC metastasized within two years of diagnosis (HR 0.5; p=0.02). Site of primary MCC is associated with a distinct pattern of initial distant metastasis, which may help inform surveillance practices. Survival outcomes vary among patients with metastatic MCC and prognosis may be more accurately estimated with stratification by timing and site(s) of initial distant metastasis. Accounting for these patient characteristics will help inform surveillance imaging intervals and define sub-staging for patients diagnosed with metastatic MCC.

Recent studies have shown that electrical stimulation of the spinal cord can manipulate spinal cord plasticity and may be effective in recovering motor function after spinal cord injury. The emerging field of optogenetics allows researchers to change a cell’s membrane potential using light. Cells are made to express light-dependent ion channels (channelrhodopsins) which cause a cell to depolarize or hyperpolarize after being triggered by specific light wavelengths. Our lab has shown that optogenetics can be used to elicit forelimb movements in rats by stimulating the cervical spinal cord. Long-term methods for providing light stimulation in vivo are needed to explore the treatment potential of optogenetics. Initial experiments by our group using a LED implant stimulator demonstrated that long-term optogenetic stimulation of the spinal cord results in increased GAP-43 staining. GAP-43 staining highlights areas of new neuronal growth, suggesting an increase in neuronal plasticity in optogenetically stimulated rats. Animals in this study, however, also exhibited unusual tissue morphology around the site of implant. Current research is working to understand the possible sources of this tissue disruption, including heat production from the LED and inflammation induced by LED stimulator implantation near a spinal cord injury. To investigate the possibility of heat production, I developed a new implant prototype that incorporates a thermistor to monitor temperature changes during long-term light stimulation. I tested this implant design in anesthetized and freely-moving rats to investigate how different light stimulation parameters affected implant temperature. I then performed histological tissue analysis at the site of the implants to assess the effects these temperature changes had on tissue condition. The results from this work will inform the next generation of light stimulation implants, and help to improve function following spinal cord injury via optogenetic activation of neural tissue.

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.

Exposure to abnormally high levels of estrogenic compounds can trigger dramatic developmental and sexual deformities in fish and wildlife. For instance, bisphenol A (BPA), found in plastics, is a common pollutant that threatens the natural environment and human consumers by acting as an endocrine disrupter. However, testing for these estrogenic chemicals requires samples to be processed at a laboratory with expensive equipment. Cell biosensors that are engineered to detect pollutants and produce a visual response are a low-cost, tunable, and highly sensitive alternative. Here, we show our work to expand access to estrogenic pollution detection by genetically engineering Saccharomyces cerevisiae, brewer’s yeast, to produce green fluorescent protein (GFP) until it detects ß-estradiol, a human estrogen. Our biosensor expresses a CRISPR transcription factor (CTF) which includes an estrogen receptor and mxi1 repression domain, coupled with a guide RNA that targets the promoter for GFP. We observed with a cytometer that upon ß-estradiol induction, the CTF-expressing yeast demonstrated heightened GFP expression and larger, possibly un-dividing cells. We hypothesize that our CTF is reducing cell growth and division rates, thus increasing the concentration of GFP molecules per cell, and show our work to investigate this prediction. In the future, we plan to tune the sensor to repress fluorescence only when reaching a hazardous concentration of  ß-estradiol. In addition, we will measure our biosensor’s ability to detect similar estrogens such as BPA, in the hopes that it can function as a universal estrogenic sensor.

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.

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.

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.

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.

According to the World Health Organization, an estimated, 780 million people do not have access to clean water sources. Disasters are another potential cause of disruption to the supply of clean water. When the safety of a water source is unknown, a cost effective method to determine water safety is necessary to reduce unsafe water related disease and death. Prior synthetic biology research has been conducted utilizing transgenic yeast as a biosensor for heavy metal detection, such as using plasmids expressing Green Fluorescent Protein (GFP) which are promoted by heavy metal binding transcription factors. While able to detect copper ions in a dose dependent fashion, the relative instability of the plasmid, reporters which are difficult to detect in situ, and limited targeting to copper and silver ions make the efficacy of the designs impractical. Using modern methods of plasmid integration and the modulation of the copper sensitive pCUP1 promoter expression we sought to produce a stable and more sensitive biosensor for the detection of copper ions in water. We successfully integrated the genetic cassette pCUP1-yeGFP into the yeast genome and produced a viable biosensor for the detection of aqueous copper. We tested this yeast strain via a cytometer after exposure to copper of varying concentrations at varied time intervals. The results showed a dose response curve that positively correlated with copper concentration in a semi-linear fashion, which indicated the pCUP1-yeGFP transgene was a viable sensitive reporter for the detection of copper. Currently, variable constitutive promoters are being altered to increase copper detection sensitivity by modulation of the expression of the metal binding transcription factor Ace1. The team is currently exploring the effect of Ace1 upregulation on the sensitivity of copper sensing. After investigating sensitivity modulation, an exploration of practical field detectable reporters, such as violacein, will be undertaken.

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.

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.

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.

MSN2 is an environmental stress response transcription factor that is activated in yeast by multiple different stresses—e.g. glucose deprivation, heat, oxidative stress, misfolded proteins among many others—and in turn activates a collection of downstream transcriptional programs. Interestingly, while MSN2 becomes increasingly activated with age, deletion of MSN2 increases the replicative lifespan of the budding yeast. We show that in a glucose-rich environment, MSN2 misreads the environment and drives a pathological glucose starvation response that limits lifespan. Our experiments are facilitated by a novel budget microfluidic/microscopy method rather than the golden standard of microdissection. This innovative system analyzes data through time-lapse images to develop replicative lifespan curves rather than picking off daughter cells with a needle. Microfluidics drastically increases the throughput of experiments and allows for whole-lifespan monitoring of aging yeast cells. The system allows us to efficiently further the research of MSN2 and other transcription factors so that we can potentially translate this to other complex organisms that have similar types of transcription.

There currently exists no device capable of accurately quantifying bone loss around dental implants. Epithelial down growth causes resorption of bone that supports the implant which can eventually result in reduced stability of the implant, and if continues unchecked, lead to mobility and ultimately implant failure. It is crucial that clinicians are able to determine implant stability using a method that is sensitive, rapid, accurate and precise. With an increasing number of dental implants being placed throughout the world, dentists need more definitive information to improve the determination of implant health and improved diagnosis. Previous studies have found that an increase in bone loss around an implant leads to decreased natural frequency of the implant in the frequency response of the system. Piezoelectric drivers are ideal to measure natural frequency shifts as they are small, sensitive, and accurate. This project proposes the use of a piezoelectric driver-sensor pair to measure the frequency response of implants to determine implant stability and bone loss. It will be divided into three sections. The first section has shown that piezoelectric driver-sensor pairs can both actuate and sense the frequency response of a model cantilever beam system and are currently being tested together. The second section consists of testing piezoelectric driver-sensor pairs in different implant conditions to determine how bone loss and other factors influence the results seen in the frequency response of the system. The final section consists of designing a prototype handpiece implementing a piezoelectric driver-sensor pair and creating a LabView Virtual Instrument that can help clinicians assess implant stability and bone loss. This project has the potential to help improve the quality of oral implants for many patients worldwide.