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

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

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

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

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

The endothelial to mesenchymal transition (EndMT) has been identified as a key part of organ development as well as many disease pathways. EndMT is characterized by endothelial cells, which make up the inner lining of blood and lymphatic vessels and are adhesive and non-migratory, gaining mesenchymal markers and invasive, migratory behaviors. This overall change in phenotype is normal in embryonic development where EndMT is linked to development of organs but has also been linked to numerous diseases in adults including cerebral malformations, Alport nephropathy, fibrosis, heart disease, and bronchopulmonary dysplasia. Whereas it appears that EndMT does not discriminate by organ, it does by sex. The diseases mentioned previously have a significantly higher incidence in males. To understand the role that sex plays on the EndMT pathway, human neonatal pulmonary cells with a gestational age of 18 to 19 weeks from three female and three male donors were routinely cultured and monitored for changes in phenotype. Using angiogenesis sprouting assays, western blot protein analysis and immunostaining, we collected quantifiable data on the reversibility of the EndMT process in each donor. We found that cells from male donors had lower plasticity, characterized as shifting between the two phenotypes, and generally existed in an endothelial state until pushed into a mesenchymal phenotype through a stressor. Female cells were more likely to shift between phenotypes regardless of conditions and exhibited more angiogenic potential, suggesting a heightened ability to transition between phenotypic states. Future experiments include placing cells in environments with differing stressors to mechanistically determine what drives EndMT processes and monitoring cells with time-lapse imaging to quantify the dynamics of the transition.

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

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

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

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

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

A DNA screening tool is being developed to allow rapid DNA testing in the field to identify endangered species of timber and wildlife. This work describes the design and testing of a chip holder and a gasket to improve the performance of this tool in the field with the consideration of usability, manufacturability and functional constraints. The chip holder is composed of a cradle with a hinge, two glass chips and one gasket. This chipset contains all reagents required to perform a DNA amplification test and features microfabricated heaters with electrical contacts to drive this reaction. Prototypes for chip holders with different hinge thickness and designs were fabricated in polypropylene using a Lulzbot Taz-6 3D printer with an Aerostruder tool head. The hinges were tested manually to ensure that the design parameters provided a stable joint. Gaskets were created by pouring Smooth-On Liquid Silicone Oomoo 25 into 3D printed molds. Silicone gaskets and chips undergo thermal testing on hot plate at 95 °C for 25 minutes to test if adhesion changes. Leakage testing for the gaskets is done through placing 0.2 g of water between gasket and chip and pressure are added between the chips to check water loss. We found the optimal material for gasket under DNA testing condition is Liquid Silicone Oomoo 25, and the best adhesive for the gasket and glass chip is silicone based adhesive GE Silicone II. Future work would be finding a more efficient method to produce the gasket, apply adhesives and adhere the gasket and glass chip. For the chip holder, the next step would be choosing the manufacturer, and select the specific polypropylene for mass production.

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

Heart rate, as an essential health indicator, can provide valuable information to evaluate the fitness level of an individual. To improve the health and wellness, an affordable, non-invasive, and robust device monitoring the condition of the heart that could track long-term physiological measurement of an individual is highly demanded. Intrinsic signal optical imaging (ISOI) technology is an innovative, simple and favorable optical technique directly used for detecting miniscule intrinsic optical signals in tissues. Here, we have developed an ISOI system and taking advantages of an intensity-sensitive algorithm to monitor blood volume change in the vasculature bed in tissues which is indicative of heart pulse throughout the day. A green (532 nm), red (650 nm) laser, and a CCD camera were implemented allowing the production of an affordable, efficient and robust heart rate monitoring device. To provide accurate evaluation for the level of health, a highly sensitive intensity-based algorithm was implemented to this device, which detect the minuscule blood volume change based on the scattered light reflected from the tissues. Parameters such as the rate of blood flow, blood pressure, blood oxygenation, and hemodynamic property were extracted from the intrinsic signal and provide information for further evaluation of the heart condition and heart diseases. Our device can be a powerful tool for medical services to track and control the progression of the disease and further lowering the cost of medical care alleviating the financial burden for the individual, communities, nations and worldwide organizations.

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

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

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

2-deoxy-ATP (dATP) is a nucleotide used in DNA synthesis and its presence has been seen to improve the magnitude and rate of contractions in heart muscle cells. However, levels of dATP are naturally low in mature cells. As an attempt to develop a novel treatment for heart failure, methods to increase the expression of ribonucleotide reductase (RNR), a key enzyme in the production of dATP are being investigated. RNR is regulated by ubiquitin-proteasome degradation of the small subunit of RNR, the Rrm2 subunit. We constructed a variant version in which two regions were changed to prevent ubiquitination. This new variant should lead to higher levels of RNR in cardiomyocytes, which also indirectly increases levels of dATP. Our preliminary results show a successful increase in levels of both RNR protein and dATP in cultured neonatal rat cardiomyocytes. Although levels of RNR and dATP were increased, the level present in our cultured samples are much higher than expected for adult rat and mice cells. Therefore, we are currently testing this RNR variant in vitro in cultured adult rat cells, as well as in vivo in aged adult mice. These models are more representative of a therapeutic use. Preliminary results have been promising toward identifying a more effective method of increasing dATP levels for improving cardiac function.

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

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

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

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