The need for effective, on-demand contraceptives and protection against sexually transmitted infections is constantly increasing in our ever-growing society. We plan to contribute to this need by utilizing the barrier and drug eluting properties of a non-woven matrix of electrospun nanofibers made from the hydrophobic polymers polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA). Our experiments will demonstrate that nanofiber technology can be effective as a barrier contraceptive. This fibrous network will serve as a platform to sustain the release of the hormonal contraceptive Levonorgestrel (LNG), providing a chemical barrier against unintended pregnancy. We quantify in-vitro via optical microscopy how effectively the fiber inhibits penetration by observing its interaction with sperm. To optimize the physical barrier properties of the fibers we investigate sperm penetration as a function or fiber thickness and pore size in order to obtain an optimized barrier contraceptive of the thinnest fiber and highest protection. We then look at how well the fibers sustain the release of LNG in-vitro such that the contraceptive is eluted over a span of 7 days. We will also investigate materials that can be encapsulated within the fiber barrier to expand and block the cervical os (cervix opening) for ensured protection against sperm. This system will swell to many times its original size instantaneously, providing immediate protection for the user upon insertion. Beyond contraception, we plan to further understand the capability of these materials to impede the penetration of HIV. By utilizing drug-eluting nanofibers we can produce a platform that acts not only as a contraceptive but also as a means of preventing the spread of HIV.

Arrhythmias kill 300,000 people annually and effective cardiac drugs are imperative in their management. However, heavy reliance on in vivo testing results in low throughput and ineffective preclinical prediction of human response. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are a promising cell source for highly predictive, high-throughput in vitro testing. Unfortunately, culture conditions in current hPSC-CM assays leave hPSC-CM tissues immature, making them ineffective at modeling human myocardial drug responses. Tissue culture conditions that mimic the myocardial microenvironment promote the maturation of hPSC-CMs with higher efficiency than non-mimetic conditions. Taking advantage of this, our lab's NanoMEA system successfully melds biomimetically nanopatterned substrates with microelectrode arrays (MEAs) that perform high-throughput electrophysiological analyses of in vitro tissues. However, greater maturation efficiency is necessary to improve NanoMEA screening throughput. Electrically conductive environments promote hPSC-CM maturation, and numerous electrically conductive scaffolds exist. Yet, overlaying MEA electrodes with these substrates results in noisy signals that bar automatic processing. NanoMEA nanopatterns are made of Nafion, a nanoporous polymer that does not negatively impact the recordings from underlying MEAs. Since Nafion exhibits robust self-assembly with electrically conductive reduced Graphene-Oxide (rGO) molecules, we hypothesize that Nafion-reduced Graphene-Oxide (NrGO) nanopatterns will convey the benefits of conducting surfaces without impacting signal clarity. To investigate this, NrGO-nanopatterns are fabricated and characterized using Raman Spectroscopy, transmission electron microscopy, scanning electron microscopy, and conductive atomic force microscopy. Then, hPSC-CM monolayers are cultured on NrGO nanopatterns of varying rGO content and differences in morphological and transcriptional maturation markers are assessed. Promising NrGO nanopatterns are then integrated into a multiwell MEA platform to examine their effects on signal clarity and electrophysiological hPSC-CM maturation. If effective, we anticipate that this enhancement to the NanoMEA system will enable faster development of safer drugs, reducing animal model reliance, and improving the treatment of arrhythmias.

Endophytes are symbionts that live in the intercellular spaces of host plants. They can be fungi, bacteria, actinomycetes and/or viruses. Initial investigations indicate that endophytes provide a number of benefits that promote plant growth including, but not limited to, dinitrogen fixation, plant hormone production, nutrient acquisition, stress tolerance and increased immunological response. In exchange, they receive domicile and photosynthates. Endophytic biological and ecological services hold great potential for bioengineering and evolutionary inquiries, particularly so for sustainable agriculture. Nitrogen has historically been the nutrient limiting factor in all crop production. Bioengineering endophytic biomes capable of alleviating input demands while ameliorating soils damaged from industrial agriculture stands to offer novel solutions. This two year student-independent research is evaluating the inoculation of three fruit species, Malus domestica, Prunus avium, and Fragaria × ananassa with an endophytic consortia isolated by the UW Doty Lab. Washington is a leading state in fruit crops with over a hundred thousand hectares dedicated to production. Sweet fruit crops require heavy nitrogen inputs to yield nutritionally and economically viable fruits. Our primary objective is to investigate the effect of inoculation on the aforementioned crop physiology and fruit production. We are examining these effects through ecophysiology metrics involving rates of photosynthesis, stomatal conductance, biomass accretion, chlorophyll content, fluorescence rate, drought response, fruit sucrose content and biostatistic analysis. We hypothesize that our inoculated fruit crops will out perform controls as indicated with greater total biomass, fruit production and quality. First season preliminary results point to greater fruit biomass with higher sucrose content in Malus domestica. With crops now well established after season one, we anticipate a continued trend of improved crop total yields, higher sucrose content and greater total biomass production in all three varieties.

Gastrulation is a stage in embryogenesis in which most progenitor cells commit to a muscle or neural fate. However, a population of bipotential progentitor cels does not differentiate during gastrulation and allows the embryos to dynamically allocate neural and muscle tissue in the correct proportion as the body axis is formed. The regulation of this process is crucial for proper neuromuscular development. The maintenance of these bipotential progenitor cells requires the repression of tbx16, a transcription factor that progenitors express when they commit to a muscle fate. Here, we examine two genes of interest termed eve1 and vent, and investigate their roles in the regulation of tbx16. While previous studies relied on Morpholino anti-sense oloigonucleotides (MOs), the credibility of MOs has recently become seriously questioned because they can cause off-target effects, generating misleading effects that don't reflect genetic mutants. Revolutionary new gene-editing techonology known as CRISPR, is hailed as the new standard for gene analysis because it allows the production of true genetic mutants in specific genes. CRISPR accurately and efficiently creates small deletions in target genes, which can result in complete loss of function. Using the CRISPR method, we will generate F1 lines of zebrafish with eve1 and vent mutations. Through high-resolution melt anaylsis (HRM) and DNA sequencing, we have thus far identified two eve1 F0 founder fish. We will then test how the absence of eve1 and vent affects the expression of tbx16 during zebrafish embryogenesis using in-situ hybridizations. By analyzing how embryogenesis in normal zebrafish embryos differs from that of eve1 or vent mutant embryos, we can identify the role that these genes play during development. Ultimately, our goal is to gain a better understanding of how serious birth defects and genetic disorders arise from complications in development. 

Recent disease models have shown that majority of sexually active individuals will acquire sexually transmitted infections (STIs) sometime in their lives. Compartmentalization of vaginal mucosal immunity, as well as the immunosuppressive immunity exhibited by the Langerhans cells in vaginal mucosal tissue, pose challenges in developing efficacious STI vaccines. To address both of these challenges, we are developing multifunctional nanoparticles for programming ex vivo dendritic cells (DCs), which subsequently can be delivered intravaginally to stimulate Langerhans cells (LCs) in the vaginal epithelial tissue. We designed our DC transfection nanoparticles using a polycationic protein called protamine to complex antigen-encoded DNA plasmids for antigen presentation, and a double stranded RNA adjuvant, poly(I:C), for provoking DC maturation. In determining the optimal formulation, nanoparticles were formulated at four different protamine to mock DNA mass ratios (P:D ratios), 8:1, 2:1, 1:1 and 0.5:1. The nanoparticles were analyzed for their size, stability and DNA loading efficiency. Integrating results from these studies, we determined that nanoparticles with P:D ratio of 2:1 is the best candidate since it is the smallest P:D ratio that yields nanoparticles with the optimal size for DC uptake, as well as high particle stability and DNA loading efficiency. Additionally, DC transfection efficiency of the nanoparticle formulated at this ratio is also discussed. Successful development of the DC programming nanoparticles will demonstrate the feasibility of developing novel DC-based intravaginal vaccine, which will enhance vaginal mucosal immunity and can potentially be translated to efficacious vaccines against various types of STIs.

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

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

The human Tumor Suppressing Subtransferable Candidate 1 (TSSC1) gene was first discovered in 1997 as a gene whose likely location was within a chromosomal region that suppressed tumor growth. A recent study reported that decreased TSSC1 gene expression results in increased metastasis of breast cancer cells to bone. However, the underlying mechanism by which the TSSC1 protein acts in cancer cells remains unknown. Interestingly, TSSC1 is a highly conserved protein among eukaryotes. We are using Drosophila melanogaster as a model organism to define the normal function of TSSC1. Previously, our lab showed that the Drosophila protein, dTSSC1, localizes near Trans-Golgi Network (TGN), the region of the Golgi that sorts and packages membrane and secreted proteins into vesicles for transport to post-Golgi destinations. We have also isolated two mutations in dTSSC1 and observed that they cause defective formation of the acrosome, a Golgi-derived organelle that is localized to the apical tip of mature sperm and is required for fertilization. Based on these observations, we propose that dTSSC1 acts in spermatids to regulate acrosomal protein and vesicular trafficking. We are expressing tagged membrane proteins to ask whether dTSSC1 is required specifically for trafficking of acrosomal proteins or if it has a more general impact on membrane protein trafficking. These studies use expression in the male germ line. Notably, dTSSC1 is expressed in many tissue types, similar to its counterpart in humans. Therefore, we are also investigating whether dTSSC1 mutations have effects on protein trafficking in somatic cells. Our results should define the normal cellular role of dTSSC1 in Drosophila and may also provide insight into the role of the human protein in normal and cancer cells.

There are different types of programmed cell death: the well-understood apoptosis, and the inflammatory process of necroptosis. The enzyme Caspase-8 has been defined as a key initiator of apoptosis and inhibitor of necroptosis from studies using knockout mouse models. While Caspase-8 is found in both humans and rodents, a close homolog of the enzyme called Caspase-10, is not found in rodents. To add on, the role of Caspase-10 pertaining to the necroptosis pathway is still undefined. The goal of this project is to understand how Caspase-10 functions in human cells, and how the apoptotic and necroptotic roles of Caspase-8 described in rodents may be divided between Caspase-8 and Caspase-10 in humans. To investigate this idea, we silenced Caspase-8 and/or Caspase-10 expression in human cells, induced Necroptosis using chemical signals and inhibitory drugs, and investigated expression levels of proteins Caspase-10 may interact with. Our results will be critical in uncovering the role of Caspase-10 pertaining to necroptosis, as well as why Caspase-10 is present in humans but not mice.

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

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