Heart disease is the leading cause of death in the United States. Due to the inability of cardiac tissue to self-heal, extreme cases necessitate heart transplants and most patients do not fully recover. A promising novel approach to create engineered heart tissue focuses on 3D extrusion-based bioprinting of stem cell-derived cardiomyocytes. Mature iPSC derived cardiomyocytes, the cells responsible for the contraction of the heart, do not proliferate. Therefore, the printed tissue construct must be created with the final desired cell-density de novo in order to mimic native cardiac tissue. Researchers need a viable method for extruding high cell-density bioinks to form functional constructs. In my research project, I am working  to generate 3D bioprinted cardiac tissues with high cell-density to measure both electrophysiological characteristics and contractile force output. The use of cell-only bioinks is atypical, and there is limited research in the literature documenting its properties. I measured the acute change in viability of NIH 3T3 cells extruded through a needle to investigate the effect of the needle’s hydrodynamic forces on the cells at high density. The data shows that cells extruded at high density maintain a high viability, and we observed strong structural cohesion in the extruded filaments. I optimized the extrusion parameters, needle diameter and flow rate, to create long-lasting filaments, and constructs remained intact over a 5 day observation period. However, we found they fail easily with agitation. Further work is needed to optimize bioink and conduct further studies using flexible posts to measure contractile force output and calcium imaging to determine the electrophysiological characteristics of our cardiac constructs. Quantifying these properties is critical to ensuring that constructs recapitulate the characteristics of native cardiac tissue. This research may aid in the development of engineered cardiac tissue for transplantation and drug discovery.

Heart disease takes an estimated 17.9 million lives each year, highlighting the pressing demand for cost-effective treatments. Melusin, a chaperone protein in the heart, holds potential as a target for heart failure therapeutics. Previous studies done in wild-type (WT) and melusin knockout (MelKO) mice discovered the absence of melusin was associated with a hypertrophic response indicative of heart failure. I plan to investigate the biomechanical role of melusin in humans using human-engineered heart tissues (EHTs) created from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that lack melusin and their isogenic controls. EHTs are a 3D in vitro model of the human heart, ideal for studying the role of melusin in humans. I hypothesize that WT EHTs subjected to mechanical stress will outperform the MelKO EHTs. The EHTs are suspended between one flexible and one rigid silicone post. The EHT displaces the flexible post as it contracts, from which the displacement can be measured to calculate various auxotonic properties of the tissue. To induce mechanical stress on the tissues, I use a brace to restrict the movement of the flexible post. I am using histology to determine if there are any morphological differences between tissue types resulting from the brace. Thus far, I have cast WT and MelKO EHTs and completed twitch force measurements two and three weeks post-casting. Overall, I found the MelKO EHTs demonstrated lower contractile force than the WT EHTs. I plan to cast and collect more EHT data with and without braces in order to provide insight into the role of melusin in humans. Furthering our understanding of the heart’s mechanotransduction properties using EHTs is important in expanding our knowledge about the various pathologies of the heart. Ultimately, studying the pressure overload pathways involving melusin can lead to the development of future therapies for cardiovascular disease.

Multiple Particle Tracking (MPT) is a powerful technique for studying the behavior of microscopic particles, such as viruses and nanoparticles, by tracking individual displacement and movement. One application of MPT is to measure microstructural changes in the brain extracellular environment (ECM) in development and aging, and in response to disease onset and progression. MPT of nanoparticle probes results in the generation of thousands of individual nanoparticle trajectories, from which geometric features, diffusion coefficients, and viscosities can be extracted. The vast array of trajectories contained within our dataset presents a good opportunity for integration into deep learning models that contains self-supervised learning, equivariant graph neural network, and Equivariant transformer. However, to enable MPT data to be trainable and predictable by deep learning models, we need to curate the data to be readable and useable by these models. To enable this, I have created a database and developed a data architecture that would allow MPT data to be passed into Deep learning models that use various techniques such as transformers. I am currently working on utilizing the data architecture on a Deep Learning model that uses transformers and self-supervised learning to predict trajectories of MPT particles. From this model, my expected accuracy of prediction of the trajectories for the MPT data is around 85%. This can allow us to learn complex features directly from raw MPT trajectory data, improve our predictions, and extract biological insights. The python package with our data architecture, the various SQL scripts, and the model will be provided as an open-source resource, allowing other researchers to expand upon my code and apply their unique modifications based on their own data and trajectories.

Repeat thrombotic events have been associated with increased levels of Von Willebrand Factor (VWF) in patients prescribed dual antiplatelet therapies (DAPT), medications designed to prevent thrombosis. VWF is a protein that regulates platelet adhesion during hemostasis, allowing platelets to aggregate at sites of vascular injury. A microfluidic device containing a rigid block that simulates vascular injury and a flexible post to measure platelet contractile force through its displacement will be used to form shear-induced thrombi. I will determine how VWF affects platelet activation by testing whole blood samples and samples doped with DAPT (0.3 mM ASA and 10 μΜ 2-MeSAMP), 50 μg/ML VWF, or both VWF and DAPT. Platelet activation is measured through the area of platelet-plug, intracellular calcium levels, and platelet-plug contractile force. Preliminary experiments have shown that high VWF levels produced the largest platelet plugs whereas adding DAPT led to opposite effects. My results present that adding both VWF and DAPT to whole blood leads to similar platelet activation and larger platelet plug size as whole blood doped solely with VWF. The data obtained from this research can provide new insights into the improvement of therapeutic agents that aim to target VWF’s interaction with platelets and ultimately prevent repeat thrombotic events.

Hypoxic-Ischemic Encephalopathy (HIE) resulting from a lack of blood and oxygen to the brain is the leading cause of mortality in term newborns. Extracellular vesicles (EVs) serve as critical transporters of biomolecules between cells, with evidence of alleviating inflammation in models after hypoxic ischemia (HI) injury. Therapeutic efficacy of EVs has only been evaluated in males because males are more susceptible to worse outcomes following HIE injury, yet knowledge about EVs and their behavior when administered to females is still needed. In this study, I aimed to address this knowledge gap by systematically comparing the efficacy of male and female neonatal brain-derived EVs (mEVs, fEVs, respectively) applied on male and female neonatal rat ex vivo brain slices. I first confirmed the purity of isolated EVs with protein assays and immunoblots, and utilized an ex vivo oxygen-glucose deprivation (OGD) model of HI injury, applying fEVs and mEVs to sex-matched OGD-exposed brain slices. I evaluated cell viability after 24h of EV exposure, and my results show that fEVs decrease inflammation and cytotoxicity in OGD models. When compared to previous results using mEV treatment, my results suggest that females have a more robust anti-inflammatory response system to injury. Ongoing work to better understand the therapeutic effect of EVs involves further observing morphological shifts in microglia through confocal imaging, as fEV application will likely result in microglia shifting towards anti-inflammatory phenotypes, similar to what was previously observed after mEV application. I am also quantifying expression levels of various inflammatory and reparative genes through reverse transcription quantitative polymerase chain reactions (RT-qPCR). Overall, I have demonstrated in these pilot studies that fEVs have a different therapeutic effect in OGD injury compared to mEVs. This research is intended to open up pathways for more personalized sex-based treatments for various injuries and therapeutics in the future. 

Although immunotherapy with T-cells is successful in treating non-solid cancers, targeting solid cancer tumors remains a challenge. Unlike T-cells, bacteria can colonize solid tumors and thrive in a hypoxic tumor microenvironment (TME). This means that bacteria could be used to treat solid tumors that T-cells cannot reach. I propose to develop new bacterial immunotherapies that can be used as an alternative treatment method to fight solid cancer tumors. Specifically, I aim to engineer E. coli that secretes therapeutic payloads upon sensing the TME. My research focused on comparing secretion efficiencies of signal peptides and secretion tags. Signal peptides are short sequences that transport cargoes, such as therapeutic proteins, to the periplasm. Similarly, secretion tags are small secreted proteins that can transport a partner cargo fused to them to the extracellular medium. From literature, I selected the signal peptide PelB and the secretion tag YebF. I compared their efficiencies in secreting the cargo human interleukin 2 (h-IL2), an immunostimulatory cytokine. To this end, I expressed h-IL2 with either the genes for the signal peptide or secretion tag fused to the N-terminus and a detection tag on the C-terminus, in an E. coli expression strain. I induced the expression of cargoes, after which I isolated the proteins that were secreted into the extracellular medium. I detected the proteins through quantitative Western blot analysis. I concluded from my experimental data that the cargoes were secreted at a higher concentration with YebF than with PelB. I plan to repeat this experiment with another secretion tag, OsmY. The next step is to use the secretion system with the highest secretion yield to secrete a variety of potential immunomodulatory cargoes. I plan to evaluate their effects on immune signaling and their ability to eliminate tumor cells.