Plasmodium vivax (P. vivax) is the most widespread malaria species. P. Vivax can lay dormant in hepatocytes and cause recurring malarial infections. Tafenoquine (TQ) is an antimalarial drug approved by the FDA in 2018 for the radical cure of P. vivax. Unfortunately, 8-aminoquinolines like TQ cause hemolytic anemia in G6PD deficient patients. Due to this contraindication, genetic G6PD screening is required before TQ administration. These additional tests pose a significant challenge for broad administration in resource-constrained countries. Our polymer prodrug conjugates (drugamers) are designed to improve TQ delivery to the liver and reduce red blood cell exposure. Using RAFT polymerization, drugamers can be optimized for delivery efficiency and avoidance of hemolytic anemia. Polymer architectures can be further enhanced by utilizing a variety of enzyme cleavable linkers, monomers, and receptor binding cofactors. We previously demonstrated increased liver-to-blood area under the curve ratios in mice using a variety of different polymer architectures. Mice received an IV or subcutaneous dose of drugamer and relevant organs were harvested at previously determined time points up to 48 hours. Pharmacokinetic profiles were created using a plate reader fluorescence assay and tandem LC/MS/MS with tissue samples. These drugamers represent an ongoing effort to iteratively improve our drug-polymer design. The improvement in TQ delivery through our drugamer vehicles could allow for more liberal administration guidelines, completely removing the need for G6PD testing for the treatment of P. vivax.

Atrial fibrillation (AFib) is the most common sustained cardiac arrhythmia, contributing to significant morbidity and mortality worldwide. Patient-specific computational models of the left atrium are currently studied to predict characteristics of reentrant activity that promotes fibrillation. However, current models’ patient-specificity is limited to anatomical structure and the distribution of disease-related remodeling (fibrosis), whereas electrical properties of cells and tissue are based on literature values. In cases where patients are clinically known to present with either AFib or atrial flutter (AFl), this lack of personalization can lead to inaccuracies in simulation outcomes (e.g., AFib-like behavior in simulations for a patient who actually had AFl, or vice-versa). My goal was to derive parameter sets that favor the initiation of one type of arrhythmia or the other (AFib or AFl). Ten fibrotic left atria were reconstructed from late-gadolinium enhanced (LGE)-MRI scans and the bioelectric parameter space (comprising ion channel expression levels and impulse propagation rates) was explored using a Taguchi L27 Design of Experiments (DoE) approach. Arrhythmias were induced by initializing four atrial regions to different phases of the action potential under each parameter permutation. I ran 300 simulations and manually classified each arrhythmia episode as either AFib- or AFl-like based on prior definitions. I pinpointed a pro-AFl parameter set – bioelectrical conditions under which 89% of all induced arrhythmias were AFl and only 11% were AFib. The pro-AFib parameter set in these preliminary simulations was comparatively less robust (61% vs. 39% for AFib vs. AFl inductions, respectively). My future work on this project will establish stronger relationships between model configurations and simulation outcomes by probing a wider array of possible parameters in a larger population of patient-specific models. Data from the present study will guide future simulations to accurately tailor models to represent the arrhythmic state in patients predisposed to AFl.

The innate immune system must have a rigorous response to many pathogens in order to successfully defend host cells against infections. During infection, inflammasome forming sensors detect pathogen-specific features which release proinflammatory cytokines such as IL-1B for immune activation. The inflammasome forming sensor NLRP1 directly detects multiple signals indicative of infection, including viral protease activity. Recently, it was shown NLRP1 is indirectly activated by bacterial toxins and UV irradiation that disrupt host protein synthesis, demonstrating it can detect environmental stimuli to cause inflammation. However, much of this activation pathway is poorly understood with only one nonpathogenic cause having been investigated. Our goal is to identify the host proteins required for NLRP1 activation and determine whether NLRP1 can broadly detect disrupted protein synthesis. We hypothesize NLRP1 detection of disruption in protein synthesis is a broad strategy to combat infection and may have an important role in causing inflammation in other diseases associated with disrupted protein synthesis, including cancer and neurodegeneration. Knockout cell lines of proteins suspected of activating NLRP1 are produced through lentiviral transduction of Cas-9, a gene editing tool which cleaves off specific nucleotides corresponding to the target gene of each protease sensor in the inflammasome activation pathway. Each cell line is confirmed to be absent of the target sensor via genotyping, and is followed by a functional assay of each knockout line which induces cellular stress targeting the activation of each cleaved protease. We predict that inflammasome activation as defined by IL-1B concentration will be significantly decreased in knockout cell lines targeting key proteases in the inflammasome signal cascade, showing that the overactivation of these proteases is sufficient for inflammasome activation. These results could provide key targets for drug discovery in the treatment of multiple diseases which cause disruption of protein synthesis, including cancer and neurodegeneration.

An estimated 5-8% of the American population have cerebral aneurysms, showing higher rates of development in patients with common risk factors like hypertension, smoking, and family history of cerebral aneurysms (CA). This study focuses on understanding the causes of aneurysmal subarachnoid hemorrhage (aSAH), where a CA ruptures, resulting in bleeding in the brain. Endovascular coiling is a minimally invasive surgical treatment method for aSAH. Unfortunately, up to 30% of endovascular coiling treatments are unsuccessful, leading to aneurysm recurrence, growth, or rupture. The risk of these outcomes can be predicted using Computational Fluid Dynamics (CFD), a tool that quantifies the hemodynamic environment by solving the equations of motion for a fluid. The CFD simulations calculate factors significant in predicting the effectiveness of coiling treatment including flow rate, wall shear stress, and pulsatility. In this project we have studied the effect of using patient-specific blood viscosity values (the resistance of the blood to fluid flow), that have typically been standardized for all patients in CFD simulations. We have analyzed the effect of using patient-specific blood viscosity on pre-treatment patient-specific computational fluid dynamics simulations of endovascularly-coiled cerebral aneurysms. Preliminary results show that there is an expected improvement in CFD simulation predictive power of treatment effectiveness when patient-specific blood viscosity values are used. We hope to improve the predictive power of CFD simulations regarding the treatment outcome of aneurysm coiling, allowing us to better predict aneurysm recurrence, and eventually guide treatment outcomes.

Cystic Fibrosis (CF) is a recessive genetic disease that affects roughly 70,000-100,000 people worldwide. CF is caused by mutations in the CFTR gene, which encodes for a channel that regulates the flow of water and ions into and out of the cell. CFTR deficiency results in mucus buildup and other defects that lead to increased bacterial burden and inflammation. However, whether or not CFTR deficiency itself is sufficient to initiate or potentiate epithelial inflammatory responses is unknown. Inflammasomes are multiprotein complexes that upon activation initiates a lytic form of cell death called pyroptosis and the release of pro-inflammatory cytokines including IL-1B, a hallmark of CF. Previous studies have shown that inflammasomes are expressed in the epithelia of mice and humans. Although CF is largely a disease that affects airway epithelium, other tissues such as the skin and gut are also affected by CFTR dysfunction. Moreover, we and others recently found that inflammasomes play an important role in host defense of epithelial barriers, including the intestinal epithelium. Thus, we hypothesize that inflammation in the gut of CF patients may be caused or enhanced by inflammasome activation. To test this possibility I used both human and mouse intestinal organoids as models and ran a Forskolin-induced Swelling (FIS) Assay to assess CFTR function in presence and absence of a CFTR inhibitor (CFTRinh-172). Having established this model system, I am now evaluating the impact of CFTR function on inflammasome activation. We anticipate that our work may reveal a link between CFTR dysfunction and inflammasome activation which may provide further insight into how inflammasomes affect inflammation in the gut epithelia of CF patients.