Lck is a lymphocyte specific tyrosine kinase involved in T cell activation in response to T cell receptor (TCR) mediated signaling. T cell activation is essential for the adaptive immune response, as it results in the proliferation of T cells after the detection of a peptide presented on a Major Histocompatibility Complex (MHC) and the production of cytokines necessary for immune response coordination. Lck activity is dependent on its global conformation, which is dynamically regulated via phosphorylation on its activation loop and C-terminus tail. Upon TCR engagement, active Lck phosphorylates the CD3ζ chains of the TCR complex, transducing the intracellular signaling events that activates T cells. Because Lck activity is dependent on its global conformation, we sought to map the conformational changes in Lck upon TCR simulation, as well as identify cysteine-reactive fragments that target and stabilize Lck in its conformational extremes. Lck has few endogenous cysteines, so we performed a yeast-growth-based deep mutational scan (DMS) of Lck–in which we utilized Lck’s toxicity to yeast to calculate the activity scores of ~5,000 Lck mutants–and identified 109 solvent-exposed, wild-type-like cysteine mutants of Lck. Expressing these wild-type-like cysteine mutants in T cells, and utilizing competition-based mass spectrometry, we can quantify changes in electrophilic reactivity of the cysteine side chains in the wild-type-like cysteine mutants upon T cell receptor (TCR) stimulation. Thus far, I have identified six wild-type-like cysteine mutants of Lck that are quantifiable using mass spectrometry and exhibit reactivity to our set of cysteine-reactive fragments, some of which show differential reactivity upon TCR simulation and fragment selectivity. Currently, I am using these mutants to map the dynamics of a hyperactive mutant of Lck. These quantifications provide insight into changes in the conformational flexibility of Lck, accessibility of the mutated residue sites, and intramolecular protein-protein interactions of Lck upon TCR stimulation.
 

The hallmark neuropathological finding of Parkinson’s Disease (PD) is the presence of intraneuronal protein aggregates, consisting of aggregated proteins and misfolded forms of alpha-synuclein. These intraneuronal protein aggregates, known as Lewy bodies, are implicated in many neurodegenerative diseases. Lewy pathology spread in a PD brain correlates with clinical disease progression. Glucosidase, beta, acid (GBA) gene mutations, the strongest genetic risk factor for PD, is also associated with accelerated disease progression and altered extracellular vesicles (EVs). EVs play a crucial role in intercellular communication and delivery of bioactive cargos throughout the central nervous system (CNS). I use a human neuronal cell culture model derived from induced pluripotent stem cells (iPSCs) to examine how GBA mutations alter EV composition, and investigate whether EVs truly act as a vehicle for the seeding of Lewy pathology in other cells, potentially accelerating the propagation of Lewy pathology throughout the CNS. To isolate and purify EVs from the conditioned media of neurons, I use centrifugation and size exclusion chromatography. I visualize and quantify the EV’s size and concentration using a ZetaView nanoparticle analyzer. I perform Western Blot Analysis for candidate cargo proteins within EVs, including alpha-synuclein, ubiquitinated proteins, and EV intrinsic proteins (CD-63 & CD-81). I isolate EVs from the media of GBA PD or WT control neurons expressing alpha-synuclein-GFP fusion protein and apply these EVs to GBA PD or WT neurons. I anticipate that EVs secreted by GBA versus control neurons will contain increased alpha-synuclein protein levels and that increased cell death, endolysosomal trafficking defects, and aggregation of endogenous alpha-synuclein will be associated with the uptake of GBA EVs by recipient neurons. This work will provide evidence supporting the role of GBA in influencing Lewy pathology propagation via EVs, which could elucidate a novel therapeutic mechanism that could be targeted to slow the progression of neurodegeneration.

Pregnant individuals infected with influenza A viruses (IAV) have higher risks of mortality, hospitalization, preterm birth, and stillbirth. The objective was to determine how the transcriptional program induced by IAV infection in the lung differs between pregnant and non-pregnant states. I hypothesized that a cluster of genes linked to aggravation of influenza disease would be upregulated in the pregnant lung early in IAV infection versus the non-pregnant lung. We used a non-human primate model [NHP; pregnant (N=10), non-pregnant (N=10); Macaca nemestrina, pigtail macaque] to investigate the transcriptional response in the lung of pregnant versus non-pregnant NHPs infected with the IAV CA/04/2009 (H1N1) strain. Maternal lung tissues were collected from the animals at necropsy 5 days after infection. mRNA-Seq was performed by first extracting mRNA from tissues, preparing mRNA libraries, and aligning raw sequencing data, using Spliced Transcripts Alignment to a Reference (STAR), to the macaque genome. I performed normalization of the raw gene count matrix using EdgeR in R Studio and alignment to the macaque reference genome. Next, I performed a single gene analysis using Limma-voom to determine differentially expressed genes (DEG). A total of 115 genes were significantly differentially expressed (>2-fold change, p<0.05) with 77 upregulated and 38 downregulated. Remarkably, genes linked to aggravation of influenza A viral disease, tissue injury, or acidification were upregulated in the infected pregnant versus non-pregnant lung 5 days after infection (MMP8, ATP12A, LGR4, NUP58, KBTBD6; log2fold change 1.28 - 2.8, all p<0.05). Next steps include gene set enrichment analysis and ingenuity pathway analysis to further investigate the gene networks linked to these upregulated genes. In summary, pregnancy was associated with upregulation of genes in the lungs 5 days after IAV infection that may predispose to greater tissue injury versus the non-pregnant lung.

Recent progress in synthetic biology has focused on utilizing probiotics as therapeutic production factories in the gastrointestinal environment to treat GI-related diseases. Although oral administration of probiotics is a convenient method for patients, a key challenge lies in the poor survival rate of probiotics in gastric and intestinal areas. Engineered living materials (ELMs), which are comprised of genetically engineered microbes embedded in a polymer matrix, present a novel formulation for orally-administered probiotics. Herein, we developed ELMs containing probiotics in a protein-based polymer matrix, aiming to enhance their viability in the GI tract. The ELMs’ photocurable polymer matrix allows us to 3D print our formulation into oral tablets. To form our protein-based polymer matrix, we functionalized bovine serum albumin with polyethylene glycol diacrylate. We then added a photoinitiator and E. coli Nissle genetically engineered to produce tryptamine (an anti-inflammatory agent) and subsequently photopolymerized this resin to 3D print probiotic tablets. We placed these tablets through a simulated gastrointestinal tract and observed cell escape using optical density measurements and cell viability through live/dead staining and fluorescence imaging. Liquid-chromatography mass-spectrometry was used to quantify the extent of therapeutic bioproduction in vitro by our ELMs over time. Overall, we found that the ELMs successfully delivered viable probiotic cells able to perform in situ therapeutic bioproduction. Furthermore, we observed that encapsulation of probiotics in ELMs yielded a higher survival rate of cells in the GI tract, suggesting that our polymer matrix formulation protected cells and allowed for extended proliferation and colonization in the colon. These findings are also supported by our observations that ELMs produced significantly higher amounts of tryptamine in the GI tract compared with non-ELM, free cells. The findings from our study can be applied to further development of orally-administered probiotic therapeutics, and show promise for future directions in drug delivery.

Malaria, a disease caused by the Plasmodium parasite, kills approximately 600,000 people per year. This disease consists of two stages, first an asymptomatic liver stage, followed by a symptomatic blood stage. The goal of our lab is to eliminate the transmission and spread of the parasite via the use of genetically modified parasites as vaccines. These parasites die in the liver, preventing blood infection and creating long-term immunity in the liver. Previous research in our group has shown that our vaccine model is effective in mice, with improved efficacy when the type-1 interferon response to the vaccine is disabled. Type-1 interferons are cytokines produced in an early immune response to a variety of pathogens. Yet, the mechanism in which these interferons are activated in a Plasmodium-infected liver cell is unknown. My project's goal is to identify and understand the mechanism in which this early immune response is turned on in response to parasite infection. To do this, I am developing an in-vitro system which can quantify the type-1 interferon response in Plasmodium-infected liver cells in culture. Utilizing various in-vitro techniques, we can identify the sensors, adaptors, and transcription factors that are most important in upregulating this early immune response. This knowledge will be used to inform methods to inactivate the type-1 interferon response, in turn improving our vaccine model. Our lab hopes to eventually use our model in humans for the goal of eradicating malaria from the modern world.

Rodents are a common model for ischemic stroke research; however, their brains are mostly grey matter while approximately half of tissue affected by stroke in humans is white matter. To study stroke in white matter, we model ischemia in the mouse optic nerve (MON), a pure white matter tract. We observe impaired axonal function and conductance in the MON after ischemia that is improved by ischemic preconditioning (IPC), a phenomenon in which a brief ischemic stimulus protects against subsequent prolonged ischemia. Our prior work demonstrates microglia are required for IPC-mediated axonal protection. Several models of injury and disease report elongation of the nodes of Ranvier (NoR) leading to reduced axonal conductance, but the role of microglia in protecting axons at the NoR is unknown. Here we investigate how NoR are affected by ischemia and microglial depletion. Based on our previous work, we hypothesize that IPC will preserve NoR lengths after exposure to ischemia and this protection will be lost when microglia are absent. Microglia were depleted with PLX5622, a colony stimulating factor 1 receptor antagonist. After treatment, a subset of animals were collected to assess baseline average NoR lengths after microglial depletion alone. Another cohort (N=5) received an in vivo IPC stimulus (15-minute transient common carotid artery occlusion) and 72 hours later experienced ex vivo oxygen-glucose deprivation (ischemic stroke) for 45 minutes. MONs were fixed overnight in paraformaldehyde and prepared for immunohistochemistry using fluorescent antibodies against Nav1.6 (nodes) and Caspr (paranodes) to identify NoRs with confocal microscopy. Nodes are measured using FIJI and the distance between Caspr+ paranodes flanking a Nav1.6+ node is calculated using MATLAB. Microglial depletion alone was found to be associated with increased NoR lengths. Our ongoing work is focusing on the impact of ischemia on NoR lengths and how this may be modulated after IPC.