Autism spectrum disorder (ASD) is a neurodevelopmental disorder marked by repetitive behavioral patterns and challenges with social interaction. Gastrointestinal symptoms are a common comorbidity of ASD, and individuals with the disorder tend to have a distinct gut microbial community composition and circulating metabolomes. Elevated levels of gut-derived metabolite 4-ethylphenolsulfate (4-EPS) are associated with ASD mouse models and children with ASD. Administration of 4-EPS to conventional mice induced atypical myelination and anxiety-like behaviors. 4-Ethylphenol (4-EP), its precursor, is produced by gut microbiota before host-mediated sulfation; however, its microbial biosynthetic pathway remains unknown. We propose a pathway involving stepwise conversion of plant-derived complex polysaccharides to 4-EP. My project aims to identify a gut microbial enzyme that completes the first step of this proposed pathway: a hydroxycinnamoyl esterase. After extensive literature review and biochemical study, candidate enzymes from resident gut microbes were identified and selected using bioinformatic tools. In vitro and in vivo experiments will be used to assess their activity towards model and dietary substrates. Remaining substrate and product concentrations will reveal species and strain specific enzymatic activity and substrate uptake. If the results are negative, this bioinformatics to experimental analysis pipeline will be repeated on new candidate enzymes. This work complements ongoing lab investigations to demonstrate the complete enzymatic pathway in a single species. Elucidating the microbial biosynthetic pathway of 4-EPS will contribute to detangling the gut’s role in ASD, regardless of if it is causal to the disorder or simply a biomarker. More importantly, studying the biochemistry and metabolism of gut microbiota supports the efforts to fill fundamental gaps in understanding the gut-brain axis. 

Assessing how organisms respond to shifting climatic conditions is crucial in the era of climate change to predict species' resilience to environmental changes. This study aimed to explore the effects of heatwaves on grasshopper development and fitness. Specifically, I investigated the reproductive potential of two grasshopper species within the framework of a common garden heatwave experiment. In Spring 2023, we reared the grasshoppers under three heatwave intensity treatments, exposing each treatment group to three heatwaves during set developmental stages. Afterward, I dissected the preserved females frozen for analysis, quantifying the number of primary and secondary oocytes in their ovaries. Oocytes develop into eggs and as such are a metric of reproductive potential. I hypothesized that increased heat stress would result in a decline in fecundity. However, we did not find a significant effect of the heatwave treatment on oocyte count, suggesting any fecundity effects of heatwaves are via a different mechanism. Understanding how organisms respond to changing environmental conditions is key to understanding how ecosystems will change in the coming years, and is important for informing conservation efforts.

Lys-R type transcriptional regulators (LTTRs) are one of the largest families of bacterial transcriptional regulator proteins with over 850,000 known members.  Many of these LTTRs are enriched in our gut microbiota, whose metabolic processes affect human health outcomes. LTTRs regulate gene expression through the binding of specific ligands to their ligand binding domain. Currently, less than 500 of them have been studied which represents a severe knowledge gap that conventional methods of characterization are unable to keep up with.  We aim to create a high throughput methodology to characterize LTTRs by their corresponding ligands that regulate gene expression. We are currently developing an assay to use chimeric LTTRs, or engineered LTTRs that share the same DNA binding domain yet a variable ligand binding domain. The use of chimeric LTTRs, which will all bind to the same DNA promoter, will potentially allow dozens of LTTRs to be tested in one assay. Our work thus far has demonstrated that chimeric LTTRs can be expressed in E.coli cells and purified using affinity chromatography and magnetic bead purification. We have also demonstrated that their ligand binding domains are functional and specific via differential scanning fluorimetry, and that their DNA binding domains are functional using an electromobility shift assay using SYBR green and SYPRO ruby dyes. Future work will explore their ability to regulate gene expression when their proper ligands are introduced with a substrate-induced gene expression reporter assay. Then uncharacterized LTTR candidates to be made into chimeras will be selected via a bioinformatic sequence similarity network analysis for assay piloting. If successful, this assay has potential to elucidate new metabolic pathways of our gut microbiota allowing for better understanding of their complex relationship with the human body.

Less than 10% of drugs successfully transition from preclinical to clinical trials, principally due to the inability of currently used 2-dimensional models to simulate the 3-dimensional structure and function of human tissues. To develop 3D in vitro models of human vasculature for more efficacious screening of anti-atherosclerosis drugs, I created a device for constructing a perfusable tissue containing a lumen by leveraging open microfluidic patterning methods developed by our group: suspended tissue open microfluidic patterning (STOMP). The device can be used to pattern tissue with a hollow luminal structure lined with endothelial cells, which can be perfused via hollow posts the tissue is suspended between. Using surface tension-driven flow, a liquid hydrogel precursor solution flows through the open microfluidic channel and around the two hollow posts. After gelling, the tissue anchors to the post, contracts away from the sides of the microfluidic channel, and the STOMP device is removed. By adding a second STOMP device that can surround the first tissue another cell-laden hydrogel can be patterned around the first tissue, encapsulating it. To form a lumen in cardiac tissue, I will pattern the inner region with human umbilical vein endothelial cells (HUVECs) in an enzymatically degradable polyethylene glycol hydrogel, surrounded by human induced pluripotent stem cell-derived cardiomyocytes in fibrin hydrogel. Enzymatic degradation of the core region will form a cavity through which HUVECs will remodel the cavity walls, forming an endothelial lining. I will assess lining formation by adding fluorescent dextran to cell media being perfused through the device and measuring fluorescence through confocal microscopy in the surrounding region over time, allowing me to evaluate the permeability of the membrane to compare with physiological values. This model can then be used to screen treatments for atherosclerosis to study how drugs interact with cells in a 3D microenvironment. 

Syphilis, caused by Treponema pallidum (T. pallidum), remains a significant global health concern, with increasing cases worldwide. Doxycycline post-exposure prophylaxis (Doxy-PEP) has emerged as a potential strategy to prevent infection. However, widespread use raises concerns about the possibility that doxycycline-resistant T. pallidum strains might emerge and spread. This issue is alarming since doxycycline is a second-line therapeutic for syphilis and is often used in patients with allergies to beta-lactams or when beta-lactams are unavailable due to shortages. If genetic resistance to doxycycline were to develop in T. pallidum, it could undermine the effectiveness of Doxy-PEP and further narrow the range of treatment options for syphilis. To address this concern, I developed a restriction fragment length polymorphism (RFLP) assay to detect potential doxycycline resistance mutations in T. pallidum. This assay analyzes the 16S rRNA gene region of T. pallidum where most likely mutations could develop based on the analysis of other resistant pathogens. The assay was optimized using three synthetic 16S rRNA gene constructs containing the resistance-associated mutations and DNA from a wild-type T. pallidum strain (Nichols) as controls. The presence of mutations in the amplified control DNA was assessed by restriction digestion with the AluI, RsaI, and SfaNI enzymes, which can selectively cut wild type and mutant sequences and reveal specific mutations. The analysis of 60 archived samples from syphilis patients collected in the US, Madagascar, Argentina, and Sri Lanka is ongoing. Results will provide data on the frequency of doxycycline resistance mutations in T. pallidum, if any are found in this selected group of specimens. Developing a rapid, cost-effective surveillance tool is essential for monitoring potential resistance and preventing treatment failures when doxycycline is used.