Caenorhabditis elegans is frequently used as a model organism for testing the effects of various compounds on longevity. A current limitation of running these experiments is the tremendous amount of work needed to collect large sample sizes of data when testing for several compounds in different genetic populations. Fortunately the Kaeberlein lab has developed the WormBot, an image capture robot used to take high resolution images of hundreds of experimental plates each containing ~30 worms. Researchers still have to rely on humans manually annotating tens of thousands of frames to extract valuable metrics for their analysis. I have developed the implementation of a neural network to automatically analyze these images so that the number of experiments and compounds that can be tested is increased exponentially. I utilize a network architecture known as Yolov3 to allow the computer to identify and track individual worms from the images. The results obtained from our new computational method extract data from the images that is equal to or even better than the human method while also requiring a fraction of the time. Using this novel platform, we are analyzing a broad spectrum of natural and synthetic compounds for their effects on longevity and health span in C. elegans.

The technique of Indirect Calorimetry (IC) allows for the non-invasive and continuous measurement of metabolically relevant functions of an animal. The Michael Schwartz lab uses IC techniques in the investigation of how brain regions, neuronal populations, and the larger neuronal circuitry that they connect with, defend either blood glucose or body weight. Taking IC measurements of treatment mice allows for quantification of glucose consumption and energy homeostasis. My project was to develop a programming pipeline for the analysis of circadian metrics from rodent populations placed on a 14-hour light and 10-hour dark cycle and placed on control or high-fat high-sucrose diets. The circadian cycling of oxygen consumption and respiratory energy ratio was quantified, daily maxima and minima were measured, and latency from dark cycle onset to peak was measured. Using C#, I generated a parsing software that could combine data from experiments, recalibrate time to the day-night cycle used, and pull out these parameters for future research.

Synapses between neurons are crucial for all of human behavior as they underlie the ability to form thoughts, make precise movements, learn skills, and form new memories. Spinules are finger like projections from one neuron that are embedded within the information-sending end (i.e., presynaptic bouton) of another neuron. While recent data suggests that spinules are abundant within synapses across the brain, we have a limited understanding of their synaptic specificity or function. It is speculated that synaptic spinules may act as anchoring mechanisms or as a novel form of neuronal communication. Here, we sought to determine how presynaptic boutons that contain spinules differ from those that do not, as a first step towards uncovering whether spinules target specific subsets of synapses in the brain. We performed extensive quantitative 3D reconstructions and analyses of 138 excitatory (i.e., releasing glutamate) presynaptic boutons within the CA1 Hippocampus (center for memory formation) of an adult rat, including 87 spinule-bearing boutons (SBBs), and 51 presynaptic boutons without spinules (Non-SBBs). We found that on average, SBB volumes were 2.6-times larger than Non-SBB volumes (p<.00001), and that the size of the synapses made by SBBs were 1.6-times larger than those made by Non-SBBs (p<.00001). Since the anatomical size of a bouton and its synapse are highly correlated with its physiological strength, these data suggest that spinules may increase the physiological strength of excitatory synaptic connections in the CA1 hippocampus, and may therefore play an important role in memory formation.

Group B Streptococcus (GBS) are beta-hemolytic, gram positive bacteria that colonize the vaginal tract of 1 in 4 pregnant women, and can cause preterm birth and neonatal infections. A key factor involved in GBS pathogenesis is an enzyme known as hyaluronidase (HylB). HylB is secreted from GBS during infection, and allows GBS to evade the host immune response by interfering with pathogen associated molecular patterns (PAMPs) recognition through toll-like receptors (TLRs) 2 and 4. The goal of this project is to use a biochemical colorimetric absorbance assay to quantify HylB activity in GBS isolates by comparing 50 maternal commensal isolates to 200 neonatal invasive isolates. Following quantification, the isolates will be stratified based on the epidemiological and clinical data to determine how HylB contributes to disease outcome. Although HylB’s role in GBS pathogenesis has been partially described by in vitro experiments and animal models, this virulence factor is not assessed clinically when patients are tested for GBS during pregnancy. Thus, this project allows us to identify new correlations between HylB activity and real-world clinical outcomes, which may be important for predicting disease progression. Currently, about 24% of invasive isolates exhibit HylB activity, though analysis for these isolates are still in progress, and we are continuing this study to include commensal isolates. We hypothesize that the commensal isolates will have reduced HylB activity compared to invasive isolates, as the ability for GBS to evade the host immune response may allow for greater dissemination. Knowledge from this study may contribute to the design of novel therapeutics, such as those targeting HylB, that may help prevent poor outcomes during human infection. 

Prior to the outbreak of SARS CoV-2, enveloped RNA viruses had been successfully detected in municipal wastewater. Since SARS CoV-2 emergence, researchers have further validated this technique. By March 2020, research suggested viral concentration via wastewater sampling was predictive, by 3-5 days of a subsequent rise in COVID-19 infections. Thus, measuring municipal wastewater quickly became a useful epidemiological tool for COVID-19 management. Despite relative validation, these data provided population level trends, leaving officials little time, or location-specificity, to make use of this warning signal. To address this problem, we partnered with the City of Tacoma Environmental Services Division and Tacoma Pierce County Department of Health (TPCDH) on behalf of RAIN, a Tacoma based biotechnology non-profit, and designed a neighborhood-level rapid response plan. By analyzing health disparities data, wastewater infrastructure and active case rates we sought to illuminate sites of active transmission. To conduct this study, we sampled weekly from two wastewater treatment plants in Tacoma, WA, and five geographically disparate neighborhood sites to monitor SARS CoV-2 levels. We collected population-specific biological samples in each neighborhood for a population-specific SARS-CoV-2 analysis. Using quantitative real-time polymerase chain reaction (qRT-PCR), we successfully isolated SARS-CoV-2 at variable levels in all sample populations between April 2020 and March 2021. Taken together, our data reveal infection trends within a municipal sub-population in absence of individual testing. Additionally, by this method we collect information on the outstanding question of asymptomatic infections. Overall, these data provide health officials geographically specific information to best mobilize limited healthcare resources.

The ability to selectively and efficiently insert DNA into the genome has been a long-standing goal to potentially fix disease associated variations and add tags to proteins for biochemical assays. The advent of CRISPR-Cas9 technology has allowed for the ability to specifically target any region of the genome with a single guide RNA (sgRNA) and lead to DNA cleavage. This double stranded break by Cas9 can be repaired either through nonhomologous end joining (NHEJ), which creates insertions or deletions, or homologous recombination (HR), which requires DNA that matches the region where the break occurred. The goal of this project is to create a tool that favors HR repair over NHEJ repair after cleaving with CRISPR-Cas9 to allow for targeted gene insertion. One way to do this is to generate large amounts of multicopy single-stranded DNA (msDNA), an RNA-DNA generated by a reverse transcriptase, which transcribes RNA back into DNA. We want to determine if combining CRISPR-Cas9 technology with a reverse transcriptase to generate msDNA will produce enough homologous DNA strands to drive HR repair over NHEJ repair in vivo. To test this method, we have replaced the tyrosine hydroxylase (TH) gene, the rate limiting enzyme that produces dopamine, with green fluorescent protein (GFP). We have generated a virus containing a reverse transcriptase with a sgRNA targeted to TH and msDNA containing homology arms flanking GFP. Four weeks after co-injection of this virus with a virus containing Cas9 into the ventral tegmental area of adult mice, we analyzed brain slices to determine the loss of TH and expression of GFP. Future studies will aim to quantify gene insertion and target other genes to insert point mutations. The findings of this study may further research in the repairment of disease associated variations in genes. 

A survey in the existing literary sources says that there are three main diet-related patterns in Blue Zone communities that contribute to their longevity. After I cross-referenced the micronutrients in Blue Zone diets with the average American diet, I found that the Blue Zone diets are all enriched for some common vitamins and nutrients, specifically: retinol, thiamin, pyridoxine, ascorbic acid, vitamin E, vitamin K, magnesium, and potassium. All of these vitamins and minerals are known to affect cellular metabolism through a wide array of factors, potentially linking them to bettering health and increasing lifespan. I initialially will confirm that these compounds indeed play a vital role in extending the functionality of the biomechanical systems in C elegans. I will then further pursue whether combinations of the nutrients increase effectiveness, and which specific areas in the body they individually impact for promoting longevity. My first step in investigating the biomechanical effects of these micronutrients will be to incorporate each one individually into the C elegans’ diet. My long-term goal, once I test the wild type model, is to focus on how these micronutrients combat age related diseases. To do this I will use my results for optimal micronutrients to test the effectiveness of the specific micronutrients in the Alzheimer’s degenerative pathway. Specifically, I will use the GMC-101 transgenic worm, (genetically modified strain to exhibit the degenerating effects of Alzheimer's disease), which expresses human amyloid- beta - a protein implicated in the progression of human Alzheimer’s Disease - in the body wall muscle and becomes increasingly paralyzed with age. I will use a similar experimental set up, looking for a delayed onset of paralysis. I hope this research will be able to shed light on how micronutrients interact and affect degenerating biochemical pathways in aging C elegans; specifically, in a strain that models Alzheimer’s disease.