Skin is a densely innervated sensory organ, populated with various somatosensory receptors that help us perceive touch stimuli. Frequent injuries to the skin cause axon damage and lead to axon degeneration. Degenerating axons leave behind debris that must be cleared before reinnervation can occur and sensation is restored. We use adult zebrafish as a model to study injury and innervation because they have homologous cells and structures to humans and have transparent skin, allowing for high-resolution microscopy. Previous experiments in our lab revealed that skin-resident immune cells known as Langerhans cells (LCs) use highly motile protrusions to engulf axonal debris in the zebrafish epidermis. How are these dynamic protrusions regulated at a molecular level? Calcium signaling regulates phagocytosis and cell motility in other cell types, but the role of calcium signaling in LCs is unstudied. Through scale pluck assays and fluorescent microscopy, I have established a model for monitoring calcium signaling in LCs. I observed transient calcium flashes in LCs that varied in frequency, intensity, and subcellular location. I perturbed calcium flux using a calcium chelator and observed decreased flash intensity and protrusion length in LCs, suggesting that calcium signaling is required for protrusion dynamics. To investigate how calcium signaling affects engulfment, I imaged calcium flux in LCs during phagocytosis of apoptotic cells after laser injury. In contrast to the transient flux normally observed, LCs exhibited sustained high concentrations of cellular calcium during corpse engulfment. Because of the effects of perturbed calcium flux on intracellular calcium and protrusion motility, I hypothesize that perturbing calcium concentrations will inhibit LC phagocytosis. Identifying the molecular mechanisms underlying debris removal, such as calcium signaling in LCs, is relevant to understanding skin repair and disease states in which axon homeostasis is altered, including diabetic and chemotherapy-induced peripheral neuropathies.

Single cell neural activity mapping is a novel experimental approach used to understand the relationship between neural activity and behavior/thought across the intact brain. The current approach utilizes immunodetection of Fos, a reliable and endogenous protein marker for neuronal activity that has unique induction and decay properties. This method combines whole-mount brain tissue clearing, IHC staining, and high speed volumetric imaging. However, whole-mount IHC is incredibly challenging due to many factors including variable antibody lots, lengthy processing protocols, and inconsistent timing. To overcome these limitations, various genetic methods including direct gene modification and replacement have been produced. However, these methods limit brain-wide expression profiles, display inaccurate signal to noise ratios, and yield low signal expression levels. Here, we have developed a novel activity-dependent tool that allows viral vector-compatible Fos-like reporting of neuronal activity. Our strategy relies upon non-promoter-based regulatory sequences that endows downstream genes with Fos-like induction profiles. Here, we present its effectiveness in ectopically labeling Fos+ cells in the mouse brain in-vivo, and report its comparison to other conventional genetic strategies. Further, we extend its range of use with the creation of multiple versions that enables a range of activity level reporting and through multiple wavelengths of fluorescence. In summary, this novel genetic tool can be used to ectopically map single cell neural activity more effectively in order to better understand the anatomical basis of neural coding driven by specific cell-types distributed across the entire brain.

 Within the field of neuroscience, optogenetics is an established experimental tool which can be used to alter specific cell function or trigger enzymatic reactions under millisecond time-scale precision. The high temporal precision of optogenetic recombinases allows for precise identification of cell populations which causally regulate specific behaviors in health and disease. Furthermore, the inducible optogenetic control specifically of recombinase activity for cell-type targeting eliminates the use of other inducible methods including exogenous chemicals that operate on lower time-scales and notoriously cause non-specific effects. Currently available optogenetic recombinases are driven by low wavelengths of light (e.g., Yao et al, 2020) which limit their in-vivo use to local and/or superficial areas of the brain in an invasive manner. Our recently engineered optogenetics-based protein pair, NOC (Near-IR Optogenetic Cre recombinase) induces Cre recombinase activity with near-infrared (NIR) light through dimerization of split-Cre fragments. We have previously demonstrated NOC’s capability for functional Cre recombinase activity under 650 nm light administration, and now aim to optimize the inducibility profile of NOC through two additional modifications. These include modifications to Cre split-site locations based off of previously designed blue light-inducible recombinases (Yao et. al, 2020), and a novel 660 nm inducible photoreceptor pair (Zhou et. al, 2022). The inducibility of these new configurations will be tested using our in-vitro fiber optic system. The optimal configuration of NOC will lay the groundwork for NOGen (NIR OptoGenetic ensemble capture), an alternative version of NOC which will include a calcium-sensing domain on one of the protein fragments. This will limit NOGen’s activity to active neurons only, offering greater precision in identifying specific cell-populations which drive particular behaviors. These improved molecular tools can be used to further our understanding of brain anatomy and function, which serve as an important catalyst for the development of improved brain disorder treatment.

Hydrogen sulfide (H₂S) impacts biological systems in multiple ways, including the arrest of aerobic respiration, and thus is mechanistically similar to cyanide. Unlike cyanide, however, H₂S can accelerate as well as end cell growth, including in plants, where it drives germination rates when administered in micromolar concentrations. However, the limited research to date leads to a need to better quantify and contextualize chemical composition changes in plant tissue following H₂S-induced plant growth. This study resulted in the ability to quantify the biophysical impacts of H₂S-induced growth in plants. The novel use of δ³⁴S and δ¹⁵N isotope ratios produced at the IsoLab in University of Washington represent the results of this sampling and its subsequent analysis. They may represent a new means to understanding the effects of H₂S on plant growth, including during the crucial phase of plant germination. The effects were observed on hypocotyl tissues from seedlings of Pisum sativum (pea), Phaseolus vulgaris (bean), and Zea mays L. (corn), all grown in hydroponic H₂S solutions, ranging from 0-100μM. These specific isotopic methods may allow comparison between modern and fossil material, because these isotopic species are known to have been preserved across a wide diversity of plant fossils. This novel application of these classic staples in the biochemical toolbox may have further implications for better understanding past events, because many major mass extinctions have now been linked to excess oceanic and atmospheric H₂S (compared to today), and may also, paradoxically, present new paths toward increased crop yields.

Synthetic promoters are increasingly being used to control and fine-tune gene expression in multicellular organisms as part of engineering novel traits. For this genetic engineering project, we aim to build and activator system that when engaged leads to a statistically significant increase in gene expression compared to the unengaged system. Previously, our lab screened for promoters that are constitutively and ubiquitously expressed in Arabidopsis Thaliana. From this screen, multiple viable promoters were found, and a subset was selected for modification by adding sequences not found anywhere else in the genome (target sites). Our first experiments used the CRISPR-mediated gene regulation system to recruit repressors to create NOR logic gates (where a reporter is only expressed when two different inputs are absent) through the use of guide RNAs (gRNAs) complimentary the engineered target site. Currently, we are working to expand the capabilities of these synthetic promoters by swapping the repressor with an activator to see if we can boost expression from these promoters. Our activator construct has an enzymatically disabled Cas9 connected to an activation domain called EDLL, and an aptamer in the gRNA that attracts a second activation domain, VPR. We are currently generating transgenic plants carrying our synthetic promoters and activator constructs. I will quantify gene expression by measuring the levels of a fluorescent reporter under the control of our synthetic promoter. The use of both activator and repressor approaches will allow for the construction of increasingly complex genetic circuits. These circuits have wide potential applications across the fields of synthetic biology and metabolic engineering.

The migratory songbird Gambel's White-Crowned Sparrow (Zonotrichia leucophrys gambelli) experiences drastic seasonal shifts in its song production and stereotypy, driven by changes in distinct song circuit nuclei, namely increased neurogenesis in the forebrain nucleus HVC and increased electrical excitability in nucleus RA. Manipulating the photoperiod and hormone levels of these sparrows in a laboratory setting results in changes very similar to the seasonally-induced behavioral and neurophysiological changes they experience in the wild. We are interested in reducing the number of birds necessary for our studies by developing a protocol to produce seasonal effects in vitro via organotypic slice culture. Organotypic slice cultures preserve the neural connections and functions involved in the seasonal changes we are interested in, so they can be more directly observed and manipulated. We maintained slices of neural tissue from white crowned sparrows, including HVC and RA, on a membrane that allowed for exchange with the media, a technique initially developed by Stoppini et al., in 1991. After the tissue was cultured, it was fixed and resectioned into thinner slices so it could be Nissl stained and imaged to test for the presence of healthy cells. Organotypic culture is an established technique for neural tissue from juvenile animals; we are attempting to use it on tissue from adult, wild caught, white crowned sparrows. There is currently no protocol for organotypic cultures of this tissue, and the nature of the tissue itself poses a challenge. Adult tissue is less plastic than the juvenile or neonatal tissue that is usually used with this technique, so it has more difficulty surviving the shift to culture. Once the protocol is developed, we plan to manipulate the hormonal environment to try to mimic changes that occur during their breeding season.

Binary systems containing a compact object may exhibit periodic brightening episodes due to gravitational lensing of the lower-mass companion as it transits the compact object. Such self-lensing systems have been discovered before by identifying this periodic brightening in light curves. We explore the possibility of using this method to detect new compact objects with data from the Zwicky Transient Facility (ZTF). Thanks to its extensive optical variability coverage of the Northern Hemisphere sky, ZTF provides an ideal dataset for this search. We present methods used in a systematic search for self-lensing signatures in Galactic binaries, and final candidates that may exhibit these signatures. This method could become a new means of discovering noninteracting compact binaries and provide a way to estimate their masses.

Over the past 50 years, breast cancer mortality rates in high-income countries have dropped significantly while low and middle-income countries (LMICs) have made few advancements towards early detection and rapid diagnosis. A lack of infrastructure in LMIC healthcare settings causes patients to receive their diagnosis several months after testing, while some never hear back due to travel and communication barriers. We have partnered with Peace and Love Hospitals in Ghana to develop CoreView Imaging on the Needle (ION), a portable, rapid point-of-care (POC) diagnostic system for use in LMIC. Our team developed ION after discovering breast core needle biopsies (CNBs) were too delicate and sticky for transport in our original millifluidic design. Our research is exploring methods of integrating the ION system into CoreView to improve the testing reliability and image quality of biopsies for diagnosis by designing, testing, and iterating fixtures to image CNBs while still on the needle. We modeled our prototypes in Solidworks and printed them using a Prusa 3D printer. Our goal is to load the CNB under a microscope, stain the tissue nuclei, compress its surface against a clear glass window, and image the biopsy surface within 5 minutes. We are using Microscopy with UV Surface Excitation (MUSE) imaging to take panoramic images along the 20 mm biopsy length. The current prototype takes 1 minute to prepare the biopsy (stain and load into ION fixture), 0.5 minutes for each MUSE image and step to the next image (total of 6 images), and 0.5 minutes to unload the biopsy, resulting in 4.5 minutes between biopsy collection and obtaining images for diagnosis. The improved image quality and expected high reliability verifies the success of our ION design for further validation testing to provide earlier breast cancer detection and rapid treatment to rural patients.

Hexagonal boron nitride (hBN) is essential for nearly all nanoscale two-dimensional (2D) devices, as its flatness and wide bandgap make it an ideal dielectric for applying electrostatic gating. At high electric fields, hBN undergoes electrical breakdown where large currents flow to the sample, damaging the device. Gating fields are therefore limited by hBN’s dielectric strength. While the dielectric properties of hBN have been studied previously, the preparation of those samples differed from that used in practice. I conducted an experiment with the help of my advisors to understand how hBN’s dielectric breakdown characteristics depend on sample thickness and temperature. I began by fabricating three hBN devices, each containing multiple regions of different thicknesses. By measuring the current and varying the voltage on a given region, I was able to locally probe the hBN’s electric breakdown characteristics with thicknesses ranging from 5 to 24 nm. I tested each device multiple times using a cryostat at a range of temperatures from 4 to 300 K. During initial measurements, I observed an increase in the breakdown voltage with temperature in hBN between 15 and 24 nm thick, conflicting with previous reports. I repeated these measurements with a finer resolution which yielded the same result. The thinnest hBN regions showed no temperature dependence, confirming the absence of systematic temperature effects. I am currently fabricating more devices to reproduce this temperature dependence and working with my advisors to find a theoretical basis for this observation. Understanding how thickness and temperature effect hBN’s dielectric strength will allow researchers to construct more resilient devices, facilitating the study of 2D materials at higher electric fields. Moreover, the study of defects in hBN remains an active subject of research for quantum information applications, and a probe of the capacitive properties of hBN may shed light on this topic.

With the sharp uptick of diagnosed depression cases since the start of COVID-19, it is likely that someone close to us suffers or has suffered from depression. The standby period when starting a new anti-depressant medication exceeds practical justification when patients must often wait two or more weeks before the efficacy of new medication can be assessed. This waiting game often leads to a loss of hope when other options have been unsuccessful. Predicting the effectiveness of a new medication based on a patient’s individualized makeup is vital to the medication onboarding process. Tests based on metabolizing properties and Pharmacogenomic capabilities provide a pathway into predictive care. The ability to assay target genes while identifying subpopulations can simultaneously reduce the likelihood of potential adverse drug reactions, as well as entice pharmaceutical companies to mass-produce medication directed to those with specific mutations to prevent adverse reactions. This research consists of a literature review detailing forms of potential adverse drug reaction prediction. Methods include the use of information from various scientific articles and the recognition of connections and disconnects between sources. It’s expected that metabolizing genes will be the primary component in efficacy prediction due to drug-gene interactions. The implications of reducing the waiting period for those suffering from depression is crucial to patient well-being. As rates of depression diagnoses increase, the implementation of personalized treatment will aid in decreasing the timespan of treatment. Particular drug manufacturers will lose money if their drug is proven ineffective ahead of time for specific individuals, though patients and physicians alike will benefit from quicker treatment options. Current tests are not mainstream due to ongoing research and strict guidelines that must be followed before clinical implementation. As innovation continues, preventative and personalized medicine will be a prominent supplementation to the treatment of individuals with depression.

This project examines developmental atypical patterns of visual attention in infants in relation to Autism Spectrum Disorder (ASD). Research in this area could help identify additional, specific risk groups or factors that could facilitate focused research that translates to real-world applications. Specifically, this project examines how cognitive development relates to visual attention to faces versus activities at 12 and 24 months of age among different birth weight groups. Developmental scores will be evaluated through data collected using: the Mullen Scales of Early Learning (Mullen), a developmental test measuring cognitive and motor development, and the Vineland Adaptive Behavior Scales (Vineland), a caregiver-interview measuring child adaptive skills. Visual attention will be quantified using eye tracking data which measured proportions of looking towards faces versus activities in social scenes. Participants in the lab were split into two groups, low birth weight and regular birth weight, and were seen by researchers at both 12 months and 24 months. Mullen, Vineland, and eye tracking tests were conducted at both timepoints. Science shows that as infants grow, they focus less on faces and more on the activities they are doing. I anticipate similar effects in eye tracking, with increasing age associated with a higher mean difference in preference for activities versus faces. Uniquely, I hypothesize that the strength of the relationship between looking at activities and developmental skills will be greater at 24 months than it will be at 12 months, and the opposite will be true for looking at faces. We will test our hypotheses on a linear regression model that predicts developmental skills from factorial effects of time point, birth weight, and region of eye tracking preference. This project hopes to seek to understand the interaction between birth weight, age, and attentiveness to faces versus activities as they relate to developmental skills.

High prevalence of antimicrobial resistant (AMR) pathogens is undoubtedly an emergent global health crisis. AMR is exacerbated by factors such as the overuse and misuse of antimicrobial drugs and a changing climate. Overusing antimicrobial drugs causes selective pressure that leads to favorable mutations of bacteria. Through mutations, bacteria acquire mechanisms that interfere with the function and effectiveness of antimicrobial drugs. AMR is a threat to global health because, with ineffective last line of defense antimicrobial drugs, we will be unable to treat the most severe infections. Our research focuses on developing pipelines that detect low abundance antibiotic resistance genes (ARGs) on a local scale. This work contributes to the broader context of AMR on a global scale because applications of AMR surveillance across the globe can inform us about the nature of AMR. In my work, I am examining the implications of seasonality in AMR alleles in Seattle. During the dry season in Seattle from July to September, we may find a higher diversity of AMR genes and in particular, unique alleles of AMR genes. The amount of rainfall influences the concentration of bacteria carrying AMR genes. I hypothesize that higher rainfall typically occurring from October to March will lead to a lower diversity of AMR genes. I am assisting in developing a workflow that uses a two-step, unique molecular identifier (UMI) PCR to enrich AMR genes in wastewater. After amplification, the PCR product undergoes long-read Nanopore sequencing and through bioinformatic analysis, I can identify what AMR alleles are present. By gathering data regarding environmental conditions such as rainfall, and wastewater flow rate, in addition to using our current workflow of amplification PCR and Nanopore sequencing, I can identify what AMR alleles are present in relation to season.

High quality surveillance of antimicrobial resistance genes (ARGs) is critical for addressing the threat antimicrobial resistance poses to global health. However, the existing surveillance systems are centered around individual-level sampling in clinical settings. Hence, they are limited in that they do not reflect dynamics in community settings and require culturing for detection. The purpose of the study is to develop a surveillance method in wastewater that provides community level detection of ARGs. We are targeting β-lactam ARGs. DNA extracted from previously collected influent samples from Seattle’s wastewater treatment plant, were seeded with known gene alleles. We then applied Unique Molecular Identifier (UMI) PCR to amplify the alleles, used Nanopore sequencing, and developed bioinformatic pipelines for genomic data analysis. The pipeline translates the nanopore sequencing output (fast5) to genomic sequences (fasta), aligns them with an ARG database to determine the allele types, and graphically represent our alignments and produce interpretable figures from the data. We successfully completed the first allele sequencing and identification of two similar CTX-M alleles (genes for β-lactam resistance) that we inserted into samples. I am evaluating and validating this method with replicates of another β-lactamase ARG - KPC. The expected result at this stage is to successfully identify multiple β-lactamases alleles and test the enrichment of two different gene targets in one reaction. This more efficient and less expensive surveillance method in wastewater will facilitate ARG detection at the community level, providing public health agencies a tool that guides effective and regional-specific monitoring and intervention program design.