The kinetochore plays a key role during the cell division process, acting as an adaptor between the mitotic spindles and the centromere. The STU1 gene codes for a microtubule plus-end-tracking non-motor protein (Stu1) and facilitates the connection between the kinetochores and mitotic spindles during cell division. It also participates in a checkpoint that ensures proper connections between the spindles and kinetochores before the cell continues into anaphase. Stu1 contains multiple MELT motifs, which are conserved sequences of amino acids that are phosphorylated and targeted by the Mps1 kinase. Mps1 plays a major role in the regulation of segregation and spindle checkpoints. The impact of phosphorylation at the MELT motif on the function of Stu1 is not known. We used a previously constructed CRISPR vector and HDR template to mutagenize the codon within the MELT motif at position 719 in the STU1 gene of Saccharomyces cerevisiae. This mutation would result in a substitution of the amino acid threonine for glutamic acid within the Stu1 protein (stu1-T719E). Threonine is a neutrally-charged amino acid and is used in S. cerevisiae proteins as a phosphorylation site, but glutamic acid mimics a phosphorylation site as it has a constant negative charge. We transformed the CRISPR vector and HDR template DNA into S. cerevisiae cells and recovered transformants. To verify the success of the mutagenesis, we purified genomic DNA from transformed yeast, amplified the STU1 gene via PCR, and then sent for Sanger sequencing. It was found that mutagenesis was successful. This will allow us to move on to the next steps, which include phenotypic tests which will allow us to see if the mutation we made impacts cell division of S. cerevisiae cells. 

Biological processes rely on the intricate functions of proteins, which drive essential biochemical reactions. Given their critical role, various methods have been developed to regulate protein functions in biomaterials and in vitro. Enhancing the precision of gene editing is crucial for advancing applications in gene therapy and minimizing off-target effects. My project focuses on integrating photoactivatable proteins with prime editors, a modified version of the widely known gene editor CRISPR/Cas9, to improve spatial and temporal control over gene modifications. By utilizing genetic code expansion, non-canonical amino acids are incorporated into human cells to express photocaged prime editor proteins and altering host genomes. This system enables optical stimulation to precisely regulate protein activity. Through the deployment of well-characterized photolabile groups, we expect to be able to render protein activity controllable in a dose dependent way. A key application of this approach is the development of a photoactivatable prime editor system to induce precise gene edits. Traditional CRISPR/Cas9 methods lack spatiotemporal control over activation. To address this, the system is adapted for use in hydrogels, where two-photon patterning allows visualization of prime editor protein activation in three dimensions. Our study aims to demonstrate the feasibility of optically controlling gene editing with high specificity, offering a novel strategy for advancing cell lineage tracing and gene therapy applications.

Linear electrochemical impedance spectroscopy (EIS) is widely used in the characterization of electrochemical systems, such as batteries, although the results of EIS are only as good as the scientist's model of their data, as it’s possible to fit multiple models to the same data. Nonlinear EIS (NLEIS) can also be helpful when characterizing batteries - as they are nonlinear devices - and reveal additional information, such as the asymmetry of the charge transfer between charge and discharge. Combining EIS and NLEIS results in multiple, interrelated data sets, which when fit together drastically reduces the set of models that fit the same data, providing a better understanding of battery physics. However, NLEIS is not as widely developed or used as traditional EIS methods. The goal of this research project is to further develop the use of NLEIS for battery characterization in order to combine EIS and NLEIS to ultimately provide a more accurate picture of battery health. To reach this goal, I plan to test fresh and aged lithium nickel manganese cobalt (NMC) pouch cell batteries with my group’s EIS/NLEIS model. Using materials and equipment from the Washington Clean Energy Testbeds, I will then deconstruct these batteries and fabricate coin cell batteries from the harvested electrode materials and run EIS/NLEIS experiments on these coin cells, comparing the results of the coin cells to the results of their parent pouch cells to assess the accuracy and usefulness of the NLEIS model. Advancing battery health testing is critical for the future development and use of batteries, as understanding battery health allows consumers and scientists to make sustainable decisions regarding battery use, recycling, and disposal.

The interaction between the kinetochore and spindle microtubule serves as a checkpoint during the transition from metaphase to anaphase in the cell cycle. Bipolar microtubule attachment and tension sensing is required for successful segregation of sister chromatids, and progression through the cell cycle. Incorrect attachment will lead to cells containing excess genetic material, or not enough; both of which will compromise the cell's survival. Proteins that make up the kinetochore, such as Dsn1, are still being investigated for their role in chromosome segregation. Dsn1 is a protein located in the MIS/MIND complex, bridging kinetochore subcomplexes involved in microtubule attachment and tension sensing. Phosphorylation is known to alter the structure and function of proteins. We were interested in whether phosphorylation events impacted the function of Dsn1. We aimed to mutate the DSN1 gene at codons that code for amino acids known to be phosphorylated. Specifically, we targeted two threonine amino acids at sites where mass spectrometry analysis has confirmed Dsn1 is phosphorylated: T380 and T386. We transformed yeast with a vector expressing the CRISPR-Cas9 system engineered to target a DNA break in the DSN1 gene, and a homology directed repair DNA molecule (HDR) that would induce mutations changing the target codons to valine (T380V and T386V) during the repair of the break. After obtaining yeast transformed with these DNAs, we amplified this region through a PCR reaction and sent out the DNA for Sanger sequencing to confirm the presence of our intended mutations. The integration of the dsn1-T380/386V mutations was not successful, however sequencing data supported the function of the CRISPR vector since an off-site mutation was present near the T380/386 site. We are repeating the mutagenesis with a longer HDR template, and hope to show the role of phosphorylation of these sites in the function of Dsn1.

Chronic pain is a public health crisis that has been clinically demonstrated to disrupt reward learning and motivation in affected individuals. Previous literature has indicated that Calca neurons in the parabrachial nucleus (PBN) play a key role in the sensory and emotional processing of pain and become hyperactive in chronic pain models. Despite this, how PBN Calca signalling impacts adaptive decision-making in a positive-reinforcement context remains unclear. This study aims to explore how chronic PBN Calca hyperactivity impacts learning and motivation. Using chemogenetics, a technique that selectively modulates neuronal activity, we chronically activated PBN Calca neurons in transgenic mice. These mice were then tested in a two-phase positive-reinforcement operant conditioning paradigm to assess how chronic PBN Calca activation altered learning rates and motivation compared to controlled animals. In phase one, mice underwent a fixed ratio schedule in which they learned to press a lever during a distinct cue to obtain a food reward. In phase two, mice underwent a progressive ratio schedule in which they had to press a lever an increasing number of times to obtain a food reward. We hypothesized that chronic activation of PBN Calca neurons would impair both learning rate and motivation. With this work, we hope to clarify the impact of centrally-mediated chronic pain on motivational and cognitive processes, which could inform the development of future therapeutic strategies.

Alzheimer’s disease (AD) is a neurodegenerative disorder that disrupts memory, thinking, and behavior. It is the most common type of dementia and occurs with increasing frequency with increasing age. Transgenic AD mouse models have not predicted clinical efficacy because neurodegeneration occurs rapidly at a young age, so an aging environment is not a factor. To address this, an adeno-associated viral vector model of AD (AAV-AD) containing a green fluorescent-induction marker (GFP) was created to deliver pathogenic proteins Aβ-42 and P301L tau to neurons of old mice. The AAV capsid was engineered to have an affinity for neurons. Analysis of the model demonstrated successful expression of Aβ-42 and P301L tau in neurons in the brains of old mice when the vector constructs were administered intravenously (IV). However, it has yet to be shown whether the AAV-AD vector has off-target effects in systemic organs like the liver. Characteristic AD pathology does not naturally occur outside the brain. Therefore, this project was designed to determine if the AAV-AD vector became established in hepatic cells. Paraffin-embedded tissues were obtained from 27-month-old C57BL/6 male and female mice infected with the AAV-AD or sham vector for 3 months. Immunohistochemistry (IHC) was used to examine expression of GFP, Aβ-42, P301L tau, MCP-1 inflammatory cytokine, and yH2AX DNA-damage response. Images were taken using digital microscope software, and quantified through an open-source digital image software. Age-related histopathology lesion scores from H&E-stained brain and liver were compared with IHC stains. The expectation is there will be little evidence of AAV-AD proteins but incremental increases in inflammatory and DNA-damage proteins proportional to histopathology lesion scores. These observations would help validate translational efficacy of the AAV-AD mouse model for preclinical testing of pharmaceuticals to treat or prevent AD.

Sleep deprivation (SD) is a pervasive issue linked to significant cognitive and neurological impairments, affecting billions of people. SD accelerates markers of aging, but some individuals exhibit resilience to its effects. SD response is indicative of resilience. Identifying factors that promote SD resilience may inform interventions to enhance resilience. Studies have shown that SD alters gene expression in rodents, yet it remains uncertain which changes are specific to homeostasis. Previous rodent studies examined the effects of single day SD. Our study increases the duration to five days and separates mice into high and low responders, providing a novel insight into SD responses. This establishes a valuable evaluation of resilience for aging interventions. Female mice in the treatment group were sleep deprived through continuously stirring them during sleep periods. Control and treated mice were then subjected to the box-maze assay to evaluate relative learning rates and cognitive impairment. High performance in the box maze was designated as a high responder, and vice versa. Mice were then euthanized, and the hippocampus was isolated. The transcriptomes of control and treated mice were analyzed via mRNA sequencing. Analyzing transcriptomes of control, high, and low responder mice showed distinct changes in expression of key physiological and biochemical phenotypes. Genes known to be associated with SD were isolated and examined separately regardless of difference. Overall, high degrees of similarity were observed in control and high responders to SD, while low responders had the greatest changes in comparison to the latter groups. These experiments provide an efficient, robust platform to study the biochemical effects of SD, offering attractive insights for frameworks to quickly evaluate therapeutic strategies aimed at enhancing resilience to aging,

Alcohol consumption is known to influence individuals' perceptions and engagement in various activities, often altering how they experience different social, recreational, and everyday tasks. Understanding the effects of alcohol on enjoyment is crucial for identifying potential risks associated with alcohol use, particularly among college students. This research study aims to explore how college students perceive their enjoyment of activities like household chores, social interactions, and leisure when alcohol is consumed, compared to when alcohol is not consumed. Given the prevalent drinking habits among university students, particularly in social settings, it is important to examine how alcohol may shape their experiences in everyday life. Participants are fraternity and sorority-affiliated college students at a large public university who are enrolled in a larger study focused on increasing the availability of and engagement in substance-free social activities. The study is currently collecting data and anticipates a sample size of N = 300.  Participants will complete an online survey of self-report questionnaires. The Substance-Free Reinforcement Survey (SFRS; Correia et al, 2002) will be used to assess participants’ enjoyment of a variety of activities with and without alcohol use. Participants were asked to rate their enjoyment on a scale from 0 (unpleasant or neutral) to 4 (extremely pleasant). Activities included social and individual activities, such as group gatherings, personal leisure activities, and household chores. We will conduct t-test to evaluate whether there are differences in enjoyment between activities experienced while use alcohol versus activities experienced without alcohol. The study’s results will provide valuable insights into the relationship between alcohol use and student engagement in everyday activities. These findings could ultimately inform interventions aimed at reducing harmful alcohol consumption while promoting healthier and more fulfilling social and recreational behaviors. Understanding these dynamics can lead to more effective strategies for addressing alcohol-related risks on college campuses.