The representation of genocide in the post-conflict period is paramount in shaping public opinion and, in turn, providing relief, aid, and justice for the victims. Two common forms of representation of violence in genocide are statistics and the memory of survivors. Data and statistics add a viewable representation in graphs, tables, and charts. However, data and statistics can be manipulated and changed to suit one's biases. One of the most prevalent forms of memory in post-genocide periods is testimony. It can be intensely personal and can provide narratives for oppressed people during the conflict. However, testimony can be altered by trauma and memory of the witness and can be influenced by an interviewer. This presentation explores the delicate balance of memory and statistical representation in a post-genocide period, primarily in the Guatemalan Genocide. It contains an analysis of data on victims that was collected by the Guatemalan police during the genocide, widely published testimonials, and personal stories about the genocide. A mix of testimony and data representation can provide additional and nuanced insight into violent conflicts and help to create a peaceful and just society. Considering testimony and data together instead of completely separate entities fits with established and proven genocide prevention efforts.

Due to the COVID-19 outbreak, in-person mental health services became, in many cases, prohibited, increasing the need for telehealth. The present study will investigate clinicians’ perceptions of the effectiveness of delivering cognitive-behavioral therapy (CBT) via telehealth. Telehealth, in this context, refers to remotely delivering psychotherapy via a variety of platforms such as on-line services, video, and/or phone calls. Previous studies found that delivering CBT via telehealth can be as effective as in-person delivery. However, there are barriers to implementing psychotherapy remotely, including lack of training support, organizational and clinician buy-in, and cost. Thus, we aimed to investigate whether clinicians found delivering CBT via telehealth effective, whether it had an impact on engagement and therapeutic relationships, and how well elements of CBT training were provided during telehealth delivery. Data came from a project leveraging a Washington State CBT+ Initiative. Clinicians and supervisors from agencies in WA state participated and got training in CBT for youth depression, anxiety, trauma, and behavioral problems. Clinicians completed pre-and post-training surveys with questions pertaining to how telehealth preparation time compared to in-person sessions, clinicians’ opinions on continued telehealth implementation, clinicians’ ability to deliver effective services via telehealth, and client and caregiver engagement levels over telehealth. We plan to run descriptive analyses to summarize clinicians’ perceptions of telehealth. Preliminary analyses suggest that most clinicians found telehealth to be beneficial and an effective way to deliver CBT to youth. Further, our analyses reveal that clinicians, on average, found engaging younger children in treatment as the most significant barrier associated with telehealth. Harnessing telehealth for the provision of CBTs may be a viable solution that addresses some barriers to access. If telehealth is found effective by clinicians, it could serve as a long-term option for delivering CBTs. Therefore, it is important to understand clinicians’ perspectives.

Pre-health undergraduate (PHU) students are often encouraged by graduate and professional programs to shadow in healthcare settings. However, shadowing opportunities can be limited and disengaging for students. A potential avenue to improve shadowing experiences for PHU students, while providing mutual benefits to health professionals who they shadow, is the active and guided incorporation of students as observers into patient- and family-centered rounds (PFCR). For this pilot study, our hypothesis was that a structured PFCR shadowing and feedback process would be mutually beneficial for PHU students and interprofessional care teams. We tested our pilot process at Seattle Children’s Hospital. There were two cycles from January through November 2019. In the first cycle, a single PHU student was trained to use the High-Value Care Rounding Tool to collect quantitative and qualitative observational data about PFCR. The second cycle expanded the approach to a cohort of six additional students. Results were presented to a convenience sample of attending physicians who afterwards completed a brief survey about the value of this shadowing process for their own practice. Additionally, PHU students shared their own experiences via an interview and survey. For the analysis, we used descriptive statistics for quantitative data and thematic analysis for qualitative data from 53 PFCR episodes, 17 attending surveys, and 7 PHU student interviews and surveys. Five major themes emerged from these results including: (1) PHU students observed PFCR from a family-like perspective, (2) students felt they had a designated role in PFCR, (3) students provided tangible and actionable feedback on communication techniques for the interprofessional team, (4) students appreciated structured experiences for their future careers, and (5) feedback was valued by attendings. This pilot study offers a promising model for shadowing programs to provide an engaging experience for students, useful feedback for health professionals, and improve patient and family-centered communication.

Hydrogen sulfide (H₂S) is a toxic gas in the environment, but it is also an important cellular signaling molecule. Our goal is to understand the genes and pathways that mediate the physiological effects of H₂S. Previous work from the Miller lab has shown RHY-1 to be a component of a pathway that mitigates H₂S toxicity. RHY-1 is an integral membrane protein with predicted acyltransferase activity that localizes to the endoplasmic reticulum. We are attempting to identify proteins that work with RHY-1 to promote survival in H₂S. In this project, we have optimized biotinylation by antibody recognition (BAR), a proximity-labeling approach, to identify proteins that may physically interact with RHY-1. In these experiments, we introduced an antibody conjugated to horseradish peroxidase (HRP) into C. elegans that expresses the epitope-tagged RHY 1::FLAG::GFP protein, so that upon addition of biotin peroxide, biotin radicals were formed only in proximity to RHY-1. These biotin radicals react with other proteins that are localized near RHY-1. We visualized biotinylation using a fluorescent label and showed that the biotinylated proteins colocalize with RHY-1, indicating a successful BAR reaction. To our knowledge, this is the first use of BAR in C. elegans. Going forward, we will use mass spectrometry to identify the proteins that are biotinylated, and then test the functional role of these proteins in mitigating H₂S toxicity. Identifying the genes and pathways downstream of RHY-1 that promote survival in H₂S will reveal new factors that can modulate H₂S signaling in cells and may lead to treatments for H₂S poisoning.

Stroke, when the blood supply to the brain is reduced or disrupted, is a leading cause of disability among adults. Brain plasticity, also known as neural plasticity, can aid in the way that the brain recovers from a traumatic injury such as a stroke by creating newer, stronger synapses (connections) between neurons and thus increasing cell functionality. This literature review explores how brain plasticity informs new bioengineering solutions to stroke rehabilitation and asks, “What type of novel stroke treatments have been developed using the concept of brain plasticity?” Preliminary findings indicate that a breakthrough in this field is using optogenetics to trigger and control the neural connections that the brain can make through promoting motor function after ischemic stroke. Other innovations in this field include repetitive transcranial magnetic stimulation, transcranial direct current stimulation, and epidural cortical stimulation, which have all been shown to make permanent changes in neural synaptic transmission. These methods partially restore brain function while being less invasive and more effective in comparison to older interventions. This literature review indicates that new bioengineering treatments informed by brain plasticity are promising and could promote better rehabilitation outcomes for those suffering from stroke and potentially other traumatic brain injuries.

Optogenetic stimulation is a technique that modulates the activity of genetically modified neurons with light of a particular wavelength. Optogenetic modulation has high temporal resolution and cell-type specificity that enables precise stimulation of cortical neurons and allows us to conduct artifact-free recording during stimulation. Using a large-scale optogenetic interface, we stimulated and recorded across the primary somatosensory (S1) and motor (M1) cortices of non-human primates (NHP). We conducted our investigation on NHPs because their cortical organization is particularly similar to that of humans. The goal of this study is to determine the effect of various spatial and temporal patterns of optogenetic stimulation on the neural response and network dynamics across the two cortical regions. Delivering stimulus pulses via two lasers placed on top of the cortical surface, we found that stimulation of one cortical region evoked neural responses across both S1 and M1, which we then classified into primary and secondary responses based on their delays. While our previous work has established that optogenetic stimulation strengthened functional connectivity between S1 and M1, we wanted to further investigate the distribution of primary and secondary neural responses after repeated stimulation. We examined two measures of neural responses, the temporal delay between the trough of evoked response and onset of light stimulation, and the distribution of power across cortical networks up to 50ms after the stimulation. Our preliminary results indicate that optogenetic stimulation changed the delay of the primary and secondary response. We also observed that different temporal patterns of paired laser pulses evoked distinct neural activity. Identifying different neural responses after complex spatiotemporal patterns of stimulation would help us predict network changes post cortical modulation and contribute significantly to the development of stimulation-based clinical therapies and rehabilitation strategies for neural disorders.

Non-human primate (NHP) research is a pivotal step in the progression of neuroscientific and neural engineering research from animal models to human trials. In most NHP neuroscience experiments, neurosurgery is required to implant devices such as head posts, recording arrays, and optical windows. Current practices for these surgeries use methods for surgical preparation that carry a degree of unavoidable uncertainty. This comes from an inability to visualize and test the physical compatibility of complex components and anatomy prior to neurosurgery. This project details methods for creating 3D printed models of a subject’s brain and skull, as well as an agarose gel model of the brain. These models can be obtained from magnetic resonance imaging (MRI) using brain extraction software for the brain model, and custom code for the skull. The preparation protocol takes advantage of state-of-the-art 3D printing technology to combine models of the brain and skull with neuroprosthesis. With the addition of a craniotomy using the custom code, the skull and brain models can visualize brain tissue inside the skull, enabling better preparation for surgeries. Using the methods outlined in the protocol, the accuracy of the 3D printed brain, skull, and craniotomy placement were successfully validated through a comparison to the original MRI scan. The gel brain was additionally used to visualize delivery of a mock viral vector through the craniotomy of a skull model. By preoperatively fitting a headpost to the physical model of the skull, we successfully shortened the implantation surgery time by 40% and greatly reduced the risk of operative complications. These methods are designed for surgeries involving neurological stimulation and recording as well as injection in NHPs, but the versatility of the system allows for future expansion of the protocol, extraction techniques, and models to a wider scope of surgeries.

Non-human primate (NHP) research has become an essential step in the translation of medical technologies from animal models to clinical trials. This is especially so in neural research, as there is a large discrepancy between rodent and human brains in both anatomy and size. For some techniques such as optogenetics, which requires viral transduction of neurons, traditional diffusion-based viral injection approaches are effective in rodent brains but are impractical for large NHP ones. Convection-enhanced delivery (CED), a large-scale injection approach, currently lacks a practical quantitative bench-side injection modeling method to guide neurosurgical preparation. We aim to develop a gel model of the NHP brain and replicate surgical injections of it in order to reduce the risks of directly injecting into a NHP without sufficient preparation. We are testing the validity of our model by monitoring the spread of the injection through the gel and comparing the data with those from MRI scans of the injections in NHP. Since CED can behave differently depending on the location of injection in the brain, we are testing bench-side injections at different depths to validate the versatility of our model. We are seeing that the injections in the gel model mirror that of the injections in NHP brains as expected. Our next steps are to test the effectiveness of smaller injection cannula sizes with our bench-side model to assess if injection results remain consistent. This would indicate that tissue damage could be minimized in surgeries while still achieving desired injection parameters.

Alzheimer’s Disease (AD) is a neurological disease that causes memory loss and neurodegeneration, and is one of the leading causes of death within the United States. Alzheimer’s is linked to the presence of neurofibrillary tangles within the brain, generated by hyperphosphorylation and aggregation of a protein called tau. However, the specific impact that tau has on biochemical pathways in neurons is largely unknown. My project attempts to fill this gap. I compared samples of wild type fruit flies, Drosophila melanogaster, with a strain which expresses the human tau gene in neurons. Despite the difference in human and fly brains, determining the affected biochemical pathway in flies could suggest similar effects in humans. I am using metabolomics, which quantifies the levels of approximately one hundred metabolites, to try to identify the biochemical pathways that are affected by expression of tau in the neurons. Head and body samples of these two fly strains were frozen at 12 days of age and assayed for a targeted set of metabolites. I analyzed these metabolome data using statistical methods in Python to search for potential differences between wild type and tau flies. Any metabolites found to be different will then be analyzed to see if they tend to represent similar biochemical pathways, allowing us to speculate about the effect of tau on those pathways, both in flies and humans. Future work aims to analyze fly brains and individual neurons, thus mapping the affected metabolic pathways more precisely. Importantly, metabolic pathways are often identical between organisms, allowing us to speculate on the effect tau has on similar pathways within humans, thus this work will inform our understanding of the mechanism of AD in humans.

  My research focuses on ‘tauopathy’—the pathological effects of misfolding of the tau protein—using the fruit fly, Drosophila melanogaster, as a model system. In particular, I am interested in how the expression of tau affects locomotive function. Tau is a protein found in both humans and flies that is associated with stabilizing neuronal microtubules. However, under certain physiological conditions, human tau proteins form neurotoxic aggregates in the brain. This neuronal damage leads to dementia, a hallmark of Alzheimer’s Disease (AD). Aging comes with a multitude of age-related functional deficits, including decline in locomotor function. Some individuals with AD pathology are never diagnosed, however, because the cognitive impairment they experience is not sufficient for a clinical diagnosis of dementia, the most common symptom of AD. Consequently, it is imperative we understand AD as more than dementia. I study the climbing abilities of transgenic tau and control flies through negative geotaxis assays. Negative geotaxis refers to the tendency of flies to move vertically upward when startled. For my project, I am measuring the climbing performance of the flies to see the effects of tau on locomotor function as flies age. I hypothesize that the neurotoxic tau aggregates that form will interfere with neural activity involved in the flies’ motor function, resulting in decreased climbing performance. This research holds an abundance of biomedical implications and a capacity to better the lives of many. By using motor function to flag at-risk individuals early in their lives, they have the opportunity to participate in preclinical AD clinical trials that may prevent them from developing AD pathology later on. It is important that we, as a scientific community, strive to better understand the effects of aging and tauopathies, as it is through this understanding we can provide elderly individuals with care that transforms their quality of life.

Aging is the most prevalent risk factor behind many common diseases such as cancer, cardiovascular disease, and various neurodegenerative diseases. The changes that take place with age are complex and affect most of the human body; age-related changes in gait, in particular, have been found to be associated with the onset of disease. While it is generally understood that there is an effect of aging on walking speed in humans, the mechanisms underlying age-related locomotor impairment have not yet been fully characterized. The focus of my research is using Drosophila melanogaster as a model to investigate such mechanisms. My hypothesis is that D. melanogaster exhibit similar age-related changes in gait as those seen in humans, which includes a decrease in walking velocity and duration, and a significant change in limb coordination over their lifespan. I followed cohorts of D. melanogaster over their lifespans and recorded videos of their walking in an arena. I then used these videos to study factors such as walking velocity and duration by analyzing the trajectories of each fly. In addition, I investigated more in-depth limb coordination of individual flies by analyzing the movement of each individual leg. Previous studies using D. melanogaster have found a significant decrease in climbing behavior with age, but have not looked at gait on a finer scale. Therefore, upon completion of this study, I expect to see a decrease in limb coordination and population walking velocity as the flies age. With the findings from this study, I hope to establish a foundation for how gait changes with age in D. melanogaster and to further use gait analysis to help predict the onset of disease and to distinguish between diseased and healthy flies.

Ovarian reserve, the pool of primary follicles available for recruitment, is a key determinant of female reproductive aging (e.g. age at menopause), but what determines ovarian reserve is not well understood. Telomeres, the non-coding DNA that cap the ends of chromosomes, may provide an explanation. Longer telomere length may establish higher ovarian reserve through three mechanisms: 1) Telomere length may limit the number of mitotic divisions, which determines the number of oogonia available to establish the ovarian reserve. 2) Oogonia with longer telomere length could be more likely to survive and differentiate into primary oocytes. 3) Follicular atresia is more likely to occur when there is an insufficient stock of granulosa cells around the primary oocytes. Telomere length may allow more granulosa cell division, maintaining the ovarian reserve. For these reasons, we expect longer telomere length to be associated with a larger ovarian reserve, which can be proxied by higher Anti-Müllerian Hormone levels. Using data from the Cebu Longitudinal Health and Nutrition Study, we conduct regressions to assess the relationship between blood telomere length and Anti-Müllerian Hormone levels among 298 women (21.70 years ± 0.35). We measure telomere length using the monochrome multiplex quantitative polymerase chain reaction assay and we assay AMH using an enzyme immunoassay. Results and conclusions have yet to be obtained, though we have the data on hand. This is due to our goal of using best practices in science. Blinding ourselves to the data when designing the study may prevent bias and overfitting in our results. We expect our results to match our prediction: that longer telomere length is associated with higher Anti-Müllerian Hormone levels. Understanding the relationship between telomere length and ovarian reserve can provide more insight into the biological basis of reproductive age, menopause, and infertility, which may help guide family planning practices.

Pseudomonas aeruginosa, an opportunistic pathogen that commonly infects cystic fibrosis patients, uses quorum sensing (QS), a form of cell-cell communication, to regulate the expression of virulence factors and public goods based on population density. P. aeruginosa QS consists in part of N-acyl homoserine lactone signal molecules that activate two separate regulatory proteins, LasR and RhlR, which in turn activates the transcription of other target genes in their respective regulons. The las and rhl regulons are hierarchical in lab strains, with LasR activating the transcription of rhlR; however, many pathogenic variants carry nonfunctional alleles of lasR and rely on rhlR as the dominant QS regulator. Two anti-activator proteins, QteE and QslA, restrict the expression of these two QS regulons; however, it is not clear how P. aeruginosa anti-activators function in many pathogenic strains. Identifying how anti-activators regulate QS in pathogenic variants could be crucial in developing therapies that do not rely on antibiotics. To investigate how QteE and QslA modulate QS, we overexpressed each anti-activator in P. aeruginosa and used transcriptional reporters to monitor the activity of rhlA, a RhlR regulated gene. Expression of both genes is reduced significantly in strains with over-expressed anti-activators. However, in a pathogenic variant, only over-expressing qteE delayed QS induction while over-expressing qslA had no effect. These results indicate that QteE can modulate QS by affecting LasR and RhlR levels, while QslA only modulates LasR levels. These experiments lay the foundation for therapeutic strategies centered on inhibiting QS rather than relying on conventional antibiotics.

Neurons in the brain are dynamical in nature, maintaining constantly changing states. Neurons modulate voltage based on input currents and produce spikes when the voltage exceeds a certain threshold. Additional dynamics after spiking, called evoked after-spike currents, are important for computation and memory over time scales. The diversity of neuronal dynamics and the variability in parameters underlying them give rise to rich and varied dynamics across networks. We hypothesize that the complexity and diversity of biological dynamics in the brain play a critical role in predictive coding of temporally complex systems, and that diverse forms of after-spike currents enable computation over variable timescales. Current artificial neural networks (ANNs), that emulate the structure of biological neural networks, successfully learn relationships between static patterns but have difficulty learning dynamic patterns that change over time. We aim to incorporate complex biological dynamics and diversity in ANNs and thereby systematically explore the function of such dynamics in network computation and learning. To this end, we construct ANNs that express biologically realistic dynamics, developing methods to learn dynamics-generating parameters, such as membrane capacitance and threshold, in individual neurons. Theoretically, diverse dynamics of individual neurons will enable even more complex dynamics when combined in networks and may improve performance on tasks requiring computation over complex timescales, such as determining actions based on temporal patterns of cues. Hence, we hypothesize that when trained on temporally challenging tasks, our networks will learn diverse dynamics across neurons. We present the diversity of parameters learned and the resulting distribution of firing patterns and compare performance between our neural networks and traditional networks that only learn connection weights. This research will inform learning methods for training novel biologically inspired neural networks and will also shed light on the physiological role of diversity in the brain.

People who spend early life at high altitude have an increased capacity for aerobic work due to developmental adaptations to hypoxic environments. Since the effects of altitude on aerobic performance became evident after the 1968 Mexico City Olympics, altitude training has been implemented to increase red blood cell carrying capacity and improve performance. However, it is unclear how early development at altitude and current training at altitude may differently advantage endurance athletes. In order to test for possible mechanisms by which altitude enhances endurance performance, this research compares personal records and biomarkers of oxygen carrying capacity among endurance athletes who experienced early development at altitude or sea-level and are currently training at altitude or sea-level. The study aims to determine if the altitude in which athletes developed and are currently training at will be associated with faster gender-adjusted personal records and greater lung capacity. I conducted a cross-sectional observational study with 23 endurance athletes in Seattle, WA and Boulder, Colorado. Participants self-collected chest circumference (CC) at maximum inhalation and completed online questionnaires about running performance, family history, and personal motivations for competing. I devised a gender-adjusted personal record percentile score for each subject’s 5k times based on the top 500 US men’s and women’s 5k times recorded during the 2019 season. This presentation discusses the results on differences in personal records and CC in relation to early development and current training altitude. I examined athlete’s motivation for competing through the qualitative analysis of open-ended interview responses to explore how motivation works synergistically with physiological biomarkers. These findings will be discussed in terms of existing research and consideration for endurance training at altitude and sea-level.

As scientists collect ever larger volumes of data, methods to deal with these data have evolved as well. One of the fields that has thus emerged is the field of network science. Network science has many applications in biological fields as it allows scientists to connect variables of any type in a quantitative way. This versatility makes network science ideal for studies on comorbidity, or the consistent cooccurrence of various diseases in individuals. By analyzing comorbidities, we gain greater insight into interactions between diseases and systems of the body. This helps us understand how potential risk factors such as age, sex, and genotype affect various disease risks as well as the risk of comorbidity. We applied these methods to data on age of diagnosis for over 300 diseases collected from more than 28,000 owner reported surveys through the Dog Aging Project. We constructed comorbidity networks from these data and analyzed these networks using quantitative network statistics which allowed us to compare nodes both in and between networks. We first constructed undirected networks with the nodes representing various diseases to establish and identify pairs of diseases with significantly higher rates of cooccurrence than expected by chance. Using this network, we assigned directions to edges based on temporal data on the relative ages of diagnoses, which allowed us to identify which diseases are precursors to others. We observed how these networks changed through stratifications based on age, sex, and size to identify disease progression through age. In doing so, we hope that we can identify how age affects comorbidity in dogs, which can help researchers identify and develop therapies for lengthening dog lifespans. This knowledge will also provide insight into the mechanisms behind certain disease connections that were previously unknown.

 Stroke is the leading cause of long-term disability in the United States. Disabilities can range from a loss of sensory function like touch to motor functions like controlling arm movements due to the damage to the brain’s network. Despite the prevalence of stroke, the underlying network dynamics that lead to functional deficits are not well understood. Due to the physiological similarities between non-human primate (NHP) and human brains, an NHP model is essential for studying the effects of stroke and developing therapies. Here we used the photothrombotic (PT) stroke technique to study network dynamics in the NHP sensorimotor cortex following an ischemic lesion. Using the PT stroke technique, we induced a focal ischemic lesion on the NHP sensorimotor cortex. We collected local field potentials from both hemispheres using an electrocorticographic  array on the cortical surface. As a measure of neural activity, we calculated ipsilesional power in the low gamma band. Channels were then organized into three clusters based on their net change in power (increase, decrease, no change). As expected, the cluster with an overall decrease in power corresponded to the lesion's physical location. We also studied the network connectivity by calculating pair-wise coherence across different frequency bands: theta, beta, low gamma, and high gamma. Overall, we saw that low frequencies were associated with decreases in coherence, while higher frequencies were associated with increases following stroke. Preliminary results from the contralesional hemisphere show similar changes. In this study, we observed local neurophysiological changes up to three hours following an ischemic lesion. The observed increases in power in the perilesional region and coherence at high frequencies suggest compensatory mechanisms immediately following an injury. We can use this study's results to guide future developments in stimulation-based therapy to alleviate the functional deficits from a stroke.

Septic cardiomyopathy is a significant heart muscle dysfunction that often occurs during life threatening systemic infection (sepsis) and correlates with higher mortality. Currently, it is unknown why some septic patients develop such cardiomyopathy, while others do not. When used as models for inflammatory responses in human patients, rodents have historically been poor predictors of clinical outcome. For the current study on septic cardiomyopathy, I have therefore created heart cells (cardiomyocytes) from human stem cells. These were derived from two groups of septic patients: one with and one without septic cardiomyopathy, representing experimental and control groups, respectively. After maturing these cardiomyocytes, we treated them with specific inflammatory signaling molecules (TNF alpha, IL1b, IL6) and recorded video footage of the cells’ contraction response. The video recordings were then processed by a Matlab script that tracks the cardiomyocytes’ contraction and calculates their contractile velocity and amplitude under each condition. In response to these specific inflammatory mediators, we anticipate a significant change in contractility in cells from septic patients with cardiomyopathy, compared to the control cells from septic patients without cardiomyopathy. If our assumption is correct, further comparison of these cells will allow us to identify molecular signals that trigger septic cardiomyopathy. Such signaling pathways could then be further targeted as novel therapeutic strategies.

Cystic fibrosis (CF) is a genetic disorder characterized by chronic lung infections involving various organisms, including the gram-positive pathogen Staphylococcus aureus. Antibiotics, such as trimethoprim-sulfamethoxazole (SMX), play key roles in treating CF infections; these drugs inhibit bacterial growth by disrupting important bacterial metabolic processes. SMX specifically inhibits folate metabolism, causing DNA damage that results in bacterial cell death. However, S. aureus is able to persist in CF pulmonary infections despite treatment with antibiotics, and evidence suggests that S. aureus does so through adaptive mutations. Our goal is to identify the adaptive mutations of S. aureus during SMX exposure in vitro, to understand how this pathogen persists and to prevent the emergence of resistance. We grew S. aureus in the presence of super-inhibitory SMX concentrations for 24 hours in Luria Bertani broth. We sampled the culture at specific timepoints and measured viable bacterial counts on chocolate agar; we evaluated all resulting colonies for SMX susceptibility and associated genetic changes. Surprisingly, we identified mutants that survived SMX treatment carrying diverse adaptive changes not associated with folate metabolism or DNA repair, suggesting previously-unknown lethal effects of SMX against S. aureus. These mutants carried mutations predicted to decrease production of reactive oxygen species (ROS) - toxic compounds produced by all cells during aerobic respiration and in response to stress. Our results indicate ROS may play a role in SMX-mediated S. aureus cell death, suggesting that treatments that augment the effects of ROS could improve antibiotic efficacy. We are now exploring the involvement of ROS in S. aureus killing by SMX using engineered S. aureus strains with knockout and overproducing mutations in ROS detoxification genes. This study will help us better understand SMX’s mechanism of action and S. aureus’ response to this drug, in order to improve the treatment of diverse infections caused by this pathogen.

Nucleic acid vaccines introduce DNA or mRNA into cells in vivo, instructing them to express antigens from a pathogen resulting in the induction of immune responses that can provide long term protection from that pathogen. They provide many advantages over traditional vaccines including lower cost, improved safety, and the possibility to rapidly update the vaccine since only the genetic sequence of a new variant is required. One drawback of DNA vaccines has been their relatively poor immunogenicity compared to traditional vaccines which has been overcome, to some extent, by using improved delivery methods and co-formulation with plasmids expressing cytokines as adjuvants. Previous studies have established IL-12, as the “gold standard” genetic adjuvant due to its ability to support differentiation of antigen specific CD4+ T cells to produce Th1 cytokines as well as expansion of antigen specific CD8+ T cells to be more cytolytic in vivo. There is growing interest in identifying other adjuvants that not only increase immunogenicity of DNA vaccines but also modulate the types of responses they induce. In this study, we sought to determine if co-administration of an adjuvant cocktail including IL-18, a pro-inflammatory cytokine, and IRF7, a transcriptional activator of type I interferons, along with IL-12 would enhance antibody responses to DNA vaccines expressing SIV and Influenza antigens in a preclinical nonhuman primate model. Plasma samples were collected at different times post vaccination and the effect of the adjuvants on immunogenicity was measured via IgG ELISA and analyzed. After 2 vaccinations, we observed a significant increase (P=0.0272) in antibody responses against SIV gp130 in the adjuvant cocktail group compared to the IL-12 group. These results indicate that combining adjuvants could provide further improvement in DNA vaccine immunogenicity. Additional studies to determine the impact of this adjuvant cocktail on T cell responses are in progress.

In response to various forms of intrinsic and extrinsic stresses such as heat shock, electrical stimulation, and viral infection, cells produce non-membrane-bound aggregates of mRNA and proteins called stress granules. These granules sequester mRNA and ribosomal subunits to halt the production of proteins unnecessary for the immediate survival of the cell, thus allowing more energy to be used in combatting the stress. Stress granules are beneficial in the short term, but the chronic presence of stress granules can be cytotoxic. If stress granules are not cleared, hyperaggregation of misfolded proteins, which is thought to play a role in neurological diseases, can occur. After myocardial infarction (heart attack), the heart experiences a lack of oxygen which is known to create free radicals and metabolic stress. Whether the stress response is involved in this process is unknown, as most research on stress granules, especially their role in disease, comes from work in neuronal and cancer cells. To test whether the stress granules response is conserved across cell types and how cardiomyocytes (heart muscle cells) specifically respond to stress, I cultured cancer cells, embryonic stem cells, and embryonic stem cell-derived cardiomyocytes and subjected these cells to various forms of stress, including sodium arsenate poisoning and heat shock. Using fixed immunofluorescence and spinning disk microscopy, I imaged each treatment and quantified the number of stress granules per cell. The sodium arsenate treatment induced stress granule formation in all three cell types, but surprisingly, the heat shock treatment only induced stress granule formation in the stem cells. It is widely believed the stress response is conserved across a wide range of cell types, but these results indicate some stress pathways differ between cardiomyocytes, cancer cells, and stem cells. Future experiments will test additional types of stress and how stress granules contribute to cardiomyocyte function.

Opioids like morphine or fentanyl have high therapeutic value as analgesics for acute and chronic pain. Unfortunately, they also have a high propensity for abuse. Previous research has indicated that the rewarding and analgesic properties of opioids may be dissociated in the brain, though how they do so remains unresolved. Endogenous and exogenous opioids act on four specialized 7 transmembrane g-protein coupled receptors (GPCRs). One of these opioid receptors is the mu-opioid peptide receptor (MOPR), which is thought to be the primary target of morphine and fentanyl. The nucleus accumbens (NAc) has long been recognized as a significant reward center of the brain that has a high density of MOPR’s. Current work in the lab by Dr. Castro and myself has shown that a circuit from the dorsal raphe nucleus (DRN) to the NAc underlies MOPR control of reward and motivation. Specifically, we have shown that MORs are activated by the endogenous ligand enkephalin on DRN terminals in the medial shell of NAc to enhance motivation. While these studies clarify the NAc MOPR motivational circuit, they leave unresolved what intracellular mechanisms are recruited by MOPRs to modulate neural activity. This is of great interest considering that various properties of opioids (analgesia vs. reward vs. respiration) may be driven by one of two different signaling pathways, the G-Protein pathway or the beta-arrestin pathway. Therefore, the primary goal of my project is to determine which pathway is specifically recruited in this circuit to drive enhanced motivation. We will use optogenetic and targeted viral modulation approaches to manipulate the DRN to NAc circuit and determine whether distinct signaling cascades mediate MOPR control of motivation. These results will be the first investigation into how specific signaling cascades contribute to motivated behaviors in a cell-type, temporally restricted, in vivo manner.

Arrhythmogenic cardiomyopathy (AC) is a devastating inheritable heart disease characterized by lethal heart rhythms and abnormal contractile function that can lead to sudden cardiac death or congestive heart failure. More than 70% of AC cases are due to mutations in desmosomal proteins, which are essential for maintaining structural integrity and intercellular junctions in the heart. Frameshift mutations in desmoplakin (DSP), a desmosomal protein, are a common cause of AC, therefore my project aims to use human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to model AC due to premature truncating DSP variants and to determine if the mechanism results from haploinsufficiency of DSP protein. From two unrelated patients with AC carrying different pathogenic DSP variants, p.Leu463Serfs*22 and DSP p.Arg941*, we generated patient-specific iPSC-CMs from each patient. With these cells, we created engineered heart tissues (EHTs) and found preliminary data that suggests DSP L463Sfs*22 EHTs generate less active twitch force compared to wild type, indicating that this human iPSC model can recapitulate the salient clinical phenotype. Protein analysis of these cells revealed that patients with premature truncating mutations in desmoplakin have lower desmoplakin levels when compared to wild type cells (DSP L463Sfs*22 iPSC-CMs have 38% less DSP protein levels compared to wild type iPSC-CMs). There was no evidence of the predicted truncated protein to suggest a dominant negative mechanism. Furthermore, we found a significant reduction in DSP transcript in the DSP L463Sfs*22 iPSC-CMs that was partially rescued by knocking down UPF1, a key regulator of the nonsense-mediated decay (NMD) pathway, suggesting a NMD-mediated clearance of DSP transcripts that results in haploinsufficiency. Should we also find that increasing desmoplakin protein rescues the phenotypes observed in patients, there is potential to find a novel treatment option for patients with desmoplakin-associated cardiomyopathy.

Human Immunodeficiency Virus type 1 (HIV-1) is currently a major cause of morbidity and mortality worldwide. HIV-1 isolates are classified into genetic groups M, N, O, and P. Group M is the most widespread and is further divided into at least nine genetic subtypes and over 100 circulating recombinant forms, the prevalence of which vary by geographic locations. Subtype B is the most common form in Europe and North America and as a result, receives the majority of funding and research. Because of this, most of what we know about antiretroviral drug resistance in HIV-1 is based on studies of subtype B. However, the majority of HIV-1 infections worldwide involve other “non-B” subtypes, and much less is known about drug resistance in these subtypes. Knowledge of resistance to integrase inhibitors (INIs) is particularly important, since INI-based regimens are now recommended as first-line treatment for HIV-1–infected individuals globally. My objective is to genetically alter a full-length HIV-1-encoding plasmid (HIV-1 pNL4-3) that can used as a “cassette” for cloning non-B, patient-derived integrase sequences. To do this, I have identified restriction sites flanking the integrase region that can be used to remove and replace the integrase region with a patient-derived sequence, but site-directed mutagenesis must be used to knock out additional restriction sites for the same enzymes within the plasmid to avoid undesired enzymatic activity. This will result in only the desired sites, without changing the amino acid sequence. This vector will enable us to generate recombinant viruses from patients who are failing INI-based treatment, which can be tested for resistance to INIs in cell culture. The resulting data will be used to improve prediction of drug resistance in non-B HIV-1 based on sequence information alone. These studies will help guide treatment recommendations for both individuals and populations infected with non-B-HIV-1.

Advances in medicine have significantly increased life expectancy for much of the population. However, aging-associated diseases such as cancer, heart disease, and hypertension amongst others continue to significantly impact the aging population. Biological aging is defined by the gradual accumulation of cellular damage and development of physiological abnormalities from repeated acute insults or chronic disease states. A major driving force in biological aging is due to generation of reactive oxygen species (ROS), natural byproducts of cellular metabolism, possessing an unpaired electron that is highly reactive with essential biomolecules. ROS generation is increased by factors like lifestyle or exposure to toxic concentrations of drugs or environmental toxins.  One marker of oxidative stress (OS) is oxidative modification of nucleic acids, in particular the modification of guanine in RNA and DNA to 8-oxo-Gsn and 8-oxod-Gsn respectively. I hypothesize that 8-oxo-Gsn is a superior biomarker to 8-oxod-Gsn for assessing transient and acute incidences of elevated OS due to RNA having a higher turnover rate than DNA. Utilizing a recently published high pressure liquid chromatography- tandem mass spectrometry (HPLC-MS/MS) method, I assessed the production of 8-oxo-Gsn and 8-oxod-Gsn in human proximal tubule epithelial cell (PTECs) 2D culture and in 3D microphysiological systems (MPS) in response to induction of OS under experimental conditions. Accessible biomarkers can serve as early indicators of cellular stress, allowing for early intervention of aging-associated diseases.

Metallic foams have long been sought after for their conductive and structural properties, but have not become widespread due to the extraordinarily difficult processes typically used to produce such materials. Utilizing conventional Fused Filament Fabrication (FFF), also known as Fused Deposition Modeling (FDM), 3D printing equipment and the viscous properties exhibited by the molten filament extruded during printing, we have established that these properties can produce fully customizable metallic foams. The material properties of these foams are configurable to produce varying degrees of density, scale, and geometry via the manipulation of standard printing variables such as print height, print speed, and extrusion speed. Preliminary results with polymeric FFF filaments have successfully produced foams demonstrating significant compressive strength in every direction despite being an open celled geometry with an extremely high surface area to volume ratio. Conversely, polymer foams printed with flexible materials allow us to tailor material properties for energy absorption over structural integrity. This work will establish the minimum and maximum limits of the fabrication process as well as the material properties of metallic foams. To date, our experiments have demonstrated this technique of manufacturing metallic foams is reliable, controllable and scalable with great potential to change the availability and usage of metallic foams, and could significantly impact fields such as structural engineering, automotive, and aerospace where these types of metallic foams are highly sought after.

Despite widespread implementation of solar energy, there are still issues with efficiency in varying conditions. For example, photovoltaic (PV) arrays function optimally at a specific temperature and have decreasing efficiencies at higher temperatures. Other factors that also impact the power production of a PV array include the amount of direct sunlight, the distribution of incident light, and the intensity of incident light. As solar energy installations continue to increase worldwide, proper modeling of PV arrays is critical for potential asset owners and power system operators. Effective simulations of a PV array’s power production require models that effectively consider the uncertain external factors that vary by geographical region and climate. In this research project, a realistic PV cell model is developed in the programming language Python. This model explores the sensitivity of a PV cell’s power production to different external variables, including ambient temperature, solar irradiance, and other weather conditions. Additionally, this PV cell model is extensible, allowing power production from PV modules and arrays to easily be considered. This model can be seamlessly integrated with other energy asset models, including those for energy storage and flexible demand resources, allowing for complex scenarios to be simulated. To display this functionality, a cost-minimizing consumer with a behind-the-meter PV array and battery is simulated.

Alzheimer’s disease, the most common human neurodegenerative disorder, is characterized by hyperphosphorylation of the protein tau, leading to the protein’s aggregation and the formation of neurofibrillary tangles. The subsequent neurodegenerative consequences of these tangles may influence the biological response and sensitivity to neurological stressors, such as a traumatic brain injury (TBI). A TBI usually results from a strong blow to the head that leads to damaged brain cells and neurodegeneration. In the Promislow Lab, we are currently examining how neuronal tau affects the mortality response to a TBI-like trauma in the fruit fly, Drosophila melanogaster. Using the UAS-GAL4 gene expression system, we are able to induce the expression of the tau gene in fly neurons from eclosion. From both an experimental fly genotype (tau expression) and a control fly genotype (no tau expression), we are currently sampling flies at various time points during their lifespan and administering a TBI-like trauma on the flies using a high-impact trauma device. To quantify the impact of tau on the flies’ response to the TBI-like trauma, we are recording the percentage of flies dead 24 hours later (24 hour mortality index). We hypothesized that inflicting a TBI-like trauma would lead to a significantly increased 24-hour mortality index in the experimental genotype compared to the control genotype due to increased sensitivity in the former. Thus far, we have observed significantly increased mortality in response to a TBI-like trauma in the control genotype compared to the experimental genotype. The decreased mortality in the experimental genotype suggests a novel positive role of tau in the response to a TBI, which holds significant implications for targeting clinical TBI treatments and therapies. Further analysis and follow-up experiments will provide useful insight into understanding the mechanisms of tau’s role and the pathways of both Alzheimer’s and TBIs.