Cardiopulmonary bypass (CPB) is required for most cardiac surgeries. More importantly, CPB is used during heart surgeries to circulate blood out of the patient's body for surgeons to operate on the heart under optimal conditions without blood obstructing their view. However, CPB has been shown to induce systemic inflammation which can lead to complications including multiorgan dysfunction. Lack of understanding, specifically not enough knowledge of the molecular mechanisms of post-CPB inflammation has been a major obstacle to improve treatment methods. To better understand these mechanisms, we performed mRNA and Assay for Transposase-Accessible Chromatin (ATAC) sequencing on total circulating leukocytes from neonatal patients undergoing CPB. Prior data identified myeloid cells, particularly monocytes, as the major cell type driving transcriptional responses to CPB. Furthermore, Interleukin-8 (IL-8) and Tuman Necrosis Factor alpha (TNF-α) were inflammatory cytokines found to be robustly upregulated in leukocytes in patients. To further explore these findings, we performed in-vitro experiments of running THP-1 human monocytic cells to CPB-like conditions, including artificial surfaces, high shear stress, and cooling/rewarming. These experiments were collected and studied at times post shear allowing us to study and contrast how the blood is affected and recovers from CPB. Additionally, we performed drug treatment experiments with egtazic acid (EGTA) to see how calcium signaling may benefit post-CPB inflammatory response. Observing the opening and closing of chromatin of the sheared and the static blood has shown positive correlation that IL-8 and potentially IL-2 contribute to patient inflammation. We hypothesize that specifically these regions, IL-8 and IL-2, are the areas to determine where the root cause and solution are to post-CPB complications. Finally, further analysis with Topologically Associating Domains (TAD) was shown to increase chromatin density and activation in post-CPB samples in IL-8 and IL-2 genomic regions. We hope this research can reduce complications with patients going through CPB.

Individuals exposed to chronic, severe, and/or inescapable stress may adopt dissociation as a coping mechanism to alleviate suffering. In dissociation, individuals report losing connection to their bodies, reality, memories, and more. Dissociative experiences are hallmark symptoms of psychiatric disorders such as Post-Traumatic Stress Disorders (PTSD) and Dissociative Identity Disorder (DID), which have a high comorbidity with personality disorders. Maladaptive personality patterns can be thought of as chronic patterns of behavior that are resistant to change, which often are heavily present in brain systems that focus on habitual learning. However, the interactions between dissociative experiences and action-learning mechanisms are still poorly understood. In this project, we review the history of dissociation and action-learning research to synthesize a model of how dissociative mechanisms may interact with action-learning mechanisms as a cause for the development of pathological behavioral patterns and even multiple personalities. We propose employing the PRISMA method to select relevant literature from PubMed or other medical databases systematically and conduct a comprehensive review across the spectrum of dissociative disorders. By utilizing the Dissociative Experiences Scale (DES) scores as a measure method, we aim to identify action-learning brain region changes and explore how these changes correlate with dissociative symptoms; thus far, we’ve found that DID, dissociative disorders, and PTSD have the highest DES mean scores. Beyond this, our findings show that in these patients with different kinds of dissociative disorders, the hippocampus and amygdala are smaller and the volume of the palladium is typically larger. When combined with future findings and discoveries, the relationships between dissociative experiences and action-learning mechanisms will be better understood.

Advances in language models have opened up endless applications in the social and health sciences. A key to fulfilling this promise is the development of rigorous practices for gathering data used for model training. This research project reports the development of standardized protocols for building databases for self-reported psychological questionnaires that can be used to fine-tune language models. These questionnaires cover a spectrum from childhood experience to altered states of consciousness and beyond. We systematically review the literature on appropriate database-building strategies, use combined data collection techniques, and instill specific criteria into our collection process to help facilitate the research’s computational pipelines on evaluating the relationship between semantics and psychological factor membership. Using a 2D semantic map of the PsycTests database as a reference, we can further evaluate the effectiveness of our protocols by developing quantitative measures to assess comprehensiveness and objectivity in our database building. Our initial explorations involve measuring data variance and density of data distribution. This research provides a general toolkit for building text databases for appropriate and effective model training purposes across various disciplines.

Every year, substance abuse, mental disorders, and psychotic behavior become more and more severe, especially in the local communities of Seattle. Our research explores the interconnection between psychology and the utilization of modern computer vision and emotion recognition tools to analyze human semantics/behavioral patterns in order to comprehend the underlying dynamics that lead to these behaviors and ultimately, spearhead efforts for prevention. As a computer science researcher, I employ advanced machine learning algorithms built upon open source programs to track and interpret gestures, facial expressions, and body language in video data. By filtering the data through a database and comparing emotions through statistical analysis in similar individuals, I am able to uncover nuanced patterns in behavior and emotion that a human researcher would otherwise not notice. While the study is ongoing, our current data illustrates correlations between specific gestures, facial expressions, and emotional states in hallucinogenic use. Though these individuals exhibit typical behavior of substance use, they have different backgrounds and contexts that lead to unique obscure changes in emotion and behavior, and our program allows us to identify and analyze these transformations that current medical knowledge overlooks. These findings hold promising implications for informing future research directions and intervention strategies across multiple domains, from understanding human behavior in social interactions to enhancing human-computer interfaces.

Individuals exposed to chronic, severe, and/or inescapable stress may adopt dissociation as a coping mechanism to alleviate suffering. In dissociation, individuals report losing connection to their bodies, reality, memories, etc. Dissociative experiences are hallmark symptoms of psychiatric disorders such as Post-Traumatic Stress Disorders (PTSD) and Dissociative Identity Disorder (DID), which has high comorbidity with personality disorders. Maladaptive personality patterns can be thought of as chronic patterns of behavior that are resistant to change. Brain systems are known to underlie these sorts of habitual learning. The interactions between dissociative experiences and action-learning mechanisms are still poorly understood. In this project, we review the history of dissociation and action-learning research and synthesize a model of how dissociative mechanisms may interact with action-learning mechanisms to develop pathological behavioral patterns and even multiple personalities. We propose employing the PRISMA method to select relevant literature from PubMed or other medical databases systematically and conduct a comprehensive review across the spectrum of dissociative disorders. By utilizing the Dissociative Experiences Scale (DES) scores as a measure method, we aim to identify action-learning brain region activity (BOLD signal) and volume changes and explore how these changes correlate with dissociative symptoms; and we’ve found that DID, Dissociative Disorders, and PTSD has the highest DES mean scores. Beyond this, our findings show that in these patients with different kinds of dissociative disorders, for the patients with different kinds of dissociative disorders, their Hippocampus and Amygdala are smaller while their Pallidum Volumes in their brains become typically larger. Despite this relationship having elementary processes, combining with future findings and discoveries, the relationships between dissociative experiences and action-learning mechanisms will be better understood.

H3K9me3 heterochromatin regulates gene silencing and plays key roles in genome stability and cellular identity. During cell reprogramming, H3K9me3 has been shown to block transcription factor binding and changes to cell identity. Recently, enhancer of rudimentary homolog (ERH) was identified as a master regulator of H3K9me3 and a repressor of reprogramming in humans. However, the roles that specific ERH protein amino acids play in its function remain unknown. We hypothesize that elucidating the role of ERH and identifying pivotal amino acids will enhance our understanding of its molecular mechanisms. We are targeting amino acids which are evolutionarily conserved as these are more likely to be important for ERH function. We have shown that the ERH knockdown phenotype of low proliferation and decreased H3K9me3 can be rescued by exogenously expressing a wild-type copy of ERH. Our approach has involved generating mutant variants of ERH, introducing them into cells where the endogenous ERH is depleted and then assessing whether they rescue the phenotype. To efficiently assess many amino acid substitutions in the ERH protein simultaneously we are developing a multiplexed alanine scanning approach with Nanopore readout in a functional screen. In this approach, multiple ERH mutants will be simultaneously introduced and integrated into the genomes of a population of cells with the endogenous ERH depleted. Mutant forms of ERH which do not rescue the proliferation phenotype are expected to decrease in abundance over subsequent proliferation. The ratio of exogenous ERH sequences will be assessed upon initial introduction and after 2 weeks by targeted long read DNA sequencing. Identifying which amino acids are important for ERH function will allow us to design further experiments, investigating their role in localization, protein-protein interactions and H3K9me3 regulation. Understanding the regulation of H3K9me3 heterochromatin will be essential to improve cell reprogramming, enabling autologous transplantation and regenerative therapies.

Neuroscience research relies heavily on cell counting to assess brain injury and evaluate neuroprotective treatments. While manual methods (i.e. hand counting) have been used traditionally, automated programs offer the potential for standardized and error-free data analysis. In the context of studying hypoxic-ischemic encephalopathy (HIE), a prevalent brain injury in infants, we aimed to assess the accuracy of an automated cell counting program in an in vitro slice culture brain injury model of injury and treatment. Code templates from ImageJ/Fiji were taken and modified using ChatGPT, and other snippets were used and modified from online forums like GitHub. The program searches through folders/subfolders for images, converts them into a binary image based on fluorescence threshold data (used to stain cell nuclei), applies the ImageJ function “watershed” that breaks larger groups into smaller groups, runs the “analyze particles” function which outputs a total cell count based on the size and circularity of the cells, and then saves the final image. Fluorescence threshold, cellular size, and circularity values were determined before data collection by adjusting the values to best fit the final image of a random slice. The settings were then kept consistent within studies. Preliminary results show the program’s high accuracy and precision, with consistent results across caffeine and Azithromycin treatments in our in vitro injury model. Despite yielding higher counts than manual methods, the program remained consistent across models. Validating this automated method represents a significant advancement in research methodology. These programs offer standardized data collection, error elimination, and faster analysis compared to manual counting, potentially saving time and resources for labs. Current limitations in our research involve differentiating between healthy and dead or dying cells, which would be an important future step for automated cell counting.

Advances in language models have made diverse applications in social and health sciences possible. With many models emerging daily, it is important to understand general principles behind how language models represent text data. The study aims to investigate the effects of text manipulation on the numerical representation of meaning, or embeddings, through language models. The research is focused on numerous text manipulations (ex. changing direction of sentiment, sentence incompleteness, object of reference, intensity of statement). The research employs SentenceTransformer for embedding generation and UMAP for the visualization of semantic shifts. A comprehensive analysis of over 50 sentence manipulations provides a preliminary basis for understanding the nuances of semantic space organization. The core hypothesis suggests that incomplete and intensity based modifications lead to greater shifts compared to negative and referencing modifications. This outcome is expected to be substantiated through measures of embedding displacement from original text, and generalized through statistical validation, with tests accounting for multiple comparisons. This research is positioned to scale towards publishable results, aiming to systematically quantify the effects of semantic modifications across a larger dataset. The anticipated contributions of this work are twofold. It offers insights into the organization of semantic space as influenced by sentence modification, providing implications for the development of more nuanced NLP (Natural Language Processing) applications. And crucially, it lays a foundational basis for understanding how large language models (LLMs) process psychological statements and terms, an area of increasing importance as these models become more integrated into applications involving psychological analysis. Overall, the outcomes of this research are poised to deepen the understanding of how semantic alterations impact sentence embeddings, with potential applications in enhancing NLP models' interpretability in processing complex linguistic constructs.

Tuberculosis remains a global public health threat due to the rising number of multi- and extensively drug resistant strains of the causative pathogen Mycobacterium tuberculosis. Development of novel drugs and an understanding of their resistance mechanisms is urgently needed. Aminothiazoles (AmT) are potent molecules with killing activity against M. tuberculosis; these compounds act as copper ionophores and target a key enzyme (enolase) by displacing its Mg²⁺ co-factor, a substance required for its activity, with Cu²⁺ imported by the compounds. Spontaneous mutations in an essential protein export system (the Esx 3 Type VII secretion system) confers resistance to AmTs. My research focuses on understanding how mutations in the secretion system cause AmT resistance. We hypothesize that copper imported by AmTs could disrupt other metallo-proteins including EccA3, a key ATPase of the of the Esx-3 secretion system that hydrolyzes ATP into ADP and inorganic phosphate, and that resistance mutations (e.g. E237K) reduce Mg²⁺ co-factor displacement by Cu²⁺. To test this hypothesis, I expressed wild-type (WT) EccA3 and mutant EccA3 [E237K] proteins in Escherichia coli BL21(DE3) expression strain and purified the proteins via Ni-NTA His-tag chromatography. Subsequently, I measured the activity of the purified EccA3 (WT) and EccA3 [E237K] proteins via an ATPase assay based on colorimetric detection of free inorganic phosphate released by ATP hydrolysis. I aim to understand whether copper inhibits EccA3 activity through this assay, anticipating that copper reduces EccA3 (WT) ATPase activity while EccA3 [E237K] ATPase activity is unaffected. Thus, my work will provide an avenue for understanding AmT resistance in M. tuberculosis.

Over 500 million adults worldwide have type II diabetes (T2D), which has caused 6.7 million deaths in 2021 alone. Yet, accessible and sustainable treatments remain limited and many address the symptoms of the disease rather than the root causes, one of which is insulin sensitivity. Recent research shows that therapies targeting glucose-regulating mechanisms in the brain that do not directly affect insulin secretion can enhance diabetes remissions in T2D rodent models. Such treatments may revolutionize diabetes treatment and public health. The Scarlett Lab’s previous studies show that exogenous intracerebroventricular administration of hypothalamic fibroblast growth factor 1 (FGF1) resulted in sustained hyperglycemic remission and increased insulin sensitivity in the mediobasal hypothalamus (MBH), suggesting that FGF1 signaling in the brain impacts glucose metabolism. FGF1 is also endogenously expressed in the MBH, but the functional role of endogenous FGF1 is still unknown. Thus, I hypothesize that endogenous FGF1 signaling in the MBH regulates glucose and energy homeostasis. To test this, I utilize adenosine-associated virus as a vector for gene editing therapy to knock out the Fgf1 gene from the MBH in adult male Fgf1-floxed mice. I then monitor food intake, body weight, and blood glucose levels; perform insulin and glucose tolerance tests; and measure insulin and glucagon levels using enzyme-linked immunosorbent assay. Finally, I use immunohistochemical analysis at the end of the experiment to validate Fgf1 knockout. Based on pilot studies, I expect to observe that MBH Fgf1 knockout significantly increases body weight and impairs glucose tolerance and insulin sensitivity. Follow-up work can elaborate on investigating the effect of a high-fat diet on FGF1 signaling and function. This project illuminates the functional role of endogenous FGF1 signaling in regulating glucose and energy homeostasis and contributes to the development of novel therapeutic strategies to treat diabetes and obesity.

Many transgender and gender diverse (TGD) people desire gender-affirming hormone treatment (GAHT) to alleviate discomfort due to the misalignment of one’s gender identity with their secondary sex characteristics, though little is known about its effects on the body. One area of interest that may be affected by GAHT is the gut microbiota (GM). GM and the sex hormones (estrogen, testosterone, and progesterone) have been shown to interact bidirectionally, referred to as the “gut microsexome.” At puberty, the commensal microbiota of males and females diverge due to circulating sex hormones. This difference is hypothesized to contribute to sexual dimorphism of disease prevalence between cisgender males and females, although little research on the effect of GAHT in the TGD population exists. Alteration of the gut microbiota, or dysbiosis, also has many adverse effects that overlap those of testosterone GAHT (THT) such as acne, weight gain, and hypercholesterolemia. Dysbiosis has also been shown to lead to intestinal and systemic inflammation by disrupting immune function. More information on the effects of THT on the gut microbiota is necessary to counsel transmasculine clients effectively. In this study, we injected mice born of the female sex biweekly with testosterone enanthate dissolved in sesame oil versus sesame oil alone. We profiled GM of mice throughout treatment using 16S rRNA sequencing and measured markers of inflammation in serum to assess the effect of THT on both the population of GM and intestinal and systemic inflammation. We expect that the mice receiving THT will have differentially abundant gut microbiota and increased concentration of inflammatory markers compared to controls. The findings of this study will serve as a basis for further studies exploring additional analysis of the gut microbiota and inflammation in both transmasculine and transfeminine people receiving GAHT.

Parental emotion socialization is how parents can use positive strategies such as modeling and discussion of emotions with their children. This skill is especially valuable for families with attention-deficit/hyperactivity disorder (ADHD), a highly heritable condition that affects one in eleven children and one in fifteen adults in the United States. Up to 50% of parents with ADHD are affected by emotional dysregulation. However, there is a lack of knowledge of how parental emotion socialization is impacted by parental emotional regulation abilities in ADHD families. The current study investigates this relationship through laboratory-based recordings of parent-child interactions to measure parental emotion-focused reactions as well as EEG recordings to detect biomarkers related to emotional regulation abilities in parents. I lead these video-recorded parent-child interactions and am trained to code them using the dyadic parent-child interaction coding system (DPICS), which systematically scores these dyadic conversations. For the EEG portion of the study, data analysis includes examining event-related potentials of interest, including the late positive potential (LPP). The LPP relates to emotional expression in response to a visual stimulus and is the variable that relates to parental emotion socialization. We measure the relationship between frequency of observed parental emotion-focused reactions and EEG-based measures of parental emotion regulation. In addition to laboratory-based measures of parental emotion regulation and parental emotion socialization, self-report measures are additionally collected. We expect parent participants with greater neural responses associated with emotion regulation to demonstrate greater frequency of parental emotion-focused reactions when interacting with their child during observed parent-child interaction. Because emotional socialization is linked to predictors of mental health, academic success, and peer relationships, understanding this relationship is critical. Establishing a better understanding of how parental emotion regulation affects parenting allows for individualization of behavioral treatments for families with ADHD.

Atherosclerosis is characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue in arterial walls, forming plaques. Plaque accumulation can lead to stenosis and potentially severe outcomes such as myocardial infarction or stroke. The gut microbiome, including Collinsella aerofaciens, is believed to play a role in the prevention or development of atherosclerosis. Gut bacteria can directly influence systemic inflammation, a factor correlated with the pathogenesis of atherosclerosis, and produce metabolites that alter the disease course. This study explores the potential link between C. aerofaciens and atherosclerosis by investigating the abundance of C. aerofaciens in the gut microbiome of individuals with and without atherosclerosis. We collected 179 stool samples from participants at the Kisumu District Hospital HIV Clinic in Kenya and conducted a comprehensive analysis of their gut microbiomes. 100 participants had carotid ultrasonography, categorized as showing atherosclerosis with visible plaque or intima medial thickness ≥ 0.7 mm. We employed bacterial 16S ribosomal RNA gene sequencing to characterize the stool microbial composition and noted that the relative abundance of C. aerofaciens was 2.6-fold less in participants with atherosclerosis (p=0.006). To validate these findings, I employed a Quantitative Polymerase Chain Reaction with a cloned plasmid control for targeted quantification of C. aerofaciens. We found 6.9-fold more C. aerofaciens copies per total 16S in Kenyan adults without atherosclerosis versus with (p=0.020). This suggests a potential protective or mitigating role for this bacterium in cardiovascular health. Future work could include assessing changes in C. aerofaciens abundance over time and its association with cardiovascular disease progression. Additionally, in vitro or preclinical studies could reveal the specific mechanisms by which C. aerofaciens influences atherosclerosis development and progression. This research contributes to our understanding of the intricate interplay between the gut microbiome and atherosclerosis, offering insights that may inform future therapeutic strategies and personalized interventions for cardiovascular diseases.

During neonatal transport, specialized pediatric transport teams closely monitor the status of critically ill newborns. Hyperspectral imaging, a method of manipulating light, can be used to measure the vital signs and video the patient’s physical appearance for remote monitoring. Appropriate light intensity is critical for clear visibility of the newborn and hyperspectral imaging accuracy, but this must be balanced with safety for sensitive eyes. My previous studies have determined the minimum range of light needed to accurately view the neonate in a transport incubator. In addition, I conducted a literature review to understand the uses of hyperspectral imaging in dermatology and surgical fields and to gain knowledge of the imaging equipment and biological signal processing. The experiment was designed to use hyperspectral imaging to gather vital sign data using a near-infrared camera with the purpose of using hyperspectral imaging in a dimly lit environment like that experienced on transport. In this experimental design, physiologic data of breathing rate and blood oxygen levels were recorded using 1-minute-long videos, while blood oxygen and heart rate was collected using a pulse oximeter placed on the fingertip of the subject. Following the data collection, the experiment is designed to extract the biological signals through filtering of two different ranges, a beats per minute range for the heart rate and a blood oxygen level range for the blood oxygen levels. Data analysis aims to compare the vital sign data collected using hyperspectral imaging and that using a pulse oximeter to understand the feasibility of the hyperspectral imaging for vital sign extraction. The expected result of this study is that the heart rate and blood oxygen levels of the patient measured using light and a pulse oximeter will be similar. In conclusion, this research will demonstrate the potential application of hyperspectral imaging to neonatal transport.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental condition characterized by differences in attention, focus, and emotional regulation. We investigate the relationship between parent temperament, including negative affect, and emotional socialization in ADHD parent-child dyads (parents and their children with ADHD).  Temperament refers to innate behavioral traits shaping one’s personality. Individuals with ADHD are known to experience higher rates of temperamental negative affect. Negative affect is a temperament factor that includes significant aversion from feelings like sadness. Emotional socialization refers to response methods of emotion regulation and can be categorized as supportive and unsupportive. Our study is impactful because ADHD has high heritability rates, yet there is little research on parental ADHD symptoms and associated temperament differences’ effect on ADHD families. We hypothesize that parents who report more ADHD symptoms and/or higher levels of temperamental negative affect use less supportive emotional socialization strategies and more unsupportive strategies when interacting with their children with ADHD. To test this hypothesis, I assist with administration of parent self-report measures and assess negative talk during laboratory-based, video-recorded parent-child interactions. Parents complete the self-report Adult Temperament Questionnaire (ATQ), which evaluates negative affect frequency. We measure negative talk by coding frequency of verbal disapproval of the child’s behavior/ attributes during parent-child interactions, where I instruct parents to perform standardized tasks with their children and code parental verbalizations into categories like negative talk using the Dyadic Parent-Child Interaction Coding System (DPICS). I then use bivariate correlation analysis to determine the likelihood that the two variables occur together and are linked. Through our anticipated findings, we hope to better inform care for children with ADHD and provide more resources for ADHD parent-child dyads. We want to identify emotional state-based targets to use in parental interventions to better support emotional regulation strategies in ADHD families.

The tumor microenvironment (TME) exhibits several obstacles to immunotherapies including suppressive signals and a lack of costimulatory ligands. The TME causes T cell exhaustion and reprogramming of immune cells (e.g. macrophages) from an anti- to pro-tumor phenotype. Therapeutic approaches such as agonist CD40 monoclonal antibodies stimulate macrophages to induce anti-tumor programming. However, enthusiasm for monoclonal antibodies has been dampened by dose-limiting toxicities and inadequate pharmacokinetics. We are developing novel adoptive cell therapies by armoring tumor-targeted T cells with fusion proteins that provide costimulatory signals that are absent in the TME. Dual Costimulatory Receptors (DCR) are composed of the CD40L ectodomain combined with a costimulatory receptor endodomain. CD40L-DCRs are engineered into CD8 T cells to provide positive signals: CD40L interacts with TME macrophages, while simultaneously stimulating CD8 T cells via the costimulatory endodomain. I hypothesize that CD40L-DCR T cells are reprogramming macrophages from pro- to anti-tumor programming, providing greater therapeutic efficacy against tumors. Our in vivo studies showed improved survival using different CD40L-DCR candidates in a murine model of acute myeloid leukemia. In the CD40L DCR-T cell-treated mice, macrophages exhibited elevated levels of costimulatory molecules, suggesting an anti-tumor phenotype. To understand how CD40L-DCR T cells induce macrophage reprogramming, I am conducting in vitro experiments, co-culturing bone marrow-derived macrophages (BMDM), tumor cells, and CD40L-DCR T cells. I predict that CD40L-DCR T cells will induce BMDM to reprogram into an antitumor phenotype and increase phagocytosis of tumor cells. I will use IncuCyte and flow cytometry technologies to determine how BMDM programming is altered and how BMDM phagocytosis of tumor cells changes over time. These studies will support the translational potential of CD40L DCR-T cell therapy, opening doors to novel therapeutic interventions against a range of cancers.

2023 was the Year of Open Science, part of a national push requiring public accessibility to the products of federally funded research. The Infant Brain Imaging Study (IBIS) Network is one such federally funded multi-site research network collecting neuroimaging data to study early brain development in infants with Down Syndrome. Data from the IBIS will be part of the INCLUDE Data Coordinating Center (DCC), a National Institutes of Health (NIH) managed open database intended to accelerate Down Syndrome research through sharable data. Understanding attitudes towards data sharing is important to ensure public trust in scientific research, and though much research exists assessing attitudes towards public sharing of genomic data, there is a lack of knowledge regarding attitudes towards public sharing of neuroimaging data. To combat this gap, I helped develop the CARDS-DS (Capturing Attitudes towards Research and Data Sharing in Down Syndrome) questionnaire, a novel parent-report measure to understand the perspectives of families participating in longitudinal Down Syndrome neuroimaging research. Currently, I am interviewing families enrolled in Down Syndrome neuroimaging research to get their feedback on the effectiveness of the questions asked in the CARDS-DS, a method called cognitive interviewing. Following this pilot study, CARDS-DS will be implemented throughout the IBIS Network. As such, CARDS-DS queries parent attitudes towards aspects of research participation that are particularly relevant to the INCLUDE DCC goals, namely: attitudes towards sharing of a child’s neuroimaging, genomic, and behavioral data, perspectives on data privacy, preferences for return of research results, and attitudes towards ongoing engagement with research teams throughout a longitudinal project. The anticipated data will reveal how attitudes may differ between parents from different sociodemographic or educational backgrounds, how attitudes may differ between the sharing of neuroimaging versus genomic data, whether attitudes are associated with study retention, and how attitudes may change over the course of longitudinal participation.

Hypoxia-ischemia (HI), or brain injury caused by a lack of blood flow and oxygen to the brain, is a leading cause of infant mortality and morbidity. Contrasting between ages, the effects of HI tend to be more severe in younger neonates. Curcumin, a dietary compound derived from turmeric, exhibits anti-inflammatory, antioxidant, and antiapoptotic properties, but is not bioavailable in molecular form, thus may serve as a neuroprotective treatment when loaded into synthetic nanoparticles to allow for effective absorption and crossing of the blood-brain barrier, forming the treatment NanoCurc. Gestational ages of 37 weeks through 42 weeks are all considered term neonates, yet their brain continues to develop and differ significantly in response to treatments against HI. Using the rat Vannucci model of unilateral hypoxic-ischemic brain injury, we studied the in vivo effects of NanoCurc in neuroprotection, in P7, P10, and P13 rats, equivalent to 34, 38, and 42 weeks’ gestation, respectively. Tissue is collected 72 hours after unilateral carotid artery ligation surgery, followed by tissue staining and analyzed by tracing the healthy tissue versus damaged tissue, to calculate the average percent area loss in treated and untreated rats. I hypothesize that in all ages, neonatal rats treated with NanoCurc will have lower injury in comparison to those treated with saline (vehicle), while the treatment will be more effective in younger rats in comparison to older ages. In the future, NanoCurc treatment may be used as a neuroprotective agent in reducing the effect of HI in preterm and term infants. If NanoCurc provides a stronger neuroprotective effect in the younger population, it may serve to target infants most severely affected by HI, potentially creating personalized treatment for gestational ages.

Our team conducted a study to understand the public attitudes towards creating human brain organoids (HBOs), a relatively new technique in neuroscience used to study brain development and disease. HBOs are structures derived from human donor stem cells and they mechanistically mimic certain aspects of the human brain. The goal of this study was to explore the values, priorities, and concerns that a sample of the general public has towards brain organoid research. An online survey was sent to 801 participants through Qualtrics. Respondents were asked to answer 51 yes or no, multiple choice, ranking, or open-ended questions, and provide their demographics. My colleagues and I qualitatively analyzed the open-ended responses to 3 questions using ATLAS.ti to code and identify themes in the general public’s opinion about brain organoid research. We found intriguing preliminary findings; 333 statements were supportive of the research and did not express concerns; for example, “it’s [brain organoids] created for research, thus it’s like hair, can be discarded”. 86 statements expressed concern about the potential future uses of brain organoids, for example: “it’s taking the first steps on the way to cloning…Dracula like”. Finally, 41 statements questioned the ethics of brain organoid research explicitly, for example: “people like to abuse things and this is no exception…[researchers] would end up using this for very harmful and unethical practices”. In open-ended responses, respondents expressed strong positions on the implications of brain organoid research, whether supportive or concerned. Further analysis of the open-ended responses is ongoing, and we plan to identify more themes. Increased knowledge of public opinions on brain organoid research could be valuable because it can help researchers improve informed consent for future studies, increase understanding of common misconceptions and concerns in the field, and influence how this type of research is regulated and conducted.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the deadliest pathogens in human history. The aim of this project is to begin probing the metabolic reprogramming that occurs in Mtb during treatment with isoniazid (INH), a commonly used frontline drug. Unpublished work from a collaborator has identified several common metabolites that change the minimum inhibitory concentration of INH needed to kill 99% of cells in a culture. The amino acid serine -- which is well known to be involved in one-carbon metabolism through interconversion with glycine -- was found to be of particular interest due to the increase in Mtb susceptibility to INH observed in serine-supplemented culture. To further explore this effect, I have generated timecourse growth curves using an avirulent Mtb (aMtb) model while under INH treatment with and without serine. I started cultures of aMtb at log-phase growth under the following conditions: without any additives, with serine, with INH, and with both INH and serine. I then plated aliquots of defined volumes on solid media to observe colony forming units (CFU) that assess how many cells were present in each culture. I repeated CFU plating 4 and 7 days after each culture was created, which gave me sufficient data to analyze the growth patterns of aMtb under these various conditions. My findings require validation but suggest serine supplementation may play a role in lowering INH tolerance in Mtb. If this result is found to be accurate, this may implicate one-carbon metabolism as a pathway with downstream effects on Mtb tolerance to INH.

Understanding the molecular mechanisms underlying the unique regenerative response of rodents from the Acomys (Spiny Mouse) kidney to injury is crucial for advancing regenerative medicine approaches. This study investigates the differential RNA expression profiles of cultured Acomys kidney cells as well as (non-regenerative) laboratory mouse kidney cells under hypoxic and normal culture conditions. The hypoxic culture environment mimics the effect of ischemic injury on kidney tissue. Comparative analysis of mRNA expression between these conditions across the two species can uncover potential factors contributing to Acomys' remarkable regenerative capacity. Observations in this model may provide valuable insights into developing novel therapeutic strategies for treating kidney diseases and promoting tissue regeneration in human patients.

Diffusible iodine-based contrast-enhanced micro-CT (diceCT) is a technique used to image soft tissue specimens using 3D x-ray microscopy. Staining soft tissues with iodine (I2KI) solution prior to scanning improves contrast for detailed visualization of internal organs, but iodine staining is also associated with tissue shrinkage which can interfere with quantitative analysis. It has been shown that stabilizing soft tissue with hydrogel can reduce shrinkage. We adopted these protocols for our lab, but, despite using hydrogel stabilization, we observed wrinkles in the external surfaces of E15.5 mouse embryos, qualitative evidence of considerable shrinkage. To quantitatively test for shrinkage, we compared the crown rump lengths (CRL) of mouse embryos measured from photos taken prior to the scanning process and then from diceCT scans. CRLs ranged from 12.4 to 20.0 mm in photos and 11.1 to 16.8 mm in scans. An average reduction of 12% resulted from the specimen preparation process and confirmed tissue shrinkage. Furthermore, the amount of shrinkage was not uniform across the specimens, complicating quantitative analysis based on diceCT. Our first hypothesis was that the iodine solution used to prepare the specimens was too acidic. We measured the pH of this solution and found a range from 4.5 - 6.4. To examine if a neutral pH reduced tissue shrinkage, we prepared specimens with a buffered iodine solution (pH 7.2). DiceCT scans of embryos in buffered iodine solution did not show reduced shrinkage compared to controls in the original solution. Further investigations will focus on other potential sources of shrinkage including the pH of other solutions and the time specimens spend in each step of the protocol. Continuing to investigate sources of tissue shrinkage in diceCT can lead to additional methods for shrinkage reduction, supporting more accurate quantitative analysis of diceCT.

Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disease that mainly affects children and adolescents. Disease monitoring is challenging as reported pain is not reliable and imaging is not always obtained at all clinic visits. To date, patient-reported outcomes used in CNO research have not yet been validated in this population. The Patient-Reported Outcomes Measurement Information System (PROMIS) questionnaires, validated in other pediatric rheumatic diseases, are administered to patients enrolled in the prospective multisite Chronic Nonbacterial Osteomyelitis International Registry (CHOIR) since 2018. Our objective was to assess the convergent and responsive validity of the PROMIS instruments in patients with CNO. Patients with CNO consented and enrolled in CHOIR. Self-reported PROMIS questionnaires of fatigue, pain interference (PI), pain behavior (PB), mobility, upper extremity (UE), physical activity (PA), and strength impact (SI) were administered to patients. External validation surveys were administered to assess patients’ perception of the difficulty of use of limb/back/jaw, fatigue, sadness, and worry on a 0-10 scale, disease status, and status change. More than 1,000 clinical visits from 184 patients were associated with self-reported PROMIS questionnaire entries. PROMIS scores correlated significantly (p<0.01) with patient-reported variables and physician global assessment (PHGA). The correlation with function and PHGA was good for Mobility, PB, and PI. All PROMIS scores, except physical activity, correlated significantly (p<0.05) with patient-reported disease status. After effective treatment when the clinical disease activity score improved by at least 2.5 points, the change of PROMIS scores from Mobility, PB, PI, and UE was significant. PROMIS questionnaires provide valuable information about the disease status of children with CNO and correlate well with self-reported functional and other psychosocial domains. Mobility, PI, and PB show sensitivity to change after effective treatment or with disease status change. These instruments are useful for CNO clinical disease monitoring and research.

Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disease that causes bone pain and destruction in children. Anti-inflammatory treatments for CNO can be effective, but there are not any FDA-approved medications specifically for CNO use, so there is interest in determining the effectiveness of different treatments for CNO. While CNO etiology is unknown, previous pathology studies indicate osteoclastogenesis plays a critical role in CNO pathogenesis. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) was shown to inhibit osteoclastogenesis derived from peripheral blood mononuclear cells (PBMCs) in adults with active rheumatoid arthritis. Our objectives are to determine whether osteoclastogenesis is altered in children with CNO and the effect of abatacept, a CTLA-4 analog, on osteoclastogenesis. We collected samples of PBMC and serum from healthy subjects and children with active or inactive CNO. We incubated patient serum samples with CD14+ cells isolated from a commercial PBMC pooled sample with MCSF and RANKL, which are necessary for osteoclast proliferation and differentiation, for 7 days before staining for osteoclasts. To determine abatacept’s effect, we incubated PBMCs from patients with MCSF and RANKL with or without abatacept (CTLA-4 analog), performed DAPI and TRAP staining, and counted osteoclasts using the computer software, Image J. Osteoclasts were defined as TRAP-positive cells with 3 or more nuclei. We intend to use multivariable linear and mixed-effects regression models to estimate the average group effect on osteoclast counts from the serum study and PBMC study and the impact of abatacept on osteoclast counts. Expected statistical results of the Serum and PBMC study include an increase in osteoclastogenesis from PBMCs of children with active CNO compared to other groups, and a decrease in osteoclastogenesis with abatacept treatment. This is an ongoing study, and the results will shed light on the effect of abatacept on osteoclastogenesis and its potential therapeutic use in children with CNO.

Asthma exacerbations often begin and increase in severity at night. Though animal models have shown molecular circadian rhythm involvement in immune and inflammatory responses, little is known about how circadian rhythms impact responses in humans or diseases such as asthma. BMAL1/ARNTL, CRY1, NR1D1, and PER2 are the genes that form the “cellular clock” by which cells tell time. Our hypothesis is that core circadian gene expression is maintained in an expected, rhythmic manner in epithelial cells from donors with asthma. We use an ex vivo model with human airway epithelial cells cultured at an air-liquid interface in a temperature cycled incubator to mimic the epithelia of the human airway. After temperature cycling to synchronize cellular circadian cycles, RNA collection occurs every four hours over a 48-hour period. After RNA isolation, I perform reverse-transcriptase quantitative polymerase chain (RT-qPCR) on a planned eight donor lines (4 healthy/4 asthmatic) to measure the gene expression of the four clock forming genes. In three asthmatic donor lines, I have found that core circadian rhythmicity is maintained in asthmatic epithelial cells and resembles the circadian rhythm expression in eight healthy donor lines previously analyzed. Shown through a preliminary study conducted by the lab, genes linked to asthma in the IL-17 signaling pathway have altered circadian rhythms of gene expression. In the future, I will use qPCR to study immune and inflammatory genes to confirm the altered rhythmicity across a wider scope of donor lines. In addition, I will analyze gene expression in different subsets of asthma to investigate whether altered circadian regulation contributes to asthma subtypes, such as T2-low which has been linked to IL-17 signaling pathway dysregulation. Investigating the differences in asthma-related circadian gene expression is essential to the development of chronotherapeutics – therapies that take into account time of day.

Hemophilia A, a severe genetic bleeding disorder affecting 1 in 10,000 people, is caused by genetic mutations inducing an inability to produce a clotting factor necessary to stop bleeding. Current treatment—replacing the missing clotting factor (FVIII)—carries risks like costly long-term injections and inhibitor antibodies in ~30% of patients. Our lab investigates base editing, a promising gene therapy solution aiming to repair the mutated FVIII gene directly to recover the natural production of the clotting factor. Our project uses a dog with a naturally occurring mutation similar to human hemophilia A, as a model for developing the treatment. In Phase 1 of this project, I will use mutagenesis and gel electrophoresis to create a plasmid containing the mutant canine FVIII gene, and other lab members will use hydrodynamic injection to apply it into mouse models. Following the verification of delivery through aPTT phenotypic examination, I will begin phase 2 by developing the CRISPR-Cas9 base editing plasmid. This will include insertion of our sgRNA into an existing plasmid, insertion of CAG promoter, maxiprep to produce a large quantity, and finally gel purification of the DNA. Following confirmed DNA sequencing of the constructs, we will use hydrodynamic injection of a mixture of the mutant canine FVIII plasmid and base editing plasmid into our hemophilia A mouse models and aPTT examination to determine the efficacy of the designed plasmid. Phase 3 will apply the base editing plasmid to treat the dog model, through collaboration with other lab members developing delivery strategies. This research holds immense potential for hemophilia A patients, offering a one-time, potentially curative solution compared to the limitations of current treatments. If successful, we expect the project to demonstrate a recovered FVIII gene and FVIII expression- first in the mouse model, then in the canine.

Birth asphyxia is the inability of a newborn to begin and maintain breathing. Twenty-three percent of neonatal deaths globally are caused by birth asphyxia. Birth asphyxia results in a neurological injury called hypoxic ischemic encephalopathy (HIE). Rapid HIE screening within six hours after birth is crucial to identify neonates at risk. Unfortunately, the diagnostic equipment is impractical for low resource settings because it is costly ($20/test and $5,000 for equipment) and requires technical staff, that are in short supply, to operate. We hypothesize that a cost-effective device can be developed for HIE analysis. pHast Cam quickly screens for birth asphyxia and HIE in infants via a paper-based blood pH sensor. The device combines an inexpensive pH sensitive dye, a smartphone camera, and a fixture that controls the imaging environment to quickly identify acidosis from samples. A low-cost paper-based strip is made with a water-soluble resin doped with a pH-sensitive dye, bromothymol blue (BTB), and a membrane to filter out red blood cells. The fixture removes lighting variation. The smartphone camera records the pH indicator image, and an algorithm captures, reduces noise, and accesses color change. pHast Cam incorporates four features: 1) accurate assessment of acidity within 0.05 pH units, 2) require only a few microliters of sample, 3) use electrical hardware and software only from the smartphone, and 4) affordability. At this stage, we have achieved a regressive linear model that predicts buffered solution acidity (y=-589.32x+4684.05 R2=0.9857), with 95% confidence interval of 0.04 pH units. In the future, we will transition from measuring buffered solutions to blood-plasma. Ultimately, we expect pHastCam to screen for birth asphyxia, and other acid-base disorders, by quantifying plasma pH in neonates so that timely therapeutic interventions and plans to address long-term complications may occur.

Pediatric patients with End-Stage Kidney Disease (ESKD) and their caregivers often face unfamiliar symptoms and complications, leading to interferences with self-efficacy and self-management, which result in poor clinical outcomes. This uncertainty leaves patients and families wondering: “Is my experience normal?” To address this unfulfilled information gap, we set out to design personal health visualizations that allow pediatric patients to assess their state of normalcy after receiving a kidney transplant. English and Spanish-speaking pediatric kidney transplant recipients and their caregivers were recruited from major transplant centers in the United States, and a standalone network striving to enhance the lives of children with kidney disease (IROC). They participated in three asynchronous Design Sessions involving sharing experiences (DS1), presenting symbolic objects (DS2), and providing feedback on personal health visualizations (DS3) to understand their perceptions of normalcy post-transplant. Data analysis comprised iteratively creating affinity diagrams using the transcripts from DS1 and DS2 to identify thematic areas about participants’ transplant experiences and normalcy. Additionally, we performed classification and statistical comparison using R to evaluate comprehension of DS3 normalcy visualizations. 14 youth (aged 10-21 years) and 16 caregivers participated in the design sessions, revealing diverse perspectives on their experiences. This highlighted the significant need for visualizations that represent normalcy, aimed at enhancing the understanding of patients and caregivers about their health. Our results show the need for normalcy visualizations to include three design principles: incorporating personal values, facilitating peer and self-comparison, and seamlessly communicating abstract concepts to help youth kidney transplant recipients comprehend and contextualize if their transplant experience is normal and what normalcy means to them. Harnessing broader aspects of patients' and caregivers' lives in personal health visualizations can enhance their awareness of progress towards normalcy. This approach helps to empower them to make informed decisions, fostering a sense of normalcy.

Perinatal asphyxia or hypoxia, where the infant brain does not receive enough oxygen or blood flow, commonly occurs in premature newborns and is one of the leading causes of neonatal mortality worldwide. Survivors often have altered white matter with cognitive impairments, motor deficits, and increased rates of cerebral palsy. There is currently no standard treatment for preterm brain injury, so there is a critical need to research neuroprotective strategies as well as ways to assess their impact. The ferret is a promising model species for studying preterm brain injury due to its gyrified brain and white-to-gray matter ratio, which are similar to that of the human brain. The gyrification index (GI) can be used to assess cortical development and is calculated using magnetic resonance imaging (MRI) images. These are analyzed using ImageJ software to perform hemispheric tracing by dividing an internal trace, including the gyri and sulci, by an external trace that excludes them. A higher GI is indicative of a larger cortical surface area. This project seeks to evaluate the effects of postnatal (P) age on post-hypoxic-ischemic (HI) gyrification in two ferret models. In both models, HI ferrets underwent bilateral carotid artery ligation and exposure to hypoxia, differing by date of surgery, with randomly assigned control animals not undergoing surgery. Model One ferrets underwent surgery at P10 (extremely preterm equivalent) and tissue collection at P70, and Model Two ferrets underwent surgery at P17 (late preterm equivalent) and tissue collection at P42. I hypothesize that GI will be affected by HI injury, with both age of injury and age of assessment altering GI relative to control animals. Contextualizing age differences in GI could help inform future therapy regimens to treat infants with premature brain injury.

Preterm birth is one of the leading causes of neurological deficits and mortality in neonates. Hypoxia-ischemia (HI), when the brain receives insufficient oxygen and blood supply, is a commonly observed condition among preterm infants. Preterm birth and HI affect the structure of the developing brain, particularly the formation of cortical folds (gyri) of the brain. This process is difficult to study in rodent models as they are lissencephalic and do not have gyrified brains. Ferrets, unlike rodents and more similar to humans, have gyrification – the folding of the cerebral cortex, creating gyri and sulci. Using the ferret as a model, examining changes in gyrification after injury can help us identify and further understand brain injuries like HI and its effects. Preterm-equivalent postnatal day (P) 17 ferrets received lipopolysaccharide intraperitoneally and underwent bilateral carotid artery ligation followed by hypoxia-ischemia plus hyperoxia (HIH). Animals were either non-HIH exposed (controls) or HIH-exposed and randomized to receive saline vehicle, erythropoietin, or therapeutic hypothermia. At P42, ex vivo (n=74) magnetic resonance images were collected. Using ImageJ software, the inner to outer hemispheric ratio was used to set the gyrification index (GI). We assessed the average GI across the whole brain and the peak GI of left and right hemispheres. Kruskal-Wallis test will be used to measure the differences in average and peak GI across treatment groups. We hypothesize that the average and peak GI of injured animals treated with Epo, TH, and saline will be lower compared to control animals. Gyrification can be an essential metric for researchers to analyze brain injuries like HI in preterm neonates. As preclinical and clinical studies for HI continue to improve, gyrification can inform the relationship of folding patterns in the cerebral cortex area, assisting us to examine the potential shared developmental processes between animals and neonates.

Group B Streptococcus (GBS) is a gram-positive bacterium that during pregnancy, can cause invasive disease including preterm-births, stillbirths, pneumonia, sepsis, and meningitis in newborns. Although GBS commensally resides in the vaginal tract, as a pathogen the bacterium can employ numerous virulence factors, including the serine rich repeat glycoprotein (SRRs) adhesins. GBS expresses one of two SRR’s (Srr1/Srr2) in a strain-dependent manner, and both can bind fibrinogen to increase GBS adherence to vaginal epithelial cells, increasing pathogenicity. Both proteins are heavily glycosylated, yet the role of glycosylation on protein function and GBS virulence remains unknown. Previously, our lab has conducted in vivo studies infecting pregnant mice with mutant GBS strains that do not express SRR or express an altered SRR glycoform. Data from these studies show that a decrease in vaginal colonization and ascending infection is seen for the SRR deletion mutant, while SRR glycomutants exhibited increased virulence compared to the wild-type strain. To further explore these findings in a more mechanistic manner, I will conduct a series of in vitro experiments to examine the host-pathogen interactions and immunological mechanisms of these mutants. Specifically, I plan to examine the role of neutrophil killing as an immune modulator of GBS pathogenesis and how susceptibility to killing changes when SRR glycosylation is altered. In addition, these GBS mutants will be used to explore whether the SRR adhesin and its glycosylation is important in inducing vaginal epithelial-mesenchymal transition – an important cellular pathway that predisposes vaginally infected mice to uterine infection and will test the susceptibility of the GBS SRR mutants to entrapment via fibrin clots. By examining how the SRR adhesin and its glycosylation impacts vaginal colonization and downstream disease-associated events, we aim to elucidate the role of SRR glycosylation in GBS virulence and identify new antimicrobial targets that can decrease GBS pathogenicity.

Genome-wide association studies demonstrate an association between the single nucleotide polymorphism (SNP) rs3184504 and many autoimmune diseases, including Type 1 Diabetes (T1D). The rs3184504*C allele encoding for arginine is mutated to encode for tryptophan in the rs3184504*T risk allele, resulting in a loss-of-function of the adaptor protein SH2B3, a negative regulator of various tyrosine kinases and cytokine receptors. The SNP is uniquely enriched in humans of European descent. Isolation of the SNP rs3184504 is challenging due to its presence in a linkage disequilibrium region. To normalize this donor variability we used a CRISPR/Cas9 system in human peripheral blood mononuclear cells (PBMCs). CRISPR guides target exon 4 of the SH2B3 gene to be cut by the Cas9 enzyme. This cut is then repaired by a green fluorescent (GFP) repair template cassette delivered by an adeno-associated virus (AAV) containing homology arms for the flanking SH2B3 sequence. The insertion of GFP permits the identification of the SH2B3 KO cells. Our previous experiments on SH2B3 KO mouse lines demonstrated heightened signaling response after IL-2 stimulation in T cells, particularly in CD8+ naive and effector cells and CD4+ effector cells. To test this in primary human T cells, we will stimulate our edited SH2B3 KO cells with IL-2 and use flow cytometry to compare differential responses between edited and unedited T-cell populations. We expect to see similar results in our edited cells. Successful gene editing of PBMCs to model the human SH2B3 disease haplotype enables us to more accurately study the biological underpinnings of T1D, ultimately providing new avenues for which to treat T1D.

Traumatic brain injury (TBI) is caused by an external force to the head, resulting in brain injury and is a major cause of death, particularly in adults 75 years and older who are at increased risk of falls which can lead to disability. Humans have a natural response to impact and strain called the Valsalva maneuver, which leads to an increased pressure in the chest and abdomen, which can result in a neuroprotective increase in intracranial pressure (ICP). However, most people are unable to anticipate TBIs and cannot perform their own Valsalva maneuver. Using a ferret model of TBI, the neuroprotective effects of externally-stimulated Valsalva-like response will be assessed. Ferrets are used to model human TBIs because the cortical thickness and layer distribution of their brains are more akin to humans in the ferret compared to rodents. The ferrets will be randomized to one of the three groups: control, TBI+sham valsalva, and TBI+valsalva. To show that the intracranial pressure of ferrets can be transiently increased, an inflatable cuff will be utilized to exert pressure on the abdomen, resulting in a partial Valsalva maneuver. TBI will be induced in the ferrets using a closed-head impact, and the neuroprotective effects of increased ICP from the inflatable abdominal cuff will be assessed using a battery of motor and cognitive tests before and after the TBI event, additionally, brain injury and neuroprotection will be evaluated using histopathology. I hypothesize that the Valsalva maneuver induced by the inflatable abdominal cuff will reduce behavioral deficits resulting from impact. If the behavioral deficits are reduced, this study can work to inform future interventions for TBI, such as environment-sensing wearable devices for high risk populations.

Protein kinases have been found to regulate cell processes such as cell growth and adaptive responses to the environment. Thus, they act as excellent targets for tuberculosis treatment. The Mycobacterium tuberculosis (Mtb) genome encodes 11 separate serine/threonine protein kinases (STPKs). One of those STPKs is PknF, which our lab has previously found to be associated with a survival deficit in Mtb. Since Mtb utilizes multiple carbon sources during its infection in vivo, my research project aims to test the effect of perturbing pknF expression levels on Mtb survival in different carbon source conditions. I grew an avirulent Mycobacterium tuberculosis (aMtb) strain that conditionally overexpresses pknF upon exposure to anhydrotetracycline (pknF OE) in different media formulations that provided different carbon sources: glycerol and acetate. I measured colony-forming units (CFU) to quantify aMtb survival in these different growth conditions. I compared survival measurements of the pknF OE strain relative to an empty vector control strain. I found that the pknF OE strain showed a survival deficit by day 7 in both media conditions, with glycerol showing a higher survival deficit compared to acetate. The extent of the survival deficit seems to be affected by which carbon source is provided by the media. Previous literature has linked pknF to the regulation of glucose transport and phosphorylation of glucose catabolism-associated enzymes in the citric acid cycle. Based on this, we hypothesize that these processes may be involved with pknF's observed survival deficit in different media. In follow-up experiments, we plan to investigate how perturbing glucose transport or catabolism influences pknF's survival deficit. These findings can be used to inform future studies on pknF as a potential target for tuberculosis treatment during infection. 

Although physical activity benefits children's health and development, most children do not meet the recommended 60 minutes of physical activity per day. Additionally, barriers to physical activity, such as lack of access to staffing or equipment at low-resource schools, can exacerbate population-level health disparities. With the goals of promoting physical activity in children and tackling health inequities, 13 schools across WA state were recruited for participation in this study with support from the Office of Superintendent of Schools (OSPI). Schools were randomized to either receiving a Physical Activity Coordinator (PAC) (n=4) or being an Active Control school (n=9). The purpose of this study is to investigate the PAC program's impact on children's physical activity at school and how children feel about physical activity. Data was collected via student surveys and observations before school, during lunch, and after school using the System for Observing Play and Leisure Activity in Youth (SOPLAY) method. Metrics collected via SOPLAY include area conditions, the number of children engaged in physical activity, the type of their physical activity, and the type of social interactions. Using our collected data, we are now working to analyze the impact of the PAC program and anticipate finding increases in some of our collected metrics. As part of the team, I worked on data collection through school observations, data verification, and data analysis. These findings are important because investing in building healthy habits in our children will pay dividends for their current and future health. Hopefully, the lessons learned through this study can be applied on a broader scale to help promote physical activity and tackle systemic health inequities.

Preterm neonates frequently undergo episodes of hypoxia, hypotension, and hyperoxia due to their underdeveloped cardiorespiratory system. As a result, preterm infants are particularly susceptible to brain injury from lack of sufficient oxygen or blood flow. In this study, we use a neonatal ferret model of hypoxic-ischemic-hyperoxic (HIH) brain injury to examine the neuroprotective efficacy of azithromycin (Az), a safe and accessible antibiotic with anti-inflammatory properties that crosses the blood-brain barrier in premature infants. Our HIH ferret surgery model produces global brain injury in Postnatal day (P)14 ferrets, which are developmentally equivalent to extremely preterm neonates. Ferret kits in the surgical group will receive a pro-inflammatory stimulus (E. coli lipopolysaccharide), followed by a bilateral carotid artery ligation and exposure to HIH. Then, surgical animals will be randomized to receive intraperitoneal injections of saline vehicle (Veh) or Az. Control animals will not receive LPS or undergo surgery. We will use a Kruskall-Wallis test to compare P21 ferret weights and brain measurements seven days after surgery to test whether Az is neuroprotective. We will assess pathology using quantitative immunohistochemistry with Iba-1 (microglia) and GFAP (astrocytes) stains. We have two main hypotheses. We hypothesize that 1) ferrets treated with Az will lose less body weight after exposure to inflammation-sensitized HIH than the Veh group, with Az-treated animals being similar in weight to non-injured controls, and 2) normalized brain measurements, corrected for body weight and sex, will show that Veh animals have smaller gyri and sulci measurements than controls, and Az-treated animals will have brain measurements that are more similar to controls. We are optimistic that our experiments will provide evidence that azithromycin is potentially neuroprotective and will offer a promising treatment option for preterm babies with brain injury.

 During gestation, a human fetus is fully dependent on the placenta which provides oxygen, nutrients, regulates waste transport, and acts as an immunological barrier. Glial Cells Missing Transcription Factor 1 (GCM1) is a transcription factor (TF) that plays a crucial role in placental development. Our goal is to understand the downstream effects of GCM1 knockdown on genes necessary for placental development. The BeWo choriocarcinoma cell line is a model of placental syncytiotrophoblasts cells which undergo a cell fusion process called syncytialization to form multinucleated cells that help exchange nutrients. BeWo cells were treated with Forskolin (FSK) to induce syncytialization to represent syncytiotrophoblast cells. RNA sequencing was conducted after GCM1 knockdown using siRNA to quantify downstream gene expression changes. BeWo cells were treated in 4 different conditions: FSK, GCM1 siRNA (GCM1 KD), FSK treatment first then GCM1 siRNA treatment (FSK + GCM1 KD), and lastly, GCM1 siRNA treatment first and then FSK treatment (GCM1 KD + FSK). In the GCM1 KD group, a total of 533 genes exhibited significant differential expression (FDR < 0.5), when compared to control BeWo cells, with 236 genes upregulated and 297 genes downregulated. Subsequently, in the FSK + GCM1 KD group, 961 genes were significantly altered, where 386 were upregulated and 575 were downregulated. In the GCM1 KD + FSK group, a notably higher number of genes, 7,165, displayed significant differential expression, with 3527 genes being upregulated and 3638 genes being downregulated. We also identified a number of biological pathways such as focal adhesion and adherens junction that may be crucial to GCM1-induced syncytialization. Overall, our results will improve our understanding of how GCM1 coordinates gene expression in the placenta during pregnancy.

The immune system and inflammatory responses to viral infections are regulated by molecular circadian rhythms in mouse models. Mice infected with influenza just prior to their active phase have a mortality rate four times higher than mice infected just prior to their rest phase. As a result, circadian rhythms are hypothesized to regulate viral replication and early immune responses in airway epithelia during viral infections. Prior work has shown circadian cycles regulate gene expression in human epithelial cells. However, the influence of time of infection on viral replication in human airway epithelia has not yet been explored. We hypothesized that circadian-synchronized human airway epithelial cells would demonstrate differential viral replication and immune responses when infected at two different times of day. To address this gap, we differentiated primary epithelial cells from healthy children at an air-liquid interface to create an ex vivo cellular model of the human airway. Airway epithelial cells underwent circadian synchronization using temperature cycled incubators and were exposed on the apical surface to human rhinovirus-16 at time 0 and 12 hours during a circadian cycle. The RNA from seven total cell lines was sequenced and viral genome copy number was quantified at hour 96 following infection using GeneSig qPCR. Infection at hour 12 led to two-fold higher viral genome copy number 96 hours after infection as compared to hour 0. Infection late in the circadian phase (time 12) leads to increased viral replication at the airway epithelium and may explain the difference in mortality in mouse models of viral infection. Ongoing work is investigating immune responses based on time of infection. In the future, we will investigate changes in circadian regulation of viral infection in airway epithelia from healthy children and children with airway diseases such as asthma.

Stress-related physiological and social factors can have a direct impact on cancer biology and patient outcomes. Exposure to a stressor can lead to downstream activation of signaling pathways that can impact cancer-related processes. Compared to older and younger patients with cancer, adolescents and young adults (AYAs) have had less improvement in clinical outcomes and report high levels of psychosocial distress. The goal of this study is to examine the feasibility and acceptability of a multimethod triangulation protocol collecting stress biomarkers along with subjective patient reported outcomes among AYAs with cancer. Eligible participants are 12-24 years old within six months of initial cancer diagnosis and undergoing treatment at Seattle Children's Hospital. Once enrolled, participants wear a sensor measuring autonomic nervous system activity [quantified as heart rate variability (HRV)] while completing validated patient reported outcome (PRO) surveys assessing psychosocial symptoms and participating in a qualitative interview querying the same domains. Interviews are recorded using Microsoft Office Dictation services. I listen to the audio files and go over the transcribed document making corrections and highlighting key quotations. Additionally, at the time of a participant's planned clinical blood dram, 5mL of blood is collected to measure a social genomics stress biomarker called the conserved transcriptional response to adversity (CTRA). We expect to find greater than 70% feasibility and 80% acceptability in this study. Feasibility is defined as the percentage of participants who complete all (100%) study procedures; acceptability is defined as the percentage of participants who agree that similar patients could complete the study. Results from this pilot study will inform future longitudinal investigations and the development of targeted biopsychosocial interventions for AYAs with cancer. 

Type 2 diabetes (T2D) is among the most common and costly chronic diseases in the United States. Nearly 10% of Americans have T2D and a further 34% have prediabetes. The brain has increasing emerged as a promising target for the treatment of T2D. For example, our group has demonstrated in rodent models of T2D, that remission of diabetic hyperglycemia lasting for weeks or months can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1). Injection of FGF1 into the brain induces sustained activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway. ERK signaling is most highly induced in tanycytes which are specialized ependymo-glial cells that line the 3rd ventricle and are implicated in the central control of energy and glucose homeostasis. However, whether MAPK/ERK activation in tanycytes is required for sustained glucose lowering to occur in response to icv FGF1 is unknown. To begin testing of the hypothesis that activation of the MAPK/ERK pathway in tanycytes is necessary for sustained diabetic remission induced by FGF1, we proposed to inhibit the activation of MAPK/ERK pathway specifically in tanycytes by microinjection of a mouse MAPK shRNA silencing adenovirus into the 3rd ventricle 1 week prior to injecting FGF1. In preliminary studies in wild-type mice, I have shown that the ability of FGF1 to induce MAPK/ERK activation is blocked by prior injection of the MAPK shRNA silencing adenovirus. I will now take C57Bl/6J mice placed on high-fat diet to induce diabetic hyperglycemia and determine if pretreatment with the MAPK shRNA silencing adenovirus blocks the ability of icv FGF1 to induce diabetes remission. I predict that successful completion of this study will deepen our understanding of the pathogenesis of T2D, bringing us closer towards a treatment for this devastating disease.

Preterm birth is one of the leading causes of infant morbidity and mortality worldwide, with a strong association between the degree of prematurity and the likelihood of death or neurodevelopmental impairment. Intracranial hemorrhage (ICH) is one of the most common neurological injuries for extremely preterm infants (born less than 28 weeks’ gestation). During the last trimester of pregnancy, neurons and glial cells develop in the germinal matrix requiring vast amounts of vascular support. In preterm infants, disturbances to blood and hydrostatic pressure are thought to rupture the immature vessels of the germinal matrix, leading to the bleeding in and around the ventricles. ICH is rated on a scale of I to IV, with severe ICH being grade III-IV. Mortality associated with ICH ranges from 30-60 percent, increasing with ICH severity, and survivors have an increased risk of cerebral palsy, seizures, and neurodevelopmental delay. From 2018-2020 the University of Washington (UW) neonatal intensive care unit (NICU) implemented an ICH Prevention Bundle, which focused on minimizing blood pressure disturbances during the first 72 hours after birth in infants born extremely premature, and appeared to result in a decrease in severe ICH. This study will evaluate the incidence rate of ICH at the UW NICU over a ten-year period. In a retrospective analysis of the UW NICU’s admissions, we will investigate extremely preterm infants born during the time periods of December 2013-September 2016 versus January 2017-December 2023 and record the incidence of ICH. Our primary outcomes will be ICH, by grades I-IV, as well as ICH complications such as posthemorrhagic ventricular dilatation with and without need for intervention, and death before discharge. We hypothesize that with improved prevention methods, such as the implementation of the ICH Prevention Bundle, we will see an associated long-term decrease in the incidence rate of ICH.

The cerebellum, which accounts for 10% of human brain volume, contains approximately 80% of all brain neurons and has long been understudied relative to the cerebral cortex. During development, the cerebellar ventricular zone (VZ) is a transient progenitor zone which generates all cerebellar GABAergic (inhibitory) neurons. This includes Purkinje cells (PCs) and PAX2+ interneuronal progenitors (PIPs). While the mouse cerebellar VZ has been relatively well characterized, there is limited knowledge about its human counterpart. In this study, we investigated organization of progenitors and birth of neurons derived from the human cerebellar VZ, uncovering several notable features. Specifically, I conducted image analysis in conjunction with immunohistology (IHC) assays to (1) identify different cell types and marker gene expression across developmental stages and (2) quantify proliferative cells at different stages of development. We found that a substantial number of PCs are generated during embryonic development, particularly within the first 50 post-conception days, within a compact two-week timeframe. This occurs well before the onset of cerebral neurogenesis, with interneuronal differentiation commencing during early fetal development. Neuronal differentiation predominantly occurs from the inner and outer subventricular zones (SVZ), zones which are completely absent in the mouse developing cerebellum, with the initial wave of differentiation occurring from the outer SVZ. Significantly, relative to mice, we observed variations in migratory patterns and the quantity of PC plates, including a subset of PCs that retain the expression of cell cycle genes several weeks after these cells leave progenitor zones. This work extends our knowledge of human-specific birth and organization of progenitors and neurons originating from the ventricular zone and cellular and molecular differences in ventricular zone progenitors, Purkinje cells, and PIPs across different developmental ages.
 

The University of Washington Neonatal Intensive Care Unit (NICU) Extremely Preterm (EP) program aims to provide outstanding care to EP infants born at 22 0/7-24 6/7 weeks’ gestation. The objective of this study is to assess the feasibility of night telerounds in a level IV academic NICU for EP neonates to enhance the support for teams caring for EP patients. This was a retrospective cohort study. Through review and analysis of electronic medical records, the EP database, and telemedicine records from July 2022 to June 2023, the frequency and duration of telerounds during the night shift (8-11pm) were examined. Comparisons of EP outcomes were made between two epochs: 6 months pre- and 6 months post-telerounds implementation. Descriptive statistics, chi square and independent samples t-test were used to compare EP outcomes between epochs. There were a total of 195 telerounds encounters, with improvements noted in network connectivity over time (fewer dropped calls lasting <20 sec). In the pre-implementation period, 9 (36%) of EPs were transferred for subspecialty/surgical care, mortality before 36 weeks’ postmenstrual age (PMA) was 14 (56%) and median length of stay (LOS) for survivors to discharge was 182 days (SD 72). Post-implementation, 10 (50%) EPs were transferred with mortality before 36 weeks’ PMA of 7 (35%) and median LOS for survivors to discharge was 139 days (SD 24). In conclusion, there was a reduction in neonatal mortality before 36 weeks’ postmenstrual age and a trend towards decreased length of stay during the post-implementation period. The findings suggest that night telerounds are feasible in supporting bedside NICU teams caring for critically ill EP neonates and may facilitate care advancement. Additional chart review is in progress to characterize orders entered during night rounds in order to evaluate the impact of this intervention.

The miR-9 family of microRNAs, known for its remarkable conservation across species, exerts significant influence over neural proliferation, differentiation, and maturation processes. However, the specific role of individual miR-9 family members is not widely known. Previous work in the Cherry Lab shows that loss of miR-9-2 in mice disrupts brain vascular development. Furthermore, previous studies have implicated the miR-9 family as important regulators of neural progenitor proliferation. Thus, I assessed the proliferative properties of brain progenitor cells and vascular development in miR-9-2 mutant and control mice from early to mid-neural development to test the hypothesis that miR-9-2 is a significant regulator of progenitor proliferation and vasculogenesis in the developing brain. To assess neural progenitor cell proliferation and vascular development, I performed immunohistochemistry and confocal microscopy to label and visualize vascular and progenitor cell populations. Images were quantified using ImageJ and AngioTool. I found that primary neural progenitor proliferation does not appear to be altered in the forebrains of E12.5 miR-9-2 mutants, while intermediate progenitor cell proliferation is increased in E12.5 mutants. However, by E16.5, both neural progenitors and intermediate progenitors display increased proliferation in miR-9-2 mutants relative to controls. The vascular vessel analyses showed no significant changes in the overall organization of the vascular network in either the E12.5 or E16.5 miR-9-2 mutants. Further work will reveal the role of miR-9-2 in regulating brain vascular functional integrity during development that may alter brain homeostasis when dysregulated and potentially serve as therapeutic targets.

Long-term neonatal intensive care unit (NICU) hospitalizations are emotionally and financially stressful for families. While family presence at the bedside and active involvement in their baby’s care have been linked to healthier outcomes, many families are unable to visit regularly. As technology continues to advance, live-streaming video platforms have been utilized in many NICUs to address the limitations of in-person presence. As the parents are able to continuously connect with their baby virtually, this allows for increased bonding with the newborn and engagement with the patient care team. The extended family and friends can interact with the infant as well. The objective of this study was to describe the utilization of the Seattle Children’s Hospital (SCH) NICU virtual visit program and its effectiveness in maintaining or increasing caregiver and patient bonding during the infant’s hospitalization. This was done through a program evaluation using mixed methodology with a parent survey and interviews. Parents and caregivers of NICU patients who used the virtual visits at least once during a patient's hospital stay were recruited using phone calls and text messages with the link to the survey. The virtual visit call frequency, time, and number of visitors were reviewed. The results showed that from July 2021 to December 2023, there were 348 virtual visits since July 2021, most of them lasting over 20 minutes (median 23.5, IQR range 3, 68.5). Most participants used the program one to two times (range min 1, max 69). Many of the calls occurred between 7 am - 7 pm with one to two attendees. In conclusion, the virtual visits program was utilized frequently and the timing of the virtual visits overlapped with the availability of the primary healthcare team which may have supported active involvement in care. The parent surveys and interviews about the program’s impact are in progress.

With the global roll-out of dolutegravir-based antiretroviral therapy (DTG-ART) for people living with HIV, emergence of DTG-resistance is of concern. People who inject drugs (PWID) are one of the key populations most vulnerable to HIV. Data on DTG-resistance, especially among key populations and in "real-world" settings are limited. To address this knowledge gap, we performed a longitudinal cohort study of PWID living with HIV in Kenya on DTG-ART to determine the prevalence of virologic failure and DTG resistance. We enrolled PWID living with HIV who had been taking DTG for ≥six months at eight clinical sites. Plasma was collected at enrollment and every six months for 24 months and plasma HIV RNA was measured. HIV genotyping for resistance was performed on any viremic (HIV RNA >200c/mL) specimens using PacBio sequencing. At each study visit, ~15-20% of the 250 enrolled participants had viremia; however, the participants with viremia at each visit were not the same. Pre-DTG genotypes were available for 61/250 participants. I compared the rates of viremia among those with vs without pre-DTG resistance to DTG-ART and found that pre-DTG resistance was not associated with virologic failure on DTG-ART. Interestingly, while 65/250 (26%) participants experienced intermittent viremia, which could potentially result in selective pressure for DTG-resistance, resistance to DTG by month-12 was rare in this cohort. One participant had detectable DTG-resistance at month-6, with the mutation G118R detected in 1/100 (1%) viral templates sequenced. Genotyping of the month-18 and -24 viremic plasmas is in-progress; however, given the frequency of DTG-resistance detected by month-12 we anticipate that DTG-resistance will still be rare in this cohort by month-24. These findings provide new data on the frequency of virologic failure and DTG-resistance among a key population living with HIV and may help inform clinical management of PWID living with HIV in resource-limited settings. 

Joubert syndrome (JS) is a rare, congenital neurodevelopmental condition diagnosed by the appearance of the “molar tooth sign” on axial brain magnetic imaging (MRI). Patients typically display hypotonia, abnormal eye movements, and ataxia. Substantial progress has been made on identifying the genetic causes of JS, which typically displays recessive inheritance. Nonetheless, the cause still cannot be identified in ~27% of our cohort of JS-affected families and the contribution of causal variants that impact RNA splicing remains unknown. Canonical splice variants impact RNA splicing by disrupting the splice site directly, whereas noncanonical splice variants affect it through alternative mechanisms, which must be validated by RNA analysis. Our goal is to evaluate the role of noncanonical splice variants in the pathogenesis of JS. We previously identified genetic causes in 582 of 714 families with JS. To identify additional causes, we used SpliceAI, a deep learning-based tool to identify variants with predicted splicing effects (SpliceAI score >0.5) for functional validation. We extracted RNA from patient cell lines then used polymerase chain reaction (PCR) and evaluated amplicons using gel electrophoresis and Sanger sequencing. Five families with candidate noncanonical splice variants that may confirm their genetic cause provided skin biopsies for further splice analysis. We confirmed the pathogenicity of three of these variants by demonstrating abnormal splicing in JS genes, AHI1 and MKS1, bringing the total contribution of aberrant splicing up to 77/714 families. The final two variants did not render conclusive results after 10+ attempts of PCR-based assays and may therefore be candidates for other methods of investigation such as mini-gene assays or long-read sequencing. By extrapolation from our data in JS, splice variants may contribute ≥11% to the genetic causes of conditions. A precise genetic diagnosis informs prognosis, avoids unnecessary work-up, guides monitoring for associated complications, and opens the door to gene-specific treatments.

Examining environmental exposures during pregnancy and their impacts on fetal and child development is vital for maternal and child health. As researchers, we have a responsibility to return these individual environmental results to participants. DERBI (Digital Exposure Report-Back Interface) is an online tool to specifically return environmental exposure results. This study examines: Are there distinguishable demographic-based patterns in those who logged in to open their results versus those who did not? What demographics are we missing in getting participants their results? PATHWAYS GAPPS is an NIH-funded study that examines environmental exposures and child outcomes in Seattle and Yakima, Washington. DERBI provided personalized biomonitoring results for environmental chemicals after over a decade of participation in research. Three hundred ninety-three participants across the Seattle (N=194) and Yakima (N=199) sites were sent an access code for their child’s return of results. I compiled demographic data on socioeconomic factors, such as race and maternal education. Within each site, I analyzed the demographics on which participants had opened their child’s return of results and those who did not open them. I then calculated and rounded percentages over the opening status for each site. At the point of data collection, mothers had five months to log in and open their results. I observed that mothers with less education were less likely to open their results, and fewer non-Hispanic Black mothers opened their results, suggesting that education and self-identified race are key factors in determining who is likely to access their results. The manner we present the return of results to participants and provide additional resources to process and take action on the results also matters. Future directions include conducting qualitative interviews and focus groups to hear first-hand from participants about barriers in accessing results and what resources would increase inclusivity and likelihood of opening their results.

Segmentation is an imaging technique commonly used to isolate an object of interest, such as an organ, from the background, or other objects in the image. When analyzing the shape of an anatomical structure, segmentation of that structure is often the first step in analysis. Precise anatomical segmentations are often created manually by subject experts, which is time-consuming, does not scale well, and can be prone to error since it is subjective. In this project, we aim to develop a machine-learning model to expedite whole-body surface segmentation from fetal mouse scans as part of an automated pipeline to detect asymmetry and abnormality in the facial region. The International Mouse Phenotyping Consortium (IMPC) has generated a large repository of three-dimensional (3D) imaging data from mouse embryos, providing a rich resource for investigating phenotype/genotype interactions. To generate segmentations required for training and validation of our deep learning model, the full body surface was manually segmented in 91 baseline scans from the IMPC’s Knockout Mouse Phenotyping Program (KOMP2) dataset. I trained a UNet with transformers (UNETR), on these segmentations that is able to estimate surface segmentations from new micro-CT mice images with an accuracy of 0.9. I am currently developing a fetal mouse full-body segmentation application powered by our deep learning model, SurfaceExtract, that will be made publicly available as an extension to the open-source image analysis platform, 3D Slicer. SurfaceExtract will be used by our lab to quickly and accurately generate segmentations of fetal mice as part of our lab’s automated facial asymmetry phenotyping pipeline.