University Of California At Davis

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT The University of California and the University of Texas Southwestern (UCaTS) Diversity Patient-Derived Xenograft (PDX) Development and Trial Center’s is a diversity-focused grant with a goal of establishing and characterizing at least 120 new PDXs from racially and ethnically diverse populations and use these PDXs as preclinical models to better understand the mechanisms of oncogenesis, to test FDA-approved and NCI-CTEP single agent and drug combination therapies and to advance cancer health disparities research. Expanding the existing PDX infrastructure from our ongoing University of California Patient Derived and Trial Center (UCaMP, U54CA233306), UCaTS is comprised of six NCI-designated Comprehensive Cancer Centers (CCC): UC Davis Comprehensive Cancer Center (UCD) [lead institution], UC Irvine Chao Family Comprehensive Cancer Center (UCI), UC Los Angeles Jonsson Comprehensive Cancer Center (UCLA), UC San Diego Moores Cancer Center (UCSD), UC San Francisco Helen Diller Family Comprehensive Cancer Center (UCSF), and the University of Texas Southwestern Harold C. Simmons Cancer Center (UTSW). Collectively, UCaTS includes a team of transdisciplinary investigators with strengths in PDX development, preclinical drug testing, cancer health disparities, clinical trial development, bioinformatics, and large multicenter administration. UCaTS will achieve its goal through two Research Projects. Project 1 – “Advancing gastric cancer precision medicine in Latinos through patient-derived modeling” focuses on developing novel, effective therapeutic regimens for most commonly druggable and genomically complex Latino GC subtype, which involves co-mutations in multiple pathway (such as cell cycle kinase, PI3K/AKT/mTOR, WNT and RTK-RAS) and to study resistance mechanisms to targeted therapies, identify response biomarkers and assess how ancestry influences response. Project 2 – “Precision targeting of disparity-associated EGFR mutant lung tumors to circumvent early resistance to EGFR inhibitors”, focuses on one of the most common lung cancer (LC) subtypes in minority populations, EGFR- mutated lung tumors. This molecular subtype represents a high proportion of lung cancer diagnosed in female patients of Latino and Asian ancestry. The project will utilize PDXNet and our UCaTS lung cancer PDX expertise and resources to develop effective targeted therapy combinations with FDA-approved and NCI-CTEP agents seeking to overcome early resistance to EGFR-TKIs.

NIH Research Projects · FY 2024 · 2023-08

Opioid drugs are essential medications for the relief of serious pain, with no substitutes currently available for postsurgical and other severe indications. Long term use of opioids, however, leads to numerous side effects, and to substantial risk of substance use disorder (SUD). SUD or “addiction” is diagnosed based on behavioral characteristics that manifest broadly as loss of control or “compulsive” drug seeking and impaired decision making or “cognitive flexibility” even after months to years of abstinence. However, the majority of preclinical research of drug abuse focuses on models of drug-taking and reward-seeking rather than on the long-lasting changes in behavioral flexibility that underlie human SUDs. In addition, preclinical studies of SUD mechanism have been limited to comparing animals that have or have not taken drug. This has made it difficult to dissociate opioid- induced changes in biology and behavior that occur merely due to drug exposure from those that actually underlie the pathology of a SUD. We have developed a unique tool to circumvent this significant confound in opioid abuse research. Specifically, we have developed a knock-in mouse that expresses a modified mu opioid receptor (MOR) with altered signaling properties. The MOR when activated by its endogenous ligands, endorphins and enkephalins, engages G protein signaling to control neuronal activity. Following G protein activation by endogenous ligand, most G protein coupled receptors (GPCR), including the MOR, then rapidly recruit arrestins that silence the G protein signal and promote receptor endocytosis and, for the MOR, rapid recycling. This mechanism thereby carefully titrates G protein signal with a precision and time course ideally suited to respond to transmitters that are released in a pulsatile manner. In contrast, MORs activated by morphine and all its derivates effectively engage G protein signaling but poorly engage arrestins. In the current vernacular of GPCR pharmacology, morphine is termed a “biased” agonist, signaling preferentially to G protein over arrestins while endorphins are “balanced” agonists, engaging both G proteins and arrestins. The RMOR receptor was engineered to effectively engage both G protein and arrestin when activated by morphine without altering signaling in response to endogenous transmitters. Importantly, in our extensive previous work, we have found that RMOR mice do not develop tolerance or dependence to morphine nor do they transition to compulsive drug seeking in a model of SUD under conditions where wild type (WT) mice do. More recently, we have found while morphine causes long-lasting changes in cognitive flexibility in WT mice, this effect is also absent in RMOR mice. Here we will use WT and RMOR mice and single cell RNAseq to pinpoint molecular mechanisms that underlie SUDs in a paradigm where all mice receive drug but only WT show pathologic morphine responses. We propose that any morphine-induced changes that occur in both genotypes is likely to reflect a response to drug exposure, whereas changes confined to WT mice likely contribute to the pathology of SUDs.

NIH Research Projects · FY 2024 · 2023-08

Project summary: Serotonin, or 5 hydroxytryptamine (5HT) is an endogenous transmitter that is broadly implicated in many modalities of human behavior including mood, libido, appetite and sleep, as well as peripheral functions in platelet aggregation, immune response and bone density. In the brain, this single transmitter binds and activates seven different families of 5HT receptors with five distinct modes of signal transduction, including all four classes of G protein-coupled receptor (GPCR) (Gi, Gs, Gq and G12/13) and a ligand-gated ion channel. Alterations in the relative balance of these diverse signaling pathways by 5HT has been implicated in a plethora of syndromes including depression, major depressive disorder, bipolar disorder, schizophrenia, obsessive compulsive disorder and attention deficit hyperactivity disorder. Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for the treatment of depression, and more than 15% of adults in the US have taken an antidepressant within the past 12 months. In theory, the efficacy of these drugs is due to their ability to increase 5HT levels by blocking reuptake of 5HT. However, while these drugs increase 5HT levels in as little as 2 hours, it takes 4-8 weeks for them to become effective at ameliorating depression. This suggests that the mechanism of action of SSRIs is more complicated than simple increases in transmitter levels. We hypothesize that high levels of 5HT, produced through use of SSRIs, can, in time, rebalance the expression levels of the various 5HT receptors and, by doing so, provide therapeutic benefit. Specifically, we propose that some of the 5HTRs, when activated by 5HT, undergo endocytosis and recycling/resensitization while others are endocytosed and degraded in the lysosome. The high levels of 5HT provided by the SSRI can, thereby, drive down expression of certain subtypes of 5HTR, while maintaining a high level of signaling through others--in effect “rebalancing” 5HT signal transduction to more closely resemble the non-depressed state. Here, we will perform a comprehensive analysis of the endocytic and post-endocytic properties of the entire family of 5HTRs. Remarkably, there is very limited knowledge regarding the post-endocytic sorting properties of the 5HTRs. Hence, regardless of outcome, these data will provide new information regarding how increased 5HT levels, due to SSRIs, could alter receptor expression and could inform new approaches to serotonin therapeutics designed to rebalance 5HT signal transduction.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Cardiac arrhythmia is a condition where the heart’s electrical signals are disrupted, causing the heart to beat in an irregular and dyssynchronous manner. While some arrhythmias may be benign, others can be life- threatening, as the ability of the heart to perfuse the vital organs with oxygen and nutrients is severely compromised. Small conductance calcium-activated potassium channels (SK channels) are activated by the increase of intracellular calcium concentrations on a beat-to-beat basis. SK channels play critical roles in the regulation of cardiac excitability, and dysfunction of cardiac SK channels causes cardiac arrhythmias including atrial fibrillation (AF). Therefore, SK channel may represent a novel therapeutic target for cardiac arrhythmias. The regulation mechanisms of cardiac SK channels have been extensively studied. Phosphatidylinositol 4,5- bisphosphate (PIP2) is an essential regulator of many plasma membrane-bound proteins, including cardiac ion channels. Hence, PIP2 may play critical roles in arrhythmia initiation and maintenance. However, whether and how cardiac SK channels are regulated by PIP2 are still unknown. We hypothesize that SK channels are regulated by PIP2 in cardiomyocytes and PIP2 plays a critical role in the trafficking of SK channels. To test the hypothesis, we will use multidisciplinary approaches including patch-clamp, total internal reflection fluorescence (TIRF) microscopy, super-resolution immunofluorescence imaging, optogenetics and genetic manipulations. Three specific aims are: (1) determine the regulation of SK channels by PIP2 using optogenetic techniques; (2) determine the molecular mechanisms of PIP2 regulation of SK channels; (3) test the physiological impact of PIP2 regulation in cardiomyocytes. These studies will reveal a new regulatory mechanism of cardiac SK channels by PIP2. The anticipated results will provide novel insights into the functional role of SK channels and PIP2 signaling in the regulation of cardiac excitability and arrhythmia. At the translational level, cardiac SK channels may represent a potential therapeutic target for the treatment of cardiac arrhythmia.

NIH Research Projects · FY 2025 · 2023-08

Abstract Novelty and surprise have long been known to facilitate learning and memory. At a functional level this makes sense; unexpected events have to be learned about so they can be predicted and responded to appropriately in the future. At a psychological level, surprising events have been shown to enhance memory because they induce rehearsal. Subjects tend to “think about” unexpected events more than familiar ones after they occur. This has been observed directly in humans (explicit rehearsal) and indirectly in animals (implicit rehearsal). In both cases, the memory enhancement can be eliminated by disrupting rehearsal with a distractor stimulus that is presented immediately after the novel event. Presenting the same distractor stimulus several minutes later has no effect. This suggests rehearsal is short-lasting and distinct from the process of memory consolidation, which stabilizes new information for several hours after learning. In addition to increasing rehearsal, novel events also trigger the release of norepinephrine (NE) and dopamine (DA), which are known to enhance synaptic plasticity. Blocking receptors for these neuromodulators in the hippocampus prevents animals from forming new spatial and contextual memories. Based on these findings, we hypothesize that surprising events enhance memory because they induce catecholamine release at the same time the hippocampus is actively rehearsing/replaying new information. Our preliminary data demonstrate that NE and DA are both released in the hippocampus during and after the presentation of an unexpected aversive stimulus. At the same time, there is an increase in sharp-wave ripple oscillations (SWRs), which are known to contain replay sequences for recently encountered stimuli. Consequently, we will test the hypothesis above by monitoring and manipulating catecholamine release in real-time during an aversive learning task while simultaneously recording oscillations and single unit activity in the hippocampus.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Drug overdose deaths continue to rise in the US due largely to opioid-related deaths, despite availability of buprenorphine, a highly effective treatment for opioid use disorder (OUD). Starting buprenorphine for OUD in emergency departments is an increasingly common practice across the US. A large clinical trial found that emergency department-initiated buprenorphine plus referral to primary care for ongoing treatment significantly increased buprenorphine treatment rates, but effects were not sustained past two months. Effective strategies to help patients start, restart, and sustain buprenorphine treatment are urgently needed to reduce overdose deaths. To increase buprenorphine treatment, California established CA Bridge, a state-funded program in >200 hospitals that offers same-day buprenorphine initiation plus referral to primary care for patients with OUD who seek care in emergency departments. However, suboptimal care coordination persists, especially for low- income patients. This study’s overarching hypothesis is that an implementation strategy that encourages coordination involving ongoing partnerships rather than one-time handoffs between emergency department and primary care teams will improve buprenorphine treatment and retention rates for patients who start buprenorphine in the emergency department. The objective is to refine an implementation strategy informed by multiteam systems theory that is focused on improving OUD care coordination between emergency department and primary care teams and then test this strategy in a hybrid type III effectiveness-implementation study using a stepped wedge design randomized at the site level. The R61 phase will involve collecting and analyzing data from two CA Bridge sites to identify core and peripheral elements of this implementation strategy and develop an implementation strategy, a preliminary implementation blueprint, and data collection protocols for the R33 phase. The stepped wedge study during the R33 phase will involve four CA Bridge sites. Implementation outcomes will include between-team coordinating activities and care transitions. Effectiveness outcomes will include rates of outpatient buprenorphine initiation (filling ≥1 outpatient prescription) and sustained buprenorphine treatment 3, 6, and 12 months after patients leave the emergency department, assessed with data from California’s prescription drug monitoring program. Factors associated with successful implementation and sustainability will be identified through pre- and post-implementation interviews with patients, staff, and managers at each emergency department and primary care clinic and through engagement with relevant stakeholders, including patients, CA Bridge statewide leadership and prospective payers. Study results will be used to develop an implementation blueprint that hospitals in California and across the US can use to improve OUD treatment outcomes for patients who start buprenorphine in emergency departments. This study will advance efforts to improve long-term buprenorphine treatment rates at scale for low-income and other vulnerable patients who disproportionately seek OUD care in emergency departments.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The rising number of children being identified as autistic has led to exponential growth in for-profit applied behavior analysis (ABA) agencies serving them, the use of highly structured approaches that may not be developmentally appropriate for young children1 and that limit use of learned skills across settings and time, and growing public health concerns regarding limited effectiveness data combined with the cost of services. Efficacy testing has led to growth in evidence-based autism interventions (EBI), especially naturalistic developmental behavior interventions (NDBIs), supported by multiple clinical trials. NDBIs integrate theory and strategies from ABA and developmental science and are considered best practice for young autistic children. The lack of effectiveness data regarding NDBI use in community-based agencies (CBAs) contributes to limited funding as payors are more likely to recognize older, structured methods. The Early Start Denver Model (ESDM) is a comprehensive NDBI shown to improve social communication and language outcomes for young autistic children in multiple controlled efficacy studies. ESDM engages social motivation as a mechanism to increase child engagement in social learning opportunities in the environment resulting in increased rate of child learning. ESDM includes assessment and data collection methods that meet funder requirements and a tested community training model. Understanding whether ESDM is effective and whether the same treatment mechanisms operating in efficacy trials are also operating in community implementation with diverse samples are critical scientific questions to determine the potential of NDBIs like ESDM to meet the public health goals of improving access to high quality, developmentally appropriate care for a broad range of young autistic children and their families. To facilitate scale up, we have partnered with autism CBAs and this study will identify factors that affect implementation of ESDM in the community. This project will use a hybrid type 1 randomized controlled design to examine ESDM effectiveness in the community and to gather data on implementation determinants. Primary child outcomes include language and social communication. Secondary outcomes include adaptive behavior, parent use of ESDM strategies, and provider fidelity to the model. Social motivation and caregiver fidelity will be measured as mediating variables. The moderating effects of maternal education, child race/ethnicity, and provider ESDM fidelity will be examined. ESDM implementation determinants will be explored. Understanding the effectiveness of an intervention like ESDM, the variables that mediate and moderate child outcomes, and engagement of its mechanisms of action in community use, has the potential to increase availability, and therefore, access to high quality intervention for all young autistic children, especially those from diverse backgrounds who depend on public services. Understanding implementation determinants will support scale-up of effective models throughout communities.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Naturally occurring osteosarcoma in dogs has been proposed as a promising translational platform to serve as an intermediary between mouse studies and human clinical trials because it develops in the presence of a fully intact immune system and demonstrates a similar clinical progression to the human disease. Furthermore, canine osteosarcoma is a naturally occurring, heterogenous disease that recapitulates the spectrum of histopathologic, immunologic, and genomic complexity of the disease in humans. However, despite the promise of the dog model, the true translational potential of this model remains unproven and important species differences in overall outcomes exist that are often not accounted for in the traditional design of translationally focused dog trials. To optimize the design and translational impact of canine osteosarcoma trials moving forward, this proposal seeks to establish the breadth and range of natural outcomes for this disease together with deep characterization of the tumor microenvironment and immune landscape. To attain these objectives, we propose the following two aims, 1) Determine natural disease progression and outcomes in a contemporaneous prospectively enrolled population of dogs treated surgically for naturally occurring osteosarcoma, and 2) Inform optimal design and establish biologically based outcome assessments for translational canine osteosarcoma studies. This work will enroll client-owned dogs with naturally occurring osteosarcoma through the NCI Comparative Oncology Trials Consortium (COTC), a well-established trials network of veterinary academic institutions. In addition to primary clinical outcome measures for dogs treated with surgery alone, deep characterization of the tumor microenvironment and immune landscape will allow us to better understand the impacts of inherent biology and chemotherapy resistance, define the role of the immune system in the natural progression of disease, and to compare molecular and immune signatures between pre- existing human and canine osteosarcoma datasets. Hence, this canine trial paired with correlative bio-marker studies are aimed to expand and improve the utility of the canine osteosarcoma model by ensuring that future dog trials can be optimally designed to provide reliable information for the benefit of human patients with chemotherapy resistant osteosarcoma.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract Fibrosis is a consequence of a myriad of skeletal muscle diseases including Duchenne muscular dystrophy. Fibrosis is the pathological accumulation of extracellular matrix (ECM) and impairs muscle function resulting in a loss of mobility and significant reduction in strength. Muscle resident fibro-adipogenic progenitors (FAPs) are the key source of ECM deposition in skeletal muscle. Pro-regenerative FAPs support regeneration by activating in response to injury, depositing ECM to replace the damaged matrix, and releasing pro-myogenic signals. However, in the context of fibrosis, there is a significant upregulation of a more pro-fibrotic FAP subpopulation. FAPs activate into myofibroblasts and remain at chronically high levels, leading to excess ECM deposition. What drives the development of a fibrotic versus regenerative FAP phenotype is not well understood. The mechanics and architecture of the ECM is altered in fibrotic muscle compared to healthy muscle, providing mechanical cues to surrounding cells. FAPs are known to be sensitive to these changes in mechanics and architecture, however, what drives this signaling pathway is not understood. Yes-associated protein (YAP) is strongly correlated with FAP activation into myofibroblasts on stiff substrates. Blocking YAP activity is a potential method to manipulate FAP-ECM signaling and reduce myofibroblast activation in the context of fibrosis. ECM signaling to FAPs may also influence the heterogeneity of FAPs, with the levels of pro- regenerative and pro-fibrotic FAPs changing during injury and fibrosis. How the subpopulations of FAPs may be contributing to ECM deposition in fibrosis and regeneration is not well understood. Elucidating the differential role of FAP subpopulations provides specific targets for anti-fibrotic and pro-myogenic therapies. Our central hypothesis is that blocking FAPs’ sensitivity to stiff substrates through inhibition of YAP will reduce the number of pro-fibrotic FAPs resulting in ECM deposition that promotes myogenesis. We will test our central hypothesis via two aims. In Aim 1, we will inhibit YAP activity in order to determine its role in FAPs’ sensitivity to engineered matrix substrates mimicking the mechanical and architectural features of healthy and fibrotic ECM. In Aim 2, we will determine the differences in ECM deposition from pro-fibrotic and pro- regenerative FAPs and its role on myogenesis to assess how the heterogeneity with the FAP population affects the development of fibrosis and regeneration. Success in these aims will identify the interactions between FAPs and the ECM in the context of fibrosis, which can be used as targets for anti-fibrotic therapies.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Adoptive cell transfer (ACT) using ex vivo expanded anti-tumor T-cells has garnered significant interest due to successes in treating melanoma and other cancers. This is a highly personalized therapy, in which autologous T-cells that can target the tumors are required. However, finding cells that specifically target tumors remains a major hurdle for the widespread application of T-cell based ACT therapies. The current methods of lymphocyte enrichment result in modest increases in tumoricidal T-cells with little control over the clonal heterogeneity. A technology that overcomes these challenges would significantly lower the barriers (e.g., reduce cost, reduce off-target effects) for broad dissemination of ACT therapies. The primary goal of this project is to develop a separation technology to enrich a population of lymphocytes with tumoricidal T-cells based on their capacity to recognize autologous tumor antigens. The premise of our microfluidic technology is that tumoricidal T-cells can be separated from a bulk leukocyte population when exposed to tumor-derived peptide-major histocompatibility complex I under optimal flow conditions. The specific aims are to: 1) Develop a microfluidic device to enrich a population of lymphocytes with antigen specific T-cells, and 2) Demonstrate the capacity of the microfluidic platform to enrich patient-derived Peripheral Blood Mononuclear Cells with tumoricidal T-cells using patient- matched tumor cells. Accomplishing our primary goal will create a potentially disruptive technology that could pave way for wide-spread application of T-cell based ACT therapies, and the agnostic feature (i.e., no a priori knowledge of tumor antigen(s) is required) of the technology would make it broadly applicable for a personalized medicine approach to a range of cancers.

NIH Research Projects · FY 2026 · 2023-08

Project summary: Optimal oxygenation in neonatal lung injury Whole-body hypothermia is currently the standard of care for moderate to severe hypoxic-ischemic encephalopathy (HIE). HIE is commonly associated with meconium aspiration syndrome (MAS) and persistent pulmonary hypertension of the newborn (PPHN). Therapeutic hypothermia can cause pulmonary vasoconstriction and exacerbate PPHN and is currently the leading indication for extracorporeal membrane oxygenation in California. The optimal target SpO2, and pulmonary vasodilator regimen that results in improvement in both pulmonary and neurological outcomes in PPHN and HIE is not known. We hypothesize that targeting 95-98% SpO2 (compared to 91-95% - current standard of care) and intravenous (IV) sildenafil will minimize brain injury and enhance pulmonary vasodilation. The first specific aim focuses on changes in gas exchange and cerebral hemodynamics with two SpO2 target ranges during 72-hours of whole-body hypothermia. The second specific aim focuses on oxidative stress and neuroprotection offered by iNO and sildenafil following birth asphyxia during hypothermia and ventilation at two different SpO2 targets. The final specific aim evaluates the impact of hypothermia on pulmonary hemodynamics and pharmacokinetics of sildenafil. Hypoxemic respiratory failure secondary to MAS and PPHN is common during whole-body hypothermia. Results from this study will inform the design of future clinical trials by defining the optimal SpO2, temperature, and pulmonary vasodilators while managing neonates with PPHN and HIE. The therapeutic potential of a commonly available and less expensive agent such as sildenafil in improving both pulmonary and neurological outcomes following birth asphyxia will be explored in this proposal.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT The objective of this research proposal project is to identify modifiable factors associated with different postoperative respiratory failure (PRF) phenotypes in adults following elective surgery and to utilize this information to develop and deploy a predictive model and electronic health record-based probability scoring system and dashboard for PRF. PRF, defined as the prolonged inability to wean from mechanical ventilation or inadequate oxygenation and/or ventilation, has an incidence of up to 7.5% and has been associated with a risk-adjusted $53,000 increase in hospital charges, 9 extra days of hospitalization, and a 22% increase in-hospital mortality. With the number of elective surgical procedures increasing annually, there is an urgent and unmet need to reduce the incidence and burden of this potentially preventable event by elucidating risk, preventive, and therapeutic factors. These factors, some of which may be modifiable, may differ between phenotypic presentations. AIM 1: To optimize and validate an automated, EHR-based, clinical prediction model for PRF. We will automate data collection and model the contributions of pre-and intra-operative factors on full model discrimination and calibration. Hypotheses: (H1.1) It is possible to automate data curation. (H1.2) A model including data from 2014-2021 and quantitative risk indices will outperform our previous model that used data from 2012-2015. AIM 2: To identify unique PRF phenotypes using clinical and biochemical markers that are readily available in the postoperative phase and determine if these markers predict PRF within 48 hours. Hypotheses: (H2.1) Readily available clinical and biochemical biomarkers (e.g., mean arterial pressure, creatinine) previously associated with hypo- and hyper-inflammatory acute respiratory distress syndrome and acute respiratory failure phenotypes are also present in PRF. (H2.2) These clinical and biochemical markers can be used to predict the probability of PRF within the next 48 hours. AIM 3: To develop and deploy a single-site, proof-of-concept, EHR-based probability scoring system, and dashboard for PRF. Hypotheses: (H3.1) Despite the benefits of the OMOP Common Data Model (CDM), data mapping into the CDM may cause information loss and decrease the predictive performance of a CDM-mapped model compared to the native, site-specific EHR model. (H3.2) The feasibility of a multisource (e.g., real-time and historic clinical and biomarker data) probability score, embedded in the EHR, will be demonstrated through successful deployment in a pre-production environment. Completing these Aims, and the five papers we foresee producing from this work will enable me to develop preliminary data for a competitive R01 proposal focused on implementing and evaluating a validated, real-time PRF predictive model in a UC-wide multi-center study. My long-term goal is to expand my existing program of research to enroll more geographically, epidemiologically, and socioeconomically diverse centers and conduct a large-scale, multisite intervention study (U grant) to validate our modeling and facilitate personalized treatment strategies to reduce the risk and burden of PRF.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Infants use their eyes to gather information from their environment and learn about the unique scenes and objects that surround them. As infants accumulate these experiences, their cortex is concurrently maturing to support more controlled attention and visual processes. However, it is unclear how much of the development of gaze control reflects cortical maturation versus individual experiences. Therefore, the proposed project seeks to evaluate three competing hypotheses regarding respective roles of maturation and experience. We will use both traditional eye tracking analyses as well as advanced computational modeling. Specifically, in Aim 1, we will record infant looking behaviors while viewing scenes and objects that range in familiarity and assess how stimulus properties (e.g., salience, meaningfulness) relate to looking patterns for familiar and unfamiliar images across the first year of life. In Aim 2, we will relate these looking behaviors to a convolutional neural network inspired by the mature brain’s visual system. This innovative approach will evaluate the relationship between infants’ looking behaviors and use of higher-level visual processing across contexts that vary in familiarity. Ultimately, results from this proposal will inform developmental theories of visual attention by characterizing the contributions of maturational versus experiential factors. The information gained from the results of this project may facilitate future assessments and interventions of clinical populations with atypical visual scanning and attention control. To successfully accomplish these aims, it is necessary that the applicant gains theoretical and methodological expertise in both infant and adult attention research. This integrative perspective will allow her to characterize infant attention and visual behaviors using sophisticated methodological and analytical approaches, which will ultimately generate a wide range of subsequent research questions using similar techniques. These aims will be most successfully completed under the supervision and mentorship of Drs. Lisa Oakes and Steven Luck at the University of California, Davis, who are experts on attention in infant and adult populations, respectively. The proposed training goals focus on expanding content knowledge of attention and gaining methodological expertise in convolutional neural networks and representational similarity analysis. Accomplishing these training goals will better prepare the applicant for a successful independent research career that pushes the field of attention development forward.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Pacemaking sinoatrial node (SAN) that orchestrates the heart rhythm can become dysfunctional with aging. Biopacemakers composed of human induced pluripotent stem cell (hiPSC)-derived pacemaking cardiomyocytes (P-CMs) can be one therapeutic strategy that can restore the sinus rhythm in patients suffering from SAN dysfunction. The development of biopacemakers is hampered by issues such as low differentiation yield of P- CMs from hiPSCs and the long-term maintenance of the pacemaking function in engineered pacemaking tissues. The function and phenotype of P-CMs can be affected by mechanical forces imposed by the extracellular matrix (ECM) forming the cellular microenvironment and the cyclic strain due to cardiac contractions. The ECM has been well demonstrated for shaping the phenotype of the working CMs. Hence, an optimal ECM should also promote and sustain pacemaking function in P-CMs. We have data demonstrating that ECM of the SAN can better sustain the pacemaking phenotype in hiPSC-derived CMs even when subjected to cyclic strain compared to the left ventricular counterpart but the exact mechanisms that induce and maintain the pacemaking phenotype are unclear. The goal of this project is to understand the mechanisms of mechanotransduction in P-CMs that can be modulated by the ECM. We hypothesize that the SAN ECM may modulate the mechanotransduction in resident P-CMs via cell-ECM junctions at the costameres and the cell-cell junctions at the fascia adherens to maintain the pacemaking phenotype. To test the mechanistic underpinnings of our hypothesis, we propose the following three aims: 1) to determine the cell-ECM-mediated mechanotransduction signaling in P-CMs, 2) to determine the cell-cell-mediated mechanotransduction signaling in the P-CMs, and 3) to determine the integrated cell-ECM and cell-cell junction network in mechanotransduction signaling in the P-CMs. A better understanding of the mechanotransduction mechanisms in P-CMs can yield a source of human P-CMs from hiPSCs with long- term pacemaking function, a set of microenvironmental criteria necessary for engineering sustainable biopacemakers and inspire future new therapeutic or preventive strategies for SAN dysfunction.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Persistent cell migration is fundamental for immune responses, development, and the dissemination of cancer cells. This migration requires the establishment and maintenance of stable cell polarity, even while a cell integrates noisy heterogeneous cues from its environment. To achieve this, Rho family GTPases act as central hubs that organize signaling cascades and cytoskeletal rearrangements into subcellular domains. Feedback and crosstalk connections are thought to be central to this pattern-forming ability. However, the wiring of this circuit is still incompletely understood, and there are major gaps in our understanding of how negative regulators limit and separate spatial domains. Determining these molecular connections in migrating leukocytes would identify new therapeutic targets for treating inflammation and would be broadly relevant for understanding Rho GTPase function in many cell types and biological processes. Major obstacles to progress have been the fast timescale and inherently spatial nature of the signaling system. To address these challenges, we have developed new molecular tools that allow us to control the activity of individual key components with light while measuring the response of a second component with subcellular resolution in live single cells. Our preliminary results indicate that in addition to acting as outputs to move the cell, different actin assemblies are intimately involved in the biochemical wiring of Rho GTPase crosstalk. We have identified an “actin-gated” crosstalk connection between RhoA and Cdc42, and we have identified the protein Arhgap30 as a previously unappreciated primary regulator of Cdc42 that is critical for polarization and migration in leukocytes. We hypothesize that different actin assemblies act as scaffolds to localize regulators of Rho GTPase crosstalk, creating subcellular zones with distinct signal wiring to promote stable cell polarity. Specifically, we aim to 1) determine how branched actin assembly regulates Cdc42 and RhoA activities in leukocyte cells, 2) determine how the local actin network structure controls crosstalk between RhoA and Cdc42, and 3) determine the regulation and role of Arhgap30 in crosstalk and polarity signaling. Our approach will combine new tool sets for optical control of signaling and cytoskeletal components with simultaneous measurement of actin assemblies and Rho GTPase activities in single cells. In combination, we will use chemical perturbations, mutational analysis, and biochemical approaches to characterize molecular connections. Our long-term goals are to determine how reciprocal regulation between actin and Rho GTPases creates robust polarity in multiple cell types, including leukocytes and disseminating cancer cells. The proposed research will advance our basic understanding of how biochemical signaling pathways both generate and stabilize subcellular domains to control behaviors such as cell migration.

NIH Research Projects · FY 2025 · 2023-07

The human auditory system transforms incoming acoustic information into distinct auditory “objects” that can be interpreted, localized, and integrated with information from the other senses. A human listener can resolve, for example, a monophonic musical recording into piano and guitar, or parse speech audio into a sequence of words. We do not currently understand how this is achieved, nor how transformations in sound processing across cortical regions contribute, especially beyond primary auditory cortex. On the other hand, we now have available another, more easily investigated system that—as of this last decade—solves such problems at human performance levels: the artificial neural network (ANN). Although crude as biophysical models, ANNs strongly resemble biological neural networks in terms of computation and representation. We propose to compare single-unit electrophysiology in macaque auditory cortex with state-of-the-art ANNs trained to solve ecologically relevant auditory tasks. This combination allows us to track how transformations in perceptual representations are distributed and instantiated in the brain; to experiment with multiple ANN architectures and tasks to test hypotheses about why the representations take the forms we observe; and to refine iteratively our stimulus protocols and models. Our objectives are (1) to evaluate these ANNs as encoding models for neurons in macaque auditory cortex, recorded during auditory discrimination tasks; (2) to use the internal structure of ANNs to generate and test novel hypotheses about the topographical and hierarchical organization of non-primary auditory cortex; and (3) to demonstrate “stimulus- based control” of neurons throughout nonprimary auditory cortex: optimizing stimuli via the ANN and then playing to the animals in closed-loop neurophysiology experiments.

NIH Research Projects · FY 2024 · 2023-07

19F MRI, has shown promising success in clinical trials for monitoring cell therapy, where small groups of cells, showing poor contrast in 1H-MRI (Magnetic Resonance Imaging), are easily tracked using 19F-MRI.1 Contrast is provided by nanoemulsions of perfluorocarbon (PFC) oils pre-loaded into cells. A major advantage of 19F-MRI is that lack of natural fluorine in the body allows for very high contrast-to-noise for 19F-MRI compared to 1H-MRI.2 Use of 19F-MRI in molecular imaging, by targeting specific biomarkers, is of great interest, but the development of probes for these applications has been an elusive goal. Typical synthetic methods produce relatively large particles, > 150 nm, which induce nonspecific uptake by phagocytic cells. While avid nonspecific uptake is advantageous for labeling T cells and other immune cells ex vivo, it is a decided limitation for imaging of biomarkers as it can create high background signal in the inflamed tissues characteristic in many diseases. 19F MRI should be an excellent platform for biomarker detection but there is a critical unmet need for suitable 19F MRI contrast agents that avoid nonspecific immune cell surveillance. Ideally, contrast agents need to be<100 nm to avoid nonspecific uptake. We propose exploratory studies to develop innovative new 19F MRI contrast agents based on nanodiscs. Nanodiscs are < 50 nm in size, avoid liver clearance, and escape immune surveillance. We hypothesize that nanodiscs, which are structurally similar to high density lipoproteins that carry cholesterol in a hydrophobic core, may be ideal for carrying hydrophobic perfluorocarbons. Our aims are to explore synthetic parameters in benchtop (Aim 1) and microfluidic (Aim 2) approaches to optimize loading of perfluorocarbons into nanodiscs. In each aim we will systematically investigate variables that influence nanodisc size and perfluorocarbon loading. New nanodiscs will be characterized for physical properties such composition and dimensions, and the top 3 products from each Aim will be evaluated for lack of toxicity and biodistribution in a mouse model. The leading nanodiscs will be modified for targeting CD204, a biomarker for tumor associated macrophages, and tested in a mouse breast cancer model. We are team composed of contrast agent (UCD) and nanodisc (LLNL) experts who are ideally positioned and well-equipped to carry out the proposed aims. The success of this project would add novel nanodiscs materials with the potential not only for targeted 19F MR molecular imaging, but for applications in photoacoustic, and radiotherapy, and tracking drug delivery.

NIH Research Projects · FY 2024 · 2023-07

PROJECT ABSTRACT Prostate cancer (PCa) is the second leading cause of cancer related death in men in the United States in 2022. A limit number of PCa cell lines and patient-derived xenograft (PDX) models hinders research to improve disease outcome. To address this unmet need, we have developed multiple PDX models, conditional reprogramed cultures (CRCs) and organoids from patients with advanced disease. AKR1C3, also named HSD17B5, is one of the most important genes involved in androgen metabolism and elevated expression of this enzyme is associated with PCa progression and failure to androgen receptor pathway inhibitors (ARPIs) treatments. We have reported that upregulation of AKR1C3, concurrent with elevated testosterone and its precursors in prostate cancer cells and xenograft tumors that are resistant to anti-androgen treatments. This proposal will use blood AKR1C3 and plasma steroid levels as biomarkers to interrogate intracrine steroidogenesis activation in ARPIs treated patients to signify drug resistance. We will also test a novel AKR1C3 inhibitor in the newly established PDX and CRC models. The inhibitor has great potential to increase efficacy of ARPIs treatments in advanced PCa which might translate into the clinical trial directly. The goal of this program is to identify novel biomarkers and develop new pharmaceutical approaches to provide co- targeting neoadjuvant with ARPIs to treat CRPC patients. Manipulation of key enzymes contributing to the production of androgen potentially offers a novel targeted therapy for the advanced prostate cancer treatment, which will have a meaningful impact on patients’ lives. The data acquired from this project will establish a clinically relevant decision making for patient treatment and pave the way to future precision medicine in prostate cancer.

NIH Research Projects · FY 2024 · 2023-07

PROJECT ABSTRACT St. Louis encephalitis virus (SLEV) is a re-emerging mosquito-borne flavivirus that causes a spectrum of manifestations in people ranging from febrile illness to encephalitis and death. Clinical disease caused by SLEV is indistinguishable from related West Nile virus (WNV). Although critical for understanding disease and evaluating countermeasures, current SLEV mouse models are limited in several ways. Prior studies used inbred or immunodeficient mice restricting assessment of the role of innate immunity, intracranial inoculation that artificially bypasses neuroinvasion, and virus strains that were serially passaged in mouse brains which artificially selects for neurovirulence. Compared to inbred or immunodeficient mice, recombinant mice better represent of human genetic diversity which can translate to similar disease to humans. Recombinant Collaborative Cross (CC) mice that were generated from a panel of 8 recombinant inbred strains represent the myriad of outcomes in people infected with the flaviviruses Zika, Powassan, and WNV. WNV-infected CC mice develop a spectrum of outcomes including asymptomatic, symptomatic with central nervous system (CNS) disease, and asymptomatic with CNS disease, where the innate immune response is the major mediator of protection from CNS disease. To circumvent limitations of existing SLEV mouse models, the goal of this project is to develop immunocompetent recombinant CC mouse models of SLEV. We hypothesize that CC mice model human SLEV including to produce clinical disease, viral tropism, and kinetics that parallel the spectrum of human SLEV outcomes, and that protection from CNS disease associates with robust innate immune responses manifest as higher expression or levels of interferon stimulated genes including the antiviral sensor RIG-I and effector cytokines including interferons (IFN)-α/β/γ. These hypotheses will be tested in two project Aims. Aim 1: Define SLEV clinical disease and viral tropism and kinetics in CC mice. We will use CC strains that manifest variable WNV outcomes and inoculate animals via the footpad with a SLEV mosquito- transmitted doses. We will assess clinical disease, infection kinetics and tropism, and viral genomic changes that associate with neuroinvasion. Aim 2: Link elevated innate immune responses to control of SLEV CNS disease in CC mice. We will measure innate responses via gene expression analyses and levels of signaling and secreted effector proteins and correlate these changes with clinical disease, virus levels in blood and target tissues, and histopathologic lesions in the brain. Augmented disease in CC mice treated with IFN- receptor blocking antibody will support IFN signaling as the mediator of protection. This project will develop new human-relevant mouse models of SLEV which can be further used to study the spectrum of pathogenesis, define virus-host interactions and host genetic determinants of susceptibility and severity, test interventions like therapeutics and candidate vaccines, and rapidly adapt to model disease for other new or re-emerging encephalitic flaviviruses.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Breast cancer is an increasing challenge globally as it became the most prevalent malignancy at the end of 2020. More than 70% of all cancer mortality now occurs in low- and middle-income countries (LMICs). Histology, critical to the diagnosis and disease management for many cancers notably including breast cancer, is currently performed using techniques that are more than 100 years old. Market forces, technological advances in optics, and innovation in care strategies are opening the door for disruptive innovation that could massively reduce costs and time and improve accessibility while provide equivalent or even superior results. We propose to contribute to the field's evolution by combining two already functioning and complementary technologies: 1) a tissue millifluidics approach (developed in PI Seibel's lab) for hands-off core needle biopsy handling; and 2) a rapid, low-cost, direct-to-digital slide-free imaging solution (developed in PI Levenson's lab). The goal is to implement a context-appropriate, automated instrument that can capture diagnostic-quality histopathology images from core-needle biopsies vital to high-quality breast-cancer diagnosis and staging, at time of procedure. Additional project goals include implementation of innovative rapid immunofluorescence methods for near-real-time therapy guidance; and development of AI tools for patient triage or even local diagnostic support, the latter under the direction of Dr. Mahmood (BWH), a leader in multiclass AI algorithms. Key to success of this project is local implementation and clinical evaluation in Kumasi, Ghana, under the direction of Dr. Addai, consultant breast surgeon and CEO of Peace and Love Hospitals (established in 2002). Her group will critically assess performance, usability, and compatibility with the service environment in both a central hospital and a remote satellite clinic; clinical validation studies will eventually encompass at least several hundred patients recruited under IRB-approved protocols. Additional guidance will be provided by Dr. Dan Milner, CMO of the American Society for Clinical Pathology (ASCP), who has extensive experience in global-health-focused initiatives, as well as by our collaborating pathologists who are familiar with issues relevant to LMIC settings. Feedback will inform the design of the second-generation automated instruments to be delivered near the end of this project.

NIH Research Projects · FY 2024 · 2023-07

A fundamental question of developmental biology is to understand how a limited number of signaling pathways direct the specification of many cell types. One such signaling pathway is the canonical Wnt signaling pathway, which is highly conserved across animals, plays a role in a myriad of developmental processes, and its dysregulation is common in disease. To direct different developmental outcomes, Wnt signaling must activate different gene regulatory networks (GRNs) in different contexts. To reveal general principles of how Wnt ligands can activate unique GRNs, I will use Hydra vulgaris to discover how two distinct cell types uniquely respond to the Wnt signaling pathway. Hydra offer several advantages for studying Wnt directed-GRNs: 1) Hydra is a relatively simple organism and we have molecularly and spatially defined all cell types and 2) the adult Hydra is in a constant state of development such that all developmental pathways, including the Wnt-directed pathways, are continuously active. Wnt signaling is high at Hydra’s oral end (i.e., the head) and directs the differentiation of multiple distinct oral cell fates. The principal effect of canonical Wnt signaling is the stabilization of the beta- catenin (Bcat) protein, which together with its binding partner TCF activates transcription of target genes. To activate target gene expression in specific developmental contexts, Bcat/TCF must work in a combinatorial fashion with other TFs. However, it is largely unknown what TFs are facilitating the activation of Wnt targets and whether these interactions are conserved across species and during disease. Based on my preliminary data, I hypothesize that ectodermal Homeobox TFs and endodermal bHLH TFs work in a combinatorial manner with Bcat/TCF to direct cell-type specific GRN modules in Hydra. Towards testing this hypothesis, I will use ChIP-seq to identify the cell-type specific direct targets of Bcat/TCF in the two oral epithelial cell types of Hydra (ectoderm and endoderm) (Aim 1). I will then use our Hydra single cell Atlas to determine the expression pattern of the direct targets. To determine if direct targets are co-regulated by Homeobox or bHLH TFs, I will knockdown these TFs in the epithelial cells to test if they are required for specification. I will then identify the Wnt target genes that also require these TFs for proper expression by conducting RNA-seq on the knockdown Hydra (Aim 2). Finally, I will perform unbiased approaches to identify additional co-regulating TFs for functional testing (Aim 3), which will also provide alternative hypotheses if needed. Upon completion of this project, I will have generated a comprehensive list of the primary targets of Bcat/TCF in Hydra and potentially have discovered a role for Homeobox and bHLH TFs in differentially regulating these primary targets. ChIP experiments conducted in mice, have shown that Bcat can bind specific Homeobox TFs to control target gene expression in different developmental contexts. Therefore, my results could suggest a deeply conserved role for Homeobox TFs in regulating different Wnt targets in different developmental contexts from cnidarians to bilaterians.

NIH Research Projects · FY 2024 · 2023-07

St. Louis encephalitis virus (SLEV) is a mosquito borne pathogen that causes febrile illness and sometimes fatal encephalitis and was a leading cause of infectious encephalitis in the US during the 1970s. After an 11- year absence in activity in California, SLEV reemerged and reestablished in CA from 2015-2021, and caused an outbreak in nearby Phoenix, AZ. SLEV reemergence is marked by increasing human cases and positive mosquito pools across more counties each year, including areas with concurrent activity of West Nile virus (WNV), a related flavivirus that invaded CA in 2003 and which shares avian reservoirs and Culex mosquito vectors with SLEV. Genetic tracing by our team showed that reemerging (‘contemporary’) SLEV in the Western US is genetically distinct from pre-2003 (‘historical’) SLEV and that it likely originated in South America. However, other than these studies, drivers of SLEV reemergence, including into WNV-endemic areas, have not been examined. Spread of WNV across the US was facilitated by augmented avian reservoir infection and vector competence. We propose that similar fitness gains may have enabled SLEV reemergence. The objective of this project is to assess the extent by which SLEV reemergence is promoted by augmented infectivity and transmissibility in mosquito and avian cells and mosquito vectors. Higher SLEV activity in different regions may be explained by differential vector competence for 3 primary Culex vectors (pipiens, tarsalis, and quinquefasciatus) that favor different environments across the state. We hypothesize that SLEV reemergence in the Western US since 2015 was mediated by augmented fitness of the introduced genotype in cells and vectors and that geographic localization is influenced by relative vector competence of 3 primary Culex species. Viral fitness can be assessed experimentally by comparing SLEV strains using vector competence and competition assays. The project hypotheses will be tested in 2 project Aims: 1) Determine transmission competence of contemporary versus historical SLEV in Culex vectors, and 2) Compare relative fitness of contemporary versus historical SLEV in avian and mosquito cells and vectors. This project is significant in that it will provide a unique opportunity to compare viral factors involved in the sequential invasion and spread of 2 Culex-borne flaviviruses, where understanding co-circulation dynamics can be applied to other sympatric flaviviruses. By defining the role Culex vector species play in SLEV transmission this project will determine whether augmented infectivity and transmissibility is a factor enhancing reemergence and spread. To reduce disease, vector control districts can allocate resources towards spatial targeting of the Culex vector species that exhibit high SLEV competence.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Lysosomes are sophisticated and dynamic cellular signaling centers that control metabolism, gene transcription, calcium (Ca2+) homeostasis, and autophagy. A key mechanism through which lysosomes communicate and receive instruction is via transfer of cholesterol at ER–lysosome membrane contact sites. At these contacts the Niemann Pick C1 cholesterol transporter (NPC1) facilitates the efflux of cholesterol out of the lysosome before it is transferred to the ER for distribution to other cellular membranes. Thus, NPC1 is a key gatekeeper in cholesterol metabolism. Further underscoring its importance, loss of function mutations in NPC1 lead to the progressive neurodegenerative disorder, NPC disease. This fatal condition has no cure and is characterized by the accumulation of cholesterol within lysosome lumen and the progressive neurodegeneration of several brain regions that are accompanied by a host of devastating symptoms including seizures, psychiatric problems, and dementia. Notwithstanding clear neuropathological consequences for cholesterol dysregulation in NPC disease, the molecular mechanism(s) linking loss of NPC1 function to disease neuropathology are unknown. Recently our group has reported that loss of NPC1 function results in (i) neuron hyperexcitability, (ii) reorganization of ER– Lysosome, ER–Golgi, and ER–mitochondrial membrane contact sites, and (iii) induces neurotoxic increases in mitochondrial Ca2+. Despite this crucial information there are critical gaps in our knowledge regarding (1) the consequences of enhanced excitability in NPC disease, (2) how lysosomal cholesterol transport alters the molecular elements and choreography at neuronal ER–plasma membrane (ER–PM) contact sites, and (3) if plasma membrane ion channels or ER–PM junctions can be targeted to reduce mitochondrial toxicity and increase neuron viability in NPC disease. Our central hypothesis is that loss of NPC1 function results in aberrant remodeling of ion channel distribution and function at ER–PM contacts to drive cytotoxic increases in mitochondrial Ca2+ leading to neurodegeneration. To test this hypothesis, we implement a multi-scale approach, including super-resolution imaging, electrophysiology, optical mapping of brain excitability, novel murine models, and animal behavior testing to rigorously investigate the mechanisms by which cholesterol efflux from the lysosome tunes neuron viability. The fundamental importance and ubiquitous expression of the NPC cholesterol transporter means we should pay particular attention to molecular elements and signaling cascades that are modified by its activity. Investigating the relationship between cholesterol homeostasis and ion channel signaling at ER–PM membrane contacts in NPC provides a testable model for examining the interdependence of lysosomal cholesterol and ion channel activity and has broad implications for several fields and other cholesterol- linked diseases such as Alzheimer’s and Parkinson’s.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Considering 20-25% of pharmaceuticals on the market today contain one or more fluorine atoms in their active ingredient (several of which appear on the WHO’s List of Essential Medicines), it is evident that fluorine chemistry continues to have a profound impact on drug development. Yet, despite the indisputable significance of fluorine atoms and fluorinated groups in modern medicinal chemistry, there has been a historical lag in their implementation in pharmaceuticals over the last century. The reason for this lag can most often be attributed to synthetic accessibility, as methods to install fluorine on molecules have been notoriously reliant on hazardous reagents, e.g., F2 and HF. While methods to incorporate, for instance, CF3, OCF3, and SCF3 groups on complex molecules have become far safer and more accessible in the last few decades (and these groups have since become commonplace in drug design), there remain a number of fluorinated groups with untapped potential that cannot be easily synthesized yet. This proposal specifically identifies the SF5, N(CF3)2, and N(CF3)(CF2H) groups as desirable motifs that have demonstrated promise yet continue to be underemployed due to the fact that there are virtually no user-friendly methods to make them. Accordingly, the proposed work is centered on addressing the synthetic chemistry bottlenecks that are preventing the realization of applications of these fluorinated groups. For one, the SF5 group has been studied as a bioisosteric replacement for a CF3 or t-Bu group, but only aryl-SF5 compounds have been made available commercially. Given the recent increase in accessibility of SF5Cl, we can now envision ways to expand the realm of possibility in C(sp3)–SF5 bond formation. Specifically, SF5 radical chemistry can be merged with strain- release functionalization to form novel “hybrid bioisosteres” that add another dimension of flexibility in molecular design. Methods are also envisioned to synthesize and examine some of the first benzylic-SF5 compounds, as well as other potential building blocks for the medicinal chemistry toolkit. The N(CF3)2 group (notably distinct from N–CF3) is arguably even less accessible/explored than the SF5 group, but it has demonstrated promise as a lipophilic NO2 group alternative. While all known methods make this group to date strictly require F2 or aHF, our approaches seek reasonable ways to circumvent these reagents altogether. This will make the N(CF3)2 group available to a significantly broader community of scientists. Lastly, the N(CF3)(CF2H) group is evidently the least accessible of them all, but it is liable to be an interesting alternative to the N(CF3)2 group with its added ability to serve as a lipophilic hydrogen-bond donor. We have envisioned ways to make this that avoid the current reliance on toxic SF4 gas. In all, safer methods to make the SF5, N(CF3)2, and N(CF3)(CF2H) groups will enable the study of their physical properties and how to leverage them in the design of potential drug candidates.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Temporal Lobe Epilepsy (TLE) is characterized by disordered neural network activity and temporal lobe seizures. As many as 3 million individuals with TLE in the United States also experience cognitive and sleep problems, resulting in poor school performance in childhood, with high risk of underemployment in adulthood, and consequent lower socioeconomic status. Individuals with TLE frequently experience sleep fragmentation, which disrupts memory consolidation and sustained attention, both of which are impaired in this disorder. While these comorbidities can be long-term consequences of repeated seizures and medications, it is now known that they also often present prior to the first recognized seizure and worsen over time even with successful seizure treatment. This suggests that an early neural network abnormality may underlie seizure development while simultaneously impairing sleep and cognitive development, even prior to the added effects of disorder chronicity. In spite of this, there has been limited research addressing mechanisms underlying these sleep and cognitive problems in TLE. This represents a critical unmet public health need and both the National Academy of Medicine and NINDS have identified this notable gap as a research priority. I will begin to address this gap with the my K23 proposal by investigating abnormal sleep architecture patterns in TLE that directly contribute to cognitive deficits using both an observational (Aim 1) and a mechanistic interventional (Aim 2) approach. In typical NREM sleep, electroencephalogram (EEG) slow wave oscillations are phase-locked and coupled with sleep spindle oscillations (SW-SSO), which facilitates memory consolidation and potentially improves attention. In TLE, disordered networks that result in interictal epileptic discharges and seizures may also contribute to altered SW-SSO coupling during sleep, resulting in memory and attention deficits. A single night of acoustic stimulation (AS) has been proven effective in enhancing SW-SSO coupling and improving cognitive performance in healthy older adults but has not been studied in TLE. My central hypothesis is that disordered networks in newly diagnosed TLE patients result in altered sleep architecture, which disrupt memory consolidation and attention capability. I will test this hypothesis by: (1) characterizing TLE sleep architecture using computational EEG – sleep spindle density, slow wave power, interictal epileptiform discharges, and SW-SSO coupling (Aim 1a), (2) linking these specific TLE-related sleep architecture patterns to cognitive processing (Aim 1b); (3) determining if AS enhances SW-SSO coupling in young adults with TLE (Aim 2a) and (4) determining if enhanced SW-SSO coupling improves memory and attention in TLE (Aim 2b). This training award will provide me the opportunity to extend my research expertise into computational sleep EEG acquisition and analysis, acoustic stimulation techniques, and clinical trial design. My long-term goal is to leverage connections between sleep, behavior and neural network activity to develop and implement tailored cognitive and sleep interventions for individuals with epilepsy.