University Of Washington

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Chromatin is a massive nucleoprotein complex that packages about three billion base pairs of genetic information in nucleated cells. Histones and transcription factors (TFs) are two important families of proteins associated with chromatin that play key roles in organizing, protecting and activating our genes. A conserved feature among histones and TFs is the critical role that post-translational modifications (PTMs) play in controlling their diverse functions. Many gaps remain in our understanding of the mechanistic roles for specific histone and TF PTMs that are either low in abundance or where the necessary molecular biological and chemical tools are unavailable for mechanistic studies. This is especially challenging when studying regulation of the tumor suppressor p53 that is rapidly turned over by the proteasomal machinery in our cells. The proposed research project seeks to overcome challenges arising from the low abundance and heterogeneity of histone and p53 modifications by applying a combination of chemical and molecular biological tools to generate site-specifically modified proteins. Specifically, sumoylated histones H2B and H4 and methylated p53 will be generated by new protein semisynthesis techniques. The semisynthetic proteins will be subjected to a range of biophysical and biochemical assays in order to elucidate the mechanistic roles for sumoylation and methylation in regulating chromatin structure and function. Results from biochemical assays will be further validated in cell- based studies to arrive at a complete picture of the molecular mechanisms underlying gene regulation by histone sumoylation and p53 methylation. The long-term goal of this project is to identify new biochemical relationships, or crosstalk, in cellular chromatin that may be controlled to engineer cellular fates and selectively therapeutically targeted in human diseases.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT Most women diagnosed with ovarian cancer are treated with many rounds of chemotherapy and often years of a an oral PARP inhibitor drug for “maintenance” therapy. These therapies have extended life for women with metastatic ovarian cancer, but at the cost of increased toxicity. One long term toxicity is the development of leukemia or other blood disorders, often called therapy related myeloid neoplasia (TMN). These secondary malignancies are a known risk of chemotherapy, and ovarian cancer survivors have one of the highest rates of TMN of any group of cancer survivors. The diagnosis of TMN is usually fatal, with survival measured in months. TMN is nearly always detectable in a pre-malignant state as a clonal expansion of blood cells years before a clinical diagnosis. Non-malignant clonal expansion of white blood cells is often termed clonal hematopoiesis of indeterminate potential (CHIP). The interval between CHIP and development of blood cancers is many years, providing an opportunity to better understand the natural progression of TMN and perhaps a window for intervention and prevention. Clonal hematopoiesis (CH) can also arise during normal aging, but only a small fraction progress to a blood cancer. A better understanding of the natural progression of CH in ovarian cancer survivors is needed to tailor safe and effective ovarian cancer therapies. Our team is co-led by experts in ovarian cancer genetics and hematological malignancies and will enroll 2000 survivors across the country with ovarian cancer, including 200 with CH. We will follow these individuals with CH with serial blood draws obtained every 6 months for at least 3 years to define risk factors for progression of CH to TMN. For a subset of patients with acquisition of TMN during the study, we will evaluate clonal dynamics and genetic and chromosomal alterations over time at the single cell level, which will provide novel data on the changes that occur in the malignant transformation of myeloid cells in response to cytotoxic therapy. In this way, we will learn who is at risk of TMN and develop strategies for the monitoring and prevention of this deadly long-term treatment toxicity. These studies will improve outcomes for patients with ovarian cancer and also will be applicable to survivors or many cancer types, who are also at risk for TMN.

NIH Research Projects · FY 2023 · 2023-05

Contact PD/PI: Qin, Qin Project Abstract Alzheimer's Disease (AD) is the leading cause of dementia in elderly population. Imaging biomarkers have been established on PET with respective tracers for mapping accumulation of amyloid and tau, as well as neurodegeneration. Furthermore, considerable overlap between cerebrovascular disease (CVD) and AD suggest additive or synergistic effects of both pathologies on cognitive decline. Vascular dysfunction has also been recognized as an important biomarker for better understanding and characterizing this multifactorial disease. Reliable mapping of neurodegeneration and vascular dysfunction could play critical roles in many clinical applications. Cerebral blood flow (CBF) is a fundamental hemodynamic parameter that characterizes brain perfusion as both a surrogate of neuro function and a marker for small vessel disease. Arterial spin labeling (ASL) perfusion MRI offers the great advantages of not requiring an exogenous contrast agent and being free of ionizing radiation. Obtaining both structural MRI and perfusion MRI in a single visit to the clinic is highly desirable for scanning the elderly subjects. Interpretation of CBF maps derived from spatially selective ASL methods need to be cautious about the artificial perfusion deficit due to the slow flow often incurred in the older subjects. Despite many efforts in this field, there remains to be an unmet and urgent need to establish a standardized, reliable, and validated ASL-based CBF mapping protocol for multi-site across-vendor neuroimaging studies for AD. Velocity-selective ASL (VSASL) derived CBF maps are ideally suited for characterization of both neurodegeneration and vascular dysfunction among the elderly population, without suffering the limitations presented by the slow flow. We have implemented the first velocity-selective inversion (VSI) based VSASL with 3D acquisition on adult brains and demonstrated its higher sensitivity to perfusion signal over conventional ASL methods. The proposed work capitalizes on the high perfusion sensitivity of VSI- ASL recognized by the first guideline paper for VSASL, as well as accelerated acquisition and reconstruction strategy, and represents its first AD application. The purpose of this study is to further optimize 3D VSI-ASL with accelerated acquisition and reconstruction, and then ensure its multi-vendor compatibility (Aim 1); to evaluate its reproducibility and validity between sessions, vendors, and modalities (Aim 2); to characterize VSI-ASL derived CBF values of various brain regions in cognitively normal (CN) subjects, and patients with MCI and AD through both cross-sectional and longitudinal comparisons (Aim 3). we will have demonstrated the optimized 3D VSI-ASL technique with high reproducibility, validity, and sensitivity to detect changes in brain perfusion and neurodegeneration, which can be readily utilized as a more practical and cost-effective imaging biomarker of neurodegeneration and vascular dysfunction for multi-center and multi-vendor clinical studies of AD and ADRD. Project Summary/Abstract Page 7

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Diabetic kidney disease (DKD) is clinically and mechanistically heterogeneous. Individual patients present with variable trajectories of estimated glomerular filtration rate (eGFR) and albuminuria that may not be concordant with severity of kidney histopathological injury, reflective of long-term outcomes, or predictive of response to therapy. Elucidating the molecular mechanisms underlying DKD pathogenesis may help explain differences in DKD’s clinical presentation and allow implementation of novel personalized diagnostic, prognostic, and therapeutic strategies. Autophagy, the process by which cytoplasmic components such as damaged organelles are delivered to lysosomes and degraded, is essential for maintenance of cellular homeostasis in the kidney and is a promising mechanism to evaluate in DKD. While kidney autophagy has been studied in extensively in experimental diabetes models, the role of autophagy in human DKD remains a major knowledge gap. The overall goal of the project proposed here is to comprehensively characterize autophagy in human DKD using clinical, histopathological, and molecular phenotyping, establishing a foundation for mechanistically- targeted diagnostic and therapeutic strategies. This project will leverage kidney molecular, clinical, and histopathologic data from the Kidney Precision Medicine Project (KPMP), a longitudinal type 2 diabetes Pima Indian cohort, and a University of Washington (UW) kidney biopsy cohort. In Aim 1, I will define clinical, molecular, and histopathological patterns of autophagy in DKD in the KPMP. Specifically, I will compare markers of autophagy in kidney tissue (autophagosome number assessed via electron microscopy, autophagy- related gene expression, autophagy-related protein concentrations) between adults with DKD and controls. I will also investigate associations of kidney autophagy markers with clinical and histopathological features. In Aim 2, I will test associations of kidney autophagy markers with long-term changes in measured GFR and albuminuria in the Pima Indian cohort. In Aim 3, I will develop urine and blood biomarker signatures, developed and internally validated in the KPMP and externally validated in the Pima Indian and UW cohorts. I aim to establish a career as a physician-scientist with a research focus on DKD and precision medicine. To accomplish my research and career development goals, I have designed a training plan supported by a team of mentors and collaborators with expertise in DKD, bioinformatics, epidemiology, and kidney histopathology. This project will generate novel insights into autophagy in DKD and urine and blood biomarker signatures for assessing kidney tissue autophagy which can be applied to other cohorts. Improved understanding of autophagy in DKD may pave the way for molecular-based subgroups with distinct prognoses and responses to existing and novel therapies.

NIH Research Projects · FY 2026 · 2023-05

Project Summary/Abstract: Lindsey Knowles, PhD is a psychologist and postdoctoral fellow in the University of Washington (UW) and VA Puget Sound Health Care System (VAPSHCS) Seattle Collaborative Fellowship in Multiple Sclerosis (MS) Rehabilitation Research. She is fully committed to becoming an independently funded investigator who develops, optimizes, and evaluates theory-driven, implementable interventions to improve symptoms, psychosocial function, and quality of life in adults with chronic health conditions/disability. Dr. Knowles is an ideal candidate for this field of research given her strong background in observational and intervention research on stress and coping and advanced fellowship training in rehabilitation research. This K23 award will provide foundational career development skills for achieving her long-term goal of optimizing evidence-based and accessible treatment for improving symptoms, function, and quality of life in people with chronic health conditions. The proposed project will develop, refine, and optimize a telehealth Cognitive Behavioral Therapy (CBT) intervention for fatigue in people with MS (PwMS). Findings will support a randomized controlled trial to evaluate the optimized CBT-based intervention for MS fatigue. Career Development and Training Plan: The proposed work will be carried out at the UW and utilize resources within the UW Department of Rehabilitation Medicine and VAPSHCS MS Center of Excellence West. Dr. Knowles’s distinguished mentorship team includes Drs. Dawn Ehde (Primary Mentor; telehealth CBT and MS expert), Aaron Turner (MS behavioral intervention expert), Linda Collins [Multiphase Optimization Strategy (MOST) expert], and Anna Kratz (qualitative research expert). Dr. Knowles’s training goals align her research and career plans via didactic and hands-on training in: (1) Intervention optimization and clinical trials methods via MOST, (2) Rapid qualitative research methods, and (3) Proficiency in grantsmanship and scientific dissemination. Research Plan: The proposed aims will address a gap in knowledge related to the active ingredients of CBT for improving MS fatigue. This knowledge will facilitate optimization of CBT for MS fatigue to maximize efficacy and efficiency, thereby improving implementation and accessibility. The study aims are to (1) Develop, pilot test, and refine three telehealth CBT components for fatigue in PwMS (Relaxation Training, Behavioral Activation, Cognitive Therapy) to maximize feasibility and acceptability; (2a) Conduct component analysis (via factorial optimization trial) to test the main and interactive effects of the three telehealth CBT components for improving fatigue and psychosocial function in fatigued PwMS (primary outcomes); (2b) Assess change in hypothesized unique and common mechanisms of the three telehealth CBT components; (2c) Evaluate the durability of outcome improvements through the 3-month follow-up. Components will be selected for an optimized intervention if they meet the threshold effect size for either primary outcome and meet the key implementation objective (defined in a preliminary study) as an optimized intervention package.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT Silencing RNA (siRNA) is of considerable current interest in medicine because it can elicit potent, target specific knockdown of virtually any mRNA, creating a useful and proven genetic surrogate tool. Three siRNA- based therapies have been FDA-approved, while another seven candidates are in phase 3 trials. A key obstacle limiting the scope of siRNA clinical uses, however, is in vivo targeted delivery, a `chronic' problem that has plagued the development of virtually all antisense therapies. Recent advances in nanotechnology have produced many nanocarriers such as cationic lipids, polymers, inorganic nanoparticles, and peptides. Although the cationic charge is important for siRNA condensation and endosomal escape, it interferes with specific targeting by non-specifically binding to most proteins and cells. To address this fundamental and chronic problem, here we propose to further develop a one-of-its-kind RNA nanocarrier for delivery of siRNA cocktails building on a recent breakthrough we made on human RNA-binding proteins (Corey & Gao Nature BME, PCT filed, US phase granted). Optimized through millions of years of evolution, these proteins are neutral or slightly negatively charged yet still binding to cargo RNA tightly and with a very high payload. The absence of excessive cationic charges is in direct contrast to conventional delivery technologies. Through orientation controlled self-assembly with siRNA-targeting ligand chimeras, the complex simultaneously achieves all the desired properties for efficient siRNA delivery and cancer treatment.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Intermediate metabolism must be finely tuned, carefully balanced, and robustly adaptable to changes in envi- ronmental conditions. While the individual enzymes that drive most metabolic processes are well understood, only have we appreciated the widespread role of metabolic enzyme assemblies in metabolic organization and control. In particular, two types of structures, metabolic filaments that assemble from single enzyme types, and metabolons that co-assemble multiple enzymes in single pathways, provide important mechanisms for regulating enzyme activity and metabolic flux. Both filaments and metabolons assemble dynamically in cells, and alter the functions of the constituent enzymes to adapt to metabolic demand. This proposal builds on recent successes from our group describing the molecular mechanisms and functional consequences of enzyme assembly in mul- tiple systems, including nucleotide biosynthesis, energy metabolism, and amino acid regulation. Recent work, including our own published and preliminary data, shows that enzyme assembly is a general mechanism of control, and that assembly itself is controlled by cells in multiple ways, including by the levels of specific metab- olites, by posttranslational modification, by expression of splice variants that alter polymerization characteristics, and by interaction with regulatory proteins. We use in vitro reconstitution and cryo-electron microscopy to deter- mine the structural basis for assembly and regulation, coupled with biochemical, biophysical, and cell biological techniques in an integrative approach to understanding the functions of metabolic assemblies. This work will provide insight into the specific roles assembly plays in modulating the function of multiple enzymes, and illumi- nate general principles of metabolic control.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY The studies proposed in this application will advance in vitro safety and efficiency testing for somatic cell genome editing in human cells in 3D organoid models. For genome editing, human organoids have the potential to be an ideal tool, as the therapeutic target is the human genome, which cannot be replicated in any other species. Organoids also have advantages for throughput and predictivity of human side effects. It is important, however, to test the utility and value of organoids in this context, for this to be demonstrated as an enabling technology for investigational new drugs. To achieve this, we will produce a set of diverse organoids representing human kidney, liver, brain, lung, retina, and/or heart as vital organ systems of great interest to gene editing applications. For each organ lineage in our 'body in a dish', we will demonstrate assays to measure editing rates as well as side effects. These assays will be optimized to establish reference standards with quantifiable measurements of assay stability, reproducibility, and analytical range. Organoid datasets will be compared with datasets produced in parallel efforts by collaborating teams using similar gene editing technologies. The objective is to demonstrate safety and efficiency assays in human organoid cultures in conjunction with complementary assessments in other systems as a tractable paradigm to support the advancement of genome editing therapeutics to human clinical trials. To maximize impact, we will focus on assays that will be broadly useful for a wide variety of genome editing therapeutics, in multiple organ systems. Organoids derived from human pluripotent stem cells possess many key features of tissues, including diverse cell types in sophisticated arrangements, and express specific disease phenotypes associated with rare populations. For regulatory consideration, there is a critical need to determine their fidelity and prediction capacity. In these studies, we will demonstrate concordance and synergy between human organoids and other preclinical models. Thus, the Specific Aim proposed is to de-risk therapeutic genome editing approaches by assessing dose-dependent efficiency with adverse events in human organoids. Collectively, these studies will produce models of genome editing in human organoids with outcomes that can be compared to orthologous models to establish a regulatory paradigm which can be applied to a range of tissues and diseases.

NIH Research Projects · FY 2026 · 2023-04

Summary Lower respiratory tract viral infections serve as the dominant trigger for the onset and progression of asthma. Although asthma management has shifted towards targeting airway inflammation at all levels of the disease, primarily with inhaled corticosteroids (ICS) and biologic therapies targeting type-2 (T2) inflammation, there is a substantial burden of disease that is not responsive to these therapies. Specifically, a substantial portion of individuals treated with ICS therapy have persistent T2 inflammation in their lower airways and a large and growing portion of individuals with asthma have inflammation of the airways that is not T2 predominant (non- T2). These related phenomena of persistent T2 (T2-high) and non-T2 inflammation in asthma are emerging as the most common phenotypes in adults, and now make up a substantial portion of children with the disease. The mechanisms responsible for persistent T2 and non-T2 inflammation are incompletely understood, but there is strong evidence that the epithelium and epithelial-derived cytokines play a major role in the immunology of asthma, and we have recently demonstrated that the epithelium is infiltrated with specific innate immune cells that interact with the epithelium to propagate and regulate inflammation. Our central hypothesis is that the airway epithelium serves as a central coordinator of the immune response to viral respiratory tract infection in asthma. Further, airway epithelial cells (AECs) from T2-high and non-T2 individuals differentially interact with immune cells to support inflammation in a manner that can be identified and targeted. We have an established program to isolate AECs from children and adults to examine the function of these cells in asthma using a combination of organotypic cell culture models, often in combination with immune cells. The primary goals of the Seattle Center are to identify primary alterations in AECs in asthma, understand how AECs differ between T2-high and non-T2 individuals, and characterize interactions between the epithelium and immune cells that are in close proximity. In Project 1, we examine the underpinnings of persistent T2 inflammation mediated through mast cells and eosinophils acting in conjunction with the epithelium and the components of this inflammation that are resistant to corticosteroids. In Project 2, we examine the basis of interactions between the epithelium and macrophages, Th17 cells, and neutrophils and how epithelial cells from each of the groups support inflammation through these cells. These projects are supported by Pediatric and Adult Epithelial Cores (Core B) to isolate AECs from well phenotyped adults and children and further examine connections between phenotype, genomics, genotype, and clinical outcome. The studies are supported by an Advanced Bioinformatics Core (Core C) using state of the art single cell and bulk RNA sequencing. These studies will facilitate the precise targeting of inflammation based on clinical phenotype and epithelial endotype and a greater understanding of the basis of interactions between the epithelium and immune cells that reside within the epithelium in asthma.

NIH Research Projects · FY 2026 · 2023-04

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Cystic fibrosis (CF) is a progressive, fatal genetic condition (median age at death was 34 years in 2020). Lung transplant (LTx) is a treatment for end-stage CF and confers 10 years median survival for adults with CF. In the U.S., among CF patients with forced expiratory volume in 1 second (FEV1) less than 30% of predicted, more patients die each year than undergo LTx. More than half of CF patients who die without LTx are never referred for consideration. Patient preference may account for 25-40% of decisions to defer referral, but choices may at times be informed by inaccurate assumptions regarding LTx. CF patients with low FEV1 have high rates of anxiety and depression, which may contribute to avoidance of LTx conversations. Increasing LTx comprehension, and promoting the process of deliberation, could reduce the number of people with CF who die without LTx. We collaborated with CF patients, caregivers, and physicians to develop Take on Transplant (ToT): a web-based, patientfacing resource for LTx education. ToT shares personal narratives of CF patients and caregivers and up-to-date, CFspecific, guideline-based medical information about LTx. The overall research objectives are to test the efficacy of ToT compared to an attention control in a multicenter randomized clinical trial incorporating mixed methods to assess preparedness for LTx discussions among all CF patients with FEV1 <50% predicted, explore the impact of ToT on patients’ psychosocial functioning, and assess patients’ and physicians’ perceptions and use of LTx education. Our research will examine patient-physician interactions and explore factors that may determine if/when physicians discuss LTx with CF patients and the impact of ToT on patients’ readiness for LTx discussions. We aim to empower all CF patients to discuss LTx. The proposed R01 will test the impact of ToT on preparedness for LTx discussions and will evaluate patient-physician dynamics that may impact access to LTx in the CF population.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) represents an emerging global health threat and is a expected to double in prevalence by 2050. AD is a disease of malformed proteins, and significant progress has been made characterizing the AD proteome with mass spectrometery. However, data missingness represents a significant barrier to the interpretation of existing AD mass spectrometry experiments. Missingness refers to peptides or proteins that are present in the biological sample but are not detected by the mass spectrometer due to various technical factors. This project will address missingness by developing machine learning methods for imputing, or estimating, missing values in quantitative mass spectrometry data. The project will develop two separate imputation methods, one using non-negative matrix factorization and the other deep neural networks. These imputation methods will increase the reproducibility and statistical power of mass spectrometry experiments and will enable new discoveries in existing proteomics experiments. These imputation methods will be applicable to virtually any kind of mass spectrometry experiment – tandem mass tag, data dependent acquisition, data independent acquisition, spectral counts, label-free quantification, etc. These imputation methods will be released as lightweight, open-source and easy-to-use software packages and may be incorporated into existing data processing workflows. I will demonstrate the utility of these imputation methods by reanalysing data from several existing AD proteomic studies. My imputation methods will identify novel differentially expressed proteins, co-expression modules and AD biomarkers in these existing datasets. I will also analyze unpublished data-independent acquisition (DIA) proteomics data derived from AD patient cerebrospinal fluid samples. Here I will focus on identifying biomarkers that differentiate between patients based on genetic background and co-morbidity status. I will also identify biomarkers of patients with asymptomatic AD. The imputation methods developed by this proposal will enable future discoveries by independent AD researchers. This proposal aligns with the NIA Strategic Direction seeking to "identify and understand the genetic, molecular and cellular mechanisms underlying the pathogenesis of AD."

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Pulmonary arterial hypertension (PAH) is a progressive and ultimately fatal disease with a median survival from diagnosis of approximately six years despite modern treatments. Up to 1 in 20,000 people are affected, and no available therapies cure or prevent this disease. PAH is characterized by pulmonary arterial endothelial cell (PAEC) and smooth muscle cell (PASMC) dysfunction leading to increased pulmonary vascular resistance and death from right heart failure. Abnormal hemodynamic forces are the primary cause of PAH in some patients, and in all cases may contribute to progression. The small pulmonary arteries in PAH are exposed to both increases in shear stress and pressure forces. Increased shear stress has previously been shown to cause EC changes mimicking those seen in PAH. Dr. Rayner has obtained preliminary data showing that PAECs from subjects with PAH have divergent transcriptomic responses to pathologically high shear stress when compared with controls. This suggests that patient abnormalities in shear-sensitive pathways may be a potential unifying mechanism in PAH that could provide targets for future therapeutics. Dr. Rayner’s overall goal is to define how shear and pressure forces combine with underlying patient factors to drive vascular dysfunction and promote PAH. Dr. Rayner has a research program focused on applying novel bioengineering techniques to the study of PAH. His research proposal will use a resistor-coupled microfluidic device to allow pressure and shear forces to be evaluated both individually and in combination. Dr. Rayner has also developed a novel pulmonary arteriole-on-a-chip (AOC) model that will be employed in this proposal to evaluate EC-SMC signaling and coordinated vascular behavior. Dr. Rayner’s research goal will be accomplished through three aims: 1) Evaluate the effects of shear and pressure on control and PAH PAECs in a resistor-coupled microfluidic platform; 2) Determine how pressure and patient factors influence cell phenotypes in PASMC-only AOCs; 3) Identify the effect of hemodynamic and patient factors on cell phenotypes and PAEC to PASMC signaling within patient-specific multicellular AOC models. These specific aims are well-aligned to the main training aims of Dr. Rayner’s Career Development Plan, which are to gain essential additional training in pulmonary vascular cell biology, bioinformatics, and vascular engineering. Dr. Rayner will gain these skills through a combination of formal didactics, experiential training, and close mentorship by a world-class team of scientists with relevant expertise. These new skills will augment his background in bioengineering and translational PAH research and facilitate his overall goal of developing into an independent physician-scientist doing basic and translational research on PAH. With his own unique engineered vascular platforms and the data generated through this research, Dr. Rayner will be well-positioned to submit a competitive R01 proposal near the end of his proposed K08 research period.

NIH Research Projects · FY 2025 · 2023-04

In natural vision, recognizing objects based on the retinal image is challenging and is often an ill-posed problem because a single image is compatible with multiple interpretations. Nevertheless, the primate brain has a remarkable ability to understand ambiguous scenes and solve difficult object recognition problems. Converging evidence suggests that this process, especially in challenging contexts—e.g., occlusion or low-visibility environments—is based on the integration of sensory information with prior knowledge built from experience. Our goal is to develop circuit diagrams at a cellular level that specify how inter-areal interactions support the integration of sensory signals related to the visual image with internal models that represent prior knowledge, thereby revealing the computations that underlie scene understanding, object recognition, and perceptual decision making in the primate brain. To achieve this goal, we have assembled a synergistic team of experts to bring together, (i) viral-based circuit tracing and optogenetic methods to identify connected neurons; (ii) multiphoton imaging and high-density electrode recordings to functionally characterize neurons and signaling motifs in the awake macaque monkey; (iii) behavioral manipulations and (iv) cutting-edge computational modeling to reveal how systems of connected neurons across brain regions interact and support complex perceptual processes. Our proposal includes four projects. In Project 1, PI Briggs will lead an effort to establish circuit tracing protocols to support dense, reliable, and long-term tracking of connected neurons in the macaque monkey. We will histologically compare lentivirus and AAVretro constructs in terms of their efficacy, toxicity, directional reliability, layering, and spread in labeling connected neurons, and we will test opto-tagging using high density neurophysiology. In Projects 2 & 3, PI Bair will lead the effort to implement multiphoton imaging in the awake monkey to identify projecting neurons in vivo during the simultaneous physiological characterization of 100s of neurons down to a depth of ~1 mm in cortex. In Project 4, PI Pasupathy will lead the effort to apply the viral methods and physiological characterization with high-density neuropixels probes and multiphoton imaging to study neurons in visual cortex (area V4), prefrontal cortex and the visual pulvinar as macaque monkeys perform shape detection in impoverished images. PI Wu will lead the effort to interpret the population dynamics in the context of communication subspace models and reveal how connected neurons in three brain regions underlie the multiplexing of sensory signals and prior knowledge to facilitate object detection and scene understanding.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Sexual assault is highly prevalent among college students and is associated with high risk for alcohol misuse and co-occurring posttraumatic stress in survivors of all genders. These conditions are associated with significant strain on campus service systems and substantial short- and long-term consequences for students. Although effective preventative interventions (i.e., implemented within the months following trauma exposure) exist for these conditions, college student survivors often do not seek or successfully access in-person care, especially among men and racial/ethnic minorities. Mobile health (i.e., mHealth) interventions are a promising strategy to increase low-barrier access to preventative interventions among college students. We developed and pilot tested a multi-component mHealth preventative intervention prototype, THRIVE, to reduce posttraumatic stress and alcohol misuse in recent survivors of sexual assault. THRIVE was effective in reducing both posttraumatic stress and alcohol misuse, making it the first post-trauma mHealth intervention with evidence of efficacy in reducing alcohol misuse. In the current study, we propose to conduct an optimization trial to identify the most effective, efficient, and low-burden version of THRIVE for college students. Prior to conducting the trial, we will revise THRIVE using the real-time user feedback collected in the pilot trial (Aim 1). Revisions will maintain the core elements of THRIVE while increasing usability. Revisions will be selected by reviewing the app and pilot user feedback in partnership with user experience/interface designers, our advisory board of experts in cultural adaptations of interventions, and our advisory board of college student survivors. This will maximize usability with attention to the needs of diverse student survivors. Using the revised app, we will then conduct a 2x2x2 factorial randomized trial to test the unique effects of the 3 core THRIVE components (i.e., in-app activity scheduling, in-app cognitive restructuring, phone coaching) on alcohol misuse and posttraumatic stress (Aim 2), as well as the added benefit of coaching to activity scheduling and cognitive restructuring (Aim 3). N=464 college students with past-12-week sexual assault, elevated drinking, and PTSD will be enrolled. All participants will receive an onboarding call and non-core app elements (i.e., self-monitoring, encouraging statements banner, referrals), and will be randomized to additionally receive combinations of the 3 core elements. Participants will complete self-report assessments at baseline, daily during the intervention, post-intervention, and 3, 6, 9, and 12 months. Results will be used to select an optimized version of THRIVE. Our design will ensure that future testing and dissemination of THRIVE reflects maximally inclusive, efficient, and effective intervention elements. If effective, THRIVE would represent a highly-scalable strategy that could be directly implemented by campus service systems to reduce the substantial burden of PTSD and alcohol misuse on students.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Enteric infection with Shigella spp. can lead to symptoms ranging from acute watery diarrhea to sudden, severe dysentery. Approximately 212,000 diarrheal deaths annually are attributed to Shigella (12.5% of total diarrheal deaths) with a disproportionate impact in low resource countries. The impact in low resource countries was illustrated by a reanalysis of the Global Enteric Multicenter Study (GEMS) which found that Shigella has the highest attributable fraction for diarrhea in children < 60 months. While recent studies have highlighted the burden of the disease, there has been a concurrent reduction in therapeutic options for the treatment of shigellosis as drug resistant strains increase in prevalence. In addition, increasing reports of drug resistant shigellosis cases in the men who have sex with men community confirm that the impact is not limited to children in low resource settings. While there is a clear need for new shigellosis treatments, it is not clear what characteristics of current therapies contribute to their efficacy and emergence of resistance. The goals of the proposed work are to determine exposure-response relationships for antibacterials and to establish a rigorous preclinical framework which can be used to identify and optimize new therapeutics for treatment of shigellosis. Our previous innovative studies on the gut localized pathogen Cryptosporidium demonstrated the importance of gastrointestinal drug exposure for in vivo efficacy. The pharmacokinetic/pharmacodynamic (PK/PD) relationship for anti-Cryptosporidium drugs was characterized with in vitro and in vivo models of cryptosporidiosis. Our central hypothesis for this proposal is that a similar approach can be used to establish the relationship between antibacterial exposure and in vivo efficacy against Shigella. In addition, we believe our models can be used to identify antibacterial concentrations associated with the emergence of resistance. Towards our hypothesis, we will use our established mouse model of Shigella infection to characterize the in vivo efficacy of WHO recommended antibacterial treatments for shigellosis. In addition, we will use an innovative hollow fiber infection model to investigate the emergence of antibacterial resistance. This crucial information will assist in rationalizing dosing regimens for current treatments and will support discovery and development of future therapeutics. We propose to evaluate the PK/PD relationship for antibacterials by undertaking the following three Specific Aims: (1) Determine in vivo exposure-response relationships for antibacterials and (2) Characterize antibacterial efficacy and emergence of resistance with a Shigella hollow fiber infection model. Taken together, these studies will help us better understand current shigellosis treatments and will provide a series of methods to identify and optimize new treatments. In addition, the results of the work will provide fundamental support for drug discovery in infectious disease, especially in the area of enteric infections.

NIH Research Projects · FY 2026 · 2023-04

The purpose of the HIV and Substance Use Cohort Coordinating Center for Emerging and High Impact Scientific Cross Cohort Studies: HIV SUCCESS is to support National Institute on Drug Abuse funded cohorts as they implement research strategies to better understand and address substance use and its consequences among people with HIV (PWH). Substance use prevalence among PWH is high, and substance use patterns, including increasing concurrent methamphetamine/opioid use as part of the 4th wave of the opioid epidemic, continue to evolve. Substantial challenges remain to improve understanding and implementation of interventions to address substance use and its impacts among PWH. Addressing these challenges is the overarching purpose of the cohorts and requires a coordinating center (CC) experienced in data integration and harmonization, health informatics, multi-site coordination, clinical care and intervention experience, and HIV and substance use research to support the cohorts and comprehensively integrate cross-cohort data. The resulting resource of comprehensively integrated data will give researchers the potential to address important scientific and public health questions that would otherwise not be possible in individual cohorts. We will work with cohorts to allow complex, careful and complete analyses of outcomes and results across diverse populations using harmonized data. We will bring in data from other cohorts and studies as needed to ensure that adequate clinical, biomarker, and/or genetic data are available to address key questions. Careful data harmonization where appropriate will improve the statistical power to identify areas or sub-groups for research focus and to understand what interventions are proving successful in the broader context of the whole population of PWH (as opposed to the target population of a single cohort). This team brings vast experience with HIV cohort data; data linkage and harmonization; methods development; statistical support including causal inference from longitudinal observational data; health informatics platform and tool infrastructure and development including data repositories and tools for efficient and accurate electronically collected patient reported outcomes and outcomes adjudication; providing overall coordination for large collaborations of cohorts and studies; a strong background in clinical epidemiology of HIV and substance use; and expertise in applying this information to clinical care and interventions. We also have a comprehensive mentoring approach to develop a new generation of HIV and substance use researchers. We will support cohorts to enhance data collection where appropriate, merge and harmonize data when feasible, and work together to address key questions on HIV, substance use, and outcomes that cannot be addressed by individual cohorts. By providing project management, mentorship, and support, as well as developing a robust data repository, and accomplishing the integration and linkage of data, we will achieve a multi-disciplinary integrated network with multi-site data with sufficient sizes needed to address substance use and its impacts among PWH.

Fonds de recherche du Québec – Nature et technologies · FY 2023-2024 · 2023-04

Volet: Bourses de doctorat en recherche; Domaine: Techniques, mesures et systèmes; Objet: Statistique informatique; Objet: Modélisation neuronale; Application: Sciences et technologies; Application: Technologies des communications et de l'information; Mots-clés: COMPUTATIONAL NEUROSCIENCE, DEEP LEARNING THEORY , NORMATIVE MODELING, ARTIFICIAL AND BIOLOGICAL NEURAL NETWORK, LEARNING AND GENERALIZATION , BIOLOGICALLY PLAUSIBLE LEARNING RULES

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract: The burden of stroke remains unacceptably high and few treatments are proven to improve stroke prevention or recovery. An estimated 70% to 90% of stroke survivors have obstructive sleep apnea (OSA). Compared to those without OSA, stroke patients with OSA have worse functional outcomes after inpatient rehabilitation (IPR), 40% longer IPR stays, a 100% increase in the risk of non-fatal cardiovascular events, particularly recurrent stroke, and a 75% increase in the risk of early death. Observational data suggest that treatment with continuous positive airway pressure (CPAP), the first-line treatment for OSA, holds promise to improve stroke outcomes, including both preventing recurrence and improving recovery. However, poor CPAP adherence has compromised pilot trials of stroke survivors with OSA, yielding mixed results and limiting the ability to test CPAP efficacy. Individualized behavioral interventions to improve CPAP adherence have shown benefit in the general population, though little is known about their effectiveness to optimize CPAP among stroke patients, who are likely to respond to behavioral interventions differently. We propose to adapt and refine for stroke patients a multicomponent behavioral intervention to improve CPAP adherence with the engagement and input of stroke survivors. Then, within a multicenter randomized controlled trial, we will test the efficacy of the intervention, initiated during inpatient rehabilitation, on CPAP use among stroke patients over the course of 3 months. The adherence intervention will include: 1) CPAP technical support, 2) motivational enhancement therapy, and 3) self-monitoring using mobile health technologies with automated support. If successful, the study will provide researchers a much-needed intervention to realize the full benefits of CPAP after stroke. It is difficult to imagine a non-invasive, relatively low-cost intervention with a similar potential as CPAP for such a common and disabling a disease as stroke.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Despite freely available HIV services and antiretroviral therapy (ART), people newly diagnosed with HIV in refugee settlements in Uganda have suboptimal rates of ART initiation, adherence, and viral suppression. Refugees face unique barriers to HIV care engagement including long distances to clinic with environmental conditions impacting travel, prohibitive transportation costs with severely limited livelihood opportunities to offset them, disrupted social networks, and guarded HIV status disclosure due to fear of anticipated stigma and reliance on other community members to meet basic survival needs. Innovative strategies to improve engagement in HIV care for this priority population are urgently needed. Community ART delivery, a differentiated ART delivery strategy offered in Uganda for stable clients, reduces time and transportation barriers, fosters social support, and improves engagement in HIV care. Individuals newly diagnosed with HIV, however, are currently excluded from participation in community ART delivery and the impact of this ART delivery strategy has not been evaluated for this group. This is a critical gap in HIV research as the time following initial HIV diagnosis is a vulnerable period with high rates of attrition from care. Preliminary findings from a pilot study in Nakivale Refugee Settlement in southwestern Uganda found that early community ART delivery, implemented at the time of HIV diagnosis, may enhance viral suppression in this setting. The overall objective of this proposal is to conduct a cluster randomized controlled trial to discern the effectiveness of “Head StART,” community ART delivery for those newly diagnosed with HIV, compared to standard care (facility-based ART delivery) to achieve HIV viral suppression. The central hypothesis is that Head StART will be an effective and affordable intervention at refugee health centers across Uganda. Guided by promising preliminary data and supported by the social ecological model, this hypothesis will be tested with these specific aims: 1) To evaluate the effectiveness of Head StART in achieving HIV viral suppression for people newly diagnosed with HIV in refugee settlements in Uganda; 2) To assess Head StART implementation across refugee settlement locations to understand the impact of contextual factors on achieving optimal HIV clinical outcomes; and 3) To estimate the programmatic cost and budget impact of implementing Head StART in refugee settlements in Uganda. The approach is innovative, in that it assesses novel timing of community ART delivery in a humanitarian crisis affected population at high risk of poor clinical outcomes. This proposal is responsive to the NIH priorities for HIV-related research in that it includes assessment of HIV viral suppression among refugees and Ugandan nationals newly diagnosed with HIV, as well as evaluation of an intervention to advance ART delivery. The long-term goal is to devise strategies to optimize HIV outcomes in refugee settlements in Uganda. The proposed research is significant, because it could improve HIV viral suppression in humanitarian contexts globally, advancing progress towards the 95-95-95 UNAIDS targets.

NIH Research Projects · FY 2024 · 2023-04

Project Summary/Abstract The goal of this proposal is to develop and validate two tools for in situ analysis of epigenetic state and chromatin organization in single cells in kidney tissue. The first tool is based on our recently developed technology SCEPTRE (Single-Cell Evaluation of Post TRanslational Epigenetic encoding) established for cultured cells that will be adapted for use with kidney tissues in order to directly resolve and quantify histone modifications at specific DNA loci in situ in single cells. The second tool is a new method called APT-FISH (Amplified Probes from Tagmentation Fluorescence in situ Hybridization) that is designed to profile changes to open chromatin. Each of these tools will be used concurrently with super-resolution expansion microscopy imaging in order to directly relate nanoscale renal physiology to chromatin or epigenetic state and they will be applied to the study of aging and senescence in kidney tissue.

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT: The integration of electrochemical biosensors with microelectronics offers a unique avenue for miniaturized, low-cost systems that merge biomolecular sensing with digital computing, programming, and communication. Here we propose to integrate electrochemical aptamer-based sensors (“aptasensors”) into a microfluidic multi-well platform to enable multi-time-point, highly parallel readouts of cell death and cytokine secretion from intact tumor biopsies during and after drug treatment. Our platform will allow for gathering the large amounts of molecular data that are needed for machine learning approaches to drug testing. Cancer drug testing – a central process in cancer drug development and personalized oncology – is often inaccurate and inefficient because it typically relies on studies in cell cultures or animals that lack the human tumor microenvironment (TME). Only <4% of cancer drugs out of the ~1,000 drugs in clinical trials each year pass the safety and efficacy tests; more than half of the failures are due to lack of efficacy. Hence, on average, bringing a drug to market takes >10 years and costs >$1 billion, often resulting in high prices for the drugs. In the last decade, technologies such as patient-derived organoids and organs-on-chips have brought some hope. However, these approaches have much lower throughput than traditional cell cultures and are generally unable to fully recreate the TME of an intact tissue. These limitations are a fundamental hurdle for the personalization of therapies which often need to be customized to the unique TME of the patient, and also for the development of combination immunotherapies, which target the TME and are exponentially increasing in number. Thus, a different paradigm for drug testing that preserves the human TME is critically needed to help transition oncology into a stage of more affordable and rapidly evolving treatments. The Folch and Gujral labs have developed an intact-tissue microfluidic drug testing platform based on regularly- sized, cuboidal-shaped microdissected tissues (referred to as “cuboids”) that are mechanically cut with a tissue chopper. In under an hour, more than 10,000 cuboids (~400 µm-wide) can be produced from ~1 cm3 of solid tumor. The cuboids are never dissociated and retain much of the native TME (e.g. immune cells and microvasculature). The platform is a user-friendly multi-well device that can microfluidically trap and selectively treat a large array of cuboids. Here we propose to integrate electrochemical aptasensors into our cuboids platform to enable the automated, multi-time-point monitoring of secreted compounds (cytokines or cell death indicators) within the wells and the straightforward implementation of electrical readouts from large cuboid arrays. As a proof of concept, we will use cuboids from mouse tumors and patient samples in a 96-well format. We will use a mouse model and patient samples of colorectal cancer (CRC) liver metastases. Using machine learning techniques, we will implement a proof-of-concept drug screen on mouse cuboids and a proof-of-concept combination immunotherapy drug evaluation on patient cuboids.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract The overarching goal of this proposal is to advance the understanding of the immune response to and protective effects of rectal Chlamydia trachomatis (CT) infections. CT is the most common bacterial sexually transmitted disease in the world and is associated with substantial reproductive tract morbidity. Rectal CT is increasingly recognized as a common infection among STI clinic patients, yet despite its high prevalence, there is no clear data describing how rectal infection affects the immune response to CT or the risk of reinfection. A better understanding of how the immune system responds to CT infection is urgently needed to inform effective chlamydia vaccine strategies. Animal studies in mice and nonhuman primates (NHP) have demonstrated rectal CT infections as non- pathogenic and persistent, similar to humans infected with non-lymphogranuloma venereum (LGV) serovars in the rectum. Furthermore, studies in the murine model have shown that rectal infection confers trans-mucosal protection against urogenital challenge, suggesting that rectal CT infection causes enhanced systemic and transmucosal immune responses to CT. It is possible the gastrointestinal tract (GIT) will be an optimal site for attenuated mucosal CT vaccines. However, more data on immune responses and potentially protective effects of rectal infection in NHPs and humans are urgently needed and will be provided in the proposed studies. Mouse and human studies provide evidence that secretion of IFN-γ by CD4+ T cells is essential for any protective immunity. The role for antibodies is less clear but appears to rely not on direct neutralization of bacteria, but rather on secondary functions of the Fc region of antibodies in activating innate and effector cells. Except for a few mouse model studies, the Fc-dependent functions of anti-CT antibodies remain largely unexplored. This project will provide informative data describing functional CT protective immunity in NHP (controlled exposures) and human (cross-sectional sampling) studies. We will conduct complementary studies in the pig-tailed macaque model and in clinical studies to address the hypothesis that rectal CT infection induces robust anti-CT immune responses that may protect from genital disease. We will directly test whether rectal infection in macaques protects against subsequent genital infection. In both macaques and humans, we will define anti-CT immune responses that may correlate with protection from reinfection and disease: secretion of IFN-γ by CD4+ T cells, presence of anti-CT antibodies in the mucosa, and functional antibody responses, using new methods to test for Fc-receptor-mediated functions of anti-CT antibodies. This project is the first step to demonstrate that rectal infection modifies anti-CT immunity in NHPs and humans, and will provide important evidence as to whether the GIT could serve as a mucosal delivery site for future vaccination strategies.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract The object of interest is not always visible at the time of movement. Visuospatial working memory plays an essential role in directing movements in such situations. This system stores the position of an object in the visual world and can be used to direct a movement to engage it. This is an internally driven movement. In contrast, we make so-called externally cued movement when the object is visible at the time of movement. To make movements accurate in both situations, visuospatial working memory and the visual signal need to be appropriately transformed to a motor command signal. The mechanism that maintains the motor accuracy is called motor adaptation. The behavioral characteristics and neural mechanisms of motor adaptation for an externally cued movement are well understood, however, little is known about motor adaptation of an internally driven movement. Do adaptations for both types of movement engage the same neural mechanism? If they are different, how are they different in their behavioral characteristics and neural mechanisms, and do they interact? We will use saccadic eye movements to study motor adaptation. Saccades, rapid eye movements that direct the gaze to targets of interest, can be induced by an external visual signal (visually-guided saccade, VGS) or by an internal visuospatial working memory (memory-guided saccade, MGS). Also, saccades are very precise. Because saccades remain accurate throughout life, despite the neural and muscular changes due to aging or injury, the saccadic system must be continually recalibrated through saccade adaptation. When we examined the VGS after MGS adaptation, VGS remained unchanged. Therefore, we hypothesize that the neural mechanism for MGS adaptation is distinct from that for VGS adaptation. In this project, we propose to investigate the adaptation of MGS and compare it with that of VGS. The main innovation of this study is that it could reveal a yet unknown motor adaptation mechanism for internally driven movement. The innovative concept is that the neural basis of motor adaptation for internally and externally driven movements could be separate but interact. Understanding the neuronal basis of motor adaptation for both externally and internally driven movements will provide a comprehensive picture of motor adaptation.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Primary cilia, or non-motile cilia, are present in almost every mammalian cell. Ciliopathies cause a spectrum of diseases in multiple organs, including the brain, kidney and liver. The most debilitating, however, are sensory neuropathies leading to deafness and blindness. Retinal ciliopathies account for one third of all inherited retinal diseases (IRDs), and are a major cause of visual impairment and blindness in the pediatric population. Usher syndrome is the most common retinal ciliopathy and presents a tremendous health burden due to congenital hearing impairment and progressive decline in vision. Usher type 2 (USH2) is the most common subtype with the USH2A gene accounting for the majority of cases. USH2 is inherited in an autosomal recessive manner; however, in approximately 20% of patients with clinical features typical for USH2, only one mutation of USH2A has been identified by exome sequencing, thereby precluding a definitive diagnosis. The second disease variant may reside in non-coding regions of the USH2A gene. Whereas the protein coding variants of IRDs have been studied in human and other model systems, the role of non-protein coding variants contributing to monoallelic disease is much less understood. Non-coding variants are difficult to classify, especially in gene products as large and complex as USH2A (spans 800 kilobases and contains 72 exons), and thus remain under-diagnosed. Genome sequencing of IRD patients can identify pathogenic non-coding variations in regulatory regions to explain causative changes and is increasingly being used to genetically diagnose patients suffering from suspected monogenic disease. Phasing genetic variation is also critical for human disease studies. We hypothesize that genomic sequencing and haplotype phasing of the USH2A locus and its surrounding regulatory regions will provide a more accurate detection of pathogenic variants in monoallelic patients. This will be accomplished by combining the base-level accuracy of Illumina short-read sequencing with the longer read lengths obtained from Nanopore based amplification-free targeted sequencing. Identification of non-coding variants and their phase information are essential first steps towards understanding their pathogenic effects in patient- derived stem cells. The pathogenicity of non-coding variants will be explored using luciferase knock-in cell culture systems as well as patient-derived induced pluripotent stem cells (iPSCs) differentiated in vitro to form retinal organoids (ROs). Use of ROs will provide clinically relevant tissue from patients that can be edited using CRISPR-Cas9 technology to determine variant pathogenicity and mutational burden. This proposal has tremendous therapeutic potential as non-coding deep-intronic variants have been the focus of gene targeting and gene editing technologies in various IRDs.

NIH Research Projects · FY 2025 · 2023-03

PROJECT SUMMARY/ABSTRACT Cannabis is the most commonly used controlled substance in the US.1,2 Young adults (YA; ages 18-25) report highest rates of use, and recent epidemiological surveys show an increase in both proportion of YA using cannabis and frequency of use among those that use.3,4 As frequent and heavy cannabis use is associated with a variety of short- and long-term unwanted physical and psychosocial outcomes (e.g., altered brain development, impaired judgement and memory, poor educational outcomes),5–7 there is a need for approaches to help individuals reduce use and/or use-related harms. One approach to mitigating substance- related harm is using protective behavioral strategies (PBS). PBS for cannabis consist of strategies an individual can use before, during, after, or instead of using to reduce use or consequence.8,9 In retrospective assessments, frequency of PBS use is associated with lower past 30-day cannabis use and consequences and mediates the relation between various risk factors and cannabis outcomes,8,10–15 highlighting PBS as a promising means of reducing cannabis use and harms. However, research on PBS-focused interventions is mixed9,16,17; this mixed support may be due to gaps in the PBS literature. Specifically, the majority of PBS research has consisted of cross-sectional, between-person retrospective designs, thus we lack understanding about which strategies work for whom under what circumstances. Emerging PBS research suggests both between- and within-person (i.e., daily) variability in whether an individual uses any PBS, and if so, which strategies they use. This suggests a need for a daily measure of cannabis PBS to increase understanding of how and when individuals utilize PBS and under what circumstances PBS are or are not effective. To address these gaps, the proposed F31 will take a novel, multimethod approach incorporating scale development work, a daily data study design, and machine learning methods. Specific Aims include (1) developing and validating a daily measure of cannabis PBS; and (2) utilizing a daily data design and machine learning techniques to develop models predicting PBS efficacy (reductions in use/consequences) for each strategy for a given individual. To complement these aims, the applicant will receive training in (1) etiology, prevention, intervention, and harm reduction methods for substance use, with a focus on cannabis PBS; (2) psychometric development and quantitative methods including multilevel modeling and machine learning; (3) daily data study design and methodology; and (4) research dissemination, including manuscript/grant writing and conference presentations. Study findings will have important implications for future PBS intervention research. Specifically, results can be used to better understand cannabis PBS on a daily level and improve future technology-based PBS interventions to reduce cannabis-related harms.