University Of Delaware

NSF Awards · FY 2024 · 2024-06

Many trace elements (TEs) such as iron are required for the growth of phytoplankton in the ocean, and their availability can limit biological production. One important pathway by which these scarce elements are delivered to surface ocean waters is through aerosol particles in the atmosphere. This project will address important questions about the variability in the atmospheric delivery of iron and other TEs to the surface waters of the North Atlantic Ocean near Bermuda through aerosol sampling, laboratory experiments, and modeling. Increased accuracy of atmospheric deposition and aerosol iron solubility representation will improve global biogeochemical models and our understanding of how changes in climate may influence the marine carbon cycle through changes in atmospheric iron deposition. The project will provide opportunities for undergraduate and graduate student involvement and support an early-career scientist. Data will be posted on the BCO-DMO website and made widely available to scientists working in similar fields by being included in the SCOR Working Group 167 (Reducing Uncertainty in Soluble aerosol Trace Element Deposition) data compilation. This project will conduct a two-year time-series of size-fractionated aerosol sampling at the Tudor Hill Marine Atmospheric Observatory in Bermuda to accomplish four goals: 1. Analyze temporal variations in the size distribution of aerosol Fe and other bioactive, pollutant, and tracer TEs, as well as major cations and anions over the western North Atlantic Ocean and link these variations to aerosol sources and transport pathways. 2. Apply a range of chemical extractions to size-fractionated and bulk North Atlantic aerosols to quantify lower and upper estimates of potentially bioavailable Fe and explain trends in bulk aerosol Fe solubility in the context of variations observed in size-fractionated aerosols. 3. Use elemental ratios, Fe stable isotopes, Fe-mineralogical partitioning and redox state, and air mass back trajectories to directly probe the chemical controls on aerosol Fe solubility as a function of aerosol particle size and source over a two-year period. 4. Parameterize concurrently measured meteorological conditions to model deposition velocities for each aerosol size-fraction during weekly sampling periods, thereby constraining supply rates of total and soluble TEs to North Atlantic surface waters. The results of this study will provide a deeper understanding of the factors influencing trace element solubility in North Atlantic aerosols and the role that aerosol size distribution plays in these variations. Rates of dry deposition calculated using size-fractionated aerosol collections will give a better understanding of the potential overestimation of bioavailable trace element supply rate that may arise from previous calculations based solely on bulk aerosol concentrations. This project will thus improve representation of atmospheric trace element solubility and deposition flux in global and regional deposition models. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-06

ABSTRACT Variants in the ABCA4 gene are a fundamental cause of several inherited retinal degenerations (IRDs), including Stargardt macular dystrophy, fundus flavimaculatus, and cone-rod dystrophy; these three ABCA4- driven diseases cause blindness in 1.4 million people worldwide. As a result, genetic testing of ABCA4 is increasingly common in clinical settings. Of the 1,485 identified missense variants in ABCA4, 50% are of unknown pathogenicity (variants of unknown significance, VUS). This genetic uncertainty leads to three key problems: (i) for IRD patients who have multiple unclassified ABCA4 mutations, it is impossible to predict which variant will cause disease in relatives who have not yet developed the disease; (ii) development of variant-specific therapies will remain limited; and (iii) these variants cannot be used to predict disease prospectively, which facilitates important life-planning decisions for patients and which is critical to direct patients to new clinical trials. Our proposed study aims to unravel the clinical significance of ABCA4 genetic variants of unknown significance (VUS) using in silico and in vitro functional analyses in combination. In this proposal, we have assembled a team of investigators with extensive expertise in complementary fields: protein biochemistry, computational biology, bioimaging, and biostatistics to create a “sequence-structure-function” workflow, whereby in silico 3D protein structural analysis of ABCA4 sequence variants will be used as a tool to predict disease severity/clinical pathogenicity in combination with high throughput functional analysis. The combined expertise of the investigators and our unique biochemical resources allow us to carry out this project. Successful completion of this study will be a significant and critical step forward in understanding the biology of ABCA4-mediated IRDs and the characterization of variants for pathogenicity risk prediction. The proposal incorporates interrelated initiatives focused on DEIA (Diversity, Equity, Inclusion, and Accessibility), which will include training opportunities for students at different stages of their careers, interdisciplinary research training, fostering inclusive and collaborative team science, and engaging with community stakeholders to raise awareness and generate interest in vision research. Together, these efforts will contribute to the creation of a transformative framework that promotes diversity within the biomedical research community while aligning with our scientific objectives.

NIH Research Projects · FY 2025 · 2024-05

Algorithms and Software for Multidimensional Vibrational Spectroscopy of Coarse-Grained Protein Models Abstract Alzheimer’s disease is age-related progressive irreversible neurological disorder which affects approximately 50 million people worldwide. It is ranked as the seventh leading cause of death in the United States with an estimated annual cost of 1 trillion USD. Alzheimer’s disease is characterized by accumulation of amyloid plaques. The failure is partially due to aggregation of A𝛽 protein. The fibrillation of A𝛽 occurs through A𝛽 oligomers which have substantial neurotoxicity. Therefore, there is much interest in understanding the mechanism by which A𝛽 aggregates because the aggregation pathway dictates the structures and populations of toxic intermediates. Amyloid aggregation is a difficult problem to study for the standard structural biology techniques because it involves kinetically evolving proteins. Two-dimensional infrared spectroscopy (2D IR) is an emerging analytical technique that probes protein dynamics with chemical bond-specific spatial and high temporal resolution. 2D IR spectroscopy is analogous to 2D NMR spectroscopy, except that it uses pulses of infrared light to measure molecular vibrations rather than pulsed magnetic fields measuring nuclear spins. New methodology improvements expand the frontiers of 2D IR spectroscopy permitting the study of amyloid aggregation and tissue imaging in native environments. Interpreting congested 2D IR spectra is difficult without simulations that connect spectral features to structural models. Computational spectroscopy advances alongside the improvements in experimental 2D IR technique. With the present algorithms and software, it is possible to calculate 2D IR spectra for a given all-atom or united-atom protein models and achieve at least qualitative agreement with experiment. There is, however, an important technology gap—methods for calculating linear and multidimensional vibrational spectra from coarse-grained protein and implicit solvent models do not exist. Such methods are highly desirable because the study of protein aggregation, especially in the membrane environment, involves large length- and timescales beyond the current capabilities of traditional all-atom molecular dynamics simulations. Instead, such simulations require the use of coarse-grained protein and implicit solvent models. The proposed work will address this gap. In Specific Aim 1 we will introduce a data-driven approach for calculating infrared vibrational spectra of all-atom protein models in a coarse-grained solvent. Specific Aim 2 will focus on an implicit solvent and coarse-grained protein models. The methods will be tested on the existing libraries of well-characterized small proteins whose vibrational spectra have been measured. Specific Aim 3 is devoted to efficient software implementation of the algorithms developed in Specific Aims 1 and 2. The new computational algorithms and software developed in the proposed work will allow interpretation of linear and 2D IR spectroscopy experiments on amyloid aggregation and will become an important technology in unravelling the fundamental molecular-level insights not only into Alzheimer’s disease but also into other pathologies associated with the aggregation of amyloid proteins such as Parkinson disease, type 2 diabetes, amyotrophic lateral sclerosis, and prion diseases.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract. With over 800 members, the largest family of human membrane proteins is the G- protein coupled receptors (GPCRs, also called seven transmembrane receptors), which account for somewhere between 30 and 40% of pharmaceutical targets. The significance of the family has fueled a flurry of structure determination work with an explosion of new structures reported in just the last few years. Over the same period, a lipidomic revolution has changed our view of the membrane environment of GPCRs — it is remarkably complex and tightly regulated, with distinct lipid compositions in different tissues and in different cellular compartments. There is abundant evidence that lipids regulate GPCRs. But how does the membrane regulate GPCR function? For example, the fully active state of a GPCR (the A2A adenosine receptor) is favored by negatively charged lipids. But, how do we quantify “favors activation?” Answering this very basic question would advance work that focuses on understanding mechanism in specific targets (like the A2A receptor), or other GPCRs, or indeed any IMP for which conformational changes couple to the lipids. It would also allow surveying such mechanisms across entire families (like the GPCRs), to understand lipid regulation across very different membranes and physiological contexts. And, it would provide a path to quantitatively compare results from well- controlled model systems and more physiological membrane environments — most GPCRs traffic to the plasma membrane, which has a complex and asymmetric lipid composition. We propose to pursue this question through a campaign of simulations and experimental measurements, tightly coordinated and organized by a thermodynamic model for lipid regulation. By comparing across different GPCRs, membrane environments, and between different receptor states, we will learn what aspects of functional lipid interactions are conserved, and also how they vary.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY This project includes both the development of advanced MRI sequences to study brain stiffness as well as clinical application of this technique to study brain health in an aging adult population. A sensitive method to establish and reliably detect the point where the progression of Alzheimer’s Disease and other dementias diverge from normal aging is crucial to developing methods for intervention. Early detection is critical in lessening the financial burden of these diseases on the US healthcare system. Magnetic resonance elastography (MRE) is a non-invasive, in vivo, MRI technique which provides information on the mechanical properties of tissues and is uniquely situated to sensitively provide insight into microstructural tissue changes that may occur prior to when structural changes caused by atrophy can be detected. The basis of the MRE technique is that displacements induced in a tissue by an external actuation source can be measured using phase-contrast MRI, and then turned into high-resolution maps of tissue mechanical properties such as stiffness. However, this technique is often difficult to widely implement because it requires use of longer scan times and then further requires specialized equipment to vibrate the tissue. We propose two major improvements to MRE acquisition and processing to remove these barriers based on a novel sampling technique and estimation scheme, EDGE (Elastography with Distributed, Generalized Encoding). EDGE utilizes non-traditional sampling directions implemented in an optimized encoding matrix to collect data much more efficiently, and be used in a novel algorithm to estimate harmonic displacement fields, together allowing room for acceleration of acquisition or redundancies to be built into the data for rejection of bad data in the post-processing step. We also extend this method to intrinsic actuation to capture motion that occurs naturally from pulsatile cerebral blood flow to provide the motion necessary for MRE mechanical property estimation (intEDGE), thus removing the need for external hardware and making the scan similar to any other for the patient experience. We will confirm the validity of these techniques by evaluating differences in brain stiffness between young and older adults using the traditional MRE method and the proposed EDGE and intEDGE methods. Success of this project will result in a novel MRE technique that removes barriers to the wide implementation of MRE as a measure of brain health in aging and aging-related neurological conditions.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Evidence-based intervention strategies are needed to address one of the most pernicious, yet least addressed, barriers to recovery from opioid use disorder (OUD): social isolation. People with OUD are trapped in a deadly cycle wherein opioid use leads to social isolation, and social isolation leads to increased risk of continued opioid use and overdose. Disclosure, which involves sharing information about one’s OUD history and/or treatment with others, can help to break this cycle by acting as a gateway to (re-)establishing social connection as individuals enter and engage in OUD treatment. Yet, disclosure is a challenging and high-stakes social process. It is challenging because it involves a series of decisions (including whether, why, what, how, and when to disclose) and requires an advanced skillset (including communication, de-escalation, and coping skills). It is high stakes because it sometimes leads to stigma and further social isolation, undermining recovery. Although there are numerous evidence-based interventions to support disclosure among people with other stigmatized chronic illnesses, none are currently available for people in OUD treatment. We have developed Disclosing Recovery: A Decision Aid and Toolkit, which is a brief, one-hour disclosure intervention designed to help people in treatment for OUD make key decisions regarding disclosure and build disclosure skills. We pilot tested Disclosing Recovery with 50 people in treatment for OUD. Participants randomly assigned to Disclosing Recovery perceived the intervention to be acceptable and feasible, and reported better decision-making quality than participants in the comparator condition. Moreover, Disclosing Recovery impacted disclosure rates and led to greater relationship closeness. In this Phase II efficacy study, we propose to test whether Disclosing Recovery results in improved treatment- and recovery-related outcomes over a 12-month follow-up period. We will randomize n=480 participants in treatment for OUD to Disclosing Recovery versus a waitlist comparator condition. We will abstract data from participants’ medical records and administer surveys every 3 months to examine the efficacy as well as potential mediators and moderators of the intervention. Our specific aims are to: (1) Evaluate whether participants randomly assigned to the Disclosing Recovery intervention versus a waitlist comparator condition experience improved treatment- and recovery-related outcomes; (2) Test whether changes in key relationship outcomes and/or social isolation mediate intervention effects (or non-effects); (3) Determine whether profiles of recovery characteristics, disclosure goals, and relationship characteristics moderate intervention effects (or non- effects). The intervention will be tested in Delaware, the state with the third-highest overdose death rate in the United States. If we find that the Disclosing Recovery intervention is efficacious, we will follow recommendations of the NIH Phase Model for Behavioral Intervention Development by progressing to real-world efficacy and/or implementation testing. By helping people (re-)establish social connections during OUD treatment, this line of research will ultimately contribute to breaking the deadly cycle of social isolation and opioid use.

NIH Research Projects · FY 2023 · 2023-09

Bone fractures are common injuries that impact millions of people each year, and poorly healed fractures cause impaired mobility, long-term nursing care, or even premature death. It is known that controlled mechanical loading can improve the quality and speed of fracture repair, but our understanding of mechano- therapeutics is still underdeveloped. Recent research has indicated that zinc may play a fundamental role in the evolution of fracture repair. Zinc is known to stimulate new bone formation, preserve bone mass, and regulate apoptosis. Importantly, intracellular zinc homeostasis must be carefully coordinated to regulate uptake, excretion, and intracellular storage/trafficking. It is believed that zinc may be mechanosensitive; however, the relationships between mechanical loading, zinc homeostasis, and fracture healing remain unclear. This project will generate preliminary data regarding the relationships between mechanotransduction in regenerative cells and establish links between mechanical load transfer and intracellular Zinc homeostasis in bone fractures. Our global hypothesis is that the combination of zinc with mechanical loading will lead to synergistic bone healing responses. In Aim 1, we will extend our existing K25 study with an in vivo rat femoral osteotomy model and determine changes in bone healing caused by zinc delivery and load transfer. Sprague Dawley rats will undergo femoral osteotomy and reconstruction. Mechanical loads across the callus will be controlled with either rigid locking plates (0-3% strain, low load across fracture) or more compliant locking plates (10-15% strain, high load across fracture). Zinc levels in animals will be manipulated locally by implantation of a non-loadbearing intramedullary nail. We hypothesize that the combinatory application of mechanical loads and localized zinc delivery will lead to synergistic improvements in bone healing that are demonstrated by faster and more robust development of callus. Changes in bone healing will be quantified with micro-CT imaging, biomechanical testing, histology, and qPCR. In Aim 2, we will define the causal relationships between zinc delivery, load transfer, zinc storage/trafficking, and osteoblast formation in an in vitro cell culture model. Here, we will use cell culture techniques to examine the fate of human mesenchymal stem cells. We hypothesize that zinc-rich cells plated on stiff, smooth surfaces will elicit improved osteoblastic proliferation and superior calcium matrix formation. These experiments will yield fundamental new understanding into the mechanisms by which cells receive and respond to mechanical stimuli and provide foundational data for long-term development of Zinc-augmented mechano-therapeutics. Characterizing these relationships may have immense implications for cellular mechanobiology and development of mechano- therapeutic approaches for bone regeneration and repair.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Post-traumatic osteoarthritis (PTOA) is an insidious consequence of joint injury, ~50% of patients with knee- injuries exhibit PTOA within 10-years of injury. Presently, no cure for PTOA exists, but the acute nature of the precipitating injuries provides for a unique approach to PTOA treatment: targeted prophylactic pharmaceutical intervention to mitigate/prevent the initiation of disease post-injury. Many pre-clinical investigations for targeted treatment have been conducted. However, due to incredibly rapid intra-articular (i.a.) drug clearance, disease- modifying drug efficiency is highly limited, requiring repeated high-dose administration of free drug for efficacy. Give the inefficiencies of i.a. administration of free drug, delivery approaches that extend drug-residence time by targeting the tissues of the injured joint could represent a cost-effective method of increasing therapeutic efficacy. We propose a novel and versatile platform for the thermally responsive, localized delivery of candidate PTOA drugs to injured joints to limit initiation/progression of PTOA. Our approach relies on our pioneering development of elastin-collagen-peptide conjugates that uniquely form cargo-laden nanovesicles that facilitate long-term passive release at body temperature and accelerated-/burst-delivery at mildly hypothermic temperatures. In addition, the collagen-like peptides comprising the vesicle’s outer ‘shell’ can target denatured collagens, allowing accumulation in tissues with elevated collagen damage/remodeling. In this proposal, we will evaluate the loading of candidate PTOA disease-modifying drugs (with a focus on dexamethasone (Dex)) in refined elastin-collagen nano-vesicles (ECnV) and monitor their stability, as well as passive and hypothermally-triggered drug release. Studies on naïve and ‘injured/activated’ chondrocytes, synovial fibroblasts, and monocyte/macrophages, and articular cartilage and synovial tissue explants, will confirm the cyto-/biocompatibility and quantify the suppression of ‘injury’ markers by Dex-loaded ECnVs. We will conduct in vivo experiments using a non-invasive repeated joint loading (overuse) model of PTOA to demonstrate the selective retention of ECnVs within injured joints after intra-articular (i.a.) injection. Multi-scale in vivo, in situ, and histological/immunohistochemical analyses will be employed to evaluate the pharmacokinetics of passively and hypothermally-triggered cargo release, tissue localization/biodistribution, and the local and systemic biocompatibility/safety of ECnVs delivered to both healthy and early-PTOA joints. Finally, we will characterize the ability of ECnV-based delivery of Dex to improve disease-modifying physiology and PTOA outcomes prophylactically in the aforementioned non-invasive, joint injury model, with standard i.a. liposomal and free-Dex treatments serving as comparators. Although the proposed work focuses on increasing PTOA therapy effectiveness, it will also lay a foundation for the use of collagen-targeting ECnV drug carriers across a broad range of diseases and pathologies characterized by aberrant collagen remodeling.

NIH Research Projects · FY 2025 · 2023-08

Project Abstract Before a newly budded HIV-1 particle becomes fusogenic (and infective) it must undergo maturation. During maturation the contents of the virion transform from a spherical shell into a conical structure after a series of cleavages of the 55 kDa Gag polyprotein by the viral protease. Proteolytic processing starts at the site between the spacer peptide 1 (SP1) and the nucleocapsid (NC) and culminates in the separation of SP1 from the capsid protein (CA). The initial cleavage event effectively separates the NC-gRNA layer from the viral membrane inducing gRNA condensation. The last event activates a molecular switch that triggers a late maturation event, namely the assembly of the mature HIV-1 capsid around the ribonucleic protein. During maturation, the matrix protein (MA), that is embedded to the viral membrane via a myristoyl group, is cleaved from CA, resulting in reordering of the MA lattice and alteration of the composition of the lipid membrane. Although several structures of immature and mature CA and MA hexamers have been solved by sub tomogram averaging, the molecular mechanism connecting the multiple events that occur during HIV-1 maturation are still unclear. Here, we propose to utilize full-scale molecular dynamics simulations, integrating shaped-based coarse- grained and atomistic methods, to determine motion-structures that reveal the mechanistic details of HIV-1 virion maturation. Results from the simulations will inform the engineering of protein mutants for structural analysis and guide the experimental design of functional analyses and testing of hypotheses derived from the molecular simulations.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Variants in the transcription factor SIX1 or its co-factor EYA1 are known underlying genetic causes of Branchio-oto-renal syndrome (BOR), an autosomal dominant disease that results in hearing loss and kidney defects. Recently, a clinical study reported craniosynostosis (CS) in individuals carrying SIX1 BOR variants, including 5’ variants (p.Q11X and p.Q22X) that are predicted to lead to haploinsufficiency; these data suggest that CS may be an undiagnosed defect in BOR. If left untreated, CS can be associated with distortion of skull shape, increased intracranial pressure, and/or brain damage. As defects in the calvarial bone osteoprogenitor cells (OPC) before and/or after birth may lead to CS via increased bone deposition in the cranial sutures, in this application, I plan to address a major knowledge gap regarding Six1 function: What is its role in the development of the calvarial bones? To detect changes in bone development caused by Six1 loss and haploinsufficiency, I will quantitatively analyze head morphology using µCT images and tissue formation using histological analyses (Aim 1). To verify if Six1 and its co-factors have a role the specification and differentiation of OPCs, I will assess gene expression in vivo using RNAscope and qPCR (Aim 1) and in vitro using neural crest-derived mesenchymal precursors and OPCs (Aim 2). Lastly, I will perform single cell RNA-seq and RNAscope to identify cell populations in the supraorbital arch mesenchyme (that gives rise to the rudiments for parietal and frontal bones) that are affected by Six1 loss and haploinsufficiency (Aim 3). Results from this application will shift the paradigm of Six1 function as a cranial placode, neural and muscle transcriptional factor by providing the first direct evidence linking it to normal calvarial development and the pathogenesis of CS. This application will establish Six1-het mice as a new model for craniofacial disease, and will help elucidate the mechanisms by which Six1 variants lead to CS. It will also provide training in soft skills, funding and collaborations required for my next career step. Finally, this training will allow me to bring my extensive knowledge of Six1 transcriptional function in mandible and otic development to a new area of clinically relevant research. Consequently, my research may ultimately prove crucial for CS patient diagnosis and care.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Many people who have experienced a stroke have language and/or speech impairments. These impairments alter how a person communicates, and significantly impacts their participation in life in both major and minor ways. Clinicians who work with individuals with communication impairments, called Speech- Language Pathologists or SLPs, often use questionnaires to learn about their clients’ participation in different communication situations. One such questionnaire is the Communicative Participation Item Bank, or the CPIB. This questionnaire helps SLPs understand the impact of the client’s communication impairment on their daily life, and they use this information to help create treatment plans and goals. However, research evidence tells us that factors other than the speech/language impairment may contribute to how a person answers questions on the CPIB. Some factors that may influence responses to the CPIB include psychosocial factors such as chronic stress, depression, resilience, and self-efficacy. If the SLP does not ask about these factors in addition to providing the CPIB, they may misinterpret CPIB scores and miss important needs of their client. The connection between each individual’s communicative participation and psychosocial factors is often neglected because SLPs have little training on measuring these factors or how they impact treatment and its outcomes. This misunderstanding about clients’ needs may influence treatment plans and goals in a way that does not help individual clients. To address this problem, this research project will examine how responses to the CPIB are related to these psychosocial factors for people with communication impairments after stroke. The project is divided into 2 studies. The purpose of Study 1 is to examine the CPIB and psychosocial factors of chronic stress, depression, resilience, and self-efficacy with individuals with post-stroke communication impairments to understand how these factors may influence their participation in daily life. The research team will ask participants to provide basic information about themselves (age, stroke date, etc.), complete speech and language tests, complete the CPIB and questionnaires that ask about the psychosocial factors, and answer interview questions about the topics presented in these questionnaires. Nine months later, the research team will ask these individuals to repeat the questionnaires and part of the interview to help understand how these factors change over time. The purpose of Study 2 is to examine how SLPs use the CPIB to understand the participation and other experiences of their clients, and how they consider psychosocial factors in treatment goals and activities. The research team will ask SLPs who use the CPIB to complete a survey and follow-up interview about how they use this questionnaire, including how they consider the psychosocial experiences of their clients in treatment planning. The results of this research project will clarify the important but neglected connection between communicative participation and psychosocial factors following a stroke, and provide critical information about how SLPs can use the CPIB to create better treatment plans for individual clients.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Blood pressure (BP)-related diseases continue to be a major public health challenge in both sexes. Men have a higher prevalence of hypertension compared to women until about age 60, after which prevalence is greater in women, reflecting the cardioprotective role of female sex hormones. The high incidence of hypertension in adults is associated with high dietary sodium intake. Thus, investigating sex differences in responses to sodium may be important in understanding these health disparities between men and women. Central sodium sensing is critical in mediating neurohormonal responses to the relative hypernatremia associated with high salt intake. The circumventricular organs (CVOs), including the organum vasculosum lamina terminalis (OVLT) and subfornical organ (SFO), lack a complete blood brain barrier and contain osmosensitive neurons capable of sensing changes in sodium concentration in the blood. The CVOs also mediate sodium-induced changes in sympathetic nerve activity (SNA), vasopressin (AVP), thirst, and BP. Studies in humans show increased activation of the CVOs during acute hypernatremia (hypertonic saline infusion), which is similar to the underappreciated relative hypernatremia that occurs with high sodium intake. However, no human studies have investigated whether there are sex differences in central neural activation during acute hypernatremia. Therefore, the focus of this proposal is to investigate sex differences in functional connectivity of sodium sensing brain regions (SFO, OVLT) and brain regions involved in sympathetic outflow (rostral ventrolateral medulla (RVLM), nucleus tractus solitarius (NTS), caudal ventrolateral medulla (CVLM)) during acute hypernatremia (via hypertonic saline infusion) using blood oxygen level dependent (BOLD) fMRI. We will also assess whether these responses are associated with changes in AVP, norepinephrine (NE), thirst, and BP. This proposal involves 2 specific aims: aim 1 will assess sex differences in change in functional connectivity between sodium sensing brain regions (SFO, OVLT); aim 2 will assess sex differences in change in functional connectivity between sympathoregulatory brain regions (RVLM, NTS, CVLM). We hypothesize that acute hypernatremia will increase functional connectivity between sodium sensing brain regions (Aim 1) and increase connectivity with the RVLM and decrease connectivity with the NTS and CVLM (Aim 2). We hypothesize that men will have greater responses since (1) in salt sensitive rodent models, male animals display larger changes in arterial BP; (2) men have greater AVP release in response to hypertonic saline infusion; and (3) men have greater MSNA, BP, and forebrain BOLD fMRI responses to various cardiovascular stressors. This proposal has the potential to offer important insight into sex-specific mechanisms of BP regulation and fluid balance and will provide the trainee with specific instruction in BOLD fMRI and technical writing, which are critical in his development into a productive research scientist.

NIH Research Projects · FY 2026 · 2023-08

Project Summary / Abstract Delivery via cesarean section (CS) now makes up roughly one third of all births in the United States. After CS delivery, newborns experience lower levels of several ‘birth signaling hormones’ such as oxytocin, vasopressin, and corticosteroids. Birth is a sensitive period for the signaling of these hormones, and so changes in their levels at birth can affect their regulation throughout development. Besides being involved in birth, these same hormones also regulate metabolism behavior in later life. We hypothesize this is why delivery by CS is associated with substantially higher rates of childhood obesity. We have begun to explore the connections between birth mode and subsequent metabolic regulation using the prairie vole (Microtus ochrogaster). The prairie vole is one of the few rodent models that allows us to examine the physiology underlying energy regulation without the burden of chronic cold stress brought on by conventional, room temperature housing. Our recent findings suggest that prairie voles delivered via CS experience changes in their thermoregulation, social behavior, and metabolic regulation sufficient to produce increased weight gain across development. In the present study, we will investigate this further to assess whether voles delivered by CS are at increased risk for visceral adiposity -one of the most dangerous aspects of obesity in humans. We will fully characterize subjects’ energy budgets as well as the brain functioning that underlies metabolism in terms of anatomy, connectivity and the regulation of the birth signaling hormones. Finally, we test whether replacing the missing hormone surge in CS newborns can avoid the metabolic outcomes typically seen in children and voles delivered by CS. In so doing, we hope to offer a simple, straightforward, and cost-effective strategy to reduce childhood obesity in this population. We hypothesize that a CS delivery represents delivery without the full complement of birth signaling hormones and as such will result differences in neuroendocrinology and metabolism throughout development.

NIH Research Projects · FY 2026 · 2023-07

Data-driven modeling of the vibrational spectroscopy of ion channels Abstract The long-term goals of this research program are (1) to develop new computational methods for accurate sim- ulations of linear and two-dimensional infrared (2D IR) spectra of proteins and use the developed methods to simulate recent and design new 2D IR experiments to (2) investigate the mechanisms of ion transport and molec- ular origins of selectivity in the KcsA ion channel and (3) elucidate the conformational and hydrational changes of the voltage-sensing domain of the KvAP channel during voltage activation. Despite decades of research, we still don’t have the direct information on ion channel dynamics and the effects of an applied voltage on ion channel structures. 2D IR spectroscopy is an emerging analytical technique that probes protein dynamics with chemi- cal bond-specific spatial and high temporal resolution. 2D IR spectroscopy is analogous to NMR spectroscopy, except that it uses pulses of infrared light to measure vibrations rather than pulsed magnetic fields for nuclear spins. New methodology improvements expand the frontiers of 2D IR spectroscopy, permitting the study of com- plex biological systems in their native environments. Particularly interesting are systems for which NMR and X-ray crystallography are difficult to apply, such as ion channels. Interpreting congested 2D IR spectra is difficult without simulations that can quantitatively connect spectral features to atomistic structural models. Currently, 2D IR spectra of proteins are modeled using model-driven, mostly empirical, spectroscopic maps that correlate solvent-induced electric field and backbone dihedral angles to vibrational frequencies and couplings. This ap- proach, however, lacks systematic improvability, has limited transferability, provides qualitative accuracy at best, and is inaccurate for peptides in heterogeneous environments. Shifting away from the model-driven paradigm, we will use ab initio-based data-driven approaches based on Graph Neural Networks to accurately model the vibrational spectra of proteins in realistic environments. The proposed methods will provide computational sup- port for the ongoing and future 2D IR experiments on ion channels. The results of the proposed studies will significantly enhance our understanding of the molecular-level mechanisms of function of ion channels. A large spectrum of neurological, cardiovascular, and muscle disorders result from defective ion channel functioning. A better understanding of the origins of these diseases will pave the way for improved therapeutics that target ion channels.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Current at-home COVID tests are not accessible to people with low vision or blindness. To interpret results, people with low vision or blindness may need a sighted assistant, an internet-based image recognition tool, or some other sort of powered implement. Instead of adapting to technologies developed for sighted people, we propose a new platform which provides a no-power tactile readout, i.e., a texture change, to interpret test results. Although COVID antigens are at relatively low concentrations in human saliva, by relying on surface chemistry effects, a relatively small amount of sample can be designed to cause a significant texture change. This project will develop a new class of antibody-conjugated polymers which, in a saliva sample, bind to COVID antigen. In conjunction, we will also develop a test surface designed to maximize tactile feedback upon antigen binding. Upon binding to the test surface, a COVID positive surface will feel distinctive from the negative control, like distinguishing between plastic and glass. To optimize polymer design and test surface design, we use a combination of materials characterization, mechanical testing, human testing, and computational techniques. The project culminates by having low vision or blind users test the device with synthetic saliva solutions containing COVID antigen, present as innocuous protein isolates. Subjects will receive synthetic saliva with and without COVID antigen, and using our platform, will be asked to determine if the synthetic saliva did contain the COVID antigen. As a platform, the technology is not limited to COVID, but could be adapted to either new variants or other use cases, such as pregnancy tests. Our team's expertise combines accessibility experts, synthetic chemists, human psychophysics, computational simulations, surface science, and mechanics. To maximize project success, the project includes people with visual impairments at all stages to ensure practicality.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Storytelling interventions for chronic disease, are described as personal narratives of living with the health condition. Previous storytelling interventions have been effective in promoting healthy behaviors and lifestyle change. For hypertension, storytelling interventions have contributed to reductions in blood pressure among and increased uptake of lifestyle and behavioral change. Groups traditionally underrepresented in health research and interventions, may not have access to innovative lifestyle interventions, but these communities often have the greatest need for access to health interventions. Our preliminary work includes the Penn State Clinical and Translational Science KL2 funded project titled Developing a Storytelling Intervention for African Americans with Hypertension. During the KL2 project, nine African Americans with hypertension receiving care at a Federally Qualified Health Center, were filmed sharing their stories of managing hypertension and sharing helpful tips for lifestyle changes to manage hypertension. Our proposed intervention will consist of three groups: 1) usual care 2) Storytelling (storytelling + educational information) accessed using a study website 3) Storytelling Plus (group storytelling sessions + peer-led educational sessions and goal setting). Peer health coaching has been a successful approach for promoting healthy behaviors and can be easily integrated into a safety-net setting. We hypothesize that incorporating Peer Health Coaches into the storytelling intervention will bolster the engagement with a storytelling intervention. Delivering the intervention using a study website increases the accessibility of the intervention and allows the participant to view study materials at their convenience. The outcomes for the proposed study are 1) to assess the feasibility of implementing the Storytelling and Storytelling Plus interventions by assessing recruitment, retention, acceptability, and fidelity and 2) the preliminary impact on self-reported medication adherence and blood pressure. The long-term goal of this project is to develop storytelling interventions that can be integrated into the clinical workflow of a FQHC and can potentially be disseminated to other FQHC and safety-net settings to promote healthy behaviors and lifestyle change among African Americans with hypertension.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Excess dietary salt intake is associated with cardiovascular disease and is a major contributing factor to the pathogenesis of hypertension. Salt-sensitive hypertension in both humans and rodent models is associated with elevations in plasma or cerebrospinal fluid [NaCl]. The resultant relative hypernatremia activates central circuits to increase sympathetic nerve activity (SNA) and arterial blood pressure (ABP). There are brain NaCl-sensors in the circumventricular organs such as the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO); activation of OVLT/SFO neurons stimulates thirst, vasopressin (AVP) secretion, and SNA, whereas interruption of neurotransmission in OVLT/SFO lowers ABP in salt-sensitive models. However, the mechanisms by which OVLT/SFO neurons sense extracellular [NaCl] are not known. Recent data suggest the Na+-K+-2Cl- co-transporter (NKCC2) is not kidney specific but is also expressed in brain regions that regulate whole body NaCl and water homeostasis. The central hypothesis of this proposal is that the ingestion of excess dietary salt elevates extracellular [NaCl] to activate NaCl-sensitive neurons in the OVLT/SFO through NKCC2. In turn, this activates descending pathways to elevate SNA and ABP. Furthermore, we hypothesize that these NaCl-sensing mechanisms are sensitized in salt-sensitive humans, since our preliminary data show hypernatremia evokes a greater increase in OVLT discharge of Dahl-salt sensitive versus Dahl-salt resistant rats fed a high salt diet. We propose 2 specific aims. Specific Aim 1 will determine the extent by which NKCC2 mediates NaCl-sensing in OVLT/SFO neurons and elevate SNA and ABP to acute NaCl loading or chronic salt- sensitive hypertension in rodents. Specific Aim 2 will test the hypothesis that an NKCC2 antagonist will blunt hypernatremia-induced central neural activation and SNA in salt resistant and salt sensitive adults with high BP, and that central neural activation will be greater in salt sensitive adults, suggesting heightened sodium sensing. Successful completion of these aims will provide needed information on the cellular elements that mediate intrinsic NaCl-sensing of hypothalamic neurons and provide novel data on central sodium sensing in salt sensitive humans. This is a translational R01, bringing two laboratories together that have a track record of successful collaboration, and these studies will provide a framework for the development of novel therapeutic treatments of salt-sensitive hypertension.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT The Day Lab engineers nanoparticles (NPs) with unique physicochemical properties to transform the treatment of various diseases and elucidates how architecture impacts function by studying nano/bio interactions from the subcellular to whole organism level. The NPs we develop enable high precision therapy by: (1) delivering antagonistic antibodies or nucleic acids to cells to inhibit genes that drive disease progression, (2) supplying heat or other payloads only to diseased cells in response to activation with tissue-penetrating near-infrared light, or (3) facilitating cell-specific cargo delivery by using cell-derived membranes as coatings that minimize immune recognition and enable target cell binding. We are applying our technologies to manage aggressive cancers, blood disorders, and maternal/fetal health conditions. Further, we are proving through rigorous studies that both what is packaged in NPs and how it is packaged dictate therapeutic potency. Much of our work advancing nanomedicine over the last five years was funded by the MIRA program. Moving forward, we will use our acquired tools and knowledge to probe unanswered questions in nanomedicine and advance the ability of NPs to surpass biological barriers. There is currently an undesired disparity between preclinical and clinical performance of nanomedicines that is driven by biological barriers that limit NP delivery efficiency, efficacy, and safety. These include immune barriers (protein corona formation leading to macrophage clearance), vascular barriers (limited extravasation), and tissue barriers (poor penetration through extracellular matrix, mucus, etc. to reach desired cells in heterogeneous populations). Over the next five years we will address these biological barriers through mechanistic studies that incorporate and adapt NPs previously developed in our lab to enhance delivery and efficacy. Specifically, we will investigate questions related to protein corona-mediated immune clearance, the role of inflammation in NP extravasation, and NP interaction with reproductive tissue barriers and the vaginal microbiome. Answering these questions will guide the development of NPs with improved clinical performance. In addition to advancing the broader field of nanomedicine, the information gained will lead into the long-term research of the Day Lab addressing both extracellular and intracellular barriers to nanomedicine. Overall, our work has both basic scientific and translational significance, and our discoveries will transform the application of nanomedicine to diverse healthcare problems by developing technologies with unmatched clinical performance.

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

Volet: Bourses de recherche postdoctorale; Domaine: Organismes vivants; Objet: Virus; Objet: Parasites; Application: Sciences et technologies; Application: Sécurité; Mots-clés: INNOCUITE, VIRUS, PARASITES, ALIMENTS, INFECTIOSITE, ENVIRONNEMENT

NIH Research Projects · FY 2026 · 2023-03

Generalized fluctuation test for deciphering phenotypic switching within cell populations The inherent probabilistic nature of biochemical reactions coupled with low-copy number components results in significant random fluctuations (noise) in mRNA/protein levels inside individual cells. How cellular biochemical processes function reliably in the face of such randomness is an intriguing fundamental problem. A long-term vision of our lab is to develop new mathematical and computational tools for studying stochastic dynamics of cellular biochemical processes, and use these tools to systematically understand how noise affects biological function and phenotype. As a consequence of noise in gene product levels, single cells within an isoclonal population can differ in their expression profile and reside in different pheno- typic states. The dynamic nature of this intercellular variation, where individual cells can transition between different states over time makes it a particularly hard phenomenon to characterize. Unexpectedly, phenotypic heterogeneity within a population can play important functional roles in diverse biological processes, from driving genetically-identical cells to different cell fates to allowing microbes and cancer cells to hedge their bets against uncertain environmental changes. The Luria-Delbrück experiment, also called the “Fluctuation Test", introduced 75 years ago, demonstrated that genetic mu- tations arise randomly in the absence of selection – rather than in response to selection – and led to a Nobel Prize. The innovation of this project is to leverage this classical experiment in conjunction with mathematical modeling to char- acterize reversible and irreversible switching between cell states. The key advantage of the proposed method is that it is general enough to be applied to any proliferating cell type, and only involves making a single endpoint measurement. This is especially important for scenarios where a measurement involves killing the cell (for example, assaying whether a bacte- rial cell is in a drug-sensitive or drug-tolerant state or doing RNA-sequencing), and hence the state of the same cell cannot be measured at different time points. The project will develop mathematical tools for characterizing phenotypic switching between an arbitrary number of states using the fluctuation test, and such techniques will for the first time differentiate between an irreversible cell-state transition via genetic alterations vs. a reversible epigenetic transition. These tools will be first benchmarked with in-silico generated data and then applied on experimental datasets investigating diverse prob- lems, including characterizing drug-tolerant states in bacterial/fungal cells, understanding differences in viral susceptibility between single human cells within the same clonal population, and uncovering the transient dynamics of stem cell states that bias individual cells to different differentiation fates. Our preliminary work reveals plasticity in drug-tolerant states in bacterial, fungal, and cancer cells with different inheritance timescales. To understand the origins of cell states, the project will develop computational tools for inferring causal interaction networks from single-cell expression data. These tools will uncover how network topologies change across cell states and modeling the stochastic dynamics of underlying biochemical networks will mechanistically capture transitions between states. Overall, tools developed through this project will result in a fundamental understanding of how single-cell difference arises from stochastic epigenetic processes without any changes to DNA, and drive translational approaches to perturb cell states for therapeutic benefit.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Key to multicellularity is the coordinated interaction of the various cells that make up the body. Indeed, patterning of embryos, establishment of cell type diversity, and formation of tissues and organs all rely on cell-to-cell communication. Thus, arguably one of the most important principles of biology involves “one group of cells changing the behavior of an adjacent set of cells, causing them to change their shape, mitotic rate, or fate”. Conventional methods of reproducing biological patterns and cell-fate in vitro suffer from multiple limitations. Previous work on understanding pattern formation has relied on delivering global stimuli and studying reaction- diffusion mediated patterning of cell fates in the cell culture. Another method has been to generate morphogen gradients using signaling molecule patterned surface or optogenetics. However, all current methods produce static patterns and give neither precise spatial nor temporal control over the cell fate. My research group aims to overcome this critical challenge, via a unique and novel cyber-bio system, in which microrobots direct the biological system, in a closed loop approach, to enable position-specific functionality and reduce noise – to direct cellular fate leading to the formation of cellular structures. Inspired by “human-in-the loop” approaches for engineering systems that must interact with complex, living individuals, we propose a “µrobot-in-the-loop” approach in which physical signaling among cells is substituted with microrobot-controlled inputs to afford excellent spatiotemporal precision and feedback control in directing cell behavior. Our efforts in the next five years would focus on designing and fabricating microrobots along with developing control algorithms for automated actuation of the microrobots. We will use these microrobots to deliver morphogens at precise positions in a cellular system which would alter cell fate at those positions only. We would also use this technology for controlling the formation of multilayer cellular structures. We would extend this to three dimensional tissues by interfacing microrobots with organoids. The proposed work is important because it would demonstrate how individual cells in a tissue volume can be spatially and temporally targeted for manipulation. This methodology applies more dynamic control over differentiation factors, which allows for increased understanding of complicated cell fate and differentiation events during cancer, development, or fibrosis as just a few of many applications.

NIH Research Projects · FY 2025 · 2022-09

PROJECT DESCRIPTION 1 Motivation Stroke is a leading cause of long-term disability in the United States. Stroke survivors now constitute around 3% of the over-20 population, with 50% of stroke-affected subjects left with impaired propulsion on the paretic side, resulting in asymmetric movement and compromised balance [1]. The hemiparetic gait observed in many individuals post-stroke is slower and more metabolically expensive than in healthy individuals [2–6], and is a primary contributor to reduced community participation and quality of life [7–11]. Contemporary approaches to gait training are based on repetitive therapy often conducted on treadmills [12], with variants including the combination of human or robotic assistance [13], body weight support [14], and functional electrical stimulation [15]. Robotic intervention enables systematic and accurate modulation of joint-level variables, such as assis- tance torques and joint angles/velocities. Robotics is an intriguing tool for gait training, but the capability of using robots as tools to support locomotor learning for rehabilitation purposes has not yet been fully demonstrated. Earlier implementations of robot-aided gait rehabilitation provided non-convincing or nega- tive results [13, 16], as extensively quantified in a meta-analysis [17]. Currently, the effects of robot-aided gait training in stroke have yet to exceed those achieved with conventional therapy methods [17]. We speculate that such limitations are mostly imputable to the controllers used for robot-aided gait train- ing. The majority of robotic devices, designed specifically to rehabilitate gait, utilize one of the various controller forms (e.g., force control, position control, or impedance control), and controller update methods (e.g., assist-as-needed control, inter-limb coordination, or finite state machine), to ultimately promote spe- cific features of gait kinematics [18]. The limited efficacy of these methods could be due to their lack of targeting specific functional mechanisms of gait, which are only partially described by joint kinematics. From an extremely reductionist perspective, walking is pushing ones' center of mass in a desired direction while not falling. Fundamentally, walking involves three main sub-tasks: propulsion, limb advancement, and balance [19]. Of these components, limb advancement may be based on kinematic control, but is the least energetically demanding. Instead, the sub-tasks of propulsion and balance require precise neuromuscular coordination, and specifically mediation of the interaction forces between the walker and ground. Despite their fundamental importance, there have been very little efforts in rehabilitation robotics in developing robot-aided methods to study and/or train propulsion and balance in post-stroke rehabilitation. The overarching goal of the proposed research Measure is to advance the science of therapeutic engineering Walking Surfac~ for gait by identifying optimal robot interventions " ., and therapies with specific functional outcomes. Stiffne.ss Perturbations Model Those interventions will be developed using a new modeling approach to target enhanced propulsion Evaluate and balance in stroke survivors. The sense-plan- act paradigm in robotics will be applied in a unique way to robot-assisted model-informed rehabilita- tion research. The proposed framework will inte- grate robotic solutions that will allow the creation of comprehensive models of sensorimotor mecha- nisms of gait. These models will then inform a set of interventions to stroke survivors, the outcomes of which will be fed back to the developed models to uncover and suggest novel patient-specific train- ing strategies. The proposed approach will enable Figure 1: Proposed integrative research framework fol- a better understanding of essential mechanisms lowing the sense-plan-act paradigm in robotics. responsible for walking and lead to the design of optimized and personalized post-stroke rehabilitation strategies. The overall framework of the proposed 49

NIH Research Projects · FY 2025 · 2022-09

Project Summary Many diseases could be prevented or treated by enlisting the immune system to recognize a specific antigen. Bacterial diseases warrant heightened attention as many bacterial pathogens lack efficacious vaccines and exhibit rising rates of antibiotic resistance. These pathogens often find many ways of evading immune system detection, including varying their most immunogenic antigens. While many virulence-related proteins can be strongly conserved across pathogen serotypes, they often exhibit weak immunogenicity that is insufficient to draw the response of the immune system. In this project, we ask: Are there strategies to shine a light on live bacterial antigens for increased recognition by immune cells? Furthermore, can we couple these strategies to shelf-stable delivery vectors that are simple to administer to patients across the world? We propose a transformational approach to expand the list of candidate antigens for use in live bacterial vaccine vectors by teaching Bacillus subtilis to produce and harness an immunogenic amino acid. This amino acid has been demonstrated to terminate immune self-tolerance when substituted on the surface of autologous proteins in mice. Site-specific introduction of nitrated residues within proteins has resulted in presentation of a neoepitope that is recognized by helper T cells for subsequent activation of B cells that produce polyclonal antibodies. It stands to reason that the immunogenicity of many weakly immunogenic foreign antigens could be increased using this strategy, though this has not yet been tested. One challenge is that prior studies also established a critical but poorly understood role of the MHC Class II locus in enabling immune response to nitrated antigens. Our project will investigate the potential of spore-displayed nitrated antigens as a transformational vaccination platform for bacterial disease, with the Shigella invasion protein antigens as a model system. We will first perform animal studies with Shigella antigens that are weakly immunogenic but strongly conserved across pathogen serotypes to determine if nitration can increase their immunogenicity. To better understand where nitrated residues should be placed for optimal recognition by immune cell machinery, we will develop a high-throughput microbial display platform to screen MHC-II preference towards unnatural peptide ligands. In parallel, we will develop tools to enable site-specific incorporation of the immunogenic amino acid within proteins fused to the spore coat of B. subtilis. Recombinant spores of this non-pathogenic organism can be orally administered and maintain immunization efficacy after exposure to harsh conditions. The spore-based platform has promise to overcome limitations in the manufacture, transport, and administration of vaccines; however, the platform has low immunogenicity. Our strategy to form nitrated residues using this platform could overcome that limitation. From this project, we will gain insights about the requirements for enhanced immunogenicity due to nitration, and we will advance towards a platform technology for immunization that features shelf-stability and oral delivery.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Due to increased demand for biologics, there is an ongoing need to scientifically and commercially advance manufacturing in both upstream cell culture and downstream purification steps. Our goal is to provide an experimental infrastructure complemented by a computational framework to investigate the continuous manufacturing of monoclonal antibodies (mAbs). The modeling approach will be based and validated using specific experimental evidence to enhance our process understanding and improve model performance and utilization during the design phase. The proposed innovations stem from the following objectives that will be delivered as the outcome of this project, namely, 1) To develop a multiscale model for perfusion bioreactor capturing the effects of operating parameters and cell line characteristics on critical quality attributes (CQAs) validated by experimental results; 2) To develop methods for optimizing continuous chromatographic operations, including primary capture and polishing steps, under a range of process conditions for optimal clearance of process- and product-related impurities; and 3) To develop predictive models that will enable determination of optimal operating conditions with direct coupling of upstream and downstream units accounting for product quality attributes. To enable process control, which is the ultimate target of advanced manufacturing, we will explore the design space and identify the relationships between critical process parameters (CPPs), critical material attributes (CMAs), and targeted CQAs. We will develop predictive models for all the important unit operations, validated by experiments, that can be used to determine the design space along with statistical analysis of experimental data to identify all critical parameters/attributes. The validated models will then be used as a virtual tool to perform risk assessment for in-plant downstream operations such as scale-up/start-up/shutdown and compare process operating scenarios. In terms of combining the developed strategies, we envision the integration with a continuous upstream facility to achieve a fully automated continuous biomanufacturing line exploring and optimizing the process interactions. This proof-of-concept line will be used to clearly quantify risk and performance-based metrics.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Tendon overuse initiates mechanical damage that leads to chronic tendinosis (degeneration) and tendinopathy (clinical presentation with pain), which are common and notoriously difficult to treat. Despite the widely accepted role of loading in tendinosis, the structural, mechanical, and cellular mechanisms by which loading leads to initiation and progressive damage in tendinosis remain unknown. We hypothesize that overload causes micro-scale structural and mechanical damage, which alters load transmission to cells, driving the multi-scale structural and molecular progression of tendinosis in a vicious cycle. To determine the mechanisms involved in tendinosis, a preclinical in vivo animal model of tendon overuse and multiscale assessments of tendon structural and mechanical damage and interrogation of cellular mechanotransduction and intracellular signaling mechanisms are all required. This is because the physiological processes in tendinosis involve tissue-scale tendon loading that is transferred to the micro-, nano-, and molecular-scale, where microstructural damage and cellular mechanotransduction signaling occurs. Our team has recently established a model of tendinosis using rat synergist ablation (SynAb), where we remove the Achilles tendon, which overloads the synergistic plantaris tendon without directly injuring it. Our pilot data exhibit hallmark features of human tendinosis (increased area, collagen disorganization, proteoglycan accumulation, collagen Type III production, and reduced tensile modulus). Our long-term goal is to enable interventions for tendon regeneration and rehabilitation to treat tendinopathy and prevent its progression. The objective of this proposal is to determine the mechanisms responsible for onset and progression of tendinosis in the SynAb model of tendon overuse in the following aims: Aim 1: Assess the multiscale structural changes following the onset and progression of tendinosis. Aim 2: Quantify the multiscale mechanical properties and damage following the onset and progression of tendinosis. Aim 3: Interrogate changes in tenocyte mechanoresponse and cytoskeleton during tendon overload. This study will determine the mechanisms responsible for the multiscale damage in overuse tendinosis and establish these in the context of key hallmarks of human tendinosis using a preclinical in vivo model. Quantifying multiscale damage and cellular mechanisms by which loading leads to tendinosis is critical for designing and evaluating interventions to prevent and treat tendinopathy.