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BIOPHYSICAL PROPERTIES ENCODE FUNCTIONAL SUBTYPES OF SINGLE CELLS in HEALTH, AGING, AND DISEASE
No full textDuring aging, cells undergo significant changes that limit their ability to maintain proper tissue homeostasis and function. One such mechanism is the age-associated accumulation of senescent cells. Senescence represents a degenerative cell state characterized by stable proliferative arrest1–3, upregulation of cyclin-dependent kinase inhibitors (e.g., p16/p21)4–6, increased secretions of pro-inflammatory molecules (SASP)7–9, and drastic changes in cell morphologies10–15. Although these senescence-associated changes are critical to our current understanding, they are limited. These limitations stem from the fact that a) there are currently no universal senescence biomarkers16,17, b) senescence is typically defined as a binary cell state15,18,19, and c) it is unclear what drives the age-associated accumulation of senescent cells. This dissertation focuses on leveraging biophysical properties of single cells to identify senescent cells and further elucidate functional differences.
Using primary dermal fibroblasts, I combined single-cell imaging, machine learning, multiple senescence induction conditions, and multiple protein-based senescence biomarkers to establish functional subtypes of senescence. Leveraging single-cell morphologies, I defined eleven unique morphology clusters, with the abundance of cells in each cluster dependent on the mode of senescence induction, the time post-induction, and the donor age. Of these eleven clusters, I identified three bona-fide senescence subtypes (C7, C10, C11), with C10 showing the strongest age-dependence across a cohort of fifty aging individuals. In the broader context of aging, I identified that baseline morphology, not chronological age, was a better predicter of response to senescence induction. Finally, to determine the functional significance of these senescence subtypes, I profiled their responses to senotherapies, specifically focusing on Dasatinib + Quercetin (D+Q). Results indicated subtype-dependent responses, with senescent cells in C7 being most responsive to D+Q.
Altogether, I provide a robust, single-cell framework to identify functional senescence subtypes with applications for next-generation senotherapy screens and the potential to explain heterogeneous senescence phenotypes across biological settings. Complementary work including understanding response modalities of lung epithelial cells to particulate matter underpin generalizability of the morphological approach. The framework outlined in this dissertation offers a powerful and generalizable paradigm to elicit complex biological phenomena nested elegantly in single cell morphology
TOWARD UNDERSTANDING THE STRUCTURAL DETERMINANTS OF C2ALPHA-LACTYLTHIAMIN DIPHOSPHATE DECARBOXYLATION ON THE ANTIBACTERIAL TARGET 1-DEOXY-D-XYLULOSE-5-PHOSPHATE SYNTHASE
No full textAs pathogens continue to evade standard antibiotic treatments, it is crucial to develop antibacterial strategies that act on new microbial processes, such as pathogen metabolic adaptation. One such target is the critical bacterial enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), which catalyzes the thiamine diphosphate (ThDP)-dependent formation of DXP from pyruvate and glyceraldehyde-3-phosphate (GAP). DXP is a bacteria-specific, essential metabolite that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate (PLP), and ThDP. This positions DXPS as a key player in pathogen central metabolism, and in addition to structural and mechanistic features, suggests DXPS could have alternative activities in vivo, supporting that it is vital for metabolic adaptations. In the DXPS mechanism, binding of a small molecule “trigger,” such as GAP, is required to induce conformational changes essential for decarboxylation of the first enzyme bound intermediate, C2α−lactyl-ThDP (LThDP), which is long lived on DXPS prior to trigger binding. This thesis aims to advance our understanding of this gated mechanism and develop inhibitors targeting unique DXPS states.
How LThDP persists on DXPS prior to trigger-induced decarboxylation and the breadth of small molecules capable of activating LThDP for decarboxylation are not well understood. Chapter 2 describes an active site network on DXPS that functions to prevent LThDP activation in the absence of co-substrate. Our findings indicate that substitution of network residues, as well as nearby residues believed to contribute to network charge distribution, predictably affects LThDP reactivity. In Chapter 3, DXPS multifunctionality is explored, where Pentose Phosphate Pathway (PPP) intermediate xylulose 5-phosphate is described as a donor substrate on DXPS, and alternate aldehyde-based small molecule triggers are identified, including PPP intermediate erythrose-4-phosphate. Finally, Chapter 4 describes studies aimed to develop selective bisubstrate triazole-based acetylphosphonate (TrAP) DXPS inhibitors. Here, we identified EcDXPS residue R99 as a new targetable feature to exploit for drug design, and discovered a low-nanomolar TrAP-based inhibitor bearing a gem-dibenzyl glycine moiety. Taken together, the results presented in this thesis work take key steps to address gaps in our knowledge of the DXPS gated mechanism and advance our understanding of how his mechanism can be targeted for the development of selective DXPS inhibitors
Modeling Cell Motility: From Single Cells in Confinement to Collective Behaviors in Tissues
No full textCell motility is crucial for physiological processes such as development, tissue repair, and cancer metastasis. In this dissertation, I employ theoretical and data-driven models to elucidate the physical principles underlying cell motility, aiming to enhance our understanding of cell movement and interactions in various confining environments.
The motility of eukaryotic cells is significantly influenced by their environment. Confined cells exhibit different motility patterns compared to those on simple two-dimensional substrates, with variations also observed between different cell types. This work explores how cells respond to environmental constraints and models these behaviors using computational phase field approaches. Notably, I develop a unified cell model that captures diverse experimental behaviors with the assumption that contact with non-adhesive substrates inhibits cell movement. Integrating data-driven techniques with simulations, I also reveal how simple quantitative changes in cell properties can lead to dramatic qualitative changes in cell behavior.
In addition to moving as single entities, cells can move together as a collective. This dissertation examines collective cell migration at two levels: cell-cell interactions in colliding cell pairs and the morphogenesis of branching tips in large cell clusters. Using a phase field model, I highlight two predictors of collision outcome -- relative cell speed or contact angle -- that remain robust to perturbations. These predictors depend on the chosen assumption of how cells select their preferred direction of motion, and since they are experimentally measurable, they can provide insights into cell decision-making processes. Additionally, by simulating branching morphogenesis in cell clusters, I suggest that cells with lower surface tension or higher sensitivity to external chemical cues can drive the formation of and localize to branching tips. These models align with recent experimental observations and offer a framework for understanding how cells navigate and interact in crowded environments
Dynamic Properties of Aging Human Dermal Fibroblasts Encode Functional Subtypes of Senescence
No full textAs humans age, the capability of cells to regulate their physiology decreases. Biophysical properties of these cells, such as morphology, biomechanics, and motility have been shown to encode information on aging in healthy donor samples. A key mechanism driving the decreased capability to regulate cellular and tissue physiology during aging is the accumulation of senescent cells. Cellular senescence is characterized by proliferative arrest, upregulation of cyclin-dependent kinase (CDK) inhibitors such as p16 and p21, excretion of senescence associated secretory phenotypes (SASPs), drastic changes in cell and nuclear morphologies, and changes in focal adhesions. Understanding the role of senescence as a functional biomarker of aging is key as our current understanding is limited due to a lack of universal senescence biomarkers. This thesis focuses on leveraging the dynamic biophysical properties of cells, namely single-cell motility, to identify senescent cells and its role in age-associated cellular changes.
Using a gender-balanced cohort of primary human dermal fibroblasts, we combined emerging data science approaches with the single-cell behavior profiling of healthy and senescent fibroblasts to classify cells based on senescent status. We identified eight unique motility clusters, each containing a combination of healthy and senescent cells from different individuals highlighting the heterogeneity within single donor samples. We show that senescence drives global changes in cell motility with a decrease in displacement based motility features and an enrichment of turning angle based features demonstrating that senescent cells lose their capacity for high displacement and loss of directional coordination (i.e., increasingly tortuosity). We additionally show that there are strong age-dependent changes in young (20-40 years) and old (65-90 years) samples for both healthy and senescent samples, while middle (40-65 years) samples exhibit higher heterogeneity, supporting the notion of a fractional-redistribution of motility states during aging even with the accumulation of senescence.
Altogether, the data presented in this thesis offers a comprehensive understanding of a mechanistic framework of aging with applications to defining high-throughput biomarkers and cheaper alternatives to understand functional aging and senescence
Regulation of Thyroid Hormone Signaling in Cone Subtype Specification in Human Retinal Organoids
No full textVision begins with the detection of light by specialized photoreceptors, rods and cones, in the retina. Humans have three types of cones: blue or S, green or M, and red or L cones, which detect specific wavelengths of light. The mechanisms underlying human cone photoreceptor specification are poorly understood. Studies in model systems have implicated two signaling pathways in regulating photoreceptor development: retinoic acid (RA) signaling and thyroid hormone signaling. However, differences in cone subtypes, patterning, and morphology of the retina between species make this an important question to understand in humans.
Human stem-cell derived retinal organoids are a model system which can be used to observe, manipulate, and interrogate mechanisms of human development. Using organoids, we previously showed that S cones are specified before L/M cones, and thyroid hormone signaling through thyroid hormone receptor B (THRB) promotes terminal L/M cone fate. The temporality of this decision suggests that thyroid hormone signaling
is intrinsically regulated in the developing retina.
We used human retinal organoids to understand how thyroid hormone signaling is regulated to specify photoreceptors. We identified DIO3, an enzyme that degrades thyroid hormone, to be a regulator of photoreceptor fates. DIO3 is expressed in retinal progenitor cells and its expression turns off as cells differentiate, intrinsically tying thyroid hormone levels to retinal development. We showed DIO3 is important in regulating the timing of
photoreceptor specification, the quantity of photoreceptors (especially S cones), and the
terminal fate of photoreceptors. A negative feedback loop, in which excess thyroid
hormone signaling results in an increase in DIO3 (degradation) and a decrease in THRB (receptor) and MCT8 (transport) expression, buffers the system. Together, these mechanisms regulate dynamic thyroid hormone signaling to ensure proper and robust photoreceptor specification
Health Service Utilization among Chinese Older Adults with Disability and Cognitive Impairment: Evidence from a Cohort Study
No full textAging is a significant global public health issue, with China home to the largest and fastest-growing aging population. In recent decades, the number of years lived with disability due to dementia has risen notably in Asia, particularly in China and India. Although healthcare systems aim to extend healthy life expectancy, a substantial proportion of older adults with cognitive impairment in China do not receive adequate treatment, and many disabled older adults express dissatisfaction with the medical care they receive. This study explores the development of vulnerability among older adults with disabilities and cognitive impairment and how these changes influence their use of healthcare services. Understanding these trajectories is essential for designing effective interventions and policy measures. Using data from the China Health and Retirement Longitudinal Survey (2011-2018), we applied group-based trajectory models to identify diverse patterns in cognitive function and physical disability among Chinese adults aged 60 and above. Three cognitive function trajectories were identified: those with stable high cognitive function, those with moderate function and rapid decline, and those with severe impairment and fluctuating deterioration. Similarly, disability trajectories were classified into low, moderate with rapid progression, and high with rapid worsening. Factors such as education, income, medical insurance, gender, and marital status significantly influenced these trajectories, highlighting the need for tailored interventions.From 2011 to 2018, outpatient service utilization declined while inpatient care increased, with higher disability levels leading to greater service use. Outpatient services were more frequently utilized by individuals with higher education, chronic diseases, and greater disability, whereas inpatient services were more common among older adults with higher education and income. To address these challenges, we recommend that the Chinese government focus on improving education, economic security, and medical insurance policies for older adults, particularly in underserved regions. Strengthening healthcare infrastructure and increasing access to quality services will be crucial in mitigating cognitive decline and disability, ultimately enhancing the quality of life for China's aging population
Continuous Health Monitoring with Distributed, Networked Wearables
No full textIn the last 20 years, the impact of diseases or injuries affecting the autonomic nervous system (ANS), specifically high blood pressure and traumatic brain injury (TBI), has increased without sufficient resolution. Nearly 50% of the adult US population has high blood pressure (BP), medically known as hypertension, and the associated mortality rate has more than doubled. It is estimated that the annual medical costs of hypertension are between 198 billion, making up more than 3% of total US healthcare expenditure. It is well established that early identification and management of hypertension can significantly reduce mortality and greatly improve the quality of life for those living with the condition, however, less than 25% have their blood pressure under control. Current approaches to measure BP for the management of hypertension suffer from inconvenient, time-consuming, and infrequent readings that result in their limited effectiveness.
TBI-related pathologies, which affect approximately 2% of the population, incur an economic burden exceeding 600,000 to $1.8 million not including the value due to lost productivity which can be a factor of magnitude higher. Current attempts to assess TBI present shortcomings such as subjective rating scales, determinations made with unmeasured data, and infrequent assessments. In fact, studies report that globally accepted methods of assessing TBI have been shown to have accuracies as low as 34%. Proper management of both these diseases has the potential to greatly reduce the personal and economic burden they incur by preventing comorbidities and pre-mature mortality as well as reducing the reliance on limited medical resources reserved for the most extreme cases.
To address these problems, the research I conducted was led by 3 major aims. 1) Demonstrating that noninvasive multisite wearable recordings of motor activity could capture underlying features predictive of cognitive progression and decline in TBI patients, 2) developing a novel noninvasive multisite multimodal wearable capable of measuring a range of biosignals moderated by the ANS continuously without manual intervention, and 3) utilizing the output of the designed wearable system to make determinations on unmeasured but related components of the ANS such as blood pressure for the evaluation of current and future health states
IMPROVING EMPLOYEE RETENTION: CHANGING ORGANIZATIONAL HIRING PROCESSES
Get PDFThe growing rate of voluntary employee turnover is having devastating effects on organizational success. By using relevant organizational models and theories, this paper is an extensive literature review supplemented with secondary data analysis of qualitative textual data. Its purpose is to identify the causes of voluntary employee turnover and evaluate recommended methods for employee retention. Across all literature and data, the main themes that support employee retention are possessing a positive organizational culture, providing comprehensive training and professional growth opportunities, and promoting a healthy work-life balance. This information was reframed and placed into a usable plan focusing on a structural change to organizational hiring processes to improve employee retention. This plan includes screening for applicants who are naturally less inclined towards turnover, assessing which applicants are the best organizational fit based on organizational culture and personal attitude, and providing a comprehensive onboarding process that includes training and outlining career growth opportunities
ACADEMIC STRESS IN ELITE HIGH SCHOOLS: PERCEPTIONS OF STUDENT WELLNESS AND POTENTIAL INTERVENTIONS
No full textStudents experiencing academic stress in elite high schools has been documented worldwide. Though students enrolled in elite high schools often have more resources available to them, they also experience greater amounts of academic stress than their peers in other institutions. Stress often manifests both somatically and psychologically, with students exhibiting multiple distinct symptoms of stress per week. Eight semi-structured interviews were conducted with student-facing faculty at Ultimate High School to determine faculty perceptions of the pervasiveness of academic stress in the student body, how students manifest stress, and how the school is supporting students. Interviews were transcribed and analyzed for codes which were consolidated into themes. Emergent themes indicate that academic stress is pervasive in the institution with students experiencing both psychological and somatic symptoms of stress. Further, though the school has put supports in place to mitigate academic stress experienced by students, those supports are not adequate. Potential interventions to mediate academic stress include parent education workshops, teacher professional development, and peer-to-peer support groups. A theory of treatment, logic model, and evaluation is included for the parent education workshop intervention
A MULTI-METHOD STUDY OF HOMELESSNESS AND DRUG USE ENVIRONMENTS IN BALTIMORE CITY, MARYLAND
No full textBackground: The United States is experiencing intersecting overdose and housing crises. The objectives of this dissertation were to (1) explore the interplay of adverse childhood experiences (ACEs), homelessness, and well-being through the lens of ontological security (i.e., a sense of security in one’s social and physical environment and identity) among people experiencing unsheltered homelessness (PEH-U); (2) examine how PEH-U perceive their micro drug use environments and how these environments shape their drug use practices and overdose risk; and (3) investigate associations of place characteristics with non-fatal overdose and whether experiencing homelessness modifies observed associations.
Methods: Data was obtained from the AIDS Linked to Intravenous Drug Experience Study. Data collection and analysis for aims 1 and 2 were guided by interpretative phenomenological analysis, and semi-structured interviews were conducted in-person. For aim 3, multilevel logistic regression models were fit to estimate associations of place-based exposures and non-fatal overdose from 2015-2019. Effect modification by status of experiencing homelessness was assessed using interaction terms.
Results: Aim 1 included data from six participants. ACEs disrupted participants’ physical and emotional security and bodily autonomy during childhood, as well as precipitated experiences of youth homelessness. During periods of adult homelessness, participants described efforts to maintain their sense of ontological security.
Aim 2 included data from nine participants. In public and semi-public settings, participants’ efforts to find secluded drug use spaces hindered their ability to engage in safer drug use practices. Private spaces enhanced participants’ sense of safety, but access was inconsistent and required the exchange of drugs and other resources. A building for private, supervised drug consumption was described as an ideal drug use space.
Data from 680 participants contributing 3,332 study visits were included in aim 3. In multivariable analyses, only census tract-level crime rate (aOR = 1.50; 95% CI: 1.01, 2.24) was significantly associated with subsequent non-fatal overdose. Null associations between place characteristics and subsequent non-fatal overdose were detected within strata of experiencing homelessness.
Conclusions: Collectively, this dissertation’s findings underscore the need for trauma-informed services; low-barrier, long-term housing; and multi-level interventions grounded in harm reduction principles for people experiencing homelessness who use drugs