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Basic Science of Cartilage Collagen Damage and Fatigue Wear Resistance, and Clinical Strategies for Repair
No full textArticular cartilage is a specialized tissue lining diarthrodial joints in the body, where itserves to reduce friction and wear, and sustains contact forces for over 100 million loading cycles in a 60+ year lifespan. Over time cartilage may degrade resulting in osteoarthritis (OA), a painful, debilitating condition. Despite being the leading cause of disability in the U.S. and affecting 52 million Americans, limited early-stage interventions exist, largely due to the complex multifactorial progression of the disease. A clear understanding of the structural contributions of healthy cartilage to its function, as well as the structural changes mediating OA progression are essential precursors to the development of early-stage diagnostic and treatment strategies. As such, the work of this dissertation seeks to improve understanding of the articular cartilage structure-function relationships through (1) investigating the contributions of the collagen to the mechanical function of cartilage (2) probing the intrinsic mechanisms by which cartilage health is maintained, and which may be disrupted in OA.
To isolate the functional significance of the type-II dominant collagen extracellular matrix(ECM) of articular cartilage, Chapters 2 and 3 utilized an enzymatic treatment to achieve near-complete (>97%) removal of proteoglycans (PGs) from the tissue. Experimental data was used to develop and validate a reactive viscoelastic model of intrinsic collagen viscoelasticity. Applying this model to rapid loading configurations, it was discovered that intrinsic viscoelasticity of type- II dominant collagen in immature bovine articular cartilage contributes non-negligibly to viscoelastic response of the intact tissue, but that other sources of flow-independent viscoelasticity (such as the contribution of PGs) are likely also needed to account for the enhanced tissue modulus under rapid loading. In Chapter 3 the curling of mature bovine cartilage strips at various ionic concentrations was measured, before and after enzymatic removal of PGs. Results showed some curvature of the sample after PGD, implying that residual stress does arise within the type II matrix of the tissue, not only due to the presence of PGs imparting a Donnan osmotic pressure. When cartilage damages under reciprocal loading, as is seen physiologically, it does so via fatigue failure or breakage of the collagen crosslinks. Fatigue failure refers to a mechanism by which a material subjected to cyclical mechanical loading weakens over time, eventually failing at a threshold of loading significantly below the failure load under a single cycle of loading. This breakage occurs below the surface of the tissue, destabilizing the ECM and causing the tissue to swell, blister, and rupture, through a process termed delamination. However, cartilage normally withstands loading without damage, suggesting the presence of a protective in vivo mechanism preventing fatigue. Studies from Petersen et al. and Sise et al. indicated that synovial fluid (SF) plays a key role in preventing delamination during sliding, which is not tied to lowering of the friction coefficient, with its effectiveness diminishing when diluted below 50%. These findings suggest that SF constituents degrade under loading if not replenished, weakening this protective mechanism and enabling fatigue failure.
To this end, Chapter 4 investigates the hypothesis that synovial fluid breaks down durationdaily reciprocating sliding frictional contact if not replenished, degrading a protective mechanism and enabling the onset of fatigue failure. Immature bovine tibial strips were subject to reciprocal compressive loading in a bath of either fresh or used SF. Results illustrated that wear testing cartilage strips in reused synovial fluid caused more wear via delamination than testing in fresh synovial fluid. A small increase in shear rate, or equivalently a reduction in shear thinning was seen at shear rates above 103 s-1, accompanied with an apparent increase in the concentration of high molecular weight (>2000 kDa) SF constituents in used SF. This could suggest entanglement or aggregation of SF components, which may have deleterious effects on the ECM-protective role of SF.
The necessitation of SF replenishment in vivo to maintain function is consistent with the 2-4day complete turnover cycle of SF documented in animal joints9. This notion also suggests the existence of a critical cellular repair mechanism by which cells address daily damage and maintain the composition of the SF. Proximity and phenotype would indicate this cell population is likely either superficial zone (SZ) chondrocytes or synoviocytes. Interestingly SZ-resident chondrocytes have been shown to die even under normal physiologic loading conditions. However, the mechanism by which these SZ cells preferentially die has not yet been explained. The SZ is known to exhibit lower compressive and shear moduli compared to deeper zones, resulting in excessive SZ compaction under physiological loading. Evidence is limited by experimental constraints that cannot examine field variables throughout the in situ cellular environment. As such, to provide access to data experiments cannot obtain, in Chapter 5, we performed multiscale finite element analysis of articular contact to understand the fate of a chondrocyte embedded in the SZ, by tracking the temporal evolution of its interstitial fluid pressure, hydraulic permeability, and volume change under physiologic loading conditions. Results showed that SZ chondrocytes can lose ninety percent of their intracellular fluid after several hours of intermittent or continuous contact loading, resulting in a reduction of intracellular hydraulic permeability by more than three orders of magnitude. These findings are consistent with loss of cell viability due to the impediment of cellular metabolic pathways induced by the loss of fluid. They suggest that there is a simple mechanical explanation for the vulnerability of SZ chondrocytes to sustained physiological loading conditions. This also begs the question of how these cells are replaced, with some evidence suggesting the most likely supply to be synoviocytes.
After much discussion of the importance of collagen crosslinks to the health and function ofarticular cartilage, particularly in the SZ, the final study of this dissertation, presented in Chapter 6, presents a clinically translation component of this dissertation, investigating the use of a femtosecond laser to (1) prophylactically strengthen the collagen to prevent the onset of damage in cartilage, and (2) repair damaged collagen at an collagenous interface (cartilage implantation, meniscal, ligament and other common musculoskeletal tears), to encourage adhesion and prevent worsening of the tear or swelling of the tissue. Results indicated the induction of additional crosslinks in the superficial zone of cartilage can help to prevent delamination wear in the tissue. Similarly, laser treatment successfully increased the interface strength by an average of 157.0% (n=6)
Toward Productive Dissonance and Rehearsing Resolutions: Talking about Literature at School
No full textWe live in an era of widening rifts: personal, social, and political. Somehow the internet, despite its potential to connect across differences, seems to have mostly served to algorithmically deepen connections that are familiar, that echo our own beliefs and suspicions. Post-COVID, we could have returned to "real life" with renewed energy for crossing thresholds, but this potential has yet to be fulfilled. Schools are microcosms of the wider environment and not immune to fractioning; in fact, intra-school group divides create additional, ever-shifting plate tectonics and guardrails.
How might reading and talking about literature at school create opportunities for people to encounter and respond to other perspectives; to also experience being encountered and responded to? Perhaps by theorizing reading as a chance to hear oneself, others, and our intertwined lives in new ways. I begin by explaining the urgency of this work and how traditional English literature studies often fail to foster transformative conversations.
I then bring several theorists together (Buber, Bakhtin, and Bhabha) to build a conceptual framework for reading literature with others — to rehearse and re-orchestrate our understanding of each other and the world — and apply this theory to a qualitative study of extracurricular reading experiences in an international school setting.
Finally, I consider how some key practices may condition students and adults in schools to use literature to improve their collective soundscape
Career Adapt-Abilities Scale - Dual Career Form - U.S. (CAAS-DC-U.S.): Psychometric Properties and Initial Score Validation in U.S. Collegiate Student-Athletes
No full textNCAA Division I student-athletes have reported difficulties in balancing their academics and extracurricular activities, including their athletic commitments (NCAA, 2020b, 2022b). Student-athlete time demands may lead to student-athletes prioritizing their athletic commitments and delaying their preparation for their professional career after sport (Brown et al., 2000; Ryba et al., 2017). A concept that can support student-athletes’ preparation for their professional career after sport is career adaptability.
This research study examined if career adaptability scores of the Career Adapt-Abilities Scale - Dual Career Form (CAAS-DC; Ryba & Aunola, 2015; Ryba et al., 2017) are reliable and valid for NCAA Division I student-athletes in the United States. Additionally, this study analyzed the relationship between career adaptability and career construction, self-esteem, resilience, and sport burnout with NCAA Division I student-athletes. The relationship between career adaptability and academic performance was also observed.
Acceptable criterion values were observed to determine that there was a good fit of the observed data to the correlated five-factor model of the CAAS-DC-U.S. Overall, significant correlations were found between the scores of the CAAS-DC-U.S. combined scale and career construction, self-esteem, resilience, and sport burnout. A significant correlation was not found between career adaptability and academic performance, and low sport burnout reliability scores should be considered when assessing the correlation between career adaptability and sport burnout in this study.
The results demonstrate that the CAAS-DC-U.S. may be used by practitioners and researchers to measure career adaptability with NCAA Division I student-athletes in the United States. By having an instrument to measure career adaptability with student-athletes, practitioners and researchers can also work to develop interventions for building student-athletes’ career adaptability resources. Increasing student-athletes’ career adaptability resources may support them with combining their academic and athletic commitments and better prepare them for their professional career after sport
Shifting Terrains of Authority: Religio-Political Governance in the Sino-Tibetan Borderlands, 1650-1911
No full textThis dissertation investigates authority and governance in the Sino-Tibetan borderlands from the mid-17th to early 20th centuries, with a focus on the intersection of religious institutions and secular polities amid Qing imperial expansion into Inner Asia. It reevaluates the prevailing Beijing-centric interpretation of the “native chieftaincy system” (Ch. tusi zhidu) by approaching the frontier as dynamic and contested spaces where Tibetan polities and Qing state forces engaged in continual negotiation over sovereignty, legitimacy, and power. Centering on the Eastern Tibetan principalities of Gyalrong, the study recasts these kingdoms not as passive subjects of imperial incorporation but as active participants who shaped the trajectory of Qing expansion. Employing the concept of empire as a web of “reciprocal relationships” between Qing authorities and Tibetan elites, the project advances a model of imperial governance as layered, negotiated, and contested—marked by strategies of resistance, adaptation, and accommodation.
Based on extensive fieldwork and research in a multilingual archive—including Tibetan, Chinese, and Manchu documents housed in repositories across Taiwan, China, and India—the study draws on previously unexamined and newly published sources, such as imperial edicts, monastic abbatial records, land deeds, and legal documents. It demonstrates that Gyalrong rulers, far from being fully absorbed into the Qing administrative structure, mobilized religio-political ideologies, monastic networks, and customary legal systems to retain de facto autonomy and resist the imposition of gaitu guiliu (the replacement of native chieftains with regular officials). Ultimately, this dissertation offers a critical intervention in the historiography of the Qing empire and Sino-Tibetan relations. It challenges state-centric narratives of frontier consolidation—whether Chinese or Tibetan—by foregrounding the agency of Tibetan actors and reconceptualizing frontier governance as a product of mutual entanglement rather than unilateral domination. In doing so, it offers a more nuanced understanding of the region’s political trajectory leading up to its incorporation into the People’s Republic of China in the 1950s
Operationalizing Symbolic Boundaries in a Multidimensional World: Estimands, Designs, and Applications of Conjoint Experiments
No full textThe empirical operationalization of symbolic boundaries—the conceptual lines people draw to classify and hierarchize individuals—has long posed a challenge for sociologists, particularly those seeking to capture the multidimensionality of the social world. The recent rise of conjoint experiments in the social sciences has opened new possibilities for cultural sociologists, as this experimental design enables them to measure symbolic boundaries across a wide range of dimensions and to identify the relative importance of each. However, two limitations constrain the use of conjoint experiments for studying symbolic boundaries: first, the quantity of interest most commonly estimated is ill-suited to accurately capture patterns of symbolic boundaries; second, standard conjoint designs can only measure perceived social distance between oneself and others, not those between others.
This dissertation addresses both limitations and develops a methodological framework for operationalizing multidimensional symbolic boundaries using conjoint experiments. It introduces a novel conjoint experimental design to capture perceived boundaries between others, defines appropriate estimands for measuring perceived social distance, and proposes a new method to uncover heterogeneous configurations of symbolic boundaries. The empirical part of the dissertation applies these methodological innovations to the study of the landscape of symbolic boundaries in the United States. It investigates how Americans perceive the structure of their society and the cleavages that divide it.
The findings show that perceived divisions span all dimensions, although their salience varies. Age, socioeconomic status, and sexual orientation emerge as the most salient overall, while immigration status (with the exception of the undocumented category), gender, family status, and locality of residence appear less important. Particularly salient boundaries are perceived between Jewish and Muslim individuals, straight and gay or bisexual individuals, those in upper- and lower-class occupations, and Republicans and Democrats.
The analysis identifies four ideal-typical configurations of symbolic boundaries in the population, which are most strongly predicted by religiosity, nativity, and educational attainment. Despite this heterogeneity, however, the results suggest a broad consensus on the dominant logics of classification in the U.S. Together, these findings offer a comprehensive map of the symbolic structure of American society and demonstrate the utility of conjoint experiments for advancing research on cultural classification, perception of social distance, and symbolic boundary formation
Constitutive and Activity-Dependent Transcriptomic Profiles of SETD1A Disruption in Cortical Excitatory Neurons of a Schizophrenia-Risk Mouse Model
No full textSchizophrenia is a complex neuropsychiatric disorder with strong genetic contributions, including rare, highly penetrant mutations in SETD1A, a lysine methyltransferase and core component of the Set/COMPASS complex. SETD1A plays a crucial role in histone H3K4 methylation, a chromatin mark associated with active transcription, yet how its disruption contributes to disease-relevant neuronal dysfunction remains incompletely understood. This thesis investigates the role of SETD1A in cortical excitatory neurons, a cell population implicated in schizophrenia, through an integrative approach combining chromatin profiling, transcriptomic analysis, and the characterization of gene regulation under both baseline and activity-dependent conditions.
In the first part of this study, I mapped SETD1A genomic binding sites in sorted cortical excitatory neurons from wild-type mice using CUT&Tag. Although Setd1a binding was predominantly enriched at promoter regions, a substantial fraction was also detected at enhancers marked by H3K4me1 and H3K27ac.
Transcriptomic analysis of Setd1a⁺/⁻ neurons revealed widespread dysregulation of gene expression, with downregulated genes enriched for metabolic processes and upregulated genes associated with synaptic signaling. These findings indicate that SETD1A orchestrates transcriptional programs related to metabolism and synaptic function through coordinated regulation at both promoters and enhancers. Notably, genes with Setd1a-bound enhancers were especially sensitive to haploinsufficiency, suggesting that enhancer-mediated regulation by Setd1a is dosage-dependent.
To investigate the full scope of Setd1a-dependent gene regulation and assess whether complete loss amplifies or alters these effects, I developed a conditional knockout strategy. Given that constitutive homozygous deletion of Setd1a leads to embryonic lethality, I generated a neuron-specific conditional knockout model enabling homozygous deletion specifically in cortical excitatory neurons.
In the second part of this study, I used this model to examine the effects of near-complete Setd1a loss. These mice exhibited pronounced hyperactivity and showed more extensive transcriptional dysregulation in excitatory neurons compared to heterozygous mutants. As in heterozygotes, promoter-associated downregulated genes were enriched for metabolic pathways, while enhancer-associated targets reflected changes in synaptic and developmental programs. A substantial subset of genes dysregulated in heterozygous neurons was also affected in homozygous mutants, supporting the idea that Setd1a haploinsufficiency is sufficient to disrupt a core set of directly regulated genes. This analysis also identifies a high-confidence set of disease-relevant targets that can guide future functional studies.
In the third part of this study, I employed a chemogenetic approach to induce neuronal activity and analyzed time-resolved transcriptional responses in Setd1a+/− and wild-type cortical excitatory neurons. In wild-type neurons, activity elicited a coordinated and temporally structured gene expression program. In contrast, Setd1a+/− neurons exhibited disrupted timing and specificity of activity-induced transcription. These neurons failed to appropriately downregulate synaptic genes and displayed exaggerated repression of metabolic genes, particularly at later time points. Mutant-specific responses were notably enriched for mitochondrial pathways, suggesting impaired energy homeostasis during neuronal activation. Furthermore, analysis of SETD1A targets dysregulated following activity induction revealed increased intergenic binding, implicating enhancer dysregulation as a key mechanism by which Setd1a deficiency disrupts activity-dependent transcriptional programs.
Collectively, this work uncovers a multifaceted role for SETD1A in regulating both baseline and activity-dependent transcription through promoter and enhancer elements in cortical excitatory neurons. Loss of SETD1A disrupts the balance between synaptic and metabolic gene programs, providing a mechanistic link between chromatin regulation and schizophrenia pathophysiology
Visible-Light-Activated Photopolymerization and Dynamic Covalent Chemistry for Adaptive Materials
No full textPhotochemistry represents one of the most powerful tools for controlling chemical reactions in materials science, offering unparalleled advantages including remote activation, precise spatiotemporal control, and operation under ambient conditions. Among various photochemical processes, photopolymerization enables the fabrication of macromolecules from monomers, providing toolboxes to engineer materials with vastly different properties from their small-molecule precursors. Photoresponsive dynamic covalent chemistry, on the other hand, provides pathways to create adaptive materials that can reversibly reconfigure their molecular architecture in response to optical stimuli.
These light-driven processes have revolutionized fields ranging from 3D printing to the development of smart materials capable of self-healing and shape-changing in response to environmental changes. However, traditional photoresponsive systems rely predominantly on high-energy ultraviolet (UV) light activation, which presents fundamental limitations including poor light penetration, photodamage, and restricted applicability in opaque materials. This dissertation addresses these challenges by systematically developing visible-light-activated photopolymerization and dynamic covalent chemistry systems for adaptive materials with enhanced control and functionality.
The first approach involves molecular engineering of dynamic covalent bonds to shift their photoactivation from UV to visible light. Carbazole-based thiuram disulfides were designed with extended conjugated systems that significantly enhance visible light absorptivity and reactivity compared to conventional alkyl thiuram disulfides. These engineered dynamic linkages enable fast photoinduced reshuffling and demonstrate controlled photopolymerization of alkyl acrylates as iniferters (chemicals that simultaneously act as initiator, transfer agent, and terminator), providing both dynamic exchange and polymerization capabilities based on a single dynamic covalent bond.
Taking advantage of these dynamic covalent bonds, we successfully developed a visible-light-induced living polymer network by incorporating these dynamic linkages into polymer backbones. This unique network showcases notable photoresponsive characteristics that facilitate fast reconfiguration, enabling photoinduced shape-shifting and multiple rounds of healing with retained mechanical properties. Additionally, utilizing the dynamic linkages in the backbone as visible-light iniferters, the polymer network can undergo chain growth, providing a robust platform for on-demand modulation of mechanical properties. Notably, due to visible-light activation and the radical exchange nature of thiuram disulfide reshuffling, photochemical control was achieved even in opaque materials.
Beyond successful modification of the singlet excited state, we discovered that extended conjugation in thiuram disulfides also significantly lowers their triplet energy levels, enabling efficient triplet energy transfer from appropriate photosensitizers. This insight led to the development of triplet-sensitized dynamic covalent chemistry using red light for activation under ambient conditions. The approach achieves fast reshuffling of thiuram disulfides with minimal sensitizer loading under red light irradiation at modest intensities without deoxygenation, with reaction rates comparable to direct blue light excitation. Most significantly, due to selective sensitization of carbazole thiuram disulfides, orthogonal equilibrium control was achieved for reshuffling between alkyl and carbazole thiuram disulfides. Thereby different photostationary states can be accessed through wavelength-selective activation, providing unprecedented control over covalent bond distributions.
Finally, triplet-triplet annihilation upconversion was employed as a general strategy to extend traditional blue-light-activated photochemistry to red light activation. This method converts low-energy red photons into higher-energy blue light to initiate free radical polymerizations within opaque objects, enabling photocuring of composite materials that cannot be processed with conventional UV-based approaches.
These advances collectively expand the accessible spectral window for photoresponsive materials from UV to red light, providing superior penetration depth, reduced photodamage, and enhanced temporal control. The resulting materials establish fundamental principles for next-generation photoresponsive materials with diverse applications including reconfiguration of polymer networks, precise control of mechanical properties, and fabrication of advanced composite materials, opening new possibilities for applications requiring deep light penetration
The Primary Care Nurse Practitioner Workforce and the Care of Adults with Serious Mental Illness in Socioeconomically Disadvantaged Communities
No full textSerious Mental Illness (SMI) is defined as mental, behavioral, or emotional disorders that result in serious functional impairments and inhibit one’s ability to perform one or more major life activities. In the United States, approximately 15.4 million people live with SMI, and the care of SMI represents a significant source of economic burden for patients, their families, and society.
Adults with SMI have an increased risk of comorbid chronic conditions, a shorter life expectancy, and higher rates of high-cost, resource-intensive healthcare utilization than the general population. Adults with SMI need comprehensive management of their mental health and physical health conditions; however, many do not receive adequate preventative care or effective management of their conditions, contributing to high rates of emergency department (ED) use and repeated hospitalizations. Mental health provider shortages nationwide have worsened access to effective chronic disease management for adults with SMI, especially in more socioeconomically disadvantaged communities. This has resulted in primary care providers, including nurse practitioners (NPs), filling gaps in care for patients with SMI. Therefore, there is a need to examine the structure of primary care delivery and the role of NP care for this population.
NPs play an important role in providing access to care in socioeconomically disadvantaged communities. As a result of the high-quality care provided by NPs and the increased distribution of NPS in socioeconomically disadvantaged communities, NPs have the ability to address health disparities for adults with SMI. However, NPs often provide care in care environments that are marked by poor organizational support for NP practice and limited comprehension of NPs’ roles. Practices that lack support staff and organizational resources for NPs inhibit NPs from working to their greatest potential and limit their ability to address health disparities.
There is limited evidence to support the role of organizational structure and community-level factors in contributing to outcomes for adults with SMI. Thus, the overall purpose of this dissertation is to produce evidence on primary care delivery for adults with serious mental illness and the role of practice and community-level factors in influencing outcomes for this population.
In chapter 1, we introduced the unique healthcare needs of patients with SMI and the role of community and organizational factors in their health outcomes.
In chapter 2, we synthesized the existing evidence on the impact of various primary care models on emergency department use and hospitalizations among adults with SMI. Our synthesis suggests that models of care that expand the accessibility of primary care, such as the medical home model, are related to reduced acute care utilization among adults with SMI.
In chapter 3, we performed a secondary data analysis using patient data from the Medicare claims of 30,425 adults with SMI, and data from the University of Wisconsin’s Neighborhood Atlas Area Deprivation Index (ADI), which measured community socioeconomic disadvantage. We examined differences in rates of all-cause and preventable ED visits based on the level of community socioeconomic disadvantage. We found greater rates of all-cause and preventable ED utilization among Medicare beneficiaries living in more disadvantaged communities compared to less disadvantaged communities. Our findings suggest that community-level socioeconomic resources play an important role in influencing outcomes for patients with SMI.
In chapter 4, we analyzed patient data from the Medicare claims of 30,425 adults with SMI, merged with NP survey data on NP care environment, and ADI data to assess the effect of the NP care environment on socioeconomic disparities in ED use among patients with SMI. We find that NP care environments moderate the association between community socioeconomic disadvantage and ED utilization (all-cause and preventable). Greater scores in one domain of the NP care environment, support for NP independent practice, was associated with reduced ED utilization in Medicare beneficiaries with SMI. Further analysis found that in more favorable care environments, socioeconomic disparities in ED utilization were mitigated. Our findings provide a modifiable factor—NP care environments, that can be leveraged to reduce socioeconomic disparities and improve outcomes for patients with SMI.
Lastly, in the concluding chapter of this dissertation, Chapter 5, we discuss the strengths, limitations, and contributions of this dissertation. We also present a summary of the findings and implications for policy, practice, and future research
Sex and the Soul: Science, Religion, and Non-binary Gender in the Nineteenth-Century United States
No full textMany scholars have explored how nineteenth-century physicians and scientists eradicated the category of “hermaphroditism” from modern medical practice, but fewer have asked why. This dissertation posits that the erasure of non-binary sex and establishment of binary sex in the nineteenth-century United States was a result of a widespread and deeply held belief in the infallibility of sex as interpreted by Protestants from Genesis 1:27 that was invisibilized by the metaphysical claims of Protestant-secular normativity.
These newly invented truths of binary sex reciprocally affected how nineteenth-century Americans imagined and practiced the body, the soul, and divinity itself. The nineteenth-century non-binary appeared in the bigender Gods of the Shakers, Oneida Community, and Thomas Lake Harris; the genderless spirit of the Public Universal Friend; the imagined eunuch and vanishing hermaphrodite; and the unsexed soul of the Transcendentalists and Julia Ward Howe.
This dissertation argues that the cultural mechanisms that foreclosed the viability of sexual ambiguity conversely provided metaphysical conditions for imagining the sexed self that defied biopolitical norms. Over the century, I argue, the discursive frictions between religion, science, secularism, spirituality, and sex caused the emergence of ways of thinking about the self’s relationship to the sexed body that anticipated and, perhaps, made possible many of our present debates on sex, gender, and sexuality. In sum, I posit that the non-binary—the haunting space between male and female—is an American tradition
Engineering Probiotics as Versatile Vehicles for Precision and Broadly Applicable Immunotherapy in Solid Tumors
No full textAdvances in synthetic biology have revolutionized the field of cell-based cancer therapeutics, enabling comprehensive genetic programming of bacteria for targeted therapy. Engineered probiotics offer a unique advantage in cancer immunotherapy due to their selective tumor-colonizing capabilities and ability to be programmed for controlled on-site therapeutic production and release.
My dissertation aims to engineer bacteria as a versatile therapeutic platform for solid tumor immunotherapy, integrating synthetic biology tools to advance both precision and broadly applicable bacterial cancer therapeutics.
Bacteria serve as natural immune adjuvants due to their intrinsic immunostimulatory properties. Leveraging this feature, we engineered Escherichia coli Nissle 1917 as a probiotic-based cancer vaccine platform for precision immunotherapy. The bacteria were designed to express tumor-specific neoantigens and immunotoxin listeriolysin O, enhancing immune activation. To further improve antigen availability, we deleted two key proteases responsible for intracellular and extracellular protein degradation, which led to increased neoantigen accumulation for delivery. These modifications also improved the safety and specificity of the bacteria by increasing their susceptibility to immune-mediated clearance, including phagocytosis by antigen-presenting cells, which in turn facilitated robust antigen presentation and subsequent activation of CD4+ and CD8+ T cells. This approach effectively remodeled the tumor microenvironment and demonstrated efficacy across multiple cancer models, underscoring its potential as a versatile and potent precision cancer immunotherapy platform.
To broaden the applicability of bacterial therapeutics beyond precision therapy, we engineered a strain with reduced immunogenicity, tumor specificity, and adaptability for diverse therapeutic payload in situ production and extracellular release. To achieve this, we first screened multiple gene knockouts to attenuate lipopolysaccharide-mediated immunogenicity, significantly improving the strain’s safety profile. To further confine bacterial growth to the tumor microenvironment, we constructed a synthetic AND-gated promoter that integrates hypoxia and high lactate as dual input signals to drive essential gene expression, ensuring bacterial survival only within tumors.
Additionally, we modified the bacterial membrane to increase permeability, enabling the passive secretion of a broad range of therapeutic payloads within a defined size limit. This three-component engineered strain demonstrated a 100-fold increase in the maximum tolerable dose compared to the unmodified strain in animal tumor models, leading to significantly improved therapeutic efficacy. To validate its versatility in payload delivery, we programmed the strain to produce four distinct cytokines that activate lymphocytes and confirmed its ability to enhance anti-tumor immunity in vivo.
Together, these findings highlight the potential of engineered probiotics as a next-generation living medicine, capable of both precision and broadly applicable cancer immunotherapies, establishing a foundation for clinically translatable and highly tunable bacterial therapeutics