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GENETIC ANALYSIS OF SOX2 FUNCTION AND CONTRIBUTION TO HAIR CELL REGENERATION IN ZEBRAFISH
Get PDFHearing loss is an escalating global health issue, with projections indicating that over 70 million people will experience some degree of hearing impairment by 2040. While not life-threatening, hearing loss significantly diminishes the quality of life and leads to broader societal challenges, including social isolation, limited employment opportunities, and cognitive decline.
The most common cause of permanent hearing loss is damage to inner ear hair cells (HCs), which are mechanosensory receptors responsible for converting sound vibrations into electrical signals that the brain interprets. In humans, damage to HCs is irreversible leading to deafness and/or balance disorders.
In contrast, non-mammalian species, such as zebrafish, can regenerate HCs after damage or loss throughout life. Due to the evolutionary conservation of inner ear auditory and vestibular organs, the zebrafish is an excellent model organism to study regeneration mechanisms with potential relevance to human therapies. Previous studies have underscored the crucial role of Sox family transcription factors in this process. This study focuses on Sox2, a transcription factor known for its involvement in early development, and its role in reprogramming supporting cells into progenitor cells that give rise to hair cells in zebrafish—a process absent in mammals.
To investigate the role of Sox2 in HC regeneration, we generated sox2 knockouts (KO) using CRISPR-Cas9 gene editing, and validated these KOs using fluorescent PCR, fragment analysis, and Sanger sequencing. Phenotypic analysis of sox2-/- fish revealed developmental abnormalities, including the absence of swim bladders, curved tails, and uncoordinated swimming. The sox2 knockouts also exhibited lethality 12 days post-fertilization. Despite these systematic defects, HC development appeared normal. Moreover, observed no significant defects in HC regeneration following copper-sulfate induced ototoxic damage. Even after conducting two rounds of HC ablation, sox2-/- larvae exhibited robust HC regeneration comparable to wild-type controls.
These findings challenge the presumed requirement of Sox2 for HC regeneration in zebrafish and suggest the presence of compensatory or redundant regulatory mechanisms that sustain regeneration in its absence. Ongoing analyses aim to identify these mechanisms and provide deeper insight into the transcriptional networks governing inner ear HC regeneration
ANALYSIS OF DNA CPG METHYLATION OF RETINAL DIFFERENTIATION FROM HUMAN BLASTOMERE-LIKE STEM CELLS
No full textHuman pluripotent stem cells (hPSCs) promise new routes to regenerative therapies, yet the epigenetic rules that steer them toward specific fates are still unclear. Here we focus on a blastomere like state produced by tankyrase/PARP inhibition (TIRN SCs)—an even earlier stage than the classic primed and naïve pluripotent states—and track how it heads toward the retinal lineage. Using multi-omics data combining RNA seq, whole genome bisulfite sequencing, and targeted ChIP seq, we mapped CpG methylation and other epigenetic changes through retinal organoid differentiation comparing TIRN and primed pluripotency state. Surprisingly, our results revealed that TIRN-SC did not exhibit global loss of methyl marks the way naïve PSCs were previously reported. Instead, TIRN-SC underwent demethylation highly selectively: more than 10,000 differentially methylated regions (DMRs) identified to cluster in CpG shores, promoters, and other key regulatory elements while the rest of the genome remained unchanged. Those putative regions coincided with the differential binding sites of the pluripotency trio — SOX2, OCT4, and NANOG (SON)— hinting that local methyl loss may regulate the chromatin accessibility for these factors to reach and maintain a blastomere like program.
Further analysis led to a more comprehensive rationale combining out data with prior proteogenomic work suggesting that reduced PARP1 and elevated tankyrase levels appeared to release SON factors from repressive complexes and to enable their redistribution to newly demethylated sites. Many of these sites were identified within or near enhancer elements that transcribe short non coding RNAs that are recognized as putative enhancer RNA/super-enhancer RNA (eRNA/seRNA) signatures based on H3K27ac chromatin binding, presumably inducing eventual blastomere-like transcriptomic dynamics. Consistent with prior work, these results implied an epigenetic and protogenomic integrated regulatory network where methylation loss, SON binding, and enhancer activation jointly amplified early developmental circuits. Despite this significant epigenomic remodeling of TIRN-SC, transcriptomics programs correlated poorly with CpG methylation status, suggesting again a multi layered indirect regulation. After retinal differentiation, most methylation and expression differences between TIRN SCs and primed cells fade, underscoring the complexity of TIRN pluripotency dynamics and the need to probe post transcriptional controls in retinal organoid development
THE STRATEGIC ROLE OF OTHER TRANSACTION AUTHORITIES: THEIR HISTORICAL EVOLUTION AND CONTEMPORARY IMPACT IN THE DEPARTMENT OF DEFENSE
Get PDFThis thesis examines the historical and legislative evolution, efficacy, and strategic implications of Other Transaction authorities and agreements (OTAs) within the Department of Defense. By exploring how ideas, exogenous pressure, and technological demands have shaped the policy environment surrounding OTAs, this research identifies the critical factors that have driven OTA adoption and adaption since their inception in 1958. Drawing on archival record, case studies, and primary and secondary interviews with policymakers and defense acquisitions experts, the study highlights the role of OTAs as a flexible mechanism for fostering innovation and accelerating the integration of critical technologies into defense ecosystems.
The findings reveal that OTAs, initially conceptualized for NASA’s experimental projects, gained traction within the DoD as a response to external threats, shifting geopolitical landscapes, and the increasing need to leverage commercial innovation. This research also assesses the comparative efficacy of OTAs against traditional acquisition methods, with a focus on cost, speed, and adaptability. It identifies key opportunities to refine OTA policies, emphasizing their potential to shape future defense innovation strategies in an era of rapid technological advancement and peer competition.
Ultimately, this thesis contributes to the broader understanding of how policy instruments evolve to address emerging challenges and provides actionable insights for leveraging OTAs to strengthen the United States’ defense industrial and innovation base
Elevating Policing Practices Through Mental Health Literacy: An Analysis of Law Enforcement Training Curricula
Get PDFLaw enforcement officers (LEOs) are often the first responders to mental health crises, yet many lack the specialized training necessary to manage these situations effectively. The intersection of trauma and mental health is particularly relevant, as adverse childhood experiences (ACEs) are strong predictors of mental health challenges and increased likelihood of criminal justice system involvement. Without adequate training, officers may struggle to recognize trauma-related behaviors, leading to ineffective responses that escalate crises, reinforce stigma, and contribute to disproportionate rates of incarceration and use of force against people with mental illness (PWMI). This study conducted a curriculum analysis (CA) of Crisis Intervention Team (CIT) training—the leading police crisis response model—to evaluate its effectiveness in preparing officers to navigate mental health crises using trauma-informed, de-escalation-based strategies. A mixed-methods, rubric-based evaluation assessed the curriculum’s content, instructional methods, and departmental readiness to implement CIT training effectively. Findings highlight key strengths, such as scenario-based training and emphasis on de-escalation, but also reveal critical gaps, including insufficient content on substance use disorders, implicit bias, and long-term recovery strategies. Additionally, the absence of structured post-training support, formalized coaching, and standardized evaluation tools limits skill retention and long-term impact. This study provides actionable recommendations to enhance curriculum design, integrate reflective practices, expand training accessibility, and strengthen community-police partnerships. By embedding trauma-informed frameworks into police education and policy, law enforcement agencies can foster safer, more equitable crisis responses that prioritize recovery over criminalization, ultimately improving outcomes for both officers and the communities they serve
Transcriptional variation in cancer
No full textThe genetic variation that underlies oncogenesis cannot explain the entirety of the variation seen in cancer. Therefore, it is important to characterize other biological factors that impact oncogenesis such as transcriptional variation. Recent RNA-sequencing technologies have enabled the comprehensive study of transcriptional variation at the gene expression level. Yet, evaluating cancer at the gene expression level alone is not sufficient for probing all relevant transcriptional variation in cancer.
Evaluating transcriptional variation in cancer using short-read and long-read RNA-seq data has resulted in discoveries relevant to oncogenesis. Evaluating transcriptional variation in cancer using short-read data is done at the level of the splice-junction or exon inclusion/exclusion to leverage the strengths of short-read RNA-sequencing data, high coverage, against one of its pitfalls, non-multi-exon spanning reads. Whereas evaluating transcriptional variation using long-read sequencing technologies is done at the transcript level since most long RNA-seq reads are multi-exon spanning reads. Using short read RNA-sequencing, it has been shown that on average there are more splicing derived neoantigens in ovarian and breast cancer than there are SNV-derived neoantigens11, that pharmacologic modulation of the splicing machinery results in the production of splicing-derived neoantigens capable of eliciting an immune response in melanoma cell lines13, and that high QKI expression recapitulates a basal epithelial cell splicing signature in PDAC108. Whereas it has been shown that long-read RNA sequencing can be used to derive translated mutations, gene fusions, and unique isoforms in ovarian cancer109,70. Altogether, this work in transcript-level variation analysis has yet to develop a continuous metric of splicing neoantigen contribution that can be correlated to other metrics and has not yet used long-read sequencing to evaluate the splicing landscape in PDAC.
In this work, I first evaluate transcriptional variation using short-read RNA-sequencing technologies pan-cancer and in a melanoma cohort treated with immune checkpoint inhibitors. In so doing, I derive the splicing antigenicity, which measures the contribution of splicing-derived antigens to the neoantigen landscape. Then I leverage single-cell long-read RNA-sequencing using a modified 10x Chromium 3’ protocol directly to Oxford Nanopore Technologies sequencing to evaluate the epithelial tumor-cell variation in pancreatic ductal adenocarcinoma patient-derived organoids. Together, this work suggests that the splicing antigenicity contributes to the response to immune checkpoint inhibitor therapy, that specific KRAS mutants show transcriptional signs of variation in tumor cell behavior, and that the basal and classical splicing signatures contribute to the epithelial stratifications. Additionally, this work helps paves the way for long-read splicing-derived neoantigen analysis
DEVELOPMENT OF A BIOSTIMULATORY NANOFIBER-HYDROGEL COMPOSITE PLATFORM FOR SOFT TISSUE REMODELING AND THERAPEUTIC DELIVERY
No full textSoft tissue loss from trauma or disease poses significant clinical challenges, as current solutions like grafts and implants face issues, including donor morbidity and poor regeneration. This thesis presents a nanofiber-hydrogel composite (NHC) combining electrospun collagen nanofibers and hyaluronic acid hydrogel to mimic the native tissue microenvironment. Engineered to balance stiffness and porosity, the NHC enables immediate volumetric restoration while promoting sustained tissue remodeling through optimized inflammatory and regenerative responses. By dynamically interacting with host tissue, it ensures the formation of vascularized soft tissues. Additionally, the NHC enables the delivery of bioactive components like extracellular vesicles (EVs) for soft tissue damage caused by radiation-induced dermatitis. This platform offers a promising off-the-shelf solution for functional soft tissue repair
Well-Being at Associate Degree Granting Institutions: Faculty and Staff Well-Being in an Actor-Network
Get PDFThe factors affecting well-being at work have been studied widely and interventions for improving well-being equally so, yet the particular context of associate degree granting institutions (often referred to as community colleges) presents a specific instance for measuring and attending to the well-being of faculty and staff. A mixed methods empirical study was conducted to acknowledge the factors affecting faculty and staff well-being; evaluate evidence of the relationship between competing demands on the institution and satisfaction or frustration of the three basic psychological needs; and create avenues through photovoice as a research methodology for illustrating well-being. Two research questions about variations in well-being by faculty or staff role and by the level of engagement with students were measured quantitatively and a question considering the demands on associate degree granting institutions in relation to well-being was addressed qualitatively and by mixed methods integration. Results from the Basic Psychological Needs Satisfaction Scale - Work Domain (BPNSS-W) were integrated with findings relevant to the context produced through serial, unmoderated focus groups relying on photovoice. The participatory research methodologies were adapted to distribute agency to the co-researchers without compromising their anonymity and a networked approach to illustrating the results was informed by actor-network theory (ANT).
The methodological adaptations led to the development of a guide for facilitators to conduct similar studies that result in maps of well-being at work. Made available as a project within this dissertation, the guide adds a novel qualitative resource to the collection of quantitative scales for measuring the satisfaction or frustration of the basic psychological needs (BPNs). The accessibility of the guide was evaluated by practitioners with backgrounds in associate degree granting institutions who articulated the improvements in form and content of the guide that make it accessible for practitioners with various levels of research backgrounds and at different size institutions to situate themselves within study. The guide was designed with adaptability in mind and emphasizes the importance of facilitators adapting it in ways that are relevant to their context at an associate degree granting institution or more broadly. The availability, accessibility, and adaptability of the guide determines its significance
AUTOIMMUNE ANTIBODIES RECOGNIZE AND CAN ELIMINATE BREAST CANCER CELLS
No full textIt has become increasingly clear that the pathologic mechanisms driving autoimmune diseases and cancer are linked, with recent findings demonstrating that a diverse autoantibody repertoire confers a protective benefit against cancer development. For this work, we sought to investigate this relationship to understand how autoimmunity can protect against cancer, and the mechanisms responsible for this effect. To do this, we studied rheumatic patient autoantibodies and their interactions with cancer cells, using both in vitro and in vivo models of cancer and metastasis. We first discovered that common autoantibodies generated during the course of autoimmune diseases are able to recognize targets on the surface of live breast cancer cells. These autoantibodies could then mediate cancer cell killing through multiple immune pathways, including the complement system and NK cell antibody-dependent cellular cytotoxicity. By analyzing and screening the autoantibodies in these sera, we identified those against the Ro52 protein as a potent mediator of these effects. Although Ro52 is normally a cytoplasmic protein, we found that interferon signaling molecules increase surface localization of an antigen containing the fourth exon of Ro52. We hypothesize that the generation and translocation of autoantigens occurs as part of a broader phenomenon during metastasis, and that the immune system has evolved to recognize these antigens as a protective mechanism to guard against cancer. Notably, surface localized Ro52 was not detected in normal mammary epithelial or mesothelial cells. This distinction may allow for the specific targeting of cancer cells using antibody-drug conjugates which could eliminate malignant cells without harming the surrounding healthy tissue. Our studies in progress have revealed that additional anti-cancer autoantibodies exist and suggest that they may be able to recognize cancer associated antigens through novel pathways. Future work will focus on characterizing these antibodies and mechanisms. Overall, we show that a conserved repertoire of self-antigens is created and expressed during both cancer and autoimmunity and can be used to specifically target cancer cells as anti-neoplastic therapies
Role of s2U tRNA modification and ribosomal protein expression in antibiotic tolerance of Yersinia pseudotuberculosis
Get PDFAntibiotic treatment of bacterial infections can fail for several reasons. Major focus centers on the problem of antibiotic resistance, which generally occurs when bacteria acquire genetic mutations that allow bacteria to grow in the presence of antibiotics. However, bacteria can also develop phenotypic changes in response to stress, typically involving a slowed growth response, that allow them to survive antibiotic treatment longer than susceptible bacteria. This phenomenon is known as antibiotic tolerance when these changes occur in the entire bacterial population or antibiotic persistence when these changes only occur in a subpopulation. Antibiotic tolerance and persistence have been postulated to contribute to relapsing infections and have been shown to directly enhance the development of antibiotic resistance, making it important to understand what phenotypic changes allow bacteria to survive antibiotic treatment. The following studies aim to understand what phenotypic changes in response to antibiotic stress and stressors in the host environment, are responsible for promoting bacterial survival.
In response to doxycycline, we find that Yersinia pseudotuberculosis downregulates tusB, a gene involved in the 2-thiolation on the wobble position of tRNAs (s2U). Whereas tusB overexpression increases susceptibility to doxycycline, tusB deletion renders bacteria tolerant to ribosome and RNA polymerase-targeting antibiotics. Our results indicate that loss of s2U results in less efficient translation of ribosomal proteins. Lower ribosomal protein levels likely globally reduce the translational capacity of the cell, inducing slowed growth and antibiotic tolerance.
In a separate study, downregulation of ribosomal proteins also seems to predict bacterial survival after antibiotic treatment. We find that expression of the type-III secretion system (T3SS) attenuates growth and induces tolerance to gentamicin. However, expression of T3SS is not the best indicator of antibiotic survival. Instead, expression of the 30S ribosomal protein S10 was a better predictor of survival wherein cells expressing lower levels of S10 preferentially survived antibiotic treatment.
These studies highlight ribosomal protein expression as an indicator of metabolic activity and potentially as a major proponent that influences the survival of bacteria in response to antibiotic treatment
WHERE ARE THE WOMEN? EXPLORING GENDER INEQUITY IN LEADERSHIP WITHIN INDEPENDENT SCHOOLS
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This paper addressed the issue of the lack of female leaders, focusing specifically on the smaller pipeline of female candidates for head of school positions in independent schools regionally and nationally. This paper reviewed the literature on the factors impacting leadership and how these factors led to a shortage of female candidates for head of school positions in independent schools. The paper focused on the factors contributing to females opting not to pursue leadership positions. At a young age, males and females demonstrated equal motivation for leadership. However, factors related to experience and exposure hindered young girls’ leadership development, including a lack of early leadership programming specifically for young girls, limited opportunities for confidence-building in leadership, a limited number of female role models in leadership positions, and social and cultural gender biases around female leadership qualities