ORCID

0009-0005-9923-9648

Graduation Year

2025

Document Type

Master's Thesis

Degree

Master of Science

Program

Biological Science

Partner Organization

Buck Institute for Research on Aging

Program Director

Patti Culross, MD, MPH

First Reader

Birgit Schilling, PhD

Second Reader

Mark Watson, PhD

Abstract

Cellular senescence is believed to be a driver in the development of osteoarthritis (OA). This cellular state leads to alterations in features such as increased cell volume, flattening of the nucleus, and secretion of inflammatory proteins known as the secretory-associated senescent phenotype (SASP). Articular chondrocytes are the cells that maintain cartilage in the knee, and with age, they can become senescent, leading to the development of OA. Our lab has previously developed a novel tool to assess and quantify senescence in vitro called fully automated senescence test (FAST). The overall goals of this study were as follows: (1) develop and characterize a senescent primary human chondrocyte model for high throughput screening; (2) determine the FAST’s ability to analyze senescence in senescent chondrocyte cells; (3) expand FAST’s capability to include the cell death assay, TUNEL; and (4) test a potential senolytic compound called Urolithin A. FAST was used to quantify the induction of senescence in chondrocytes and IMR90 cells with different inducers, including irradiation (IR), doxorubicin, and MIDAS (antimycin A with rotenone). Using these conditions, we successfully established TUNEL staining assessment as an addition to FAST. Senescence-induced cells were treated with Urolithin A to test for any senolytic capability. Higher doses of urolithin A in human chondrocytes appeared to enhance senescence signatures, whereas lower doses resulted in an increase in nuclear areas. FAST was optimized for IR-induced senescence. Here, we applied FAST to analyze MiDAS-induced senescence, which resulted in a marked increase in senescence signatures, demonstrating the successful incorporation of MiDAS into the FAST platform and TUNEL. This study used methods of automated fluorescence microscopy to assess different senescence inducers like IR, doxorubicin, and MiDAS. FAST could assess up to three stains in a single cell allowing for a comprehensive senescence evaluation. This study lays the foundation for a high-throughput senolytic screen in senescent human chondrocyte cells that could identify a potential therapeutic for osteoarthritis (OA).

The musculoskeletal system undergoes progressive decline with age, often leading to slower fracture repairs in older individuals. To investigate the molecular basis of this delay, we applied spatial proteomics through MALDI mass spectrometry to unfixed tibial fractures in young versus aged mice. Because MALDI workflows currently lack robust tools for unbiased protein-to-library matching, we developed a custom script to annotate spectra against a comprehensive reference database. Using this pipeline, we identified a suite of fracture-associated proteins, with type I collagen chains (COL1A1 and COL1A2) dominating the dataset. Analysis of their fibrillar states uncovered distinct assembly patterns between young and aged samples, implying age-related alterations in collagen organization. Additionally, we detected elevated levels of calreticulin in both young and fully healed bone, offering novel insights into the mechanisms that may underlie impaired bone remodeling in aged fractures.

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Available for download on Wednesday, May 31, 2028

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