Doctor of Philosophy, The Ohio State University, 2020, Biomedical Engineering

The extracellular matrix is extensively reorganized throughout breast cancer progression. This reorganization contributes to cancer cell invasion and intravasation and is an independent prognostic factor for breast cancer patients. Cancer-associated fibroblasts appear to play a major role in this reorganization but the cellular signaling pathways contributing to this reorganization remain unclear. We show here that loss of the tumor suppressor phosphatase and tensin homolog (Pten) in fibroblasts promotes extracellular matrix alignment both in vitro and in vivo through increasing cell traction forces. Furthermore, low stromal PTEN expression correlated with high mammographic density, one of the major risk factors for breast cancer development. Matrix reorganization was concomitant with a marked increase in collagen deposition within the mammary gland. We therefore investigated the mechanism of collagen deposition and showed that loss of PTEN upregulated SPARC, which mediated both collagen and fibronectin assembly without modulating cell traction force. To further determine how Pten loss connected to matrix alignment, we designed a novel screening platform to examine matrix alignment in vitro using fibroblast-derived matrices, automated microscopy, and automated image analysis through MATLAB. We discovered a number of novel matrix alignment modulators, including protein kinase C (PKC), dual specificity tyrosine regulated kinase 1b (DYRK1b), platelet-derived growth factor receptor β (PDGFRβ), and Janus kinase (JAK), among others. The effect of these markers on patient survival was examined using publicly available patient datasets. Finally, we examined the effects of hypoxia and matrix metalloproteinase activity on matrix organization.

Committee: Jennifer Leight (Advisor); Samir Ghadiali (Committee Member); Jonathan Song (Committee Member) Subjects: Biomedical Engineering

Bachelor of Science (BS), Ohio University, 2023, Biological Sciences

Fibrosis, a pathological process characterized by excess extracellular matrix (ECM) deposition, can occur in many internal organs and tissues in response to various stimuli. As fibrosis progresses, scarring occurs, which ultimately leads to tissue dysfunction and organ failure. Patients with acromegaly, a rare disease usually caused by a benign, GH-producing pituitary tumor, have been reported to have prominent ECM deposition and scarring in certain tissues, which is indicative of fibrosis. In bGH transgenic mice, which express high levels of bovine growth hormone, several tissues [white adipose tissue (WAT), heart, intestine, and kidney] demonstrate a fibrotic phenotype. However, there is no previous research that investigates various bGH tissues – particularly from mice derived from a single cohort – for fibrosis. Additionally, WAT fibrosis is associated with obesity and lipodystrophy, and seems to be particularly associated with excess GH. This study aims to investigate the role of different cell types and genes involved in the development and progression of WAT fibrosis and determine if fibrosis is increased in BAT, liver, quad, kidney, lung, and spleen of aged bGH mice. Results of this thesis included a striking observation of increased fibrosis in all bGH tissues examined. For WAT, decreases in fibrosis-associated RNA expression in 3-month-old bGH mice via qPCR analysis was only observed in the perigonadal depot and not the subcutaneous depot that has more prominent collagen deposition. Interestingly, we observed an intriguing increase in fibrosis-associated RNA expression in a population of adipose stem and progenitor cells in 6-month-old mice within subcutaneous bGH WAT. These results indicate a potential common GH-induced mechanism of fibrosis across bGH tissues and pave the way for future research into WAT fibrosis.

Committee: Darlene Berryman (Advisor) Subjects: Biology; Biomedical Research

Master of Sciences (Engineering), Case Western Reserve University, 2017, Biomedical Engineering

Patient-derived xenografts (PDX) are living ex situ tumor models that aid clinicians in selecting potent therapies for cancer patients. Unfortunately, a liver PDX is difficult to develop due to poor liver tumor engraftment in mice hosts. Tissue engineering studies have suggested that beneficial factors may exist in extracellular matrix (ECM) that can enhance tumor viability after transplant. We conducted comparative analysis of three published decellularization protocols for efficient cell removal. ECM samples produced by Freeze-thaw with Triton X-100 (TX-100), Sodium Dodecyl Sulfate (SDS) with TX-100, and TX-100 alone, were analyzed with nuclear labeling and structural analysis. SDS with TX- 100 was efficient and caused minimal alterations to the matrix structure. Additionally, we demonstrated with developed assays that liver cell lines can respond to isolated decellularized matrix and mimic in vivo liver activity. The work completed provides confidence to study the effects of decellularized liver matrix on patient-derived liver tumors.

Committee: Samuel Senyo PhD (Advisor); Eben Alsberg PhD (Committee Member); Analisa DiFeo PhD (Committee Member); Anirban Sen Gupta PhD (Committee Member) Subjects: Biomedical Engineering

Master of Science in Biomedical Engineering, Cleveland State University, 2014, Washkewicz College of Engineering

Vascular diseases such as atherosclerosis and aneurysms are characterized by the over-proliferation and migration of aortic SMCs, and degradation of ECM within the vessel wall, leading to compromise in cell-cell and cell-matrix signaling pathways. Recent tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency and matrix deposition yields. In this study, the benefits of exogenous nitric oxide cues, delivered from GSNO, to cell proliferation and matrix deposition by adult human aortic SMCs within 3D biomimetic cultures have been explored. The first experiment utilized a microfluidic platform with two adjacent, permeable 3D culture chambers, to enable paracrine signaling between vascular cell cocultures. Healthy HA-SMCs were cultured in these devices within 3D collagen hydrogels, either alone or in the presence of human aortic ECs cocultures, and exogenously supplemented with varying GSNO dosages (0-100 nM) for 21 days. Results showed that EC cocultures stimulated SMC proliferation within GSNO free cultures. However, with increasing GSNO concentration, SMC proliferation decreased in the presence or absence of EC cocultures, while EC proliferation increased. GSNO (100 nM) significantly enhanced the total protein amount synthesized by SMCs, in the presence or absence of EC cocultures, while lower dosages offered marginal benefits. On a per cell basis, multi-fold increases in the synthesis and deposition of elastin, GAGs, hyaluronic acid and LOX were noted at higher GSNO dosages, and coculturing with ECs significantly furthered these trends. The matrix yields of these proteins reached almost 40 - 51 % within selective cocultures receiving GSNO. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages, although MMP-2 levels remained attenuated. These quantitative assay data were strongly supported by immunofluorescence image (open full item for complete abstract)

Committee: Chandra Kothapalli PhD (Committee Chair); Nolan Holland PhD (Committee Member); Moo-Yeal Lee PhD (Committee Member) Subjects: Anatomy and Physiology; Biochemistry; Biomedical Engineering; Biomedical Research; Cellular Biology; Engineering; Health; Health Care; Medicine; Pharmaceuticals

Doctor of Philosophy, The Ohio State University, 2007, Animal Science

Failure to terminate the inflammatory response results in chronic inflammation that may lead to disease or cancer, especially in epithelial cells. We explored mammary epithelial cells as a model to identify mechanisms of anti-inflammatory activity in epithelia alone in the absence of immune cells. Bacterial endotoxin (ET) added to SCp2 mammary secretory epithelial cells: (1) induced both interleukin-6 (IL-6) secretion and nitric oxide (NO) production, but with unexpected delay in expression of mRNA for iNOS compared to IL-6; and (2) NFκB activation by 1 h after ET application (post-ET) that was transient for NFκB/p65 but persisted for NFκB/p50. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in ET-induced IL-6 and iNOS regulation via NFκB activation. Serum supplementation but not soluble extracellular matrix (EHS) enhanced ET-induced IL-6 mRNA expression and protein secretion without affecting iNOS mRNA expression or NO production, confirming the different modes of regulation of IL-6 and iNOS expressions. Culturing SCp2 cells on a confluent monolayer of SCg6 mouse mammary myoepithelial cells increased IL-6 secretion dramatically even in the absence of ET, with ET treatment further increasing IL-6 secretion but having little effect on induction of NO production over that for SCp2 cells alone; showing importance of microenvironment and cell-cell interaction in regulation of inflammation and likely its link to cancer in epithelia. ET-induced inflammation in SCp2 cells was used to screen and identify anti-inflammatory fractions of methanol extracts of wild Lebanese Centaurea ainetensis, used in Lebanese folk medicine to treat inflammatory diseases. A partially purified solid phase (SPE columns) methanolic elution fraction of C. ainetensis followed by methanol gradient elution on reverse phase HPLC chromatography (RP-HPLC) strongly inhibited ET-induc (open full item for complete abstract)

Committee: Floyd Schanbacher (Advisor); Charles Brooks (Other); James DeWille (Other); Joy Pate (Other) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2011, Biomedical Engineering

Rotator cuff tendon injury is a debilitating health concern that affects more than 40% of the aging population. Despite advances in surgical treatment, the failure rate of rotator cuff repairs ranges 20-90%. Naturally-occurring extracellular matrices (ECMs) have been recently investigated as augmentation scaffolds, but none has yet demonstrated both the appropriate biological and mechanical properties. This dissertation proposes to enrich fascia ECM with high molecular weight tyramine substituted-hyaluronan (TS-HA) for rotator cuff repair. The central hypothesis is that TS-HA treatment will decrease chronic inflammation without decreasing the time-zero or post-implantation mechanical properties of fascia. The specific aims are to develop a TS-HA treatment method and to evaluate the host response and concomitant mechanical properties of treated fascia in a rat abdominal wall model. TS-HA treatment increased the HA content of fascia by an order of magnitude to ~1% tissue weight. The incorporated HA was distributed throughout the ECM and, upon cross-linking, was retained as a hydrogel network. Cross-linked TS-HA treated fascia exhibited an increased macrophage and giant cell response and a lower density of fibroblast-like cells than water treated controls. Treated fascia, with or without cross-linking, exhibited a predominantly M2 pro-remodeling macrophage profile similar to water controls, which is suggestive of constructive tissue remodeling. All grafts exhibited a chronic lymphocytic response that is suggestive of an immune response to the fascia xenograft. Fascia samples in all groups demonstrated time-dependent decreases in mechanical properties. Cross-linked TS-HA treated fascia exhibited a lower toe-region elastic modulus and trended towards a higher transition strain than water treated controls not only after implantation, but also at time zero. These findings demonstrate that HA augmentation can alter both the host response and the mechanical properties of f (open full item for complete abstract)

Committee: Kathleen Derwin PhD (Advisor); Roger Marchant PhD (Committee Chair); Eben Alsberg PhD (Committee Member); Thomas Bauer MD, PhD (Committee Member); Vince Hascall PhD (Committee Member) Subjects: Biomedical Engineering