Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / Typescript. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 146-162).

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references.

The objective of the presented research is to examine the relationship between the cellular microenvironment and biochemical response of metastatic cells. Clinically recognized as a trait of cancer progression, the cellular microenvironment can have variable and distinct mechanical properties that are processed via cellular mechanosensing, resulting in a cellular biochemical response. A range of studies investigating the interactions between the cellular micromechanical environment and the cell's molecular response during disease progression have been made, yet remain absent of quantitative characterization of many of these coordinated responses. The fundamental inquiry that drives the following research attempts to elucidate how a cell perceives the physical microenvironment and converts that signal to a biochemical response. With the goal of providing insight to such responses, the presented research seeks to elucidate the following questions: (1) What are the integrated effects of ECM stiffness, ECM architecture, and breast cancer cell metastatic potential on cell migration? (2) How does endogenous tissue transglutaminase (tTG) cross-linking of the ECM scaffold effect ECM mechanical properties? (3) How does the architecture and stiffness of the extracellular matrix (ECM) effect the systems-level cellular migration and signaling response? (4) What are the integrated effects of ECM architecture and the targeted knockdown of integrin [beta]1 and MT1-MMP on cellular metastatic potential? The presented research utilizes an interdisciplinary approach, integrating experimental mechanics, biochemical analysis, cellular biology techniques, covalent chemistry, and various microscopy techniques, to investigate these events. In short, cancerous cells are cultured atop or within synthetic collagen type I ECMs of varying mechanical stiffness and structure. These cells are subsequently analyzed by molecular analysis and immunoassays, including quantitative PCR, Western blotting, and gelatin zymography, to acquire measures of the cellular response to perturbations of micromechanical environment. Time-lapse microscopy experiments and subsequent image analyses enable observations of cellular migratory potential through synthetic ECMs. Results indicate that cooperative synergy between ECM properties, cell-matrix adhesion, and pericellular proteolysis drive cell migratory potential of highly invasive tumorigenic cell populations. Collectively, these findings contribute to the cancer biology and mechanobiology fields by systematically extending current insights of matrix mechanics, cellular signaling, and cellular migratory potential in cancer. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

One of the challenges in tissue engineering is to fabricate scaffolds which can mimic the natural microenvironments of cells. In a cell niche, biophysical and mechanical cues are crucial factors influencing cell functions. Given the complexity of natural extracellular matrix (ECM) engineered ECMs providing controllable biophysical and mechanical cues are appealing both in enhancing the understanding of cell-matrix interaction and in controlling cell fates in vitro. The ultimate goal of our study is to establish a platform as an engineered ECM by fabricating customized solid protein microstructures from solution using two-photon photochemical crosslinking, a novel laser-based freeform fabrication technique. In this study, protein structures varying from submicron lines, 2D micropatterns and microporous matrices, to 3D micropillars were successfully fabricated, demonstrating freeform fabrication capability with two-photon photochemical crosslinking. Two-photon fluorescent imaging and scanning electron microscope (SEM)-based microstructural characterization revealed that power, scan speed, total exposure time and concentrations of protein (bovine serum albumin) and photosensitizer (rose Bengal) in the solution were crucial processing parameters in this fabrication technique. Quantitative imaging analysis showed that porosity of protein matrices was highly dependent on processing parameters including power, scan speed, number of cycles in time series scan and protein concentrations in the solution. An atomic force microscopy (AFM)-based step change nano-compression test was used to measure the reduced elastic modulus of 3D viscoelastic protein micro-pillars fabricated, as a pilot study. Microporous protein matrices and 3D micropillar arrays fabricated with two-photon photochemical crosslinking can be used as engineered ECM for future study in cell-ECM interactions. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

The mission of tissue engineering is to recapitulate the natural process of tissue formation by assembling cells into synthetic scaffold. This relies on the understanding of the functions and properties of the tissue microenvironment (TME), the specific extracellular environment within endogenous tissues. Although existing studies demonstrated the effect of each of the topographical, mechanical and biochemical properties on cell behaviors in isolation, the effect of these properties within the native TME are complicated and ill defined. This thesis aims to investigate how topographical, mechanical and biochemical features of natural TME contribute to the modulation of the biochemistry, morphology and functions of cells, and to translate this knowledge into the fabrication of biomaterials. Tissue cryosections as a cell culture model system was established. It allowed robust assessment of cell phenotypes in a near-natural TME. Mesenchymal stem cells (MSC) cultured on bone, cartilage and tendon cryosections adopted different morphology, supporting the idea that tissue cryosections forms a robust platform for cell-TME studies. Then, Achilles tendon TME was chosen for proof of concept. This tendon cryosection induced different cell types to adopt different morphologies, indicating that the effect of TME is cell type specific. The proliferation of MSC cultured on cryosection was suppressed, however it was instructed to commit tenogenic differentiation. Then, the necessity of TME topographical properties in forming this instruction was delineated by seeding MSC onto cross-sectional tendon cryosection. Although this surface contained native biomechanical and biochemical cues, it could not promote differentiation. This highlighted the necessity of topographical cues within the TME. Next, nano-grooved titanium surface that resembles the topographical cues of tendon TME was used to replicate the function of TME. This surface successfully promoted morphogenesis of MSC but not differentiation. This implicated that biomechanical and biochemical cues are both necessary for instructing desired cell phenotypes. The proteomes of MSC cultured on nanogrooved and planar surfaces were then studied using quantitative proteomics. This revealed some expected changes such as up regulation of cytoskeleton and cell-adhesion proteins, suggesting mechanotransduction events might have been induced by nano-grooved surface. However, expressions of RNA-binding proteins were also regulated, representing novel findings. These proteins were also found in the proteome of cellmicroenvironment interface identified through the use of subcellularfractionation and proteomics. This consolidated their involvement in cellmatrix interactions. The topographical and mechanical properties of cryosection were replicated by using bioimprinting. This imprint induced the morphogenesis of MSC, but tenocytic differentiation was induced only when collagen 1 was coated. However incorrect mechanical properties would abolish such phenotypic guidance. This suggests that topographical, mechanical and biochemical information in a TME are individually indispensable, and it is possible to functionally reconstruct a TME by bioimprinting and ECM protein coating. In summary, this study investigated the topographical, mechanical and biochemical properties in tendon TME and their combined effect on controlling cell phenotypes. It illustrates that biomimetic approach that mimics these three properties of a tissue can effectively control cell phenotypes. Further investigation on better biomimetic methods and its molecular mechanisms will help establishing strategies for constructing functional tissues. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Matrix metalloproteinase (MMP) induced extra cellular matrix (ECM) degradation is a crucial process involved in the development of many chronic inflammatory diseases, including cardiac remodeling and cancer metastasis. In cardiac remodeling, the presence of pathological stimuli leads to elevated MMP-9 expression and impairment of cardiac performance, which subsequently develops into heart failure. While in tumorgenesis, MMP-9 has been found to play key roles in metastasis, as it can break physical barriers for the tumor. Therefore, searching for agents targeting MMP-9 is a new direction for the treatment of cardiac remodeling and cancer metastasis. Chinese herbal medicine is becoming increasingly used worldwide in recent decades. In the past twenty years, as many highly selective and sensitive bioassays were introduced into the bioactive compounds screening from herbal medicine, more than one hundred new drug candidates have been identified. Therefore, herbal medicine is a potential source of bioactive compounds. Panax notoginseng (PNG) is one of the most common traditional Chinese medicines to treat cardiovascular diseases, and it was also reported to have anti-cancer effect. We hypothesized that it contains bioactive compounds that could inhibit MMP-9 activity in cardiomyocytes and cancer cells. In order to examine the effect of PNG on cardiac remodeling and cancer metastasis, we employed TNF-α induced MMP-9 in H9c2 cell (a rat cardiomyocyte) and HepG-2 cell (a human hepatoma cell) as an in vitro assay, respectively. PNG was first extracted by four different extraction methods according to the polarity of the solvent. The most effective fraction in suppressing MMP-9 activity in TNF-α induced H9c2 cell was chosen for further separation by silica gel column chromatography and high performance liquid chromatography (HPLC) until a single compound was isolated. According to the result of spectroscopic analysis by NMR, the compound was identified as ginsenoside Rb1. For the bioactivity assays, real-time quantitative polymerase chain reaction (QPCR) and Enzyme-linked immunosorbent assay (ELISA) were used to measure the mRNA and protein expression of MMP-9, respectively. We also examined the MMP-9 activity by gelatin zymography. The results showed that both of the PNG extract obtained from 10% ethanol extraction method (PNG-3) and purified Compound P (ginsenoside Rb1) showed significant inhibitory effect on MMP-9 expression and activity in H9c2 cells and HepG-2 cells. We further examined the molecular mechanisms of the inhibitory effect of PNG-3. H9c2 and HepG-2 cells were pretreated with different kinase inhibitors followed by the activation by TNF-α. The results showed the protein kinase R (PKR) inhibitor could inhibit TNF-α induced MMP-9 in both of the two cell lines. Furthermore, the results of Western blot showed the PNG-3 suppressed the phosphorylation of eIF-2α which is a down-stream effector of PKR in TNF-α stimulated H9c2 and HepG-2 cells, respectively. Therefore, PNG-3 may act through PKR to regulate TNF-α induced MMP-9 activity. In summary, bioactivity guided fractionation is an effective way of isolating bioactive compounds from medicinal herbs. In addition, PNG containing ginsenoside Rb1 may be a potential candidate of MMP-9 inhibition for the treatment of cardiac remodeling and cancer metastasis. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

abstract / Mechanical Engineering / Doctoral / Doctor of Philosophy