Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 132-157). Also available online through Digital Dissertations.

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Thesis (M.S.)--University of Michigan, Ann Arbor, 1969. / Typescript (photocopy). Includes bibliographical references (leaves 68-71). Also issued in print.

Thesis (M.S.)--University of Michigan, Ann Arbor, 1969. / Typescript (photocopy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 68-71).

Peritoneal adhesions pose a considerable postoperative burden. The mechanisms underlying adhesion formation remain poorly understood but relate principally to inflammation and fibrinolysis. The peritoneum may coordinate the inflammatory response that leads to adhesion formation but human adhesion formation cannot be studied directly so most research has employed animal models and individual transformed cell lines. Furthermore, the specific roles of the visceral or parietal peritoneum in adhesion formation are unclear. The aims of the studies undertaken in this thesis are to investigate the regulation of a gut bacteria-driven inflammatory response in human visceral peritoneum. A novel ex vivo model was designed using human small bowel visceral peritoneum, cultured with E. coli derived LPS, for 18 to 48 hours. ELISA and rtPCR were used to analyse supernatant protein concentration and gene expression respectively for key inflammatory mediators associated with adhesion formation including cytokines (IL-6, IL-10, TNFalpha), transforming growth factor-β1 (TGF-β1) and fibrinolytic factors (tPA and PAI-1) as well as the signalling receptors thought to be involved in LPS-mediated inflammation (TLR2 and 4). LPS exposure resulted in stimulation of IL-6, IL-10 and TNFalpha production from human visceral peritoneum at 18 hours. These increases correlated with increased gene expression for IL-6 and TNFalpha but not IL-10. TGF-β1 production and gene expression were unchanged at 18 & 24 hours but increased at 48 hours when co-incubated with IL-1β. PAI-1 was increased but tPA was unchanged following LPS stimulation, suggesting a reduction in peritoneal fibrinolytic activity. Peritoneal tissue expressed the signalling receptors TLR 2 and 4. However, inhibition of TLR 2 or 4 failed to abrogate production of cytokines in cultured primary mesothelial cells or peritoneal tissue post-LPS exposure. TLR 4 inhibition did reduce mesothelial cell production of chemokines, CCL2 and CXCL1. These novel findings demonstrated that human visceral peritoneum actively participates in LPS-induced peritoneal inflammatory responses and reduced fibrinolysis which may account for the visceral peritoneum's affinity to form adhesions. Ex vivo human peritoneal culture is a novel, feasible and reproducible method of investigating the role of the peritoneum in inflammation.

Postoperative adhesions form as the body's natural response to injury in an effort to temporarily protect and supply nutrients to these tissues. However, adhesions can remain permanent, and fail otherwise successful surgeries by tethering tissues together that are normally separated. An ideal anti-adhesive device reduces unwanted adhesions and leaves the patient in a state most similar to before surgery. This dissertation introduces a novel, robust hydrogel film consisting of two hydrophilic polydisaccharides, hyaluronic acid (HA) and alginate. To address the challenge of retaining HA in alginate-rich hydrogels, we methacrylated the HA (GMHA), creating hydrophobic moieties. These hydrophobic interactions shift the percolation threshold, allowing for greater concentrations of GMHA to be retained in resulting films. UV crosslinking retains GMHA beyond the percolation threshold and widens the possibilities of usable films. To enhance the mechanical properties of these alginate/GMHA films, we employed a previously developed method for creating thin, branched, interconnected fibers using urea crystal templating. Templated films are softer and, yet, tougher than films that have not been templated. This toughness is a result of increased density of polymer in the fibers. These films were selected as most conformable and most robust by surgeons in a blinded handling study. In a rat peritoneal abrasion model for adhesion formation, the films successfully prevented adhesions with statistical equivalence to the leading anti-adhesion device commercially available. Finally, future recommendations are suggested for the development of a bilayer construct with a collagen/alginate blend bound to an alginate/HA layer for an anti-adhesive and regenerative strategy. This construct addresses the need for opposing strategies in the dynamic environment of wound healing. Further research is needed to develop the usefulness of this bilayer system, as preventing unwanted adhesions is merely a first step in achieving a blemish-free healed wound. / text

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Serine phosphorylation of FAK. Includes bibliographical references (p. 209-231). Also available online through Digital Dissertations.

Focal adhesions are dynamic multiprotein structures connecting cells to their surrounding microenvironment. Cells receive critical mechanical signals from adhesions that control many cellular processes including wound healing, differentiation, development, and cancer. Proteins that form adhesions are called adhesion proteins and some of these proteins can be mechanosensitive, meaning that they respond to mechanical stimuli. During spreading and migration, cells mechanically test extracellular matrix rigidity by contracting matrix to a constant displacement. Transmission and processing of such mechanical signals rely upon the dynamic regulation of the adhesions, which is tightly coordinated with activation of intracellular signaling cascades involving various adhesion molecules. However, the molecular mechanisms of mechanical signals that are transmitted through the adhesions to control cell behavior are poorly understood. In this thesis, we discovered novel phenomenon and mechanisms to elucidate roles of mechanical signals for multiple key aspects of basic cell behavior, especially cell growth. We performed live cell imaging of cells spreading on fibronectin coated micropillars to understand adhesion formation, adhesion regulation, and their impact on cell behavior. One of the earliest molecules to arrive at an adhesion formation site is a mechanosensitive protein called talin which binds to several other entities to form the backbone of focal adhesions. We found a novel role of talin cleavage, which previously was thought to play a role only in focal adhesion turnover. We found that talin cleavage is a force dependent process that regulates proper adhesion formation, thereby governing several critical cellular processes. In the absence of this talin cleavage, cells formed abnormal adhesions and showed inhibited growth. Further, we found that upon inhibition of talin cleavage, one of the key cellular behaviors of increased cellular motility upon stimulation by epidermal growth factor seemed to disappear. Epidermal growth factor receptor is a transmembrane protein and has previously been shown to play important role in various cancers where cells exhibit altered rigidity sensing. Surprisingly, we found that epidermal growth factor receptor was required for cellular rigidity sensing only on rigid substrates, highlighting the importance of the interplay between mechanical and biochemical signals in determining cell behavior.

The fundamental principle of tissue engineering is to use appropriate cell source, combined with scaffolds and bioactive factors to develop tissue constructs which restore, maintain or improve tissue function. There is increasing data emphasizing the importance of mechanical signals and extracellular matrix (ECM) proteins presented by the scaffold in determining stem cell fate/functions which are critical to tissue construct maturation and success of stem cell-based therapies. Cell-matrix adhesions are one of the major mechanosensing machineries cells use to convert information provided by ECM ligands and mechanical signals presented by scaffolds into intracellular biochemical signaling cascades which lead to particular functional responses. Therefore, understanding how ECM ligands and mechanical signals regulate cell-matrix adhesion formation and activation of associated intracellular signaling proteins is fundamental to rational design of biomaterial and loading protocol for optimal cell functional responses in tissue constructs. In this study, we attempted to understand the regulatory effects of external mechanical signal and exogenous ECM protein on cell-matrix adhesion formation and associated intracellular signaling proteins of human mesenhymal stem cells, and in particular, to test the hypothesis that mechanical stimulation or exogenous ECM protein can lead to adhesion maturation into 3D-matrix adhesions in 3D collagen environment. We used microencapsulation technique to embed cells in 3D collagen environment, forming disc-shaped hMSC-collagen constructs. By immunofluorescent staining and confocal microscopy, we visualized changes in size, morphologies and molecular composition of the adhesions. First of all, 2D adhesions of hMSCs were characterized. We showed that hMSCs form well-organized αv integrin-based focal adhesions and fibrillar adhesions in 2D culture. To investigate the regulatory effects of mechanical signals on adhesion signaling and maturation, we used micromanipulator-based loading device to impose dynamic compression to hMSC-collagen constructs. We found that dynamic compression lead to enlargement of integrin αv adhesions which recruit focal adhesion kinase (FAK), vinculin and extracellular signal-regulated kinase (ERK). In addition, FAK was activated at enlarged integrin αv adhesions and translocated to peri-nuclear region after compression, suggesting that loading induces activation of FAK signaling pathways through increased integrin αv clustering. Moreover, we demonstrated that dynamic compression can induce 3D-matrix adhesion formation, indicating the role of external force in integrin α5-based adhesion maturation in 3D collagen environment. We explored the effect of exogenous ECM proteins on adhesion maturation of hMSCs by adding fibronectin into cell-collagen mixture during fabrication of collagen constructs. Our results demonstrated that the exogenous fibronectin can induce α5 integrin-based adhesion maturation into 3D-matrix adhesions in our collagen constructs in a dose-dependent manner. This study demonstrated that the effect of external mechanical signals and exogenous ECM ligands on adhesion signaling and maturation of hMSCs in 3D collagen environment. Our findings contribute towards mechanobiology of hMSCs in 3D context. In particular, our results showed that exogenous proteins or external loading can lead to 3D-matrix adhesion formation, which may serve as a potential way to enhance biological functions of hMSCs in collagen constructs, facilitating stem cell-based therapies. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Abstract FAP52 (focal adhesion protein, 52 kDa) is a focal adhesion-associated protein composed of a highly α-helical NH2-terminus containing a poorly characterized FCH (Fes/CIP4 homology) domain, unstructured linker region and the COOH-terminal SH3 domain. FAP52 is also known as PACSIN 2 or syndapin II. Together with other PACSINs and syndapins FAP52 shares a common domain architecture. The aim of this study was to characterize FAP52 in structural and functional terms. The function was pursued by identifying binding partners for FAP52, and by overexpressing the recombinant FAP52 in cultured cells. For the structural studies, various physico-chemical methods, such as chemical cross-linking, gel filtration chromatography, circular dichroism and X-ray crystallography were applied. In addition, the histological distribution of FAP52 in chicken tissues was explored. FAP52 binds filamin, a protein that regulates the dynamics of the cytoskeleton by crosslinking actin filaments. The binding site in FAP52 was mapped to the NH2-terminal 184 amino acids, of which the residues 146–184 form the core of the binding. In filamin, the binding site resides in the repeats 15–16 in the rod-like molecule encompassing 24 such repetitive domains. Overexpression of FAP52 or its filamin-binding domain in chicken embryo heart fibroblasts induced the formation of filopodial extensions on the cell surface and reduced the number of focal adhesions, suggesting a role in the organization of the cellular cytoskeleton and in cell adhesion machinery. Experiments utilizing surface plasmon resonance analysis, size exclusion chromatography and chemical cross-linking showed that FAP52 self-associates in vitro and in vivo. The region responsible for the self-association was mapped to the amino acids 146–280, which is predicted to fold into a coiled-coil arrangement. FAP52 was crystallized by using the hanging-drop vapor-diffusion method and ammonium sulfate grid screen. Native dataset was collected from two crystals, which diffracted to 2.8 Å and 2.1 Å resolution. For one form of crystals, phasing was performed using the native dataset and the datasets from two xenon-derivatized crystals. X-ray crystallography studies revealed a dimer in asymmetric unit. Histological and in vitro studies showed that, in liver, FAP52 is preferentially expressed in bile canaliculi. In other tissues, FAP52 showed a specific staining pattern in gut, kidney, brain and gizzard. Together, these data show that FAP52 self-associates in vivo and, probably via its interaction with its binding partner filamin, participates in the organization of the cytoskeletal architecture, especially of the cell surface protrusions, such as filopodia and microvilli of bile canaliculi.