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FIBRONECTIN INFLUENCES THE RATE OF ASSEMBLY AND STRUCTURAL CHARACTERISTICS OF THE FIBRIN MATRIX AND A MAP OF LYSINE PEGYLATION SITES IN FIBRONECTIN
Title
FIBRONECTIN INFLUENCES THE RATE OF ASSEMBLY AND STRUCTURAL CHARACTERISTICS OF THE FIBRIN MATRIX AND A MAP OF LYSINE PEGYLATION SITES IN FIBRONECTIN
Creator
Ramanathan, Anand
Date
2015, 2015-07
Description
Fibronectin serves multiple roles during tissue formation and wound healing, functioning through interactions with cells and extracellular...
Show moreFibronectin serves multiple roles during tissue formation and wound healing, functioning through interactions with cells and extracellular molecules. The overall objective of my research was to investigate fibronectin biochemistry on responses associated with wound healing. My approach was to engineer relevant in vitro models highlighting fibronectin functionality in tissues and link this work to more complex wound healing systems. My research goals were accomplished through the following three specific aims: (1) Determine the role of fibronectin on the kinetics of formation and structure of a fibrin-fibronectin matrix, (2) Determine the effect of protease on the activity of fibronectin in decellularized extracellular matrices and (3) Map the sites of polyethylene glycol conjugation or PEGylation to lysine residues in fibronectin. Aim 1: I demonstrated that fibronectin increased the initial rate of fibrin matrix formation and altered the fibrin matrix structure. These findings are novel because they link results from light absorbance studies to microcopy analyses and demonstrate the influence of fibronectin on fibrin matrix structural characteristics. Aim 2: I demonstrated a link between fibronectin proteolysis and reduced cell adhesion in decellularized extracellular matrices. This study demonstrates the susceptibility of fibronectin to proteolysis in the extracellular matrix and the resulting loss of matrix functionality, placing weight on bioengineering strategies to stabilize fibronectin against proteolysis. Aim 3: I examined proteolytic fragments of native and PEGylated fibronectin to map fibronectin lysine residues that are conjugated PEG. From four key chymotryptic fragments that span fibronectin and are recognized by specific monoclonal antibodies, I provide a map of lysine PEGylation sites for fibronectin. Moreover, I show that lysine PEGylation of fibronectin occurs asymmetrically on the dimer arms. Knowledge of the lysine PEGylation sites can be used to plan future experiments for investigating fibronectin biochemical interactions in complex in vitro and in vivo models. In accomplishing these specific aims, I identified key biomolecular mechanisms involving fibronectin and created relevant in vitro models to study these interactions. The work detailed in this thesis lays the foundation for future experiments to investigate fibronectin functionality and develop therapeutic strategies targeting fibronectin biochemistry in tissue development.
Ph.D. in Chemical Engineering, July 2015
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