The effects of Botulinum neurotoxin A on the passive mechanical properties of skeletal muscle have not been researched but may have significant clinical effects in the treatment of neuromuscular disorders including spasticity. Single fiber and fiber bundle passive mechanical testing was performed on muscles treated with Botulinum neurotoxin A. Myosin heavy chain and titin composition of single fibers was determined by gel electrophoresis. Muscle collagen content was determined using a hydroxyproline assay. Botulinum neurotoxin treated single fiber passive elastic modulus, stiffness, and slack sarcomere length was reduced from the contralateral side. Single fiber myosin heavy chain composition shifted from faster to slower isoforms after treatment. The average titin molecular weight in a fiber also increased after treatment. Fiber bundle passive elastic modulus and stiffness increased while collagen content per mass of muscle tissue increased 48 percent. The passive mechanical properties of muscle change after injection with Botulinum neurotoxin and may be clinically beneficial to patients with spastic muscle

Heparan sulfate structural heterogeneity is driven, in part, by the placement of sulfate groups at various positions in the polysaccharide. While many ligands bind to heparan sulfate without strict requirements for the positions of sulfate groups, binding of a small number of known ligands is influenced by the presence of a sulfate at the C3 position of a glucosamine residue. In mammals, seven enzymes can catalyze the addition of 3-O-sulfate groups, suggesting the possibility of other, previously unidentified, ligands whose binding is influenced by 3-O- sulfated sequences. Chapter 1 contains a comprehensive review of the synthesis, biochemistry and physiology related to 3-O-sulfate. Chapter 2 describes the development and use of heparan sulfate affinity matrices to identify ligands that rely on 3-O-sulfate. Neuropilin-1, a modulator of vasculogenesis and axonal guidance, is one of the 3-O-sulfate dependent ligands identified. Chapter 3 demonstrates that 3-O-sulfate is required for high affinity binding of neuropilin-1 to heparan sulfate. Furthermore, 3-O-sulfation influences a neuropilin- 1 dependent process, namely neuronal growth cone collapse. Chapter 4 elaborates on the implications of these findings and the future work needed to fully characterize 3-O- sulfation. This work indicates the existence of many proteins that rely on 3-O-sulfate and outlines the methodology needed to identify and characterize their interaction with heparan sulfate