Magainin and melittin are 2 members of a class of small amphipathic helical peptides which act as potent antibiotics or toxins. It has been shown that the peptides in this class interact directly with the lipid bilayer rather than with protein targets within the membrane. Using oriented circular dichroism, lamellar x-ray diffraction and small angle in-plane neutron scattering we have determined the structures formed by these peptides on association with the bilayer. We have found that magainin forms 'wormhole' channels in the lipid bilayer and melittin solubilizes the membrane by the formation of peptide bounded discoid micelles. This is compared with our earlier results for alamethicin which forms channels using the barrel-stave model. The discovery that three ostensibly similar peptides form completely different structures is quite surprising, and emphasizes how much work remains to be done in this field. Hopefully the methods used and the motifs identified in this thesis will provide a good basis for continuing studies of similar peptides.

Macromolecular dynamics is important for both understanding biological processes and practical applications such as MR imaging data interpretation. We have used the QNS method to study the dynamics of trypsin chain segments. Trypsin powder and trypsin in D$\sb2$O were studied for temperatures from 100K to 300K. Energy spectra of the scattered neutrons were measured for various neutron momentum transfer. Diffusive type motion of chain segments are observed for trypsin solutions at temperatures above the freezing point, while powder and frozen samples display minimum chain motion. The motion of trypsin chain segments can be fitted by a general "jump-diffusion" model which describes the conformational changes of a macromolecule as transitions between its substates. The diffusion coefficient of the trypsin chain segment is 2.4 $\times$ 10$\sp{-6}$ cm$\sp2$/sec and the average residence time of trypsin in its substates is 1.3 $\times$ 10$\sp{-11}$ second when trypsin is in D$\sb2$O at 300K. We measured the average mean square thermal vibration amplitude of trypsin (0.65 A$\sp2$) which is slightly larger than the results from computer simulations and X-ray diffraction studies. We also have tested macromolecular dynamics theory on poly-acrylic acid. We measured the frequency dependence of proton T$\sb1$ relaxation rates of poly-acrylic acid in D$\sb2$O solutions with different pDs and salt concentrations. Frequency dispersion data analyzed using both a flexible chain model and a stiff chain model give a maximum correlation time of 1.5-2.3 $\times$ 10$\sp{-7}$ second depends on the model. No significant change due pD and salt concentration difference was found.

A model of $\beta$-adrenergic and muscarinic cholinergic stimulation of the bullfrog atrial myocyte has been developed that mimics the dose-dependent effects of isoprenaline (ISO) on the action potential duration (APD); i.e., low doses of ISO lengthen the APD, while high doses shorten the ADP. This reduction in APD is modeled as the result of (1) calcium-dependent inactivation of $I\sb{Ca}$ resulting from the enhancement of $I\sb{Ca}$ by ISO and (2) an enhancement of $I\sb{K}$ due to both an ISO-induced increase in the rate of activation of $I\sb{K}$ and an increase in peak action potential height. The effect of acetylcholine (ACh) is to reduce the ISO-induced increase in $I\sb{Ca}$ and $I\sb{K}$ through a reduction in relative (cAMP) as well as to stimulate the ACh-sensitive $K\sp{+}$ current $I\sb{K,ACh}.$ At low (ISO) levels or high (ACh) levels, the muscarinic cholinergic effect dominates over the $\beta$-adrenergic effect. However, for a large (ISO) and a small (ACh), this pattern of changes in transmembrane currents is different; in this case the model predicts that ACh can actually increase APD. A distributed parameter model of an idealized bullfrog atrial trabeculum is developed. Individual cardiac cells are resistively coupled end to end via intercalated discs to form an idealized cylindrical cardiac strand encased in a resistive-capacitative trabecular sheath which, in turn, is located in a finite cylindrical volume conductor. A second-order implicit finite numerical integration method is used to calculate the time-varying potentials within the intracellular $(V\sp{i}),$ interstitial $(V\sp{e}),$ and the outer volume conductor $(V\sp{o})$ media of the concentric cylindrical structure. 'Reduced' cell membrane models which lack the complete complement of transmembrane currents are compared with regard to their accuracy in representing the foot, upstroke, and plateau regions of the propagated action potential in the complete model. A reduced cell membrane model should contain the sodium current $I\sb{Na},$ the calcium current $I\sb{Ca}$ and the background rectifying $K\sp{+}$ current $I\sb{K1}.$ A cell membrane model which contains a linear background $K\sp{+}$ current $I\sb{L}$ instead of $I\sb{K1}$ produces a poor approximation to the upstroke, plateau and conduction velocity of an action potential. The trabecular sheath reduces the extracellular resistance seen by the cell by shunting current away from highly resistive interstitial medium into the volume conductor medium which is of low resistance, and thereby increases conduction velocity. Finally, the effects of the cholinergic neurotransmitter, acetylcholine (ACh), on both the passive and active properties of the trabeculum are investigated. The addition of ACh to the extracellular medium reduces the space constant and input resistance of the trabeculum, as well as the conduction velocity of electrical activity propagating through it.

The distribution of light in a tissue is determined by it's optical properties. Several techniques are available for determining a tissues optical properties, and models are available for predicting the distribution of light within a tissue of known optical properties. The accuracies of these models were compared. The optical properties of neonatal skin were determined in the visible region from 450-750 nm. The reduced scattering coefficient, $\mu\sb{\rm s}$(1-g), increases directly with gestational maturity of the infant. The increase in the reduced scattering coefficient with gestational maturity is due to the accompanying increase in size and density of the collagen fibers. In neonatal skin, the optical density perceived by reflection, the depth probed by photons escaping from the surface, and their pathlength in the tissue are dependent on wavelength and on collection geometry. The penetration of visible light into neonatal skin is strongly dependent on wavelength and on gestational age.

Bilirubin, a yellow pigment, is formed by the breakdown of hemoglobin. Neonates are susceptible to high bilirubin levels in their blood which places them at risk of neuronal damage, and monitoring of the bilirubin levels in these neonates is clinically required. Transcutaneous optical monitoring of the bilirubin will provide a non-invasive, inexpensive measurement of bilirubin in the skin. The optical properties of skin are important for interpretation of the reflected light from the skin. In this report, the optical properties of neonatal skin were measured in the visible range on twenty in vitro skin samples. The scattering of the skin is dominated by the collagen fiber bundles in the dermis. Scattering in the dermis increases linearly with gestational maturity due to the accompanying increase in the size and number of the collagen fiber bundles. Scattering in the dermis was modelled by Mie and Rayleigh scattering. The collection efficiency of an optical patch used for reflectance measurements at the skin surface varies with the skin's optical properties. The collection efficiency of the optical patch as a function of optical properties was determined by measurements in phantoms, and by Monte Carlo computer models. Dermal absorbers and epidermal melanin affect the reflected signals differently, and have to be analyzed separately. In addition to bilirubin content of neonatal skin, other sources of variation include skin maturity, skin thickness, melanin content, blood depth, and blood content. Each of these factors affects the reflected spectrum. Each source of variation was analyzed individually and an algorithm was developed to determine the absorbances of bilirubin and blood in the dermis from optical patch reflectance measurements. The algorithm was applied to analyze reflectance measurements performed on a heterogeneous clinical population consisting of 47 neonates. The algorithm was then adjusted to minimize a score designed to ensure that the determined in vivo cutaneous bilirubin concentrations were invariable with skin melanin and blood content. Consideration of optical transport in the skin has enabled the determination of cutaneous bilirubin concentration in heterogeneous neonatal populations.

Osteoarthritis and the subset of post-traumatic osteoarthritis both represent the end-stage of a degenerative process that can result from an initial tissue insult, particularly from a single mechanical impact. No current treatment has been shown to slow or stop its progression. Here, two approaches are taken to understand the physiology and pathology of articular cartilage. A cellular approach develops and uses a novel imaging technique for single cells and bioactive surfaces, while a tissue approach consists of understanding the acute and temporal effects of mechanical impact. Thus, the goals of this study are two-fold: (1) to develop vertical scanning interferometry (VSI) to obtain all salient features of chondrocytes and characterize bioactive surfaces, and (2) to develop methodologies for protecting diarthrodial joints from pathologic impact loading. VSI was validated and developed to obtain three-dimensional chondrocyte and fibroblast geometries, as well as to characterize protein-coated surfaces. VSI can now be applied to an array of studies involving single cell biomechanics, surface characterization, and cell adhesion and spreading. To examine pathology, an impact instrument was built and validated to apply repeatable impacts to articular cartilage. An explant model was characterized to understand the physiologic changes articular cartilage tissue experiences in culture over four weeks. Then, the acute and temporal effects of two levels of impact were characterized, consisting of a low level impact that did not show initial gross damage and a high level impact that caused immediate surface disruption. These studies illustrated that clinically undetectable impact injuries immediately show some subtle changes in extracellular matrix (ECM) glycosaminoglycan release and gene expression, but otherwise resemble the culture controls, while the high impact level caused gross damage. However, over a four week culture period, the subclinical impact proved to have started a degeneration cascade that significantly affected the biomechanical integrity, gene expression profile, biochemical makeup of the ECM, and chondrocyte viability. Therefore, impact injuries may account for a substantial proportion of the primary osteoarthritis cases. Further, if the start of the degeneration cascade of the low impact level can be stopped or reversed when only subtle changes are occurring, osteoarthritis prevention would be possible.

This thesis work presents a multi-scale mathematical modeling framework to investigate issues regarding vascular smooth muscle contraction. The overall structure of this thesis consists of (1) a model of single vascular smooth muscle cell membrane electrophysiology, contractile system kinetics and cell mechanics; (2) a model of signal transduction of nitric-oxide induced vascular smooth muscle relaxation, and (3) a cellular-based model for myogenic responses of isolated arteries. These models present a general and comprehensive description of VSM contraction and its associated signal transduction, which integrates information from both microscopic (cellular and subcellular interactions) and macroscopic (isolated vessel) levels. These models were utilized to investigate many underlying principles of vascular smooth muscle contraction mediated by multiple signaling pathways and can be applied to study functions of vascular smooth muscle under a variety of physiological and pathological conditions.

Spine disorders are one of the most prevalent and costly problems facing modern medicine, with an estimated annual cost of over $95 billion. Improved understanding of the biomechanical properties of the normal and pathological spine is essential for prevention of such disorders and treatment of traumatic injury, disc degeneration, or other ailments. However, the complex structure of the spine makes experimental testing that is relevant to physiological behavior difficult. Hence, new testing methods are needed to provide more accurate descriptions of in vivo behavior. Therefore, three principal aims are pursued in this work. The purpose of the first aim was to investigate the biomechanical performance of the human cadaver spine by applying a pure bending moment in conjunction with a range of compressive axial loads. This study showed that a minimum of 500 N compressive axial preload along the spinal curvature produces more comparable results to in-vivo studies, providing an important guideline for experiments investigating range of motion and spinal stability. The purpose of the second aim was to determine the motion response of the spinal joint to pure bending in any plane around its circumference. Towards that goal, the three-dimensional motion envelope of two-level spinal segments was analyzed. Since the posterior elements were intact, restriction in motion caused by zygapophyseal joints resulted a smaller displacement for extension than in flexion. Characterization of the motion envelope can provide a better understanding of the complex joint kinematics and assist in the design of new implants with the goal of restoring joint function. The purpose of the third aim was to investigate the strength of a single vertebral body during compression fracture by following the path of least resistance. In vivo, the spinal column continuously maintains equilibrium and minimizes stress via a complex system of muscles, tendons and ligaments such that each vertebra experiences a predominantly axial load. In a typical experiment, compressive loads are also applied axially but unwanted shear forces and moments are generated as well. Using a novel testing approach, identification of the weakest region of a vertral body, and following this path of least resistance, a true measure of vertebral strength, we were able to demonstrate the effect of current experimental limitations in overpredicting bone strength.

Biofilm formation, a common cause of medical device failure and tissue infection, often follows bacterial adhesion to proteins present in the tissue or adsorbed on the implant surface. Certain species of gram-positive bacteria have covalently anchored transmembrane molecules called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) which mediate adhesion. Developing a greater understanding of the interactions between MSCRAMMs and their ligands can lead to improved methods of combating bacterial adhesion. The goal of this research was to use optical tweezers to quantify and characterize the forces of staphylococcal detachment from surfaces coated with extracellular matrix (ECM) molecules. An optically trapped bacterium was brought in contact with an ECM-coated polystyrene microsphere, and the force required to separate the cell and microsphere was determined. The forces required to detach S. epidemidis from fibronectin occurred in a series of clusters whose means were integer multiples of an 18-piconewton (pN) base value depending on the number of bonds formed. For S. aureus binding, this estimated single-bond force was 25 pN for fibronectin and 20 pN for fibrinogen, respectively. In S. aureus, we have found that varying degrees of mutation of the fibronectin-binding MSCRAMM may cause reduction or inhibition of binding depending on the degree of mutation. However, multiple mutations are required before any reduction in binding is observed, which confirms that multiple regions of the S. aureus fibronectin MSCRAMM may substitute for one another in the binding process. We have also tracked the force required to detach S. aureus from fibronectin using the signal generated by the trapped cell on a quadrant photodiode throughout the detachment process for a series of loading rates. The magnitude of the force increased in an approximately linear fashion until the point of bond rupture. The peak single-bond rupture forces ranged from 10 to 29 pN for loading rates spanning three orders of magnitude from 101 to 103 pN/s. Bond lifetime increased with loading rate, which suggests the presence of a catch-bonding mechanism. This work provides additional insight into the specific binding mechanisms of staphylococci and is a step toward developing improved methods of preventing or treating infections.

The compelling evidence that platelets play a contributory role in hematogenous metastasis raises a need to elucidate the molecular mechanisms by which tumor cells activate and adhere to platelets. To model the in vivo interactions of platelets with arrested tumor cells, heparinized mepacrine-labeled whole blood was perfused over an A875 melanoma cell monolayer in a parallel plate flow chamber at shear rates characteristic of the microvasculature. Epi-fluorescence video microscopy utilizing a cooled CCD camera and digital image processing were used to quantify the dynamic adhesion and aggregation of platelets. Varying the exposure time of the monolayer to the 445 nm excitation wavelength revealed that photoactivation was stimulating the tumor cells and that the cells were relatively nonthrombogenic in absence of the light over the time scale of the flow experiments. Preactivation of the tumor cells was attempted with 12(S)-HETE, thrombin, and TNF-alpha and resulted in close to no platelet deposition.