The control of redox state of free thiols on ryanodine receptor (RyR) has been implicated as an important mechanism to regulate RyR channel activity and tune its responses to the physiological modulators. Both the skeletal and cardiac RyRs have been shown to be activated by S-nitrosylation of free thiols on them by a chemical process analogous to the oxidation of ¡°critical¡± or ¡°hyperreactive¡± thiols on RyR proteins. Inositol 1,4,5-triphophate receptors (IP3Rs) that control Ca2+ release from internal stores in non-excitable cells were found to be activated by oxidation, which emphasizes the redox reaction as a common mechanism to regulate intracellular Ca2+ channels. Therefore, the study on nitric oxide-mediated regulation of these ion channels will be important to understand the regulation of Ca2+ homeostasis in all cells including excitable and non-excitable cells since it regulates internal Ca2+ stores via RyR and /or IP3 receptors. The aims were to investigate the chemical reaction underlying the thiol-oxidation and activation of ryanodine receptors (RyRs) by various types of NO donors namely authentic NO¡¤, S-nitrosothiols and other NO¡¤ species such as nitroxyl anions (i.e. HNO). The different actions of these various NO¡¤ species were used to better evaluate the physiological significance of RyR activation by biologically relevant forms of NO, to investigate the role of oxygen on these chemical reactions and to identify the critical cysteine residues involved in redox mediated regulation of RyRs. The main findings are that RyRs are direct targets of S-nitrosothiols which trans-nitrosate hyper-reactive thiols and activate RyRs at biologically relevant concentration. In contrast, NO¡¤ gas cannot modify RyRs at biological circumstances found in cells. HNO is considerably more potent activator of RyR1 than NO¡¤, activates RyRs at nM concentrations. The study with truncated RyR1 indicated that all of transmembrane domains are located close to the C-terminus of the protein and the ¡®critical¡¯ regulatory thiols are part of conserved cysteines residing in it. Further studies will be required to elucidate the interplay of oxidants and reductants found in the cytosolic milieu of all cells and how these activators and inhibitors act to regulate the opening and closure of Ca2+ release channels.

The knee joint is a sophisticated biological mechanism involved in locomotion at the lower extremity. Despite its apparently simple motion during gait, the knee actually features complex 6-DOF kinematic patterns and 3D force distributions, stemming from the biomechanical interdependence of its component tissues, that become upset during injury and are difficult to restore with existing clinical treatments. In the interest of studying and characterizing the mechanics of the knee, a robotic/UFS testing system, capable of recording the complexity of joint kinematics and of the forces transmitted by the soft-tissues in response to meaningful loading conditions, has been used by various laboratories to obtain quantitative data with which to evaluate injury mechanisms, prevention, treatment and rehabilitation. This system has been successfully used to quantify the mechanical behavior of ligaments and their reconstruction grafts, menisci and cartilage, in response to a variety of experimental conditions. The effort of this work is to modernize the robotic/UFS testing system by upgrading its software control to manage more general and realistic loading conditions. The resulting software system, named the biomechanical testing platform, is expected to ultimately integrate the operation of the robotic/UFS testing system with that of other valuable experimental and computational approaches aimed at the study of the human knee joint. The biomechanical testing platform is designed with the use of state-of-the-art development technologies and comprises the mathematical formulations, control algorithms, and data abstractions specialized to a clinically relevant description of the kinematics and kinetics of the human knee. The system accommodates logical choices of hardware, motion description, iterative algorithms, as well as the use of automatic regression verifications. The biomechanical testing platform is demonstrated with a homologous experiment to that of the robotic/UFS testing system: the measurement of in situ forces in the ACL of a cadaver specimen, in response to anterior-posterior (translation) and varus-valgus (rotation) tibial loads. Furthermore, an application with concurrent interoperability between the robotic/UFS testing system and a computational analysis method is proposed.

The interaction between tumor cells and dendritic cells (DC) is a critical event for both initiation and regulation of specific antitumor immune responses. Based on the unique ability to present tumor antigen to and stimulate clonal expansion of naïve T cells, DC have been widely used to induce antitumor immunity in both preclinical animal models and human clinical trials. However, a growing body of clinical data and experimental evidence demonstrate that tumor exhibits a variety of inhibitory effects on the immune system and the DC system, in particular. It is well documented to date that tumor-derived factors influence DC generation, maturation, activity, and survival both in vitro and in vivo. Therefore, DC dysfunction in the tumor-bearing hosts could be largely responsible for the ability of tumor cells to escape from immune recognition or induce tumor specific tolerance. However, understanding the suppressive effect of tumor on the immune system, in particular on DC, and the protection of DC from tumor-induced dysfunction has not been studied. Therefore, the goals of this research project were (i) the understanding of DC immunobiology in the tumor microenvironment, revealing cellular and molecular mechanisms involved in tumor escape from immune recognition and elimination, (ii) the generation of novel antitumor vaccines which protect DC from tumor-mediated suppression, and (iii) the identification of primary mechanisms involved in vaccine-mediated antitumor immunity. The Tumor Necrosis Factor Ligand (TNFL) family is a group of cytokines, which through the interaction with corresponding receptors, regulate cell functions and activities. In this project we had examined three proteins which belong to the TNFL family: CD40L, RANKL, and 4-1BBL. Importantly, the receptors for these cytokines are found on activated DC. CD40L, RANKL and 4-1BBL were shown to influence DC activation and cytokine secretion. It also has been demonstrated that CD40L and 4-1BBL have a strong antitumor effect, whereas, the role of RANKL in antitumor immunity has not yet been examined. CD40L is a well studied cytokine, shown to play an important role in the development of both humoral and cell-mediated immunity. However, the mechanisms of action of all these three cytokines on immune effectors are not fully understood. Here, we have evaluated the ability of local adenoviral gene transfer of CD40L, RANKL and 4-1BBL to elicit an antitumor immune response to established tumors in mice. Adenoviruses encoding these genes (Ad-CD40L, Ad-RANKL and Ad-4-1BBL) were constructed and tested in murine MC38 colon and TS/A breast adenocarcinoma therapy models. Our results demonstrate that intratumoral administration of all three tested vectors resulted in a significant inhibition of MC38 and TS/A tumor growth when compared with control groups treated with either saline or control adenovirus. In addition, a single intratumoral injection of DC transduced with the adenoviral vectors also resulted in a significant inhibition of MC38 and TS/A tumor growth. Furthermore, treatment of TS/A tumors with DC transduced with Ad-CD40L induced a complete tumor rejection with the generation of a tumor-specific immune memory. Thus, these results demonstrate that DC genetically modified to express CD40L immunotherapy displays the strongest antitumor effect compare to Ad-CD40L or RANKL- and 4-1BBL-based therapeutic approaches. Next, we have observed that DC generated from tumor-bearing mice, in addition to expressing low levels of CD80 and CD86 and producing decreased amounts of IL-12, exhibit decreased expression of CD40 molecules. Moreover, we have detected that MC38 tumor suppressed CD40 expression on DC isolated from spleens of tumor bearers. These data suggest that tumors induce suppression of CD40 expression on DC, and, thus, result in dysfunction and inhibition of maturation of these cells. Subsequently, we have demonstrated that CD40L, in addition to generating a strong antitumor immunity, was able to protect DC from tumor-induced dysfunction. CD40L stimulates DC to express higher levels of co-stimulatory molecules, produce significantly higher levels of IL-12 protein, survive longer in cultures, efficiently stimulate T cells, induce high cytotoxic T lymphocyte activity, present tumor antigens to T cells more efficiently, and migrate to the lymphoid organs faster then control DC. We have shown that by up-regulating DC activity and function, CD40L rescues DC from tumor-induced suppression. In summary, our data demonstrate that CD40L-based immunotherapy is an effective approach for inducing antitumor immunity and rescuing DC from tumor-induced dysfunction. These results should guide the development of novel therapies for prevention of immunosuppression in cancer patients and design of novel effective immunotherapeutic strategies for cancer.

A possible approach to stimulate proteoglycan and collagen synthesis for treating intervertebral disc degeneration (IDD) is introduction of growth factors. The objective of Part I of this study was to screen the effect of human recombinant bone morphogenetic protein (BMP)-2 and BMP-12 on nucleus pulposus (NP) cells and to investigate the effect of Ad/BMP-12 on NP and anulus fibrosus (AF) cells. Cells were isolated from degenerated human discs and cultured in monolayer. RhBMP-2 (25, 50, 100, 200, 300, ng/ml) and rhBMP-12 (25, 50, 100 ng/ml) stimulated NP cells in serumless media (1% ITS) for 2 days. Ad/BMP-12 (50, 100, 150 MOI) transduced NP and AF cells, then pellets (150,000 cells/pellet) were formed and incubated in serumless media (1% ITS) for 6 days. Proteoglycan, collagen, and non-collagenous protein synthesis were measured. RhBMP-2 had a more substantial effect on upregulating matrix synthesis than rhBMP-12. Ad/BMP-12 significantly increased matrix synthesis. Total DNA content was increased pellets stimulated by Ad/BMP-12 when compared to control. The increase in matrix synthesis was attributed to both an increase in cell number and in matrix synthesis per cell. Intervertebral disc (IVD) is the largest avascular organ in the body, it has been suggested that lack of nutrition may be one of the cause of IDD. The goals of the second part of this study were to develop a rabbit disc organ culture method to study the effect of FBS concentration in disc metabolism and to attempt gene therapy in the organ culture. Twenty-seven rabbit lumber intervertebral discs were harvested and cultured for 2 weeks in serumless media (1% ITS) or F-12/DMEM (5%, 10%, 15% FBS). NP and AF wet weight, dry weight, % hydration, glycosaminoglycans (GAG), DNA, and lactate content were measured. In 25 discs, 6x106 PFU of Ad/lacZ or Ad/Luciferase was injected into the NP 2 days after the culture to determine the effect of gene transfer. Histology and viability staining of 10 discs were used to show cell morphology and viability. Due to low cell viability, we did not observe successful gene transfer in the organ culture. NP and AF lactate content and AF DNA content were significantly higher in the 15% FBS group than serumless group.

Several studies have examined the influence of optic flow presented in the central and peripheral fields of view on postural sway, but they have yielded diverging results. Early reports suggested that movement in the periphery was associated with increased sway. Later studies have argued that central movement is equally important. The primary objective of this set of experiments was to further investigate the influence of central and peripheral optic flow on postural equilibrium. The postural sway of 20 healthy subjects (ages 21-30 years) was measured while they viewed moving visual stimuli in a computer-generated immersive environment encompassing their entire horizontal field-of-view (FOV). Two main studies were conducted. In the first, optic flow was presented at different FOVs, but the motion contained only one frequency component during each trial. In the second, the central and peripheral regions of the visual stimulus moved simultaneously but at different frequencies. In the first experiment, the location of the optic flow stimulus significantly affected the number and size of significant postural responses. There were twice as many significant postural responses to the peripheral optic flow condition compared to the central optic flow. In addition, the magnitude of the response to the peripheral optic flow was 6.5 times greater than the response to the central flow. The number and magnitude of significant responses were also greater for higher-frequency stimuli, after accounting for quiet-stance sway components. In the second experiment, there were 3.5 times as many significant postural responses to the peripheral optic flow stimulus as there were to the central optic flow. Likewise, the amount of sway was greater in response to the peripheral stimulus. The results of both experiments suggest that the postural system is more sensitive to anterior-posterior optic flow in the peripheral FOV, regardless of stimulus frequency. These findings have implications for the use of virtual environments, showing that head-mounted displays with a limited central FOV do not provide the visual information most important for postural control. Furthermore, the results suggest that rehabilitation therapists should incorporate peripheral movement cues in treatment to enhance balance retraining.

Biologically active replacement heart valves (tissue engineered, recellularized xenograft) offer enhanced function compared to current valve therapies by possessing the capacity for remodeling and growth to meet the hemodynamic needs of the patient and eliminating the need for chronic medication. However, many fundamental questions remain as to how these valves will function in vivo, and new in vitro tools need to be created to address these questions. Traditional in vitro heart valve testing devices (mock flow loops) are designed to subject valves to physiologic and pathologic hemodynamic conditions. These devices offer a heart valve designer a useful tool with which to evaluate the mechanical functioning of their device in a variety of well-controlled hemodynamic situations. Unfortunately, these devices have not been designed for testing valves built of biologically active materials which require proper nutrient and waste exchange, pH, temperature, and freedom from attacks by microbial organisms in order to function. Pulsatile bioreactors have been developed to provide the aforementioned biological requirements to developing tissue engineered valves [1, 2], but these systems offer very limited hemodynamic control in comparison to mock flow loops. Therefore, in order to better understand the role of hemodynamics in the function of biologically active heart valves (BAHV), and to thereby create better BAHV designs, a new type of pulsatile bioreactor should be created that also incorporates more of the hemodynamic control found in mock circulatory loops. This thesis details the both the development of such a device and evaluating its functionality.

For more than 40 years, replacement of diseased natural heart valves with prosthetic devices has dramatically extended the quality and length of the lives of millions of patients worldwide. However, as in many medical therapies today, replacement valves are never as good as natural, healthy valves. Bioprosthetic heart valves (BHV) continue to fail due to structural failure, a result of both poor tissue durability and faulty design. Clearly, an in-depth understanding of the biomechanical behavior of the BHV at both the tissue- and functional prosthesis levels is essential to improving BHV design and the mechanisms of failure. The goal of this research effort was to develop and evaluate a complete process for biomechanical simulations of heart valve biomaterials, with an emphasis on numerical stability and experimental validation. This process started from the collection of appropriate experimental data, formulating and validating a constitutive model, obtaining and refining material parameters, finite element implementation and validation of a constitutive model, and finally finite element simulation of valve deformation. The results of this study indicated that explicit expression of shear behavior was required for proper computational implementation of the exponential Fung pseudo-elastic model and thus, biaxial testing with extension only did not provide sufficient information to constitute a strain energy function for computational implementation. This study also demonstrated that a set of model constraints imposed by the convexity of strain energy function and condition number of elasticity tensor were necessary for numerical stability. When applied to an intact valve, the finite element model demonstrated an overall discrepancy of only 0.0187 strain when compared to experimental validation data, which was within the experimental error. This result underscored the need for rigorous experimentation and constitutive modeling to allow a close match between FE and experiment output. The present study is, to our knowledge, the most rigorously developed and validated model available to date for characterizing valve deformation. It is hoped that the developed approaches will be a valuable tool for evaluating various valve design parameters and will greatly facilitate optimal BHV design.

Throughout the course of daily activities, wheelchair users are subjected to a variety of whole-body vibrations that are suspected to cause rider discomfort and a number of harmful physiological effects. In efforts to improve comfort and prevent secondary injuries, manufacturers of manual wheelchairs have integrated suspension systems into their designs. The purpose of this research was to provide a thorough evaluation of currently available suspension manual wheelchairs and to determine the advantages, if any, of wheelchair suspension. The evaluation was composed of two sections: 1) a durability and cost analysis of three selected suspension manual wheelchairs; and 2) a pair of functional tests comparing suspension manual wheelchairs to standard folding- and rigid-frame models. The durability and cost analysis revealed that integrated suspension did not significantly improve wheelchair fatigue life; in fact, in some cases, the modifications reduced wheelchair integrity. In addition, their increased expense considerably lowered their value in relation to the other types of wheelchairs. Altogether, little evidence was found to suggest that suspension manual wheelchairs provide advantages in terms of durability or value over non-suspension, folding-frame wheelchairs. The second section evaluated the ability of suspension manual wheelchairs to reduce the transmission of vibrations to the rider during various height curb descents and while traversing a level, uneven surface. In addition, impact force was measured during curb descent trials and used in the comparison. The results suggest that while the suspension manual wheelchairs provided significant (p = .0002) reduction in seat accelerations over both types of standard wheelchairs, this was due to the superiority of one wheelchair, the Sunrise Medical Quickie XTR. Furthermore, few significant improvements were found in terms of impact force and vibration dose value, which was calculated from seat accelerations measured during uneven surface testing. Overall the results indicate that suspension manual wheelchairs are not suited to suppress the shock vibrations or repeated low-level vibrations transmitted by curb descents and uneven terrain, respectively. The results of this research should be used to develop a more adequate wheelchair suspension system, and more importantly, should be considered by clinicians and wheelchair users when selecting a wheelchair for everyday use.

Radiological imaging of the liver is an important medical problem. The ever increasing amount of data acquired when imaging the liver makes integration of information desirable and crucial in building up a comprehensive diagnostic picture of the patient. The foundation of all such image integration is image registration. </br></br>Image registration is the process of aligning images so that corresponding features can easily be related, including: (1) landmark-driven methods, (2) surface-based methods, and (3) voxel similarity-based methods. A challenge with registering the liver is that the liver moves within the abdomen with respiration. Therefore any effective alignment of the liver must first separate the liver from the remainder of the image. With this as a constraint, the goal of this research effort was to determine the feasibility and efficacy of surface-based and voxel similarity-based schemes in registering abdominal CT and MR images with and without contrast. </br></br>A multi-scale surface fitting technique was implemented based on the Head and Hat algorithm. Equivalent surfaces from the in vivo images were extracted manually. The hand segmentation approach was validated by ensuring the volume of the liver of each image from the same patient was consistently within +/- 7% of one another. The registration transformation was determined by iteratively transforming the hat with respect to the head surface, until the closest fit of the hat onto the head was found. </br></br>In addition, registration of in vivo CT and MR images was performed using a multi-resolution mutual information scheme distributed with the ITK Insight software package (National Library of Medicine, Bethesda, MD). As an independent measure of registration accuracy, the mean displacement of automatically selected point landmarks was evaluated. For the multi-resolution mutual information approach, mean misregistrations were in the range of 7.7-8.4mm for CT-CT intramodality registration, 8.2mm for MR-MR intramodality registration, and 14.0-18.9mm for CT-MR intermodality registration. For the Head and Hat surface registration scheme, mean misregistrations were in the range of 9.6-11.1mm for CT-CT intramodality registration, 9.2-12.4mm for MR-MR intramodality registration, and 15.2-19.0mm for MR-CT intermodality registration.

The urethra serves a dual function by maintaining continence during bladder filling and aiding the release of urine during micturition. Within the urethra, a sphincter region containing both smooth and striated muscle layers normally prevents involuntary leakage of urine. However, patients with stress urinary incontinence lose this ability upon sudden increases in intravesical pressure (i.e. from coughing, straining, etc.). This condition has been associated with a decline in striated muscle, which may be susceptible to direct muscle or associated nerve damage. Cellular uromyoplasty proposes to augment this muscle layer through the transplantation of myogenic progenitors. The goal of this work was to address current deficiencies regarding the isolation and identification of efficient progenitors, and the urethral biomechanical consequences of striated muscle restoration. Both issues are essential for effective clinical implementation of this therapeutic approach. The ability of various progenitor populations to regenerate skeletal (striated) muscle was assessed in a dystrophic mouse model. Both cell surface protein expression and behavioral characteristics were investigated for their potential use as indicators of regenerative efficiency. The results demonstrate the limited utility of surface proteins due to fluctuations in expression and lack of regenerative consistency between directly-isolated and cultured cell populations. Behavioral characteristics related to the ability of cells to maintain a proliferative phenotype under differentiation-inducing conditions appears more promising in this regard, and indicates that in vivo expansion of transplanted cells may be a critical variable in the regeneration process. A new ex vivo method to assess the regional biomechanical function of the intact urethra, under physiologic loading conditions, was introduced and validated. Quantitative characterization and comparison of tissue responses to applied intralumenal pressures was performed in the presence or absence of selected muscle activity. The dominant smooth muscle influence observed suggests that a large degree of striated regeneration may be necessary to impart functional changes in urethra mechanics. Importantly, these results also indicate that muscle fiber orientation may significantly impact urethra closure function. Together, this information will be useful in progressing uromyoplasty therapy toward clinical utility, and aid the broader scientific community investigating myogenic cell transplantation and lower urinary tract function.