Molecular motors perform diverse functions in cells, ranging from muscle contraction, cell division, DNA/RNA replication, protein degradation, and vesicle transport. The majority of molecular motors use energy from the ATP hydrolysis cycle, converting chemical energy into mechanical work in cells. All ATP-dependent molecular motors have a similar ATP binding site, although the functions can be drastically different. Myosins comprise a large group of ATP-dependent molecule motors. The structure-function relationship governing different functions for different myosin families remains elusive. Hypothesizing that members of each family possess conserved residues for their consensus functions and residues distinctive from those of other families to differentiate their functions from functions of other myosin families, we developed an algorithm for comparative sequence analysis in a phylogenic hierarchy to identify family-specific residues for 38 myosin families/subfamilies that comprise human myosin members. We found a number of family-specific residues that have been reported, such as residues in β-cardiac myosin associated with hypertrophic cardiomyopathy and residues in myosin 7A associated with hereditary deafness. We also identified distinct features among myosin families that have never been reported, including a unique signature of the SH1 domain in each of the myosin families, residues differentiating α- and β-cardiac myosins, and a unique converter domain of myosin VI. We further examined myosin VI to understand why it moves toward the (-)-end of actin filaments, opposite to the direction of all other myosins and to shed light on their links to prostate cancer and ovarian cancer, where myosin VI is over-expressed. We found that many of myosin VI specific residues locate in or adjacent to the converter domain, including a cluster of unique residues at the interface between the motor domain and the converter. Using molecular dynamics (MD) simulation, we found mutations of M701 on the SH1 helix and F763 on a helix of the converter caused the separation of the motor domain and the converter, indicating their important roles in linking the converter and the motor domain in the pre-power stroke state structure, potentially critical for positioning of lever arm. Using the location of the unique residues at the interface of the motor domain and the converter as the site of drug docking, we identified a set of candidate small molecules binding to this unique binding site selectively, potentially blocking the converter rotation of myosin VI. A benzoic acid (C15H17N3O3) was found to have the best score in docking, binding to both the converter and motor domain stably in a 200 ns MD simulation run. This molecule can be a good lead to be optimized to inhibit myosin VI functions in cancer patients. We have also applied our algorithm to other ATP-dependent molecular motors, including hepatitis C virus NS3 helicase and DEAD box helicase Mss116. We found an important residue, T324, in NS3 helicase connecting domains 1 and 2 acting as a flexible hinge for opening of the ATP-binding cleft and an atomic interaction cascade from T324 to residues in domains 1 and 2 controls the flexibility of the ATP-binding cleft in NS3 helicase. We also found a conserved flexible linker for Mss116, and the tight interactions between the Mss116-specific flexible linker and the two RecA-like domains are mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

Diabetes Mellitus is one of the most common diseases in the world. Cardiovascular diseases account for ~80% of deaths amongst diabetic patients, primarily through coronary artery disease (CAD). However, a new clinical entry, termed Diabetic Cardiomyopathy (DC), may lead to heart failure in diabetic patients independently of CAD or hypertension. In DC, hyperglycemia and hyperlipidemia associated with diabetes produce structural and biochemical alterations at the cardiac cell level. Early stage cell alterations include hypertrophy, calcium mishandling, cell apoptosis, excessive ROS production, and increased collagen production by fibroblasts. Eventually, major structural and functional changes can appear in myocardial tissue, characterized by diastolic and systolic dysfunction, and eventually heart failure. While specific changes associated with DC are well characterized, the mechanism underlying disease development and progression as a whole remain to be elucidated. The ability of researchers to develop general treatment options for this disease is thus limited. Currently, a majority of DC studies focus on either in vitro molecular pathways, or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease of changes in structural and functional properties, these studies can not precisely quantify what conditions (such as hyperglycemia and hyperlipidemia) are producing specific biomechanical changes such as increased myocardial stiffness or diastolic dysfunction. To address this, we developed an in vitro approach, based on culturing cardiac cells in elevated glucose and fatty acid, to examine how structural and functional properties may change as a result of a diabetic environment. Increased myocardial stiffness is associated with increased collagen production in the heart. However, diastolic dysfunction is found to occur in DC prior to significant collagen accumulation. We hypothesized that increased cardiac cell stiffness could contribute to early stage diastolic dysfunction. To test this hypothesis, we developed and used contemporary biomedical engineering tools to characterize the biomechanical properties of cardiac myocytes and fibroblasts under a variety of hyperglycemic and hyperlipidemic conditions. We showed that our in vitro model of DC exhibits increased stiffness in myocytes, but not fibroblasts. We then developed an assay to measure cardiac myocyte contractile force, as well as assess systolic and diastolic function. This assay was then used to determine the role of N-acetyl-cysteine (NAC), towards regulating reactive oxygen species (ROS) and reversing cellular-level changes associated with our DC model. We found that DC model cardiac myocytes exhibited greater incidences of diastolic, but not systolic, dysfunction, and that treatment with NAC reduced dysfunction to a normal level. In terms of structural properties, we additionally determined that treatment with NAC attenuated increases in myocyte stiffness found in our DC model, and that NAC reduced myocyte hypertrophy for certain diabetic conditions. Overall, treatment with NAC attenuates the maladaptive mechanical and functional changes found in our DC model.

The menisci are fibrocartilaginous tissues of the knee that specialize in load-bearing and stabilization of the joint. Though once believed to be "the functionless remains of leg muscles," and therefore routinely removed after injury, it is now understood that loss of meniscus integrity leads directly to degenerative changes in the knee, inspiring new pursuits to overcome the intrinsic limitations to meniscus repair. However, many components of the meniscus environment and their roles in regulating the cellular response of injured tissue remain unclear. This thesis presents novel strategies to enhance integrative repair of the meniscus, via control of the cell environment from the micro- to macro-scale. The unifying hypothesis of this dissertation was that the application of chemical, physical, and environmental factors can significantly influence the meniscus cell environment and contribute to healing. Specifically, we studied the direct role of vasculature on the meniscus, and identified angiogenic factors that regulate cell migration and tissue repair between the inner and outer regions. We analyzed the effects of pulsatile direct current electrical stimulation on meniscus cell migration and repair, and describe the mechanisms of electrotransduction in the meniscus. Finally, we developed a decellularized meniscus extracellular matrix hydrogel to deliver and dictate the behavior of stem cells for meniscal repair. By rigorous study of each element at the micro-scale and integration at the macro-scale, novel therapies for meniscus repair will emerge towards clinical applications.

Cardiac conduction abnormalities and arrhythmias are associated with stroke, heart failure, and sudden cardiac death, and remain a major cause of death and disability. However, the imaging tools currently available to the physician to guide these treatments by mapping the activation sequence of the heart are invasive, ionizing, time-consuming, and costly. In this dissertation, Electromechanical Wave Imaging (EWI) is described with an aim to characterize normal and abnormal rhythms noninvasively, transmurally, at the point of care, and in real time. More specifically, the methods to map the electromechanical wave (EW), i.e., the transient deformations occurring in response to the electrical activation of the heart, are developed and optimized. The correlation between EW and the electrical activation sequence during both normal and abnormal rhythms is demonstrated in canines in vivo and in silico. Finally, EWI is shown to noninvasively detect and characterize arrhythmias and conduction disorders in humans. Novel ultrasound imaging methodologies were developed to track the EW. Radio-frequency (RF) frames acquired at high frame rates were used in conjunction with cross-correlation algorithms to map the onset of the small, localized, transient deformations resulting from the electrical activation and forming the EW. To validate the capability of the EW to characterize cardiac rhythm, it was compared against the electrical activation in vivo and in silico. A high correlation between the electrical and electromechanical activations was obtained in normal canines in vivo during various pacing schemes and sinus rhythm. An in vivo-in silico framework was also developed to demonstrate that this correlation is maintained transmurally and independently of the imaging angle. EWI was also validated in abnormal canine hearts in vivo during ischemia, left bundle branch block, or atrio-ventricular dissociation. In a clinical feasibility study, we demonstrated that EWI was capable of noninvasively mapping normal and abnormal activation patterns in all four cardiac chambers of human subjects using a readily available clinical ultrasound scanner. Specifically, EWI maps were generated for three heart failure patients with cardiac resynchronization therapy (CRT) devices and for three patients with atrial flutter who subsequently underwent catheter mapping and radiofrequency ablation. Preliminary validation of EWI maps against invasive transcutaneous electroanatomical cardiac mapping was also demonstrated. EWI has the potential of becoming a noninvasive and highly translational technology that can serve as a unique imaging tool for the early detection, diagnosis and treatment monitoring and follow-up of arrhythmias and conduction disorders through ultrasound-based mapping of the transmural electromechanical activation sequence reliably, at the point of care, and in real time.

The overall goal of this dissertation is to describe the development of a dynamic diffuse optical tomographic (DDOT) imaging system for the diagnosis and monitoring of peripheral arterial disease (PAD) within the lower extremities. PAD affects 8-12 million individuals in the United States and is associated with significant morbidity and mortality. Early detection and monitoring of disease progression is crucial, but remains difficult. This is especially true for diabetic patients, as roughly 30 percent of all diabetic patients over the age of 50 are diagnosed with PAD. Diabetic patients have calcified arteries, which renders them incompressible. This falsely elevates blood pressure readings and causes false negative readings using traditional diagnostic techniques. DDOT offers an attractive opportunity to overcome current shortcomings in assessing PAD. This technology uses harmless near-infrared light to create three-dimensional, time-dependent images of biological tissues. Using DDOT to measure blood-perfusion in the foot should help diagnose and monitor the PAD. To test this hypothesis, I adapted an existing optical tomographic imaging system for the particular application of vascular imaging in the foot. In particular I design and tested various measuring probes that can accommodate different foot sizes and shapes. The result was a patient friendly interface that can be employed in a clinical setting. Using this modified DDOT imager, which we called vascular optical tomographic imaging (VOTI) system, I conducted a 40-subject pilot study to quantify its ability to diagnose PAD. The subjects were recruited into three cohorts, non-diabetic PAD patients (N=10), PAD Patients (N=10) and healthy volunteers (N=20). With this data in hand, I performed a comprehensive data analysis, in which I found imaging features that led to a good separation between the healthy and affected cohorts. In particular I demonstrated that statistically significant difference exist between the amount of blood pooling in the leg during a 1-minute, 60mmHg thigh cuff occlusion within healthy subjects and both affected cohorts (P=0.006, P=0.006). In addition, using receiver operating characteristic (ROC) curve analysis, I identified that the new VOTI system could diagnose PAD with a sensitivity and specificity of over 80%, even within the diabetic patients. This imaging modality was also capable of identifying the severity of the disease with similar accuracy to the existing diagnostic methods while not being inhibited by arterial calcifications. Furthermore, the VOTI system provided spatial information, helping identify which regions of the foot suffered from mal-perfusion. When combined with angiosome theory, the spatial information could help physicians in deciding how to intervene in PAD patients. After completing this first clinical study, I developed a dedicated VOTI system by entirely redesigning the hard and software. This new system has many novelties over its predecessor. First it employs a contact-free patient interface that allows to imaging patients with ulcerations. The illumination fibers used do not need to make physical contact with the patient. Second, instead of using individual silicon photodiodes as detectors, a highly sensitive CCD camera is use to detect transmitted light intensity. The system has two wavelengths of light (660 and 860 nm), which can be illuminated at up to 20 different positions along the surface of the foot. The system is built for dynamic imaging and is capable of imaging at a multispectral-volumetric frame rate speeds of 1 Hz. This set-up allows us to create three-dimensional images of large portions of the foot. This imaging system was tested on phantom studies and healthy volunteers and was shown to be able to image blood flow dynamics within a three-dimensional volume of the foot.

Adipose tissue is traditionally regarded as a source of energy storage and cushion for skeletal functions. Soft tissue injuries take place in war and peace time, as a result of tumor resection such as breast cancer, and in rare disorders such as lipoatrophy. Adipose tissue reconstruction is one of the key challenges in medicine. Here we report the robust differentiation of human adipose derived stem cells using a small molecule into adipocytes. This thesis describes the biological effects and actions of novel unknown small-molecule inhibitor of BMP signaling--Pyrintegrin, which was previously found to promote human embryonic stem cells survival. The overall objective of this thesis is to test the hypothesis that this novel drug, Pyrintegrin treatment will induce, promote and accelerate adipogenic differentiation of stem cells in vitro and in vivo. We found that Pyrintegrin promotes the adipogenesis-dependent transcriptional changes of multiple gene products involved in the adipogenic process, including peroxisome proliferator-activated receptor (PPARgamma), CCAAT/enhancer-binding protein alpha; (C/EBPalpha), adiponectin and leptin secretion, and total triglyceride secretion. When transplanted into mice, Pyrintegrin treated adipose cells/progenitors gave rise to ectopic fat pads with the morphological and functional characteristics of white adipose tissue. This was further confirmed by higher expression of human PPARgamma gene in Pyrintegrin treated cells group than any other group. We further tested the presence of human nuclear staining that confirmed the presence of human cells in all the transplanted groups. However, the number of positive human cells was substantially low in all the groups, which was likely due to transplanted cell death because of lack of vascularization. Hence, we further tested the Pyrintegrin adsorbed scaffolds capacity to regenerate better soft tissue compared to Ptn-free scaffold. We found that Ptn adsorbed scaffolds was positive for adipocytes as evident by positive Oil Red O staining. We further investigated the signaling mechanism of Pyrintegrin. Using a human PPARgamma reporter assay system, based on non-human mammalian cells engineered to express human PPARgamma protein, we found that Pyrintegrin is not a PPARgamma agonist as witnessed by lack of any luciferase activity. In contrast, Rosiglitazone, a known PPARgamma agonist demonstrated significant amount of luciferase activity in these reporter cells. We also found that Pyrintegrin selectively inhibits the BMP pathway and thus blocks BMP-mediated SMAD1/5 phosphorylation, target gene transcription and osteogenic differentiation. In vitro studies showed that Pyrintegrin inhibited the differentiation of stem cells into putative osteoblasts, as evident by decreased alizarin red staining. A striking finding was that Pyrintegrin up regulated markers of adipogenesis and stimulated lipid droplets accumulation in stem cells undergoing osteoblastic differentiation in vitro. This came at the expense of down regulating markers of osteogenesis and osteoblastic differentiation of stem cells, as compared to the cells undergoing osteogenic differentiation in the absence of the drug treatment. These findings show that the novel small-molecule Pyrintegrin is a potent promoter of adipogenesis and thus may have therapeutic potential for soft tissue reconstruction.

In multicellular organisms, one-cell-thick monolayer sheets are the simplest tissues, yet they play crucial roles in physiology and tissue engineering. Cells within these sheets are tightly connected to each other through specialized cell-adhesion molecules that typically cluster into in discrete patches called cell-cell junctions. Working together, these junctional organelles glue cells to their neighbors, integrate the cytoskeletons into a mechanical syncytium and transduce a variety of mechanical signals. Human bodies offer many vivid illustration of how a cell sheet physiology changes considerably during development and diseases, as shown in epidermal blistering and certain cardiomyopathy. Despite the extensive molecular and clinical work on cell junctions, relevant in vitro experimental data are often masked by cell-substrate interactions due to a lack of suitable experimental methods. It is therefore important to develop novel in vitro methods for characterizing how junctional proteins, as well as tightly associated cytoskeletal proteins, may modulate various cellular behaviors, such as viability and apoptosis, cell-cell adhesiveness and tissue integrity. Control over cell viability is a fundamental property underlying numerous physiological processes. Cell-cell contact is likely to play a significant role in regulating cell vitality, but its function is easily masked by cell-substrate interactions, thus remains incompletely characterized. In the first part of this thesis, we developed an enzyme-based whole cell sheet lifting method and generated substrate- and scaffold-free keratinocyte (N/TERT-1) cell sheets. Cells within the suspended cell sheets have persisting intercellular contacts and remain viable, in contrast to trypsinized cells suspended without either cell-cell or cell-substrate contact, which underwent apoptosis at high rates. Suppression of junctional protein plakoglobin weakened cell-cell adhesion in cell sheets and suppressed apoptosis in suspended, trypsinized cells. These results demonstrate that cell-cell contact may be a fundamental control mechanism governing cell viability and that the plakoglobin is a key regulator of this process. The study also laid groundwork for subsequent characterization and manipulation of viable cell sheets for cell sheet engineering purpose. Cell sheet engineering, characterized by harvest of cultured cell monolayer as a scaffold-free sheet, was recently developed. Particularly, cell sheet engineering based cardiac tissue engineering has emerged as an alternative method for the repair of damaged heart tissue. Such an engineered cell sheet offers a new way to study cell junctions when substrate interactions are no longer dominant. While this method is promising, it is limited by the fragility and shrinkage of the sheets as well as the lack of information regarding the characteristics of such sheets. In next part of the thesis we pursued two related research projects by developing a novel partial-lift method to generate strong, unshrunk substrate-free and scaffold-free cell sheets, first using skin cells and then refined and expanded to cardiac cells. The rationales for this approach are the ease with which skin cells can be manipulated, the similarities in cell junctions between skin and cardiac cells, and their potential clinical applications. These partially-lifted cell sheets engage primarily in cell-cell interactions, yet are amenable to biological and chemical perturbations and, importantly, mechanical conditioning. This simple yet powerful method was then deployed to test the hypothesis that the lifted cells would exhibit substantial reinforcement of key cytoskeletal and junctional components at cell-cell contacts, and that such reinforcement would be enhanced by mechanical conditioning. Results further demonstrate that the mechanical strength and cohesion of the substrate-free cell sheets strongly depend on the integrity of the actomyosin cytoskeleton and expression of the junctional protein plakoglobin. Moreover, our results showed that dissociating cell-substrate interactions and implementing mechanical conditioning enhances contraction, calcium signaling, alters viscoelastic property, and thus improves the functional cell-cell coupling in the cardiac sheets. In sum, this thesis represents a first systematic examination of junctional regulation of cell viability and mechanical conditioning on cells with primarily cell-cell interactions. The information gained from this study will help advance our understanding of cell-cell interactions and improve cell sheets biomechanical properties. For tissue engineering purposes, our dispase-based partial-lift cell sheet harvesting method has the advantage of being biocompatible, easily applicable, rapidly collectable and stretchable, compared to currently available techniques. This simple yet powerful partial lift technique has enormous potential for fabricating clinically applicable skin and cardiac tissues.

Magnetic resonance imaging (MRI) is a medical imaging technique which can provide fine tissue contrast with relatively high image resolution in human. Besides the image quality, imaging speed is the other major concern in modern MRI, especially in human experiments where sufficient volumetric coverage is necessary. One approach to increase imaging speed is increasing image acquisition speed so that the same amount of volumetric coverage can be achieved within shorter time under conventional experiment paradigms. In this dissertation, the application of pseudo-random amplitude modulation (PRAM) in MRI was explored to increase imaging speed by designing more efficient experiment paradigms for the human brain. Two relatively slow MRI studies were investigated. The first study was measuring longitudinal relaxation time. A novel method "Relaxation by Amplitude Modulation" (RLXAM) was invented. The RLXAM modulation code can be chosen from a large family of binary sequences. PRAM is a specific implementation using the maximum length sequence, also known as pseudo-random sequence. The other study was measuring transit time distribution in arterial spin labeling. The application of PRAM in transit time measurement was reported before on a 3T Philips Acheiva scanner using a single-slice protocol with standard gradient echo acquisition. The original theory was extended and multi-slice sequences with two different acquisition strategies were developed on a 3T Siemens Trio scanner. Both methods were applied to both phantom and human to demonstrate the theories and evaluate their performance.

Osteoarthritis (OA), the most prevalent form of joint disease, afflicts nine percent of the US population over the age of thirty and costs the economy nearly $100 billion annually in healthcare and socioeconomic costs. It is characterized by joint pain and dysfunction, though the pathophysiology remains largely unknown. The progressive loss of cartilage followed by inadequate repair and remodeling of subchondral bone are common hallmarks of this degenerative disease. Due to its avascular nature and limited cellularity, articular cartilage exhibits a poor intrinsic healing response following injury. As such, significant research efforts are aimed at producing engineered cartilage as a cell-based approach for articular cartilage repair. However, the knee joint is mechanically demanding, and during injury, also a milieu of harsh inflammatory agents. The unforgiving mechanochemical environment requires constructs that are capable of bearing such burdens. To this end, previous work in our laboratory has explored the application of stimuli inspired by the native joint environment in attempts to create tissue with functional properties similar to native cartilage so that it may restore loading to the joint. While we have had success at producing these replacement tissues, there is little evidence in the literature that the biological functionality (i.e. response to in vivo-like conditions) of engineered cartilage matches native cartilage. Therefore, in an effort to provide a more complete characterization of the functional nature of developing tissues and facilitate their use clinically, the overarching motivation of the work described in this dissertation is two-fold: 1) characterize the response of engineered cartilage to chemical and mechanical injury; and 2) develop strategies for enhancing the performance and protection of engineered cartilage for in vivo success. Studies in the literature have extensively characterized the effects of wounding to native articular cartilage as well as the effects of an inflammatory environment. For mechanical injuries, cell death is immediate and progressive, ultimately leading to failure of the tissue. Chemical insult has been shown to promote degradation of the matrix components, also leading to failure of the tissue. Under a controlled application of injury (mechanical and chemical), it was found that engineered cartilage, in contrast to native cartilage, has the potential to repair itself following an injury event, as long as there is no catastrophic damage to the matrix. Additionally, when this matrix is intact and well-developed, engineered cartilage constructs exhibit a resistance to degradation, highlighting the potential utility of engineered cartilage as replacement tissues. Enhancing functionality in engineered cartilage was also explored, with the aim of developing strategies to improve, repair, and protect engineered cartilage constructs for their use in vivo. For these purposes, the studies in this dissertation spanned both 2D migration studies to influence the limited wound repair potential of cells as well as 3D culture studies to explore the possibility of protection effects at a tissue level. Together, these models allowed us to capture the complexity needed to fully develop approaches for cartilage repair. Though it has previously been found that applied DC electric fields modulate cell migration, we have developed a novel strategy of employing this technique to screen for desirable populations of cells (those with the greatest capacity for directed migration) to use in cartilage repair. We also found that the AQP1 water channel plays a key role in mechanosensing the extracellular environment, highlighting the potential for its use in therapeutic strategies. For tissue engineering efforts at creating functional cartilage replacement, we uncovered novel strategies to foster better tissue development via co-culture systems and promote the resistance of engineered cartilage to catabolic factors. These findings motivate their potential use in therapeutics and in tissue engineering efforts at creating clinically relevant tissue-engineered constructs for the treatment of OA or following injury. The research described in this dissertation has characterized the biological functionality of engineered tissues and identified strategies for enhancing their use in vivo by modulating the subsequent response to injury, laying the foundation for their use in clinical applications.

Mathematical formulation and physical models are the foundation of scientific understanding and technological advancement. Our ability to design experiments effectively is heavily dependent on our physical understanding of the system under investigation, and careful mathematical analysis is required in order to effectively progress from scientific concepts towards viable technologies. With increasing system complexity, the focus of mathematical formulation has shifted from simple, elegant models which rely on basic physical concepts to tailored, increasingly complex solutions using high-powered simulations and numerical solutions. While these methods may provide insights into specific systems, adapting these models to different systems is generally difficult, even when the systems under question operate according to the same physical laws. This is especially evident in nanobiotechnology, where the complexity of the systems studied has given rise to experiment-driven focus. Our aim is to focus on the mathematical modeling of transport processes in nanoscale systems, and to construct generalized, conceptual models for three model systems, which in turn could be applied to many biological and engineered systems. The three model systems we use - enzyme cascades, coupled molecular motors and self-assembling molecular shuttles provide a broad basis for generalized transport systems in nanoscale systems. These systems combine diffusive and active transport, as well as diverse assembly conditions and multi-scale systems with size scales spanning nano- to millimeter sizes and system complexity ranging from isolated two-component systems to multimolecular, highly-coupled systems. By applying and adapting these basic models to increasingly complex systems, we can both understand the physics behind nanoscale systems, as well as design these systems with increased robustness, scalability and repeatability.