Mutations in myosin may lead to severe muscle disorders that greatly reduce the quality of life. For example, the embryonic skeletal myosin (MYH3) mutation R672C leads to Freeman Sheldon Syndrome (FSS), a rare inherited disorder that causes severe contractures at birth. A comprehensive understanding of the relationship between protein structure and function is urgently needed to treat diseases such as FSS. Computational methods, such as molecular dynamics simulations, can be used to examine the effects of mutations on protein structure and function. However, the Protein Data Bank (PDB) is missing most human skeletal myosin heavy chain structures. We employed homology modelling to construct structures of human MYH3. To inform homology modeling, I generated a multiple sequence alignment of 7 human myosin genes. The extent of sequence identity was used to identify the optimum myosin isoforms to use as templates for model generation. For example, MYH3 and MYH7 were the most distinct at 78.93% similarity, which was expected as they are embryonic skeletal and adult cardiac myosin respectively. Specific sequence consensus at each position in the sequence was used to determine the most and least conserved regions of myosin. The cleft region was the most conserved; the N-terminal Domain was the least conserved. I used MYH7, adult cardiac myosin, as a template structure to derive a homology model of the ATP state of MYH3. A structure of MYH3 R672C was generated via in silico mutation of the wild type structure. Molecular dynamics of the resulting structures will be used to explore how R672C, which is located near myosin’s converter domain, alters myosin structure and function. This computational platform will model all phases of the cross-bridge cycle, potentially reveal new drug binding pockets, guide and be validated by in vitro experiments using human induced pluripotent stem cell derived myocytes (hiPSC-Ms).

Metastasis, or the spread of cancer to a secondary site, is responsible for most cancer-related deaths. The tissue-specific environment that disseminated tumor cells experience influences whether they will proliferate and colonize the tissue, remain dormant, or die. Skeletal muscle (SkM) is one of the rarest sites for metastasis, despite making up nearly half of human body mass. What makes SkM so resistant to metastasis? We set out to test the hypothesis that the mechanical nature of SkM is responsible for the lack of metastases at this site. To do so, we used the mdx mouse model of Duchenne muscular dystrophy to probe if the destruction of SkM structure and function would make it a more hospitable host to disseminated tumor cells. Wild type and mdx mice were intramuscularly injected with EO771 murine mammary tumor cells and monitored for tumor outgrowth using bioluminescent imaging. Preliminary results suggest that tumor growth is increased in dystrophic (e.g. dysfunctional muscle) versus wild-type mice. A complementary and more reductionist approach to test whether mechanics influences tumor colonization of muscle is to employ a culture model that allows tumor cells seeded on top of a SkM layer to experience mechanical stretching akin to the contraction/relaxation movements of muscle. To accomplish this, we constructed a device that applies a cyclic stretch to a 3D organotypic SkM culture model on a flexible silicone plate. We predicted that stretching would reduce tumor cell survival, when compared to no stretching. While these experiments are in progress, we believe that these data may elucidate a relationship between mechanical activity and suppression of tumor outgrowth in SkM. This work will contribute to a more complete understanding of how SkM avoids tumor colonization and could inform future approaches that leverage tissue mechanics to treat or prevent metastasis.

Transcription factors such as TCF-1, as well as inflammatory cytokines such as IL-12, have been shown to play a role in cytotoxic (CD8) T cell differentiation during an immune response. Here, we build a stochastic computational model of the canonical CD8 T cell immune response encompassing experimentally hypothesized transcription factor and inflammation control mechanisms. We hypothesize that the transcriptional factor control of CD8 T cells can be quantified into concrete model parameters that control various aspects of the immune response such as cell effector cell expansion, proliferation, and memory cell formation in various immune challenge conditions, such as inflammation. With a fully developed model, effects of inflammation levels and gene regulation kinetics on CD8 T cell differentiation can be measured in situ, and new hypotheses surrounding cellular differentiation kinetics can be generated. Understanding the factors that control CD8 T cell differentiation is crucial in the development of vaccines and immunotherapies, such as CAR T cell therapy.

In the United States, 1.5 million individuals suffer a fracture due to bone disease each year. In addition, there are many unknown mechanisms behind how muscular disorders and mechanical load adversely affect bone development, such as in the disease distal arthrogryposis. Disease research in human cell models has greater translational potential compared to animal models but have faced challenges when constructing highly-specialized tissues such as bone. We propose a novel, three-dimensional bone tissue model as a platform for musculoskeletal disease modeling that allows for compressive loading. By seeding induced pluripotent stem cell (iPSC) derived osteoblasts and osteoclasts in a 3D, porous, hydroxyapatite-coated poly-L-lactide scaffold, we propose to generate a bone tissue model that replicates human tissue in a laboratory. By applying compression to the novel 3D bone tissue model, we expect to observe phenotypes of bone disorders and bone development under mechanical loading. We propose to induce osteoblast and osteoclasts lineage from mesenchymal progenitor cells and hematopoietic progenitor cells, respectively, and co-culture to identify optimal conditions for cell growth. Preliminary experiments have found success in culturing active osteoblasts from iPSC-derived mesenchymal progenitor cells. By screening for markers of cell proliferation, calcium deposition, bone resorption and secretion, the cultures can be assessed for their robustness. In parallel, a porous scaffold will be fabricated by dissolving poly-L-lactide in chloroform and molding over sodium chloride particles. Coating said scaffold in fibronectin and hydroxyapatite will improve cell adhesion and uptake bone secretion. Seeding osteoclast and osteoblasts cells in a porous scaffold will allow for improved cell diffusion and 3D growth, mimicking the human microenvironment. We expect that combining robust, osteogenic tissue culture on a bioactive scaffold that allows 3D bone growth with mechanical loading will reveal phenotypes of distal arthrogryposis. Thus, this method has significant applications in accelerating laboratory findings to clinical research.

The purpose of a newly developed foot prosthesis is to improve frontal plane foot adaptability for over 1 million adults living with lower extremity amputation in the US. On uneven ground, side slope, and turning corners, the anatomical foot can adapt but most prosthetic feet cannot. The innovative prosthesis mimics biomechanical aspects of anatomical joints that use ligaments in tension throughout the range of motion while the joint surface acts as a specialized load-bearing structure similar to that of a cross four-bar linkage. In the current prototype cam linkage, we found deviation between the instantaneous center of rotation (ICR) and the point of contact (POC) as it rotated through the frontal plane. The error in this distance could cause unexpected wear on the prosthesis. The goal of this project was to reduce the error caused by the difference in location of the ICR and POC during rotation and translation of the four-bar linkage. In a crossed four-bar linkage, the ICR is found at the intersection of the crossing links. To find the path of the ICR, we plotted a series of points by rotating the linkage about a fixed link in space. The continuous ICR path was estimated by smoothing the ICR points. The curved shape developed becomes the load-bearing surface of the linkage. To create the upper and the lower load-bearing surface, the upper and lower link must be fixed and the opposite rotated, respectively. Using the method described, we built linkage assemblies that showed an error of 0.96 mm per 20 degrees of rotation compared to 2.17 mm of error in the original prototype. The International Organization for Standardization (ISO) requires that foot prostheses undergo wear and fatigue testing. Reducing potential wear on the prosthesis is advantageous because engineers can design for optimal product durability for the foot.

The dystrophin protein protects cardiac and skeletal muscle from damage during contraction and relaxation. Mutations in dystrophin lead to Duchenne muscular dystrophy (DMD), an incurable X-linked recessive disease affecting 1 in 3500 boys. Previous work has shown that several cardiac symptoms of DMD can be traced to calcium handling defects. To that end, a preliminary drug screen by our lab identified several L-type Calcium Channel blockers (CCBs) that were able to provide a cardioprotective effect. To conclusively determine the effectiveness of these CCBs, a platform that can accurately replicate physiological cardiomyocytes and screen these CCBs at semi-high throughput is needed. A major limitation with current drug screening platforms is that they use cardiomyocytes equivalent to the fetal heart. This is due to the limitations in current differentiation protocols, which fail to induce further maturity. Because symptoms of most inherited cardiomyopathies are exhibited in mature cardiomyocytes, these platforms are unable to predict drug efficacy accurately. Additionally, microelectrode array (MEA) systems - a system for high throughput drug studies - require highly accurate cell plating to provide good quality results, which requires extensive and costly training. Here, we addressed these issues by developing a novel platform that uses ComboMat, a technique used to enhance cardiomyocyte maturity, and designing an assistive device to plate cardiomyocytes in MEA plates. We showed that our platform with ComboMat-treated cardiomyocytes can give a more physiologically relevant response compared to platforms that use untreated cardiomyocytes. A MEA-based drug study is currently being performed to validate the CCBs identified in the preliminary drug screen. We expect to successfully validate a subset of the CCBs analyzed and further test them in animal models. This project will culminate in creating a novel and cost-effective platform that offers superior prediction of drug efficacy for DMD and potentially other cardiomyopathies as well.

Fluorescent biosensors are a vital tool in the goal to decipher the complexity of neural networks. Genetically encoded fluorescent indicators (GEFIs) are protein-based sensors that increase in fluorescence upon ligand binding and allow for passive monitoring of neuronal signals. However, the development of such sensors is limited by the slow throughput of traditional protein engineering which has long engineering cycles of new plasmid variants. Our project aims to tackle this problem by developing a high-throughput sensor engineering platform that can effectively generate and screen unbiased genetic libraries of GEFIs in mammalian cells. Our platform can identify high performing sensor variants on a custom microarray and effectively isolate and recover their genetic material. Our new platform will be used to develop a sensor for the μ-Opioid receptor (MOR), which is a G-protein coupled receptor that is involved in opioid addiction. Our experiments have already developed a MOR sensor that surpasses the standard in the literature and we will continue to optimize it for maximum spatial and temporal precision. The development of a MOR sensor through this iterative process allows researchers to further investigate the molecular mechanisms underlying the pathology of addiction and provides a novel platform for protein engineers to more efficiently develop a wide variety of biosensors.