Theses and Dissertations

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Open Access
Integrating discrete stochastic models with single-cell and single-molecule experiments
(Colorado State University. Libraries, 2019) Fox, Zachary R., author; Munsky, Brian, advisor; Stargell, Laurie, committee member; Wilson, Jesse, committee member; Prasad, Ashok, committee member
Modern biological experiments can capture the behaviors of single biomolecules within single cells. Much like Robert Brown looking at pollen grains in water, experimentalists have noticed that individual cells that are genetically identical behave seemingly randomly in the way they carry out their most basic functions. The field of stochastic single-cell biology has been focused developing mathematical and computational tools to understand how cells try to buffer or even make use of such fluctuations, and the technologies to measure such fluctuations has vastly improved in recent years. This dissertation is focused on developing new methods to analyze modern single-cell and single-molecule biological data with discrete stochastic models of the underlying processes, such as stochastic gene expression and single-mRNA translation. The methods developed here emphasize a strong link between model and experiment to help understand, design, and eventually control biological systems at the single-cell level.
Open Access
A comparison of tri-polar concentric ring electrodes to disc electrodes for decoding real and imaginary finger movements
(Colorado State University. Libraries, 2019) Alzahrani, Saleh Ibrahim, author; Anderson, Charles W., advisor; Vigh, Jozsef, committee member; Rojas, Don, committee member; Abdel-Ghany, Salah, committee member
The electroencephalogram (EEG) is broadly used for diagnosis of brain diseases and research of brain activities. Although the EEG provides a good temporal resolution, it suffers from poor spatial resolution due to the blurring effects of volume conduction and signal-to-noise ratio. Many efforts have been devoted to the development of novel methods that can increase the EEG spatial resolution. The surface Laplacian, which is the second derivative of the surface potential, has been applied to EEG to improve the spatial resolution. Tri-polar concentric ring electrodes (TCREs) have been shown to estimate the surface Laplacian automatically with better spatial resolution than conventional disc electrodes. The aim of this research is to study how well the TCREs can be used to acquire EEG signals to decode real and imaginary finger movements. These EEG signals will be then translated into finger movements commands. We also compare the feasibility of discriminating finger movements from one hand using EEG recorded from TCREs and conventional disc electrodes. Furthermore, we evaluated two movement-related features, temporal EEG data and spectral features, in discriminating individual finger from one hand using non-invasive EEG. To do so, movement-related potentials (MRPs) are measured and analyzed from four TCREs and conventional disc electrodes while 13 subjects performed either motor execution or motor imagery of individual finger movements. The tri-polar-EEG (tEEG) and conventional EEG (cEEG) were recorded from electrodes placed according to the 10-20 International Electrode Positioning System over the motor cortex. Our results show that the TCREs achieved higher spatial resolution than conventional disc electrodes. Moreover, the results show that signals from TCREs generated higher decoding accuracy compared to signals from conventional disc electrodes. The average decoding accuracy of five-class classification for all subjects was of 70.04 ± 7.68% when we used temporal EEG data as feature and classified it using Artificial Neural Networks (ANNs) classifier. In addition, the results show that the TCRE EEG (tEEG) provides approximately a four times enhancement in the signal-to-noise ratio (SNR) compared to disc electrode signals. We also evaluated the interdependency level between neighboring electrodes from tri-polar, disc, and disc with Hjorth's Laplacian method in time and frequency domains by calculating the mutual information (MI) and coherence. The MRP signals recorded with the TCRE system have significantly less mutual information (MI) between electrodes than the conventional disc electrode system and disc electrodes with Hjorth's Laplacian method. Also, the results show that the mean coherence between neighboring tri-polar electrodes was found to be significantly smaller than disc electrode and disc electrode with Hjorth's method, especially at higher frequencies. This lower coherence in the high frequency band between neighboring tri polar electrodes suggests that the TCREs may record a more localized neuronal activity. The successful decoding of finger movements can provide extra degrees of freedom to drive brain computer interface (BCI) applications, especially for neurorehabilitation.
Open Access
Force spectroscopy and dynamics in biological systems
(Colorado State University. Libraries, 2019) Schroder, Bryce William, author; Krapf, Diego, advisor; Bark, David, committee member; Popat, Ketul, committee member; DeLuca, Jennifer, committee member
Communication is key to any process involving the transmission of information or some sort of signal. For communication to occur, a signal must be created that can be detected. Cells communicate through cues transmitted in the forms of chemical and mechanical signals. The most fundamental means for transmitting chemical cues is through the process of diffusion. A single particle undergoing diffusion is considered to undergo Brownian motion, which can be modelled as a random walk. The random walk behavior is characteristic of both the particles properties and the fields in which it is occurring. An unbiased walk will be completely random without outside influence. A biased walk will be random within the confines of a potential influencing its direction. Both are Stochastic processes characterized through probabilistic models with known solutions. The work herein presents the development of single molecule experiments and the associated particle tracking tools targeting particles undergoing biased random walks within a trapping potential on or near a cellular membrane. In the first set of experiments, the trapping potential, an optical tweezers setup, has been developed and employed in measuring cellular membrane biophysical properties as well as blebbing forces. The optical trap was also used to directly measure flow driven forces in live embryonic zebrafish, the first known measurements of this kind. In the second set of experiments, synthetic lipid bilayers provided a trapping potential in a single dimension for protein binding experiments leading to exchanges between free, 3-dimensional diffusion and bound, or biased, 2-dimensional diffusion. In all cases, stochastic models have been used in conjunction with image-based particle tracking tools to better characterize the biophysical properties and forces associated with the cellular membrane and its means of signal transduction. These measurements are key to understanding both the chemical and mechanical signaling means by which the cellular membrane transduces an external signal into an internal response.
Open Access
Hemocompatibility of hyaluronan enhanced linear low-density polyethylene for heart valve leaflet applications
(Colorado State University. Libraries, 2018) Simon-Walker, Rachael, author; Popat, Ketul C., advisor; Reynolds, Melissa, committee member; Orton, Christopher, committee member; Chicco, Adam, committee member
Heart valve disease is a major concern in both developed countries with advanced ageing populations and undeveloped countries which experience a high incidence of rheumatism leading to valvular disease. To reduce mortality and improve quality of life, heart valve implantations have been widely used to assist in improving function of the native cardiovascular system. While mechanical heart valves and tissue-based heart valves have been successfully used to improve quality of life compared to untreated valvular disease, draw-backs are inherent. Mechanical heart valves are prone to thrombosis and require life-long supplemental anti-coagulation therapy. Tissue-based valves are more hemocompatible, but lack the durability required for long-term implantation. To address these issues, polymeric heart valves have been highly sought after due to polymers' abilities to enhance durability and be manufactured to be similar to the native heart valve leaflet. In addition, their surfaces can be modified to increase hemocompatibility. In this work we explore the hemocompatibility and immune response to a novel polymer for use in heart valve leaflet applications; hyaluronan enhanced linear low-density polyethylene. It is proposed that the combination of linear low-density polyethylene with hyaluronan will create a highly durable material that will reduce thrombosis and inflammation due to the anionic and hydrophilic nature of the glycosaminoglycan.
Open Access
Spinal cord and meningeal mechanics: viscoelastic characterization and computational modeling
(Colorado State University. Libraries, 2018) Ramo, Nicole Lauren, author; Puttlitz, Christian M., advisor; Troyer, Kevin L., advisor; Heyliger, Paul, committee member; James, Susan, committee member
Suffering a spinal cord injury (SCI) can be physically, emotionally, and financially devastating. With the complex loading environment typically seen in SCI events, finite element (FE) computational models provide an important economical and ethical option for investigating the mechanical etiology of SCI, evaluating prevention techniques, and assessing clinical treatments. To this end, numerous research groups have developed FE models of the spinal cord using various degrees of material and structural sophistication. However, the level of model complexity that is necessary to achieve accurate predictions of SCI has not been explicitly investigated as few studies have reported applicable tissue behavior. What are reported in the literature as "spinal cord mechanical properties" are most commonly based on ex-vivo tests of the spinal-cord-pia-arachnoid construct (SCPC). The pia and arachnoid maters are fibrous meningeal tissues that closely envelope the spinal cord, and together are referred to as the pia-arachnoid-complex (PAC). Currently available data demonstrate the PAC's importance in the overall SCPC stiffness and shape restoration following compression. However, only one previous study has reported mechanical properties of isolated spinal PAC, and therefore, conclusions about its contribution to SCPC mechanics are largely unknown. Additionally, it has been shown that SCPC material properties begin to degrade within 90 minutes of death. Considering the experimental difficulties and ethical concerns associated with in-vivo mechanical testing of the SCPC, determining the relationship between in-vivo and ex-vivo viscoelastic properties would allow researchers to more accurately analyze existing ex-vivo data. Therefore, the overarching goal of this work is to address the current gaps in knowledge regarding spinal cord and meningeal tissue mechanics and incorporate the developed material models into a FE model. Comparisons of ex-vivo and in-vivo porcine SCPC non-linear viscoelastic behavior revealed significantly different acute behaviors where the ex-vivo condition exhibited a higher stress response but also relaxed quicker and to a greater extent than the in-vivo condition. Although it only made up less than 6% of the ovine SCPC volume, the PAC was found to significantly affect the non-linear viscoelastic behavior of the SCPC which supports the conclusion that it plays an important protective mechanical role. Examining the fitting and predictive accuracy of linear, quasi-linear, and non-linear viscoelastic formulations to SCPC, cord, and PAC stress-strain data, non-linear formulations are recommended to model the SCPC and cord response to arbitrary loading conditions while the QLV is recommended for the PAC. This work provides researchers with novel insights into the complex mechanical behavior of the spinal cord and PAC. The experimental results represent an important addition to the limited literature on in-vivo versus ex-vivo neural tissue viscoelastic properties; they are also the first to quantify the non-linear elastic behavior of spinal PAC and the non-linear viscoelastic properties of the isolated spinal cord. Finally, the computational portion of this work provides a detailed report of the effects of viscoelastic formulation complexity on FE model prediction accuracy and computational time allowing researchers interested in modeling SCI to make informed decisions about the balance of accuracy and efficiency necessary for their specific modeling efforts.
Open Access
Development of microsystems for point-of-use microorganism detection
(Colorado State University. Libraries, 2018) Wang, Lei, author; Dandy, David S., advisor; Tobet, Stuart A., committee member; Henry, Chuck S., committee member; Geiss, Brian J., committee member; Bailey, Travis S., committee member; Marchese, Anthony J., committee member
To view the abstract, please see the full text of the document.
Open Access
Meniscal root tears and repairs
(Colorado State University. Libraries, 2018) Steineman, Brett Daniel, author; Haut Donahue, Tammy L., advisor; LaPrade, Robert F., committee member; Goodrich, Laurie R., committee member; Heyliger, Paul R., committee member
Meniscal root tears are defined as radial tears of the meniscal insertions and lead to an inability for the menisci to transmit compressive loads into circumferential hoop stresses. These are common among the posterior meniscal insertions due to acute or chronic conditions. Anterior root tears have also been shown to occur from iatrogenic injury during anterior cruciate ligament reconstructions; however, the relationship between anterior insertions and the anterior cruciate ligament are understudied. Root tears of the posterior insertions lead to measurable osteoarthritis within a year if left untreated. Despite this, changes to tissue characteristics due to anterior root tears are unknown. If untreated anterior roots result in tissue degeneration, then it is important for both anterior and posterior root tears to be repaired to prevent, or at least delay, the onset of osteoarthritis. Meniscal root repair techniques have been developed to prevent joint degeneration following meniscal root tears; however, clinical studies of root repairs show that meniscal extrusion and joint degeneration are not completely prevented. This limited repair success may be due to inaccurate placement of repairs during surgery or from repair loosening postoperatively as early as during rehabilitation. The goals of this work are to better understand anterior root tears and to investigate potential causes for insufficient meniscal root repairs. Thus, the aims are to: 1) Quantify the overlap between the anterior cruciate ligament and the anterolateral meniscal insertion in the coronal and sagittal planes. 2) Assess early in vivo degeneration after untreated anterior meniscal root tears. 3) Determine the extent of repair loosening and recovery due to short-term rehabilitation. 4) Develop finite element knee models to determine the effect of repair placement and loosening on knee mechanics. The completion of this project will improve clinical practice and basic scientific knowledge of current issues facing meniscal root tears and repairs.
Open Access
Modeling effects of microvilli on somatic signal propagation
(Colorado State University. Libraries, 2018) Aldohbeyb, Ahmed A., author; Lear, Kevin, advisor; Vigh, Jozsef, committee member; Bailey, Ryan, committee member
The electrical behavior of small compartments in neurons such as dendritic spines, synaptic terminals, and microvilli has been of interest for decades. Most of these fine structures are found in the dendrite, where most excitatory inputs are received, or in the axon where the action potential is generated and propagates. However, a recent study has shown expression of sodium voltage-gated channels (VGCs) in the soma of intrinsically photosensitive retinal ganglion cells (ipRGCs). Confocal imaging locates these sodium VGCs outside the main soma membrane, which implies that the VGCs occur in structures that protrude from the soma but are too small to be resolved with conventional optical microscopy. An investigator has hypothesized the voltage-gated sodium channels are positioned in microvilli. The microvilli hypothesis raises the question of the role of voltage-gated sodium channels on microvilli and more specifically what effect they would have on propagation of signals in the soma. The nanoscale dimensions of the microvilli, which are much smaller than patch-clamp probes, prevent conventional electrical studies that isolate individual compartments. In the absence of direct, high-spatial resolution measurements, computational models are valuable tools for developing a better understanding of the electrical behavior of the neuronal compartments. Well known models such as Hodgkin–Huxley models and cable theory have been the foundation of many advances in neuroscience. In this work, initial insights about the role of somatic microvilli are being generated using an equivalent circuit model based on the cable equation. For the circuit model, microvilli stubs containing resistor-capacitor networks and sodium channels are treated as branches off the main soma membrane. Circuit models of the soma membrane without microvilli serve as controls. The circuit models were simulated using Simulink. The results show that voltage-gated sodium channels placed on the main soma membrane or on the microvilli increase the amplitude of somatic signals as they propagate to the axon initial segment. Moreover, restriction of the VGCs to the somatic microvilli reduces the probability of misfires originating from spontaneous ion channel opening while still enhancing above threshold depolarizations propagating in the main soma membrane. For comparison, simulations of somatic signal propagation were also performed using the NEURON software as it readily incorporated the Hodgkin and Huxley model, including both sodium and potassium voltage-gated channels. The dendritic input signal was generated using the current clamp technique. The results show that the presence of VGCs on the main soma membrane lower the threshold for triggering the AIS to generate action potential. However, restricting sodium VGCs to the microvilli only did not initiate an action potential at the AIS. The ability of the microvilli Na+ VGCs to serve as excitatory inputs directly to the soma in the absence of the dendritic input was also investigated using NEURON. Using a current clamp, current was injected at the tip of the microvilli and the signal was recorded at the AIS. The results show that the signal at the AIS increases linearly with the injected current. However, the amplitude of the AIS potential was lower than the microvilli signal due to the high microvilli neck resistance. The results support the view that the microvilli act as electrical compartments that attenuate the microvilli VGCs' signals.
Open Access
The effect of repetition on force platform metrics during the bilateral bodyweight squat
(Colorado State University. Libraries, 2018) Sirkis, Jillian Rose, author; Reiser, Raoul F., advisor; Tracy, Brian L., committee member; Rosecrance, John C., committee member
INTRODUCTION: Functional Movement Assessments (FMAs) have been gaining popularity for screening athletes to determine weaknesses that could increase risk for injury. A common movement among many FMAs is the bilateral bodyweight (BWT) squat. Currently, FMAs typically require only 3-5 repetitions of the squat when unfatigued. However, it is known that fatigue affects performance and increases injury risk. It is possible that performing more repetitions might simulate the effects of fatigue, increasing the sensitivity of the FMA. PURPOSE: The goal of this investigation was to analyze the vertical ground reaction forces (GRFvs) and center of pressure (CoP) stability metrics during the down phase, up phase and whole movement across the course of a 20 repetition set of the BWT squat in a recreationally competitive group of young adult women. It was hypothesized that on average, due to changes in some individuals, there would be an increase in asymmetries and normalized values from the early repetition ranges to the later repetition ranges of the bilateral BWT squat in the down phase, up phase, and in the whole movement, that there would be evidence of fatigue in the later repetitions, and that the measures would be repeatable from day-to-day. METHODS: Fourteen recreationally active women (mean ± SD age: 20.5 ± 2.1 yrs; height 167.2 ± 7.3 cm; mass 66.9 ± 10.6 kg) with competitive sport backgrounds performed 20 bilateral BWT squats to a thighs parallel to the floor position while force platform data were recorded under each foot. Asymmetries and normalized metrics were compared across the early repetitions (2-7), middle repetitions (8-13), late repetitions (14-19), and entire set (2-19). Six subjects were reassessed for repeatability. RESULTS: Although not all measures analyzed produced group level changes from the early repetition ranges to the later repetition ranges, there was evidence of some people expressing different force platform metrics in the middle and later repetitions compared to the early repetitions in both GRFv (absolute average GRFv asymmetries during the up phase (p=0.008) and whole movement (p=0.025), relative minimum GRFv asymmetries during the down phase (p=0.018), normalized net minimum GRFv during the up phase (p = 0.005)) and CoP (relative AP sway asymmetry during the down phase (p=0.006) and whole movement (p=0.006), relative AP path length asymmetry during the down phase (p=0.002) and whole movement (p=0.002), normalized net AP sway (p=0.031)). High repeatability (Cronbach's alpha > 0.8) was found in the majority (107 out of 168) of metrics from day-to-day. CONCLUSION: Evidence of change, but limited evidence of fatigue, was found from the early repetition ranges to the later repetition ranges of the BWT squat. Furthermore, the high Cronbach's alpha values show that many of the metrics are repeatable from day to day. Differences also existed in response to repetitions between the up and down phases. Therefore, it is important to further investigate beyond the typical 3-5 repetitions as well as within each phase of the movement in order to determine if greater sensitivity for risk of injury while performing the BWT squat can be obtained.
Open Access
Development of a hierarchical electrospun scaffold for ligament replacement
(Colorado State University. Libraries, 2018) Pauly, Hannah Marie, author; Haut Donahue, Tammy L., advisor; Easley, Jeremiah, committee member; Kelly, Daniel J., committee member; Palmer, Ross, committee member; Popat, Ketul C., committee member
The anterior cruciate ligament (ACL) is a dense collagenous structure that connects the femur to the tibia and is vital for joint stability. The ACL possesses complex time-dependent viscoelastic properties and functions primarily to prevent excessive translations and rotations of the tibia relative to the femur. It is estimated that 400,000 ACL tears occur in the United States annually and the monetary burden of these injuries and their subsequent treatment is approximately $1 billion annually. After injury allografts and autografts are commonly implanted to reconstruct the torn ACL in an attempt to restore joint stability, prevent pain, and limit damage to surrounding tissues. However surgical reconstructions fail to completely restore knee functionality or prevent additional injury and regardless of intervention technique radiographic osteoarthritis is present in 13% of patients 10 years after ACL rupture. Drawbacks to traditional treatments for ACL ruptures motivate the development of a synthetic ACL replacement. Tissue engineering is the use of a scaffold, cells, and signaling molecules to create a replacement for damaged tissue. The goal of this work is to develop a polymer scaffold that can be utilized as a replacement for the ACL. A tissue engineered ACL replacement should replicate the hierarchical structure of the native ACL, possess reasonable time zero mechanical properties, and promote the deposition of de novo collagenous tissue in vitro. Additionally, the scaffold should be implantable using standard surgical techniques and should maintain in situ tibiofemoral contact mechanics. Thus, four specific aims are proposed: 1) Fabricated and characterize an aligned 3-dimensional electrospun scaffold for ACL replacement. 2) Assess the in vitro behavior of ovine bone marrow-derived stems cells seeded on the scaffold in the presence of conjugated growth factor. 3) Evaluate the performance of the electrospun scaffold using uniaxial mechanical testing. 4) Assess the effect of the electrospun scaffold on ovine stifle joint contact mechanics. Development of a tissue engineered ACL replacement that mimics the structure and function of the native ACL would provide a novel treatment to improve outcomes of ACL injuries.
Open Access
Orthogonal pair-directed codon reassignment as a tool for evaluating the factors affecting translation in E. coli
(Colorado State University. Libraries, 2018) Schwark, David, author; Fisk, John, advisor; Ackerson, Chris, committee member; Kennan, Alan, committee member; Peebles, Christie, committee member; Snow, Chris, committee member
Proteins are polymers of amino acids that are essential for life, central to cellular function, and have applications in fields ranging from materials science to biomedicine. Proteins in nature are composed of 20 amino acids with limited variability in size and chemical properties. Expanding the genetic code to contain non-canonical amino acids (ncAAs) that contain functionalities not contained in nature is a powerful strategy for probing and extending the properties of proteins. Current in vivo systems for expanding the genetic code have focused on using an engineered orthogonal aminoacyl-tRNA and aminoacyl tRNA-synthetase pairs (tRNA/aaRS) to direct incorporation of ncAAs at amber stop codons. In order to further expand the genetic code to 22 or more amino acids, additional codons must be targeted for reassignment to ncAAs. The genetic code is degenerate; 18 of the 20 canonical amino acids are encoded by more than one codon. Breaking the degeneracy of the genetic code by orthogonal pair directed sense codon reassignment is one pathway to genetic codes of 22 or more amino acids. However, orthogonal pair directed sense codon reassignment is hampered by a limited understanding of the relative importance of the factors that affect the translation of proteins. Here, we describe the repurposing of two commonly used orthogonal pairs from Methanocaldococcus jannaschii (M. jannaschiiI) and Methanosarcina barkeri (M. barkeri) to measure the in vivo reassignment efficiency of 30 different sense codons to tyrosine in E. coli with a simple fluorescence-based screen. The suite of sense codon reassignment efficiencies identified multiple promising codons for reassignment to ncAAs that have not been previously identified. Importantly, every sense codon was partially reassigned to tyrosine when either orthogonal tRNA/aaRS pair was used. Sense codons reassigned to tyrosine with high efficiency may be used directly for reassignment to ncAAs, and any sense codon with measurable reassignment to tyrosine may be improved through directed evolution. The sets of in vivo sense codon reassignment also revealed that E. coli are broadly tolerable to a large number of amino acid substitutions to tyrosine throughout the proteome. The codon reassignment efficiency measurements also enabled an analysis of the in vivo importance of local codon context effects, tRNA abundance, aminoacylation level, tRNA modifications, and codon-anticodon binding energy in determining translational fidelity. Quantitative sense codon reassignment efficiency measurements showed that the process of translation is highly balanced and both tRNA abundance and aminoacylation efficiency do not appear to be dominant factors in determining translational fidelity. Furthermore, quantitative measurements of amber stop codon reassignment efficiencies to tyrosine with the orthogonal M. jannaschii pair revealed that local codon context is an important factor for orthogonal pair directed amber stop codon reassignment.
Open Access
Microfluidic culture of human hepatocytes and endothelial cells with applications in drug toxicity screening
(Colorado State University. Libraries, 2018) Ware, Brenton R., author; Khetani, Salman R., advisor; Gustafson, Daniel L., committee member; Henry, Charles S., committee member; Twedt, David C., committee member
Drug-induced liver injury (DILI) continues to be a major problem for patient health and pharmaceutical expenditures, partially due to inadequacies of current model systems for predicting hepatotoxicity prior to clinical trials. In the drug development pipeline, many platforms are implemented depending on the stage of development, the number of compounds in question, and the specific hypothesis being studied. Primary human hepatocytes (PHHs) are considered the 'gold standard' for in vitro screening, as they retain a full complement of drug metabolizing enzymes and transporters. However, PHHs are in limited supply and lack the genetic diversity representative of the human population. In this dissertation, we explore alternative cell sources to PHHs such as iPSC-derived hepatocytes, mouse hepatocytes, and the hepatocarcinoma cell line HepaRG in an engineered liver platform. We found that each of these cell types showed a high level of hepatic functions when incorporated into a micropatterned co-culture (MPCC) of the hepatocyte type in question with 3T3-J2 murine embryonic fibroblasts. MPCCs of PHHs and 3T3-J2 fibroblasts were then challenged with known hepatotoxins and their non-toxic structural drug analogs before undergoing global gene expression analysis. These analyses revealed that hepatotoxins caused a differential expression of significantly more genes than the non-toxic analogs, and the corresponding pathways could reveal underlying mechanisms of drug toxicity. Next, these in vitro models were supplemented with endothelial cells to give a more complete representation of liver physiology. We found that co-cultures of hepatocytes and endothelial cells rapidly lost functionality, but tri-cultures of hepatocytes, endothelial cells, and 3T3-J2 fibroblasts were stable for multiple weeks. However, endothelia in the body experience shear stress from fluid flow, a phenomenon not mimicked with traditional in vitro cultures. Thus, we developed an in vitro platform for perfusing cultures with a physiologic level of shear stress. This system, constructed of tissue culture polystyrene with polydimethylsiloxane, was modeled using computational software and compared alongside static controls. Ultimately, we believe these platforms can be incorporated as the liver compartment into a "body-on-a-chip" platform used to understand multi-organ effects of drugs and diseases that impact the liver including diabetes, hepatitis B/C, and malaria.
Open Access
Development of a novel block copolymer hydrogel for meniscal replacement
(Colorado State University. Libraries, 2018) Fischenich, Kristine Marie, author; Haut Donahue, Tammy, advisor; Bailey, Travis, advisor; Easley, Jeremiah, committee member; Palmer, Ross, committee member; Goodrich, Laurie, committee member
Menisci are C-shaped fibrocartilaginous tissues responsible for distributing tibial-femoral contact pressure and are crucial for maintaining healthy joints and preventing osteoarthritis. Meniscal damage can be caused by age-related degradation, obesity, overuse from athletic activities, and trauma. Due to their primarily avascular nature, once damaged there is limited healing capacity and surgical intervention is often required. Limited technologies exist to replace damaged menisci, and standard treatment is to leave asymptomatic damage alone or perform partial meniscectomies, however, these treatment options lead to increased risk of OA. Attempts at tissue engineered meniscal scaffolds, and replacements have had mixed results due to design limitations and inability to recapitulate native tissue's material properties, shape, and pressure distribution. This project strives to create an artificial meniscus from a polystyrene-polyethylene oxide diblock copolymer. It is hypothesized that this hydrogel can be tuned to have material properties similar to those of the native meniscus. Furthermore, it is hypothesized this hydrogel can be molded into a 3D meniscal construct, implanted into the joint, and have similar pressure distribution properties as the native meniscus. Thus, the aims of this project are: 1) Mechanical comparison of a polystyrene-polyethylene oxide diblock copolymer TPE hydrogel to native meniscal tissue. 2) Develop a 3D meniscal construct which can be implanted into an ovine model and assess load distribution properties including contact area, mean pressure, and max pressure in both the medial and lateral hemijoints. If the goals of this project are met, there would exist a 3D TPE hydrogel construct that mimics the mechanical and functional properties of the native human meniscus. This meniscal replacement could provide a revolutionary addition to the field of osteoarthritis and meniscal injury.
Open Access
Engineering effective fibrocartilage replacement technologies using nanostructure-driven replication of soft tissue biomechanics in thermoplastic elastomer hydrogels
(Colorado State University. Libraries, 2018) Lewis, Jackson Tyler, author; Bailey, Travis S., advisor; Haut Donahue, Tammy L., advisor; James, Susan P., committee member; Popat, Ketul C., committee member; Li, Yan, committee member
Synthesis of hydrogel networks capable of accurately replicating the biomechanical demands of musculoskeletal soft tissues continues to present a formidable materials science challenge. Current systems are hampered by combinations of limited moduli at biomechanically relevant strains, inefficiencies driven by undesirable hysteresis and permanent fatigue, and recovery dynamics too slow to accommodate rapid cycling prominent in most biomechanical loading profiles. This dissertation presents a new paradigm in hydrogel design based on prefabrication of an efficient nanoscale network architecture using the melt-state self-assembly of amphiphilic block copolymers. Rigorous characterization and preliminary mechanical testing reveal that swelling of these preformed networks produce hydrogels with physiologically relevant moduli and water compositions, negligible hysteresis, sub-second elastic recovery rates, and unprecedented resistance to fatigue over hundreds of thousands of compressive cycles. By relying only on simple thermoplastic processing to form these nanostructured networks, the synthetic complexities common to most solution-based hydrogel fabrication strategies are completely avoided. Described within this dissertation are a range of efforts, broadly focused on refining synthetic and post-synthetic processing techniques to improve the modulus, surface hydrophilicity, fatigue resistance and cytocompatibility of these thermoplastic elastomer hydrogels, with the ultimate goal of producing a material viable as a meniscal replacement.
Open Access
Engineering complex liver models for drug screening and infectious diseases: a biomaterials and co-culture perspective
(Colorado State University. Libraries, 2018) Lin, Christine, author; Khetani, Salman R., advisor; Gustafson, Daniel, committee member; Kipper, Matthew, committee member; Pagliassotti, Michael, committee member
In vitro liver models have many applications in disease modeling and drug screening. Micropatterned cocultures (MPCCs) of primary human hepatocytes (PHHs) and supportive stromal cells have been shown to display high hepatic functions for long-term drug and disease studies. However, MPCCs lack liver non-parenchymal cells (NPCs) and the proper microenvironmental cues that can play important roles in conditions such as drug-induced liver injury (DILI), which is the leading cause of the prelaunch attrition and post-market withdrawal of pharmaceuticals, or diseases such as viral hepatitis. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are major health problems that affect >250 million and ~130-170 million people worldwide, respectively, and the development of therapeutics has been hindered due to the lack of models in which to study human response to virus and drugs. Thus, long-term in vitro models that can be used to study the progression of viral infection and drug pharmacodynamics are required to develop safe and efficacious therapeutics. These models must also be human-relevant due to the narrow host tropism of hepatitis B and C and differences in liver pathways across species. Thus, the goal of this dissertation is to augment the MPCC model to include the relevant substrates and cell types for the study of cell-cell interactions in diseases such as hepatitis and DILI. Biomaterials can present important microenvironmental factors that interact with cells. Chitosan and heparin polyelectrolyte multilayers (PEMs) were utilized as a substrate to present extracellular matrix (ECM) proteins and growth factors (GF) to hepatocyte cultures. Liver biomatrix (LBM) derived from human and porcine sources were also assessed as substrates since LBM contains both soluble and insoluble cues that are usually found in the liver in vivo. In addition to improving the MPCC substrate, liver NPCs, such as primary human Kupffer cells (KCs), were incorporated into the MPCC model since KCs play key roles in immune responses and inflammation. This work will be used to establish models that integrate multiple liver cell types on a physiologically-relevant substrate to study disease states such as hepatitis and DILI towards creating effective therapeutics.
Open Access
Engineering a biomimetic periosteum on cortical bone allografts for the reconstruction of critical-sized bone defects in mice
(Colorado State University. Libraries, 2017) Romero, Raimundo, author; Kipper, Matt J., advisor; Ehrhart, Nicole P., advisor; Kisiday, John D., committee member; Schenkel, Alan R., committee member
Load bearing bone allografts suffer from clinical failure due to low allograft-host tissue integration. Removal of the periosteum, a thin tissue layer on bone with a high regenerative capacity, is responsible for bone allografts' decreased clinical performance. This interdisciplinary project addressed this problem by creating multiple engineered periostea on mice bone allografts. Using a polysaccharide biomaterial, chitosan, tissue engineering scaffolds constructed on these bone allografts were modified with the glycosaminoglycan, heparin, and a chitosan derivative, trimethyl chitosan, to create periostea with different scaffold morphologies yet similar surface chemistries. Cell instructive cues such as growth factors fibroblast growth factor-2 (FGF-2) and transforming growth factor-β1 (TGF-β1) were adsorbed onto the engineered periostea and found to release up to 14 and 7 days in-vitro, respectively. Engineered allografts were found to support adipose-derived stem cell (ASC) adhesion and proliferation. FGF-2 and TGF-β1 delivered from the engineered allografts to ASC supported an osteoprogenitor phenotype in ASC and did not inhibit alkaline phosphatase and receptor activator of nuclear factor-kappaB (RANKL) protein expression. From in vitro results, the nanofiber engineered periosteum was found to be the most cytocompatible scaffold and was selected for subsequent implantation in a pre-clinical mouse critical-sized femoral defect model. We assessed the engineered periosteum's efficacy on modulating allograft healing and incorporation. We confirmed the engineered allografts successfully delivered ASC, FGF-2, and TGF-β1 to the femur defect and found ASC persisted in the femur defect for at least 7 days, similar to other reports in the literature. At week 6, microcomputed tomography results of excised femurs showed no statistical difference in new bone volume formation between experimental groups. However, treatment groups containing ASC showed a trend of at least 24% more bone volume compared to their respective cell-free controls suggesting possible therapeutic effects of ASC. Union ratio and histological analysis both confirmed the nanofiber engineered periosteum did not degrade at 6 weeks and inhibited allograft incorporation. Subsequent relative gene expression experiments showed that ASC maintained an undifferentiated phenotype in response to FGF-2 and TGF-β1 delivered from chitosan nanofibers. Overall, this project developed a novel polysaccharide-based engineered periosteum for delivering growth factors and progenitor cells to a bone defect for regenerative medicine applications.
Open Access
Effect of rear wheel suspension on tilt-in-space wheelchair shock and vibration attenuation
(Colorado State University. Libraries, 2017) Hischke, Molly, author; Reiser, Raoul F., II, advisor; Gilkey, David, committee member; Tracy, Brian, committee member
Suspension systems are designed to reduce shock and vibration exposure. Prior to the QuadshoX LLC suspension kit (Fort Collins, CO), manual tilt-in-space wheelchairs did not have rear wheel suspension available for use. Furthermore, it was anticipated that rear wheel diameter would have an independent effect on shock and vibration transmitted to the wheelchair. The aim of this study was to investigate the shock and vibration reducing capabilities of the newly available aftermarket rear wheel suspension system and wheel diameter for manual tilt-in-space wheelchairs. Ten healthy non-wheelchair users volunteered for the study (7 men, 3 women: age 22.1±3.36 yrs, height 1.75±0.067 m, weight 73.9±8.87 kg (mean±SD)). Subjects were pushed by the same trained investigator over four different obstacles while using a Quickie IRIS® Tilt-in-Space manual wheelchair (Sunrise Medical, Phoenix, AZ) with two different diameter solid wheels, (0.381 m and 0.508 m), Primo Cheng Shin Tires (Cheng Shin Rubber, Yuanlin, Taiwan). Surfaces included a/an 1) exterior door threshold, 2) truncated domes, 3) 2 cm descent, and 4) 2 cm ascent. The subjects traversed the obstacles with the wheelchair as manufactured, and followed ~ 2 weeks later with the QuadshoX suspension kit installed. A tri-axial accelerometer, (Model339A31, PCB Piezotronics, Depew, NY), was mounted to the rear of the wheelchair seat pan with signals sampled at 2000 Hz. Peak resultant accelerations were analyzed from surface 1, 3-4, root mean square (RMS) resultant accelerations were analyzed from surface 2, and vibration dose value (VDV) and total power were analyzed from all surfaces 1-4. Unweighted and ISO 2631-1 frequency weighted (FW) accelerations were analyzed. The use of suspension decreased the un-weighted peak acceleration at the rear wheel when it impacted the door threshold, and when the rear wheel traversed the 2 cm descent and ascent (p=0.043, p=0.001, p=0.001, respectively) and FW peak accelerations at the rear wheel when it impacted and left the door threshold, and when the rear wheel descended 2 cm (p=0.049, p= 0.001, p= 0.005, respectively). With suspension, RMS and total VDV significantly decreased 14% and 10- 22% respectively (p=0.011, p=0.004). There were no significant differences between the rigid and suspended chair in total vibration power in frequency octaves most harmful in human exposure (4 – 12 Hz). The results of wheel diameter were not evaluated because there were significant differences in time spent over the obstacles between the two diameters (door threshold p= 0.018, truncated domes p= 0.028, 2 cm descent p= 0.029, 2 cm ascent p = 0.024). However, there were not differences in time spent over the obstacles between rigid and suspended conditions (p ≥ 0.064). The results indicate the aftermarket rear wheel suspension reduces some aspects of shock and vibration exposure, specifically at the rear wheel. While low back pain, neck pain, discomfort, and muscle fatigue correlate with shock and vibration exposure there is no set threshold of reduction in shock and vibration exposure to decrease the health risks with exposure. Considering how much time tilt-in-space users spend in their wheelchairs, we expect the observed reductions in shock and vibration with the use of the aftermarket rear wheel suspension may decrease the health risks, such as pain and muscle fatigue.
Open Access
Mathematical and experimental studies in cellular decision making
(Colorado State University. Libraries, 2017) Lyons, Samanthe Merrick, author; Prasad, Ashok, advisor; Medford, June, committee member; Kisiday, John, committee member; Snow, Chris, committee member
The biological sciences are undergoing an epistemological revolution. Mathematical modeling, quantitative experiments and data analysis, machine learning and other methods of "big-data" modeling are slowly but surely changing the way the biological and biomedical sciences and engineering are being carried out. This thesis presents work that seeks to advance understanding of biological processes using mathematical modeling as well as experiments coupled with sophisticated quantitative analysis. The central theme of the research presented is cellular decision-making. A cellular decision is defined here as a transition from one cell state, or phenotype, to another, based upon information received from an external or internal signal. This work explores the mechanisms behind cellular decisions with three specific systems and a variety of mathematical and modeling techniques. This dissertation begins with a brief survey of the use of mathematical modeling in cellular biology, utilizing specific example of various approaches. This reviews the diversity of techniques available from detailed mechanistic models to simplified phenomenological representations, and notes some applications demonstrating the utility of such models. The first exploration of cellular decisions is concerned with the question of how cells can make decisions in the face of cross-talk from multiple signals. The real cellular environment is noisy, with stochastically varying levels of external signals and cellular decisions required in spite of this noise. In Chapter 3 the ubiquitous bacterial two-component signaling system and the similarly structured mammalian TGF-β pathway are modeled with stochastic simulations of the chemical master equation. Information theory is utilized to quantify the amount of information transmitted by these signaling systems in the presence of competing signals from cross-talk, revealing that the mammalian TGF- pathway was able to transmit information accurately despite high levels of cross-talk, while the bacterial two-component system, due to a smaller system size and the structure of phospho-transfer rather than phospho-relay, was poor at discriminating from competing cross-talk. This work presents a novel thesis: many signal transduction systems suffer less from cross-talk than was commonly imagined, and may actually make use of cross-talk for cross-regulation. The second system of cellular decisions studied in this work is a bistable synthetic toggle switch network motif composed of mutually repressible promoters in Chapter 4. This motif has been widely studied in isolation for its dynamical and static properties. However, the behavior of these switches has never previously been analyzed when coupled with a downstream binding partner, termed a "load". Real toggle switches, whether synthetic or natural, always have loads connected with them. The toggle-switch system was modeled mathematically with ordinary differential equations as well as using stochastic simulations of the chemical master equation to determine the effect of a load. The quasi-potential energy landscape of the bistable switch was calculated utilizing a novel method which revealed that, in some parameter spaces, a downstream component can significantly alter the stability of the switch; addition of a positive feedback loop could provide for a tunable switch. Chapter 5 is concerned with developing methods for identifying a complex cellular transition from less metastatic to more metastatic cancer cells. The importance of metastatic disease in the pathology of cancer cannot be understated as it is the cause of 90% of deaths from cancer. The process by which cancerous cells become metastatic is complex, but requires specific cellular mechanical conditions in order to occur. The use of cancer cell shape to predict metastatic behavior in pathology samples is a key component of prognostication, however in vitro cancer cell shape is less commonly studied. This work developed a mathematical algorithm to extract shape parameters from images of cancer cells and applied multiple statistical techniques to elucidate differences between metastatic and non-metastatic cancer cells. While both simple and complex statistical techniques including t-tests, principle component analysis (PCA) and non-metric multidimensional scaling (NMDS) revealed distinct changes, the population of cells from highly metastatic and less metastatic paired osteosarcoma cells showed significant overlap. Machine learning algorithms were, however, able to successfully classify samples of cells to high or low metastatic lines with high accuracy. The concluding chapter presents a brief analysis of the new questions that this research has elucidated, and delineates some future tasks to address them.
Open Access
Engineering in vitro models of non-alcoholic fatty liver disease
(Colorado State University. Libraries, 2017) Davidson, Matthew David, author; Khetani, Salman R., advisor; Chicco, Adam J., committee member; Donhue, Seth W., committee member; Kota, Arun K., committee member
Decreased resources and a scarcity of affordable, healthy food is contributing to rising obesity rates throughout the world. Consequentially, non-alcoholic fatty liver disease (NAFLD), which is highly correlated with obesity, rates are also increasing with greater than 30% of the US population currently diagnosed. NAFLD starts as a benign state of fat accumulation within liver hepatocytes but often progresses to more detrimental conditions such as non-alcoholic steatohepatitis (NASH), fibrosis/cirrhosis and hepatocellular carcinoma (HCC). There is no cure for NAFLD or its downstream complications and questions still remain about what factors contribute to disease progression. Specifically, the cause(s) of insulin resistance, lipid accumulation, inflammation, and fibrosis are not completely understood. Many of these questions cannot be elucidated in animal models due to confounding contributions from other organs, differences in animal disease pathology (relative to humans) and dietary restrictions. Additionally, if therapies are to be identified for NALFD, human-relevant systems will need to be used due to species differences in drug metabolism enzymes. Primary human hepatocytes (PHHs) are the gold standard for assessing drug metabolism in vitro, but these cells rapidly lose their liver phenotype in vitro. Here we show that micropatterned co-cultures (MPCCs) of PHHs and stromal cells maintain glucose and lipid metabolism in hepatocytes, which suggests their utility for in vitro disease models of NAFLD. Major advances in culturing methods were developed to increase the insulin sensitivity and overall health of hepatocytes in MPCCs prior to carrying out studies regarding NAFLD-related insulin resistance. The highly insulin sensitive MPCC model was then used to develop models of fatty acid-induced NAFLD and hepatic stellate cell induced NASH phenotypes. Potential disease mechanisms and treatments for fatty acid-induced insulin resistance and NASH disease progression were identified using these models.
Open Access
P300 wave detection using Emotiv EPOC+ headset: effects of matrix size, flash duration, and colors
(Colorado State University. Libraries, 2016) Alzahrani, Saleh Ibrahim, author; Anderson, Charles W., advisor; Vigh, Jozsef, committee member; Gavin, William, committee member
Brain-computer interfaces (BCIs) allow interactions between human beings and comput- ers without using voluntary muscle. Enormous research effort has been employed in the last few decades to design convenient and user-friendly interfaces. The aim of this study is to provide the people with severe neuromuscular disorders a new augmentative communication technology so that they can express their wishes and communicate with others. The research investigates the capability of Emotiv EPOC+ headset to capture and record one of the BCIs signals called P300 that is used in several applications such as the P300 speller. The P300 speller is a BCI system used to enable severely disabled people to spell words and convey their thoughts without any physical effort. In this thesis, the effects of matrix size, flash duration, and colors were studied. Data are collected from five healthy subjects in their home environments. Different programs are used in this experiment such as OpenViBE platform and MATLAB to pre-process and classify the EEG data. Moreover, the Linear Discriminate Analysis (LDA) classification algorithm is used to classify the data into target and non-target samples.