Theses and Dissertations

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Open Access
316L stainlesss steel modified via plasma electrolytic oxidation for orthopedic implants
(Colorado State University. Libraries, 2022) Michael, James A., II, author; Popat, Ketul C., advisor; Li, Vivan, committee member; Sampath, Walajabad S., committee member
316L stainless steel (SS) is widely used biomaterial for implantable devices and is estimated to the base material for 60% of implantable devices. However, one challenge of the material is the inhomogeneity of the surface morphology which may influence the adhesion process of host cells and bacteria. One method to create a uniform surface of 316L SS is plasma electrolytic oxidation (PEO). PEO creates an oxide layer on the outer surface thus changing the surface topography on the microscale. PEO process on SS functions by anodizing the surface via direct current in electrolyte solution. Preliminary research found that a continuous direct current over a time manufactured undesirable samples, to overcome this challenge the use of pulse timings was utilized during fabrication. This research aimed to answer the questions how do PEO modifications effect cellular adhesion and viability, and how do PEO modifications affect bacteria adhesion and viability. PEO modified 316L SS surfaces were characterized and its effects on the adhesion, morphology, and differentiation of adipocyte derived stem cells, along with the adhesion and morphology of Staphylococcus aureus was investigated.
Open Access
A Haversian bone model of fracture healing in a simulated microgravity environment
(Colorado State University. Libraries, 2015) Gadomski, Benjamin C., author; Puttlitz, Christian M., advisor; Browning, Raymond, committee member; Donahue, Tammy, committee member; Heyliger, Paul, committee member
Ground-based models of weightlessness and microgravity have provided valuable insights into how dynamic physiological systems adapt or react to reduced loading. Almost all of these models have used rodent hindlimb unloading as the means to simulate microgravity on isolated physiological systems. Unfortunately, results derived from rodent studies are significantly limited when one tries to translate them to the human condition due to significant anatomical and physiological differences between the two species. Therefore, it is clear that a novel animal model of ground-based weightlessness that is directly translatable to the human condition must be developed in order for substantial progress to be made in the knowledge of how microgravity affects fracture healing. In light of this, four specific aims are proposed: (1) develop a ground-based, ovine model of skeletal unloading in order to simulate weightlessness, (2) interrogate the effects of the simulated microgravity environment on bone fracture healing using this large animal model, (3) develop a computational model of weightbearing in ovine bone under different experimental conditions in order to characterize the loads experienced by the fracture site, and (4) develop countermeasures that enhance bone fracture healing in the presence of simulated microgravity. Successful completion of this project will substantially elevate the understanding of how fracture site loading affects the subsequent healing cascade in the presence of microgravity and will form the foundation for designing future rehabilitation protocols to facilitate bone healing during long-duration spaceflight.
Open Access
A nanoparticulate-reinforced hyaluronan copolymer hydrogel for intervertebral disc repair
(Colorado State University. Libraries, 2011) Yonemura, Susan S., author; James, Susan P., advisor; Bailey, Travis S., committee member; Kisiday, John D., committee member; Wheeler, Donna L., committee member
Degenerative disc disease (DDD) is an inevitable consequence of aging, commonly resulting in low back pain (LBP). Current clinical treatment options for disc degeneration exist at two extremes: conservative management or extensive surgical intervention. Given the economic impact of lost productivity and disability associated with low back pain, there is significant interest in earlier, less invasive intervention. Biomimetic disc replacement and regenerative therapies offer an attractive alternative strategy for intervertebral disc repair, but materials employed to date have not exhibited a successful combination of mechanical and biological properties to achieve viable solutions. The composite material developed and characterized in this work consisted of a novel hyaluronan-co-poly(ethylene-alt-maleic anhydride) (HA-co-PEMA) hydrogel matrix reinforced with nanoparticulate silica; the hydrogel matrix provided a compliant hydrated matrix conducive to integration with the surrounding tissue while the nanoparticulate reinforcement was manipulated to mimic the mechanical performance of healthy ovine nucleus pulposus (NP) tissue. HA-co-PEMA was formed via an esterification reaction between a hydrophobically-modified HA complex and PEMA, and candidate formulations were characterized by chemical, thermal, and physical means to select an appropriate base hydrogel for the reinforced composite. Three grades of commercially-available fumed silica, varying by degree of hydrophobic surface modification, were evaluated as nanoparticulate reinforcement for the composite materials. Mechanical testing of two reinforced composite formulations (620-R and 720-R) emphasized dynamic shear properties and results were directly compared to ovine nucleus pulposus (NP) tissue. The complex shear modulus (G*) for 620-R ranged from 1.8±0.2 KPa to 2.4±0.3 KPa over the frequency range 0.1 Hz < f < 10 Hz, while G* for 720-R varied from 4.4 ± 0.5 KPa to 6.1 ± 0.6 KPa over the same frequency range. Ovine NP tissue tested using identical methods exhibited G* of 1.7 ± 0.2 KPa at 0.1 Hz up to 3.8 ± 0.5 KPa at 10 Hz. Thus, the complex shear moduli (G*) for 620-R and 720-R effectively bracketed G* for NP over a physiologically-relevant frequency range. Subsequent in vitro cytotoxicity and biocompatibility experiments suggest that the 720-R formulation warrants consideration for future in vivo modeling.
Open Access
A novel approach for critical bone defect repair
(Colorado State University. Libraries, 2022) Schneiderhan, Adam, author; Prawel, David, advisor; Popat, Ketul, committee member; Séguin, Bernard, committee member
Critical bone defects are defined as defects that will not naturally heal over a patient's lifetime, even with surgical stabilization. When these occur in the long bones of the axial skeleton (secondary to trauma, tumor resection, etc.), limb-sparing surgery can be performed to avoid amputation of the limb. This procedure typically involves the installation of a steel locking plate over the defect, along with an endoprosthesis or allograft to fill the void of resected bone. Much progress has been made in the natural bone regeneration using tissue engineering (TE) scaffolds in place of these grafts. Porous hydroxyapatite (HAP) is a well-established bone TE scaffold biomaterial but lacks sufficient mechanical strength when fabricated at porosities shown to best induce osteogenesis. To remedy this, polymers such as polycaprolactone (PCL) are often mixed with HAP to fabricate scaffolds with increase load-bearing capacity. However, the addition of PCL makes the scaffold less osteogenic and dramatically slows the degradation rate of the scaffold. This translates into reduced new bone volume where the PCL cannot be remodeled as new bone is formed. This project involves a pilot clinical trial of a novel method that augments the gold-standard limb-sparing procedure by implanting a 3D printed endoprosthetic "sleeve" device that attaches to the locking fixation plate and contains and protects the brittle HAp scaffold. The PCL sleeve alleviates the dependency on scaffold strength which enables use of the most osteogenic possible biomaterials at ideal porosities to maximize the rate and density of new bone formation. The purpose of the study is to clinically validate the construct design and surgical procedure. Thus far, pilot limb-sparing surgeries have been performed on 4 client-owned dogs, in which sleeve-scaffold devices were installed in the critical defects caused by the removal of osteosarcomas in distal epiphyseal radii. Recombinant human bone morphogenic protein-2 (rhBMP-2) was added to the scaffolds to further encourage osteogenesis. Mechanical tests were performed on both the sleeves alone and the full construct installed in canine cadaver limbs. Results from this testing demonstrate the sleeve's ability to prevent mechanical failure of the HAp scaffolds. Similarly, no scaffold failure has been observed in clinical trial patients, with some having the device installed for greater than 24 weeks. Additionally, pressureometry and gait analysis confirmed excellent return of limb function in these animals. However, to date, no new bone formation has been observed within the scaffold devices, which has likely been inhibited by anti-cancer treatment. Regardless, results from ex vivo testing and the clinical trial validate the construct design and the viability of our novel method for protecting and maintaining brittle bone tissue engineering scaffolds, while aiding in restoration of normal limb function.
Open Access
A pharmacokinetic investigation of chloroquine analogues in cancer autophagy modulation
(Colorado State University. Libraries, 2020) Collins, Keagan P., author; Gustafson, Daniel, advisor; Prasad, Ashok, committee member; Yao, Tingting, committee member; Tham, Douglas, committee member; Thorburn, Andrew, committee member
Hydroxychloroquine (HCQ) is currently being investigated for safety and efficacy as an autophagy inhibitor in Phase I/II cancer clinical trials. It is the only clinically-approved autophagy inhibitor for use in cancer clinical trials in the United States. HCQ is used in combination with other chemotherapeutics to augment their efficacy and has shown moderate success in treating patients with late stage cancers. While HCQ has a good safety index and shows promise as an addition to standard of care treatment regimens, it suffers from several critical pharmacologic shortcomings which we take steps to address herein. The primary issues with usage of HCQ addressed in this work are the metrics used to predict patient tumor concentration of the drug following various dosing regimens. HCQ pharmacokinetics (PK) are highly variable in patients, with no correlation between traditional plasma:tumor concentrations. The first step taken to address this problem is to characterize likely sources of interindividual variability in HCQ PK. To do this a physiologically-based pharmacokinetic (PBPK) model was developed to investigate absorption, distribution, metabolism, and toxicity (ADMET) factors relating to HCQ in a mathematical system representative of the human body. This model was developed based on physiological and biochemical parameters relevant to HCQ ADMET in mice and scaled to represent humans. The model was capable of simulating single and multiple dosing regimens in humans, that would be characteristic of a cancer clinical trial. PBPK modeling addressed variability that would be associated with the macrophysiologic scale, but intrinsic and extrinsic factors on the cellular scale needed to be further defined to strengthen the understand of HCQ PK. To investigate factors that affect cellular uptake and sub-compartment localization of HCQ, a base PK model of lysosomotropic agents like HCQ was applied. Model specific parameters were identified for a panel of four human breast cancer cell lines, and a majority of differences in cellular uptake of the drug could be attributed to differences in the relative lysosomal volume fraction of each cell line. The model was able to characterize HCQ PK under different extracellular pH conditions, and identified a positive-feedback loop, related to transcription factor EB (TFEB) activation. This feedback loop caused the cell to increase its lysosomal volume over time of exposure to HCQ, resulting in a continuous increase of HCQ concentration within the cell. Through sensitivity analysis of the model, acidic extracellular pH was identified as a critical limiting factor of HCQ uptake into cells – which is particularly important as the tumor microenvironment is physiologically acidic. HCQ concentrations in cells cultured in an acidic microenvironment are decreased up to 10-fold, which cannot be overcome without the aid of agents that neutralize this pH. Dimeric analogues of HCQ, Lys05 and DC661, have been reported to maintain potency in acidic conditions and so were investigated in a comparative context to HCQ. Lys05 and DC661 were found to behave similarly to HCQ pharmacokinetically – i.e. highly dependent on the lysosomal profile of the cell. These drugs exhibited similar kinetic uptake curves as HCQ, and also induced the lysosomal biogenesis PK feedback loop. Unlike HCQ, Lys05 and DC661 uptake was not completely inhibited by acidic extracellular pH, and they were able to maintain activity under these conditions. PK of these drugs was characterized in a murine model to investigate their potential as in vivo agents, suggesting they could maintain high concentrations for a longer duration than HCQ. Lys05 and DC661 share many pharmacologic similarities to HCQ, while not sharing significant shortcomings such as inactivity under acidic extracellular conditions suggesting they should be investigated for further application as next generation autophagy inhibitors.
Open Access
Action potential initiation mechanisms: analysis and numerical study
(Colorado State University. Libraries, 2022) Aldohbeyb, Ahmed A., author; Lear, Kevin L., advisor; Vigh, Jozsef, committee member; Prasad, Ashok, committee member; Venayagamoorthy, Karan, committee member
Action potentials (AP) are the unitary elements of information processing in the nervous system. Understanding AP initiation mechanisms is a fundamental step in determining how neurons encode information. However, variation in neuronal response is a characteristic of mammalian neurons, which further complicate the analysis of neuronal firing dynamics. Several studies have associated the variation in AP onset with the type and densities of voltage-gated ion channels, diversity in synaptic inputs, neuron intrinsic properties, cooperative Na+ gating, or AP backpropagation. But the mechanisms that underlie the response variability remain unclear and subject to debate. Even though all these studies tried to answer the same question, the definition of AP onset and rapidity differs between them, highlighting the need for a more systematic and consistent method to quantify AP onset features, and hence analyzing the variation in AP onset. Two novel methods were developed to quantify AP rapidity. The proposed methods have lower relative variation, higher ability to classify neuron types, and higher sensitivity and specificity to voltage-gated Na+ channels parameters than current methods. AP rapidity was used to analyze different factors impacting the AP activation mechanism. However, the prior rapidity quantification methods are subjectively based on the researcher's judgment, which complicates the comparison between different studies. Thus, we proposed a more systematic and consistent method based on the full-width or half-width at half the rising phase peak of the membrane potential's second-time derivative (Vm). First, using an HH-type model, we showed that the peak width methods are sensitive to changes in the Na+ channel parameters and conductance and minimally impacted by changes in the K+ channel parameters compared to the phase slope, the standard quantification method. Second, we compared the peak width methods to the two prior methods, phase slope and error ratio, using recordings from cortical and hippocampal pyramidal neurons, hippocampal PVBCs, and FS cortical neurons found in online databases. The results showed that the new methods have the lowest variation between neurons within a specific type while significantly differentiating several neuron types. Together, the two studies showed that the peak width methods provide another sensitive tool to investigate the mechanisms impacting AP onset dynamics and provide a better tool to study Na+ channels kinetics and AP onset features. A conductance-based model that includes dynamics of ion concentration and cooperative Na+ channels was developed to investigate the mechanisms responsible for observed neuronal response variation. Random response variability has previously been observed in spike trains evoked from individual neurons by the same DC stimulus, but we observed systematic variation. The first APs' in a burst had attributes that were comparable regardless of the stimulus strength, while the subsequent APs' attributes monotonically change during bursts, and the magnitude of change increases with stimulus strength. These two spike train features were observed in three different neuron types (n = 51), indicating a shared mechanism is responsible for the spike train pattern. Various existing computational models fail to replicate the monotonic variation in AP attributes. We proposed incorporating ion concentration dynamics and cooperative gating to account for the missing behavior. A model with dynamic reversal potential but without cooperative Na+ channel gating reproduces the AP attribute's variation during bursts, but not the first APs' attributes. The first APs' attributes were reproduced only in the presence of a fraction of cooperative Na+ channels. Cooperative gating also enhanced the magnitude of modeled variation of some AP attributes to better match the electrophysiological recordings. Therefore, we conclude that changes in ion concentration dynamics could be responsible for the monotonic change in some AP's attributes during normal neuronal firing, and cooperative gating can enhance this effect. Thus, the two mechanisms contribute to the observed variability in neuronal response, especially the variation in AP rapidity.
Open Access
Advancing impulsive Raman spectroscopy and microscopy for biological applications
(Colorado State University. Libraries, 2024) Smith, David R., author; Bartels, Randy, advisor; Wilson, Jesse, advisor; Tobet, Stuart, committee member; Jost, Dylan, committee member
Chemically sensitive, label-free spectroscopy and microscopy is a critical tool for the study of many complex and dynamic biological systems. The development of the impulsive stimulated Raman scattering (ISRS) techniques in this thesis represent important steps forward in addressing the ability to interrogate Raman vibrations in complex and scattering samples, particularly low frequency Raman modes.
Embargo
Anomalous diffusion of mRNA in the cytoplasm of HeLa cells
(Colorado State University. Libraries, 2024) Roessler, Ryan, author; Krapf, Diego, advisor; Stasevich, Tim, committee member; Prasad, Ashok, committee member
Information about the diffusive motion of RNA would provide insights into intracellular structures and functions, as well as gene expression and genetic regulation. We study the motion of individual messenger RNA molecules in the cytoplasm of HeLa cells. RNAs are imaged in live cells via total internal reflection (TIRF) microscopy. In order to visualize individual RNA molecules expressing the MYH9 gene, they were labeled via MS2 stem loops bound to coat proteins tagged with the HaloTag-JF646 fluorophore. We then used single-particle tracking to obtain trajectories of individual molecules. Trajectories were analyzed in terms of their mean-squared displacement (MSD) and power spectral density (PSD). We observed non-ergodic, subdiffusive behavior, with statistics that depend on observation time, i.e., aging. Additionally, we observe stochastic switching between two mobility states with an order of magnitude difference in diffusivity. This switching process is responsible for the aging nature of the system. When compared to the cytoplasmic motion of synthetic nanoparticles, the analysis of RNA trajectories gives rise to discrepancies that raise questions about specific intracellular interactions.
Open Access
Atomic force microscopy: more than surface imaging
(Colorado State University. Libraries, 2020) Bishop, Terrance Tyler, author; Krapf, Diego, advisor; Prasad, Ashok, committee member; Van Orden, Alan, committee member
Atomic Force Microscopy (AFM) is a powerful imaging tool that has capabilities that go beyond the abilities of most other microscopes. Here, three examples of these capabilities were considered. First, the AFM was operated in an image generating mode to determine the surface heterogeneity of polysaccharide membranes. Second, the AFM was used to record force-indentation curves, these curves were fit with a Hertzian model to determine the stiffness of murine smooth muscle cells. Finally a approach for attaching 10 µm and 2 µm polystyrene beads to tip-less AFM cantilevers was proposed, and a viscoelastic contact model was tested to determine the viability of the created probes.
Open Access
Biomimetic and antimicrobial surfaces for orthopedic implants
(Colorado State University. Libraries, 2021) Wigmosta, Tara, author; Kipper, Matt, advisor; Popat, Ketul, advisor; Giess, Brian, committee member; DeLong, Susan, committee member; Schenkel, Alan, committee member
The number of total knee and hip replacement surgeries is expected to continue to rise in the United States. As such, the number of revision surgeries is also expected to rise. The two most common causes of failure for these implants is aseptic loosening, caused by incomplete osseointegration, and infection. Therefore, preventing infection while increasing the osteogenic properties of the surfaces used in orthopedic implants could reduce the number of revision surgeries. It is the goal of this work to create nanostructured surfaces that both increase mineralization and antimicrobial properties of titanium surfaces commonly used in orthopedic implants. To accomplish this, chitosan/heparin polyelectrolyte multilayers (PEMs), with the addition of either bone morphogenetic protein 2 (BMP-2) or gentamicin, were adsorbed onto titania nanotubes. BMP-2 has been used in clinical applications to increase osseointegration in spinal fusions, and gentamicin is effective against the most common pathogens found in infected orthopedic implants. Both heparin and chitosan are biocompatible and have antimicrobial properties. BMP-2 has a binding site for heparin that increases BMP-2's half-life in vitro. The first chapter summarizes the motivation and previous strategies used to increase osseointegration and antimicrobial properties of nanostructured biomimetic orthopedic implant surfaces. The first chapter concludes with a shift in hypothesis testing, outlining three different hypotheses: 1) surface modification(s) increase cytocompatibility and the osteogenic properties of mammalian bone cells; 2) surface modification(s) reduce bacterial adhesion, proliferation, and infection rate, without decreasing cytocompatibility; and 3) surface modification(s) provide a favorable environment in which mammalian cells can beat bacterial cells and colonize the surface first, thus increasing the osteogenic and antimicrobial properties of the surface. The testing of these hypotheses are explored in chapters 2 through 4. The second chapter explores hypothesis 1) by testing if BMP-2 released from chitosan/heparin PEM coated titania nanotubes surfaces induce an osteogenic response from rat bone marrow cells. Chapter 3 explores hypothesis 2) by testing if iota-carrageenan/chitosan and pectin/chitosan PEMs have antimicrobial properties against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), and support rat bone marrow cell adhesion and proliferation. The last chapter explores hypothesis 3) by testing if gentamicin released from titania nanotubes coated with chitosan/heparin PEMs influences the "race to the surface" in favor of mammalian cells.
Open Access
Characterization of chromatin remodeling in mesenchymal stem cells on the application of oxidative stress
(Colorado State University. Libraries, 2022) Kabi, Neda, author; Ghosh, Soham, advisor; Popat, Ketul, committee member; Goodrich, Laurie, committee member; Johnstone, Brian, committee member
Chromatin is a highly dynamic entity of the eukaryotic cell nucleus. Contrary to previous belief that chromatin maintains a well-defined permanent architecture in the interphase nucleus, new evidences are emerging with a support of the notion that chromatin can locally and globally rearrange itself to adapt with the cellular microenvironmental changes. Such microenvironmental changes can be related to biophysical such as change in the stiffness of extracellular matrix or the force applied on the cell as well as biochemical such as change in the oxidative stress, osmolarity or the pH. It is not well understood how the chromatin architecture changes under such environmental changes and what is the functional significance of such change. Characterization and quantification of chromatin remodeling is therefore a first step to understand the chromatin dynamics for elucidating complex subnuclear behavior under the influence of single or multiple environmental changes. Towards that end, in this work, human bone marrow derived mesenchymal stem cells were used to characterize such chromatin level changes under the changing oxidative stress on the cells. Oxidative stress was applied using hydrogen peroxide treatment. After validation of the application of oxidative stress, a series of experiments and subsequent analysis was performed to understand the hallmarks of chromatin remodeling at high spatiotemporal resolution. Specific chromatin remodeling pattern was observed in the heterochromatin, euchromatin and the interchromatin regions. Finally, a key component of chromatin remodeling complex called ARID1A was identified which is critical for the chromatin remodeling process.
Open Access
Characterization of the unique biomechanical behavior of right ventricle using experimental and constitutive modeling approaches
(Colorado State University. Libraries, 2022) Liu, Wenqiang, author; Wang, Zhijie, advisor; Puttlitz, Christian, committee member; Bark, David, committee member; Chicco, Adam, committee member
Ventricle dysfunction leads to high morbidity and mortality in heart failure patients. It is known that right and left ventricles (RV&LV) are distinct in their embryologic origins, anatomies and functions, as well as the pathophysiology of ventricular failure. However, how exactly the RV is distinct from the LV in their biomechanical properties remains incompletely understood. Furthermore, the prevalence of RV failure is significantly increased in the later stages of diseases such as pulmonary hypertension (PH) and heart failure with preserved ejection fraction, and the clinical management and treatment of RV failure are persistently challenging. This calls for a further understanding of the mechanisms of RV failure including the biomechanical mechanism. In addition, ventricular tissues are viscoelastic, which means both energy storage (originated from elasticity) and energy loss (originated from viscosity) are present during the deformation. However, the investigation of ventricular tissue viscoelasticity is much less than that of the elasticity, and it is largely unknown how the RV viscoelastic behavior changes during RV failure progression and impacts on the physiological function of the chamber. To fill these knowledge gaps, the overall goal of my study was to investigate the unique biomechanical properties of the RV in its physiological and pathological functions using experimental and constitutive modeling approaches. The Specific Aims are: 1) Develop the experimental protocols and characterize ventricular tissue passive static and dynamic mechanical properties in both large and small animals; 2) Adapted and performed constitutive modeling of ventricular tissue static and dynamic mechanical behaviors; 3) Quantify the changes in RV biomechanics during the maladaptive remodeling induced by pulmonary hypertension. At the completion of my study, I established the ex vivo testing protocols and provided fundamental data regarding static and dynamic mechanical differences between the healthy left and right chambers to delineate the unique biomechanical properties of the RV. I also adapted the constitutive models to capture static and dynamic mechanical behaviors of the RV. Finally, I quantified the biomechanical changes of the RV during the RV failure development and offered new insights in the contributions of the RV tissue biomechanics to the organ function. The findings were obtained from both large and small animals' species, which are translational to human diseases and a strong addition to the current literature of RV failure. More importantly, the investigation on the viscoelastic (dynamic) mechanical properties of the RV and the changes of viscoelasticity in RV failure progression is highly novel. The constitutive modeling of the RV biaxial viscoelastic behavior is pioneering and unique in the computational study of the RV. In summary, this study will deepen the understanding of the biomechanical mechanisms of RV failure and assist with the development of new computational tools for diagnosis and treatment strategies.
Open Access
Clinical importance of autophagy dependency and inhibition in cancer treatment
(Colorado State University. Libraries, 2021) Van Eaton, Kristen M., author; Gustafson, Daniel L., advisor; Munsky, Brian, committee member; Thamm, Douglas, committee member; Thorburn, Andrew, committee member; Yao, TingTing, committee member
Autophagy, a lysosomal degradation recycling process, has a complex and context-dependent role in cancer. Certain cancers have been found to be inherently dependent on autophagy for survival regardless of the environment. Autophagy is also implicated as a mechanism of resistance to many chemotherapies. More autophagy dependent tumors are generally more sensitive to autophagy inhibition genetically and pharmacologically. Therefore, determining what tumors are autophagy dependent is important for selecting patients that are viable candidates for autophagy inhibition. Currently, autophagy inhibition is being tested in over 90 clinical trials using FDA- approved hydroxychloroquine (HCQ) alone or in combination with other therapies. However, responses have been variable, especially in trials where HCQ is used as a monotherapy. Further, the relationship between HCQ pharmacokinetics and pharmacodynamics is not well understood in patients. Pharmacokinetics of HCQ and one of its active metabolites DHCQ was assessed in non-tumor bearing mice. Both parent and metabolite were observed at clinically relevant concentrations after 72 hr and this corresponded with evident autophagy inhibition in various tissues, although autophagy inhibition was inconsistent across the mice. The pharmacokinetic data established 60 mg/kg as the human equivalent dose observed in patients based on HCQ exposure. Cellular responses to HCQ were assessed in 2D cell culture, 3D tumor organoids, and in vivo tumor xenografts using autophagy dependent and autophagy independent tumors. Overall, cellular responses were similar across the in vitro and in vivo methods. Autophagy was inhibited regardless of autophagy status, but autophagy dependent tumors had increased cell death and decreased cell proliferation at earlier time points and lower doses of HCQ, suggesting autophagy dependency matters for optimal results. Since autophagy inhibition was inconsistent in vivo, it is still important to determine better biomarkers and possibly consider using more potent autophagy inhibitors in the clinic. Since there have not been any major advancements in osteosarcoma survival over the past four decades, autophagy dependency was assessed in osteosarcoma. Osteosarcoma was found to be intermediately to very dependent on autophagy following a genetic screen. Further, initially autophagy dependent tumor cells were able to survive and adapt to autophagy loss. Not all tumor cells adapted in the same way nor were these autophagy deficient tumor cells more sensitive to standard osteosarcoma chemotherapy, highlighting the difficulty of determining what context autophagy inhibition should be used in the clinic. Since some autophagy inhibitors like HCQ are lysosomal inhibitors and do not specifically target autophagy alone, the results of these studies also emphasized the importance of understanding whether autophagy inhibition via lysosomal degradation or autophagy inhibition of the autophagic pathway itself is superior. Overall, these results indicate targeting autophagy in osteosarcoma is a promising therapy.
Open Access
Compartmentalization of membrane proteins by the actin cytoskeleton
(Colorado State University. Libraries, 2013) Higgins, Jenny, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Bamburg, James, committee member; Azimi-Sadjadi, Mahmood, committee member
Acting as the point of contact for the outside world, the plasma membrane is crucial for cellular signaling events. Proper organization of membrane components is necessary to accomplish this task. Although a number of experiments have demonstrated the compartmentalization of lipids and proteins on the plasma membrane, direct observation of the mechanisms by which the organization occurs has been challenging, in part due to the imaging restrictions of a diffraction-limited system and the dynamic nature of the membrane compartmentalization. Using photoactivated localization microscopy (PALM), a superresolution technique, we have captured the dynamics of compartments formed by the cortical actin cytoskeleton. Live human embryonic kidney (HEK293) cells were imaged with a temporal resolution of 2 s and a spatial resolution of 40 nm. The actin cytoskeleton forms compartments with a mean area of 2.3±0.3 μm2 that are partially outlined by actin bundles. When the PALM images of actin were combined with single particle tracking of membrane proteins, we directly observed the cytoskeleton acting as a barrier to the diffusion of Kv2.1 and Kv1.4, two voltage-gated potassium channels. In addition, we used a novel compartment detection and tracking algorithm to show that Kv2.1 and Kv1.4 channels avoid actin when changing compartments. This work represents the first direct observations of individual membrane protein interactions with barriers formed by the actin cytoskeleton.
Open Access
Computational approaches to predict drug response to cytotoxic chemotherapy
(Colorado State University. Libraries, 2020) Mannheimer, Joshua D., author; Gustafson, Daniel, advisor; Prasad, Ashok, advisor; Krapf, Diego, committee member; Thamm, Douglas, committee member
Cancer is the second leading cause of death in the United States. Statistically, within a lifetime there is slightly above a one-third chance of developing some form of cancer and a one in five chance of dying from the disease. Thus, it is no hyperbole that the understanding and treatment of cancer is one of the most pressing issues in medical research of the current era. Cytotoxic chemotherapies are a class of anti-cancer drugs that are widely used to treat a number of cancers. While cytotoxic chemotherapies are extremely effective in treating a subset of individuals for some cancers, drug resistance resulting in failure of treatment is a prominent obstacle in many cancer patients. Precision medicine, a novel concept to the 21st century, is the application of disease treatments that are specifically tailored to an individual and the specific attributes of their disease. In oncology, precision medicine particularly refers to the use of gene expression and other biological factors to inform an individual's treatment. Because cancer and its response to treatment result from many complex biological interactions, computational methods have become an essential tool to identify the molecular signatures that are the basis for precision treatment. In this thesis, a systematic analysis of the computational approaches is performed to gain insight necessary for the development of novel computational approaches in precision medicine in cancer. Statistical learning models are a class of computational modeling methods that identify and extrapolate complex patterns from large amounts of data. Specifically, this involves applying statistical learning approaches on in vitro data from cell lines and patient tumor data to predict drug response, particularly for cytotoxic chemotherapies, with an emphasis on understanding the fundamental modeling principles and data attributes driving model performance. The first chapter serves as an introduction to chemotherapy and the advancements that have driven computational approaches to precision applications in cancer. The second chapter serves as a technical introduction to statistical learning models and approaches. In the third chapter a systematic assessment of linear and non-linear modeling approaches are applied to in vitro cell lines panel including the National Cancer Institute's 60 cancer cell lines (NCI60) and cell lines of Genomics of Drug Sensitivity in Cancer (GDSC) to predict drug response in several cytotoxic chemotherapies. With in-depth analysis it is shown that the relationship between tumor tissue histotype and drug response is the major driver of model performance and can be maintained in as little as 250 random genes. The fourth chapter utilizes statistical models to explore the influence of drug induced gene perturbations on drug response models in comparison with basal gene expression. The findings indicate that drug induced changes in gene expression are superior predictors of drug response. Second, it is demonstrated that Boolean network representation of gene interactions show distinct topological differences between drug induced changes in gene expression and basal gene expression. Finally, in the fifth chapter, drug induced gene changes demonstrating high levels of connectivity in the previously developed networks are applied to derive a basal gene expression signature to predict response to combined gemcitabine and cisplatin chemotherapy treatment in patients with bladder cancer. These models show that this derived signature performs better than a random cohort of genes and in some situations genes derived directly from basal gene expression.
Open Access
Constitutive modeling of the biaxial mechanics of brain white matter
(Colorado State University. Libraries, 2016) Labus, Kevin M., author; Puttlitz, Christian M., advisor; Donahue, Seth, committee member; Heyliger, Paul, committee member; James, Susan, committee member
It is important to characterize the mechanical behavior of brain tissue to aid in the computational models used for simulated neurosurgery. Due to its anisotropy, it is of particular interest to develop constitutive models of white matter based on experimental data in order to define the material properties in computational models. White matter has been shown to exhibit anisotropic, hyperelastic, and viscoelastic properties. The majority of studies have focused on the shear or compressive properties, while few have tested the tensile properties of the brain. Brain tissue has not previously been tested in a multi-axial loading state, even though in vivo brain tissue is in a constant multi-axial stress state due to fluid pressure, and data from uniaxial experiments do not sufficiently describe multi-axial stresses. The main objective of this project was to characterize the biaxial tensile behavior of brain white matter via experimentation and constitutive modeling. A biaxial experiment was developed specifically for testing brain tissue. Experiments were performed at a quasi-static loading rate, and an Ogden anisotropic hyperelastic model was derived to fit the data. A structural analysis was performed on biaxially tested specimens to relate the structure to the mechanical behavior. The axonal orientation and distribution were measured via histology, and the axon area fraction was measured via transmission electron microscopy. The measured structural parameters were incorporated into the constitutive model. A probabilistic analysis was performed to compare the uncertainty in the stress predictions between models with and without structural parameters. Finally, dynamic biaxial experiments were performed to characterize the anisotropic viscoelastic properties of white matter. Biaxial stress-relaxation experiments were conducted to determine the appropriate form of a viscoelastic model. It was found that the data were accurately modeled by a quasi-linear viscoelastic formulation with an isotropic reduced relaxation tensor and an instantaneous elastic stress defined by an anisotropic Ogden model. Model fits to the stress-relaxation experiments were able to accurately predict the results of dynamic cyclic experiments. The resulting constitutive models from this project build upon previous models of brain white matter mechanics to include biaxial interactions and structural relations, thus improving computational model predictions.
Open Access
Design and evaluation of an instrumented microfluidic organotypic device and sensor module for organ-on-a-chip applications
(Colorado State University. Libraries, 2020) Richardson, Alec Evan, author; Henry, Charles, advisor; Tobet, Stuart, advisor; Bark, David, committee member; Abdo, Zaid, committee member
Organ and tissue-on-a-chip technologies are powerful tools for drug discovery and disease modeling, yet many of these systems rely heavily on in vitro cell culture to create reductionist models of tissues and organs. Therefore, Organ-on-chip devices recapitulate some tissue functions and are useful for high-throughput screening but fail to capture the richness of cellular interactions of tissues in vivo because they lack the cellular diversity and complex architecture of native tissue. This thesis describes the design and testing of 1) a microfluidic organotypic device (MOD) for culture of murine intestinal tissue and 2) a microfluidic sensor module to be implemented inline with the MOD for real-time sensing of analytes and metabolites. The MOD houses full-thickness murine intestinal tissue, including muscular, neural, immune, and epithelial components. We used the MOD system to maintain murine intestinal explants for 72 h ex vivo. Explants cultured in the MOD formed a barrier between independent fluidic channels perfused with media, which is critical to recapitulating intestinal barrier function in vivo. We also established differential oxygen concentrations in the fluidic channels and showed that more bacteria were present on the tissue's mucosal surface when exposed to near-anoxic media. The sensor module is a reversibly sealed microfluidic device with magnetic connections that can withstand high backpressures. Further, electrodes housed in commercial finger-tight fittings were integrated into the sensor module in a plug-and-play format. Future work will include developing electrochemical/optical sensors for various biological compounds relevant to intestinal physiology. Ultimately, the MOD and sensor module will be implemented in long-term microbiome studies to elucidate the relationship among microbial, epithelial, neuro and immune components of the gut wall in health and disease.
Open Access
Design and fabrication of bioactive coatings to catalytically generate nitric oxide on the surfaces of extracorporeal circuits
(Colorado State University. Libraries, 2022) Wick, Tracey V., author; Reynolds, Melissa M., advisor; Kipper, Matthew, committee member; Olver, Christine, committee member
Blood-contacting medical devices suffer from biofouling caused by proteins, platelets and other cells adhering to the surface which often leads to severe complications and eventual device failure. In particular, extracorporeal membrane oxygenation (ECMO) is a life support treatment that is highly prone to coagulation issues due to a large blood-contacting surface area and turbulent blood flow. The ECMO circuits are constructed from catheters, tubing, and an oxygenator which all come into contact with blood and have several connections that alter the blood flow. The standard therapy to decrease thrombotic complications is to administer a systemic anticoagulant, usually unfractionated heparin. While this reduces clotting, harmful and potentially fatal hemorrhagic complications arise. Researchers have looked to nitric oxide (NO), a common biomolecule produced by the endothelium, as an alternative to locally inhibit clotting. Previous work has shown a reduction in thrombotic activity using NO-releasing substances, but these substances only last for a short period of time. An approach explored herein takes advantage of a catalytic mechanism to generate NO from endogenous NO-donors, S-nitrosothiols (RSNOs). RSNOs have been shown to catalytically generate NO through copper catalysis and in particular, with a copper-based metal-organic framework, H3[(Cu4Cl)3(BTTri)8-(H2O)12]·72H2O where H3BTTri = 1,3,5-tris(1H-1,2,3-triazole-5-yl)benzene] (CuBTTri). Importantly, CuBTTri has been shown to be stable under biological conditions and compatible with human cells; therefore, it is a promising candidate for biomedical applications. This report explores the addition of CuBTTri on the surfaces of ECMO. In Chapter 2, a CuBTTri-doped composite is coated onto the extracorporeal circuitry tubing. The fabrication method to apply CuBTTri to the tubing is reported, and the coating was shown to actively generate NO when exposed to a RSNO and no adverse effects were noted during hemocompatibility testing. In Chapter 3, CuBTTri is immobilized on the surface of an ECMO oxygenator using polydopamine. The morphology of the coating was evaluated and the CuBTTri on the surface of the oxygenator was catalytically active, generating NO when exposed to a RSNO. The incorporation of CuBTTri on the surfaces of these components could improve the hemocompatibility of the device, providing a safer and more effective life support system.
Open Access
Development of fluidic devices to facilitate more accessible monitoring of human health
(Colorado State University. Libraries, 2024) Cherwin, Amanda E., author; Henry, Charles S., advisor; Tobet, Stuart A., advisor; Snow, Christopher, committee member; Abdo, Zaid, committee member
In December of 2023, the World Health Organization (WHO) Director-General Tedros Adhanom Ghebreyesus outlined the 'Five P's' of global health priorities: Promoting health, Providing health, Protecting health, Powering health, and Performing for health. Despite the mantra of 'prevention is better than cure,' many countries still prioritize treating the sick over proactive health promotion, leading to inadequate prevention of non-communicable diseases (NCDs). Access to healthcare services poses a significant barrier to early recognition and treatment of health issues, particularly in low-income communities. To address these challenges, harnessing the power of science and technology becomes imperative. Powering health involves leveraging scientific research and collaboration to understand disease mechanisms better. Physiologically relevant models, such as microfluidic systems, offer insights into disease progression. Microfluidics, especially when combined with 2D and 3D culture systems, enhances functionality by mimicking physiological conditions. These devices provide cost-effective solutions for diagnostic challenges, bridging the gap between in vitro and in vivo studies. Protecting health requires a deeper understanding of organ systems. Chapter 2 examines a microfluidic model of the gut, an organ that plays a critical role in maintaining overall health. Two devices are discussed, an organotypic device for maintaining ex vivo gut tissue explants, and an electrochemical sensor module for monitoring relevant molecules such as oxygen or hydrogen peroxide within the tissue media. Dysbiosis in the gut microbiome has been linked to various pathologies, emphasizing the need for accurate models for studying gut barrier integrity. Ex vivo models using microfluidic devices offer promising avenues for studying disease mechanisms. The devices described in Chapter 2 serve as an effective model of the intestinal barrier that can be closely monitored in real-time. Providing health involves making effective healthcare solutions universally accessible. Point-of-care (POC) diagnostics, facilitated by microfluidics, enable rapid and cost-effective disease detection. Capillary-driven flow microfluidic devices enhance accessibility by eliminating the need for bulky external pumps, making POC testing feasible even in resource-limited settings. Combining the concepts of Powering and Providing health leads to the development of innovative diagnostic devices. Capillary-driven flow microfluidics enables the development of portable devices for diagnosing conditions from viscous sample matrices like blood and saliva. These devices offer less invasive and more accessible alternatives to traditional diagnostic methods, potentially revolutionizing healthcare delivery. Chapter 3 describes a capillary flow device used to quantify levels of two salivary biomarkers (Galectin-3 and S100A7) correlated to Heart Failure (HF) outcomes. This rapid, noninvasive, accessible POC test can drastically improve the quality of life for HF patients, particularly in rural and resource-limited areas. Using an electrochemical detection method, we demonstrate successful multiplexed detection of both biomarkers in spiked buffer solutions. Chapter 4 focuses on microfluidic devices probing rheological properties of whole blood related to Sickle Cell Disease (SCD) and clotting using capillary flow. For the SCD device, our goal was to develop a low-cost Point-of-Care (POC) multiplexed device for rapid and accurate identification of SCD phenotypes using three key reagents tied to altered sickle cell blood rheology: calcium chloride, sodium metabisulfite, and adenosine diphosphate. We developed an integrated device where whole blood reacts with reagent pads, enabling rapid assessment of a patient's SCD phenotype to inform appropriate treatment. We also introduced the Paper-based Clotting Analysis Test (PCAT) for efficient, low-cost analysis of primary hemostasis. Current methods for monitoring hemostasis are expensive and slow. Our capillary flow device uses whole blood moving at high flow rates for sustained durations to induce thrombus formation. This dissertation bridges the gap between effective health monitoring and accessibility through fluidic devices using either pump-driven or capillary-driven flow. Chapters detail the development of microfluidic systems for monitoring intestinal barrier function, detecting biomarkers in saliva for Heart Failure prognosis, and processing blood samples for Sickle Cell Disease phenotyping and clotting analysis. Ultimately, these devices hold the potential to transform healthcare management, particularly in underserved communities.
Open Access
Development of novel mechanical diagnostic techniques for early prediction of bone fracture healing outcome
(Colorado State University. Libraries, 2021) Wolynski, Jakob G., author; McGilvray, Kirk, advisor; Puttlitz, Christian, advisor; Heyliger, Paul, committee member; James, Susan, committee member; Wang, Zhijie, committee member
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