2020-

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Open Access
A new approach to addressing two problems in pharmacokinetics and pharmacodynamics using machine learning
(Colorado State University. Libraries, 2020) Habib, Sohaib, author; Reisfeld, Brad, advisor; Munsky, Brian, committee member; Shipman, Patrick, committee member
In this work, machine learning was applied to develop solutions for two problems related to drug pharmacokinetics (PK) and pharmacodynamics (PD). The first problem was finding a way to easily predict important pharmacological measures accurately representative of those from simulation results computed via a sophisticated model for drug absorption via oral dosing. This model (OpenCAT: Open source Compartmental And Transit model) comprises a system of differential equations describing the absorption of drugs into the gastrointestinal tract, including such factors as drug dissolution and spatially-distributed absorption, metabolism, and transport. For this problem, a machine learning framework was built to develop a self-contained random forest representation of the model predictions that could be queried for critical PK parameters such as maximum plasma concentration (Cmax), time at which the maximum concentration occurs (tmax), and the area under the concentration-time curve (AUC). The random-forest representation was able to generate predictions for the targeted PK parameters close to the solution of the original OpenCAT model over a wide range of drug characteristics. The second problem involved predicting the pharmacodynamics (cholinesterase reactivation) of antidotes for nerve agents. In this case, a machine learning framework was built to use experimental data and corresponding theoretically-derived chemical descriptors to predict the pharmacodynamics of new candidate antidotes against both tested and untested nerve agents. Overall, this project has demonstrated the utility of machine learning approaches in the fields of drug pharmacokinetics and pharmacodynamics.
Open Access
An evaluation of yield and quality characteristics for vegetable crops grown under organic management in Fort Collins, Colorado
(Colorado State University. Libraries, 2020) Mason, Tyler, author; Uchanski, Mark, advisor; Bartolo, Michael, committee member; Johnson, Sarah, committee member; Prenni, Jessica, committee member
To view the abstract, please see the full text of the document.
Open Access
Effects of Lubabegron supplementation on carcass traits, muscle fiber type, proteome profile and meat quality attributes of finished feedlot steers
(Colorado State University. Libraries, 2020) Corona, Ashley, author; Nair, Mahesh N., advisor; Belk, Keith E., committee member; Scanga, John A., committee member; Prenni, Jessica, committee member
Two thousand one hundred and sixty (2,160) British and Continental crossbred steers were supplemented (1, 4, 3.2 or 5.0 g/ton (DM basis) Lubabegron and a control diet (Experior; EX, Elanco Animal Health) for the last 28, 56, or 84 d of the finishing period resulting in twelve treatment combinations. Fifteen pens (12 hd/pen) were allocated to each treatment combination consisting of a dose and feeding duration. A total of five harvest cycles were conducted, consisting of 432 head per cycle. Each harvest cycle consisted of 3 blocks, each block contained all dosages and each block was associated with a specific feeding duration. Hot carcass weights (HCW), marbling scores (MS), adjusted fat thickness (aFT), longissimus muscle area (LMA), kidney pelvic and heart fat percentage (KPH), and USDA calculated yield grade (YG) were evaluated for all carcasses (N = 2160). No dose x feeding duration (FD) interaction (P > 0.05) was present for any of the characteristics measured. Supplemented cattle produced heavier (P < 0.05) carcass weights, larger (P < 0.05) LMAs and decreased (P < 0.05) YGs. As feeding duration was extended from 28 to 56 and 84 d, carcass weights were increased (P < 0.05). Control cattle produced MS that were significantly higher than those that were supplemented EX at the highest dose; nonetheless MS remained within USDA Premium Choice (MT00-99). Whereas, EX supplementation did not affect aFT and KPH. A subset of carcasses (N= 540) (3 carcasses/pen) that graded USDA Low Choice (SM00-99) were selected for the purpose of objective color, muscle fiber typing, proteome analysis, and the evaluation of the effect of postmortem aging on tenderness and palatability. As dose increased (P < 0.05) to 3.2 and 5.0 g/ton steaks became less (P < 0.05) red (a*), less (P < 0.05) yellow (b*), and less (P < 0.05) saturated than the controls. Striploin steaks collected during fabrication (before aging) were analyzed for muscle fiber typing (N = 96, n = 8). No detrimental shifts (P > 0.05) were observed for muscle fiber type as it relates to meat quality. The muscle fiber type IIX cross sectional area remained similar across the majority of treatment groups, except for decrease in CSA seen in cattle fed 5.0 g/ton for the final 56 and 84 d of feed. Meat quality attributes were measured using trained sensory panels, slice shear force (SSF) and Warner-Bratlzer shear force (WBSF). Striploins from the right side of each carcass were collected, fabricated into 2.54-cm steaks, and aged for 0, 7, 14, 21, and 28 d postmortem. Steaks for all postmortem aging periods were evaluated using SSF and WBSF, whereas, only those aged for 14 d were evaluated by trained panelists. Non- supplemented cattle produced striploin steaks that were juicier and more tender (P < 0.05) than those from EX supplemented cattle regardless of dose, and no differences (P > 0.05) were observed as a consequence of FD. All steaks (supplemented and non-supplemented) subjected to a minimum 7 d of PM aging produced WBSF that were less than 3.9 kg, and therefore eligible to be labeled as "Certified Very Tender." Once 21 d of postmortem aging was reached, no differences (P > 0.05) in tenderness were observed between the treatments. Based on meat quality attributes, six samples each (N = 24, n = 6) from four treatments (control, low dose for 28 days, high dose for 28 days, and high dose for 84 days) were selected for proteome analysis using a chemical labelling approach know as tandem mass tag (TMT). Experior supplementation influenced expression of proteins involved in muscle contraction, calcium signaling, transport, growth factor, and proteasome activation. Myosin light chain 3 (MYL3) was associated with an improved tenderness and carcass grading, which could be reflective of the increased intramuscular fat content. The proteins identified such as hemoglobin subunit α (HBA), hemoglobin subunit β (HBB), and alpha-1-acid glycoprotein (ORM1) were suggestive of increased vascularization in muscles as a response to EX supplementation.
Open Access
The impact of time-restricted eating on circulating factors, insulin sensitivity and circadian rhythms
(Colorado State University. Libraries, 2020) Kennedy, Devin, author; Broussard, Josiane, advisor; Braun, Barry, committee member; Stephens, Jaclyn, committee member
Purpose: Obesity has been steadily increasing over several decades. In 2008, prevalence rates of obesity were reported at over 300 million people, defined as a body mass index of >30kg/m2. For years, scientists have tried to find "solutions" to obesity. While obesity prevention measures taken in childhood might result in decreased adulthood obesity, childhood prevention measures are not common, and obesity is often a health issue in adulthood. Negative energy balance and caloric restriction is most effective for reducing body weight, and studies have reported beneficial effects such as reduced fasting glucose and insulin, reductions in body weight [1], significantly higher insulin sensitivity, significantly lower BMI [2], reduced β-cell sensitivity [3], and reduced fasting glycemia and fasting insulinemia [4]; however, long-term adherence to caloric restriction is low. Certain fasting practices are emerging as promising possible solutions to help combat obesity. Fasting practices have resulted in improvements in cardiometabolic health including but not limited to protection from obesity [5], improved LDL and HDL cholesterol, reduced HbA1c and c-reactive proteins, [6], cell proliferation, and body weight [7]. Intermittent fasting is one method by which an individual can reduce body weight but also improve numerous cardiometabolic factors. However, research exploring intermittent fasting (IF), specifically time-restricted eating (TRE), as a method of improving cardiometabolic health is limited. Circadian rhythms might be the reason that aligning feeding windows to earlier in the day is showing these benefits. Currently, a gap in the knowledge exists as to whether circadian rhythms play a role in contributing to the metabolic benefits that are conferred by TRE, or if the timing of the food intake/duration is what results in the benefits. Therefore, our objective was to examine the effects of TRE on 24-hour glucose homeostasis and nighttime patterns of circulating factors (glucose, insulin, free fatty acids, triglycerides, and glycerol) as well as insulin sensitivity and the central circadian clock. Methods and results: This study employed a consecutive design. Eight healthy adults (6F; 27±4 y; 22.6±2.1 kg/m2; mean ± SD) completed a 2-week protocol. During Week 1 participants were instructed to consume their daily calories over a 13h period (control condition). In Week 2, participants were instructed to consume their daily calories over an 8h period (TRE condition). Specified mealtimes were pre-determined based on the habitual sleep and wake time for each individual participant. At the end of each week, participants were admitted to the Sleep and Metabolism Laboratory for an overnight stay that involved hourly blood samples. Plasma samples were analyzed for glucose, insulin, free fatty acids (FFA), lactate, triglycerides, and glycerol. The plasma analyses indicated that TRE decreased glucose variability during sleep (p=0.03), reduced nighttime insulin concentrations (p=0.005), increased nighttime FFA levels (p=0.04), increased nighttime triglycerides (p=0.006) and increased nighttime glycerol (p=0.02). TRE did not impact glucose variability during wakefulness (p = 0.49), nighttime glucose (p = 0.39), insulin sensitivity (MATSUDA-ISI, p = 0.38), or central circadian rhythms. Conclusion: The observed changes in nighttime glucose variability and insulin levels could represent mechanisms by which TRE can improve metabolic homeostasis in healthy lean individuals. Future studies are warranted to determine whether TRE can improve metabolic homeostasis in people at risk for diabetes such as people with overweight and obesity, and impaired glucose tolerance.
Open Access
Integrated techno-economic analysis and life cycle assessment of emerging technologies with temporal resolution
(Colorado State University. Libraries, 2020) Sproul, Evan, author; Quinn, Jason C., advisor; Marchese, Anthony J., committee member; Jathar, Shantanu H., committee member; Denning, A. Scott, committee member
Techno-economic analysis (TEA) and life cycle assessment (LCA) are analytical tools used to quantify the economic and environmental performance of emerging technologies. TEA and LCA help guide the development of these technologies by identifying areas where additional research will significantly reduce economic costs and environmental impacts. Although often used in tandem, TEA and LCA output separate results that rely upon disconnected metrics. When considering the impact of time, the disconnect between TEA and LCA methods is critical and can significantly impact results. In this dissertation, three phases of research are conducted to illustrate and reconcile the disconnect between TEA and LCA. In the first phase, standard TEA and LCA methods are used to evaluate the economic and environmental performance of natural rubber derived from guayule (Parthenium argentatum). This evaluation is used to identify the strengths and weaknesses of interpreting disconnected TEA and LCA results. In the second phase, two new methods are created to overcome this disconnect by integrating temporally resolved TEA and LCA. These methods are applied to electric power and guayule rubber production to highlight the impacts of integrating temporally resolved TEA and LCA. In the third phase, integrated TEA and LCA is used to perform a deep-dive evaluation on low-emissions technology options for natural gas combined cycle power plants. In this phase, TEA and LCA with temporal resolution are used to identify cost targets for biomethane, carbon capture and storage (CCS), and bioenergy with CCS (BECCS) under different emissions pricing scenarios. Taken together, the three phases of research in this dissertation represent a wide range of applications and methodologies, each with varying objectives and complexity. Understanding the details of these approaches will help guide future analysis where economic costs, environmental impacts, and time are important considerations in technological development.
Open Access
Cloud property retrievals using polarimetric radar: untangling signals of pristine ice and snow
(Colorado State University. Libraries, 2020) Kedzuf, Nicholas J., author; Chiu, J. Christine, advisor; van Leeuwen, Peter Jan, committee member; DeMott, Paul, committee member; Chandrasekaran, V., committee member
Ice and mixed phase clouds are critical components of Earth's climate system via their strong controls on global precipitation distribution and radiation budget. Their microphysical properties have been characterized commonly by polarimetric radar measurements. However, there remains a lack of robust estimates of ice number concentration, due to the difficulty in distinguishing embedded pristine ice from snow aggregates in remote sensing observations. This hinders our ability to study detailed cloud ice microphysical processes from observations. This thesis presents a rigorous method that separates the scattering signals of pristine ice and snow aggregates in scanning polarimetric radar observations to retrieve their respective abundances and sizes for the first time. This method, dubbed ENCORE-ICE, is built on an iterative ensemble retrieval framework. It provides number concentration, median volume diameter, and ice water content of pristine ice and snow aggregates with full error statistics. The retrieved cloud properties are evaluated against in-situ aircraft measurements from a UK field campaign. For a stratiform cloud system with embedded convective features associated with observed ice number concentration of 0.1–10 L–1 and ice water content from 0.01–0.6 g m–3, the retrievals are mainly in the range of 1.0 –15 L–1 and 0.003–0.6 g m–3. To investigate the ice property evolution in a Lagrangian sense, the retrieval method is also applied to along-wind scanning radar measurements from an Atmospheric Radiation Measurement (ARM) campaign in Finland. For the cases presented, snow aggregates are typically of 5–10 mm size in diameter, which is ~10 times larger than pristine ice and thus dominates radar reflectivity. However, the partitioning in ice water content between pristine ice and aggregates varies and largely depends on ice number concentration. More importantly, the retrieved pristine ice number concentration exceeds the predicted concentration of primary ice nuclei at a mid-cloud temperature of –15°C by two orders of magnitude, suggesting possible secondary ice production, one of the outstanding issues in cloud physics. This highlights the potential of using ENCORE-ICE to identify secondary ice production events and understand their trigger mechanisms.
Open Access
The microtubule-associated protein She1 regulates dynein-mediated spindle positioning in budding yeast
(Colorado State University. Libraries, 2020) Ecklund, Kari, author; Markus, Steven, advisor; DeLuca, Jennifer, committee member; Peersen, Olve, committee member; Krapf, Diego, committee member
Microtubules are polar filamentous proteins part of a complex cytoskeletal network within cells that provides an organized interface with which motors use to transport vesicular cargoes and organelles, and mediate positioning of the mitotic spindle during cell division. There are two groups of molecular motor proteins that use microtubules as a track: (1) kinesins, the predominant anterograde motors and which are represented by six distinct different motors in budding yeast and (2) dynein, the predominant retrograde motor to which there is only one, cytoplasmic dynein, in budding yeast. Regulation of motor proteins is paramount to ensure that these various functions are achieved efficiently in a time and space-sensitive manner. There are many ways microtubules regulate their track, including through a class of highly diverse proteins called microtubule-associated proteins (MAPs), one of which in budding yeast is She1. In budding yeast, the only currently known role of cytoplasmic dynein is positioning the mitotic spindle during cell division. To direct the polarized movement of the spindle towards the daughter-cell, dynein relies on the MAP She1. To understand the mechanism by which She1 may regulate dynein-mediated spindle positioning, we first characterized the effects of She1 on dynein motility using recombinant protein. Our results demonstrated that She1 affects dynein motility by enhancing dynein-microtubule binding through simultaneous interactions with the dynein microtubule binding domain (MTBD) and the microtubule. From our in vitro data, we suggested a model where She1 assists dynein force generation to pull the large nucleus into the narrower bud neck connecting mother and daughter cells. However, we tested this model in vivo and found no such effects on nuclear translocation success, leaving us to investigate an alternative model where She1 polarizes spindle movements towards the daughter cell through inhibiting dynein activity in the mother cell. We explored this model in vivo using a comprehensive analysis of dynein-mediated spindle movements which revealed She1 ensures dynein in the daughter cell maintains bud neck proximity by inhibiting dynein activity and the initiation of dynein-mediated spindle movements in the mother cell. Moreover, we find that this process depends on She1 binding to aMTs in the mother cell and not spindle microtubules where She1 also localizes. Finally, we provide evidence that She1 requires the MTBD of dynein for some aspects of this inhibition, reconciling, in part, our in vitro and in vivo data. Our data provides a fascinating new mechanism of regulation by a MAP and suggests a new angle to approach future exploration of MAP-mediated regulation in higher eukaryotes.
Open Access
False Bakken' interval- sediment patterns and depositional architecture at the facies boundary between siliciclastic mudstones and carbonates, Lodgepole Formation, Mississippian in the Williston Basin, ND
(Colorado State University. Libraries, 2020) Spansel, Joel, author; Egenhoff, Sven, advisor; Sutton, Sally, committee member; von Fischer, Joe, committee member
The lateral facies transition on deep shelves between carbonates and siliciclastic mudstones is largely enigmatic. Based on detailed facies descriptions and interpretations, this study explores which processes have shaped the sedimentary rocks on both sides of this lithological divide, and adds to our understanding of processes operating on deep shelves in general. Both siliciclastic and carbonate rocks of the 'False Bakken' and 'Scallion' intervals of the lower Lodgepole Formation in the Williston Basin, ND, can be grouped into twelve facies: these facies are graded argillaceous mudstone (F1), massive siliciclastic-argillaceous mudstone (F2a), massive calcareous-argillaceous mudstone (F2b), bioturbated pyrtitized bioclast-bearing mudstone (F3), lenticular mudstone (F4), bioclast-rich wavy mudstone (F5), siliciclastic siltstone (F6), glauconitic siltstone (F7), calcareous siltstone (F8), massive to bioturbated carbonate mudstone (F9), nodular skeletal wackestone (F10), and laminated skeletal packstone (F11). These facies are here presented in order of increasing grain size, carbonate content, and bioturbation from F1 to F11. They are arranged in three fining- and coarsening-upward units that can be identified throughout the basin within the succession. These twelve facies are interpreted to represent distinct processes on a low-inclined shelf system with carbonate occupying the proximal, and siliciclastic mudstones the distal portions of this transect. An overall decrease in energy is reflected from the proximal carbonate to distal siliciclastic facies in this sedimentary system. Nevertheless, most of the mudstone facies still reflect high energy processes operating within the distal portions of the basin; in fact, only one mudstone facies is interpreted to reflect suspension settling under tranquil conditions. Therefore, this study suggests that storm wave base is best placed within the distal siliciclastic mudstones instead of in the proximal carbonates. Carbonate mudstones, deposited above storm wave base but lacking tempestite deposition are therefore interpreted as having been subject to intense degradation of storm-derived bioclasts. A decrease in oxygen concentration is inferred from proximal carbonates to distal siliciclastics as indicated by the decrease in size and type of burrows; yet, the presence of burrows within the most distal facies belt indicates that at least dysoxic conditions prevailed throughout the Williston Basin during the deposition of the 'False Bakken'. Three transgressions and regressions are identified within this succession based on laterally correlated facies patterns and indicate an overall increase in sea level from the beginning to the end of 'False Bakken' times. Sediment starvation occurred in the northeastern and/or southwestern portions of the basin as indicated by the presence of glauconitic siltstones and/or lenticular mudstones at various locations within the succession. However, a source of sediment input is interpreted to be located in the northwestern part of the basin based on a high abundance of detrital silt. In addition, a shift in the basin depocenter southwards from Bakken to lower Lodgepole times is reflected in this succession most likely mirroring an increase in subsidence south of Mountrail County during 'False Bakken' deposition.
Open Access
Vulnerability of a coastal industrial community to sea level rise, hurricanes, and climate change
(Colorado State University. Libraries, 2020) Abdelhafez, Mohamed, author; Mahmoud, Hussam, advisor; Ellingwood, Bruce R., advisor; Arneson, Erin, committee member
Approximately 10% of the world's population live and work in low-lying coastal regions that are less than 10 meters above sea level, and this percentage is likely to increase during the remainder of the 21st century. Along with this growth is the potential for increasing economic losses due to hurricanes, storm surge and sea-level rise (SLR) in an era of climate change. More than 80% of global commodities are traded by sea. In the United States ports contributed $5.4 trillion to the U.S. economy, or approximately 26% of the GDP, and provided employment, either directly or indirectly, to over 30 million individuals. In this study, the potential impact of SLR on coastal communities are reviewed and critically appraised. A new model for quantifying the functionality of the seaports subject to various hurricanes, storm surge and sea level rise due to a changing climate is implemented in a fault tree analysis. A hydrodynamic analysis to enable the impact of tropical cyclones and SLR on port facilities is validated using data from Hurricane Katrina. A series of plausible hazard scenarios is identified and their impact on the Port of Mobile, AL is assessed. The key findings show that if a Katrina-like hurricane were to occur late in the 21st Century, the damages to the Port of Mobile, AL would be increased by 5.5 times and 6.5 times by SLR under RCP 4.5 and RCP 8.5 scenarios when compared to the damages caused by historical Hurricane Katrina alone. Furthermore, the immediate post-disaster functionality of the port under the RCP 8.5 scenario would be reduced to virtually zero.
Open Access
Systems of uncertainty: acting and undergoing
(Colorado State University. Libraries, 2020) Faherty, Lauren, author; Faris, Suzanne, advisor; Harrow, Del, committee member; Moore, Emily, committee member; Kissell, Kevin, committee member
For most of my life, I have sought to understand how systems within the body function and engage with one another — how a healthy and organized structure can undergo rapid deterioration stemming from networks failing to communicate properly. The body is supported by an abundance of systems that are introduced to aging, disease and other biological effects throughout our lifespan. The transformation that takes place in the physical self when introduced to a biological disruption is the basis of my body of work Acting and Undergoing. The confrontation of my body's mortality was spurred by my family's genetic predisposition to autoimmune diseases. The organized structure of systems in our bodies lacks the security or stability many people enjoy. In my sculpture, Acting/Undergoing, thin, precarious wood structures work to support plush fabric pieces that are actively overtaken by black forms. Viewers looking at my unpredictable structures are confronted with their own bodily relationships — as one that is intimately familiar yet shrouded by the unknown.
Open Access
Studying age-related changes in white matter microstructure in healthy aging using noninvasive MRI techniques
(Colorado State University. Libraries, 2020) Mendez Colmenares, Andrea, author; Thomas, Michael L., advisor; Burzynska, Agnieszka Z., advisor; Rojas, Donald C., committee member
Age-related deterioration of the white matter (WM), such as demyelination, is an important mechanism of cognitive decline in healthy aging. Lifestyle factors can influence the course of WM aging. Most evidence have used diffusion tensor imaging (DTI) metrics, but these are not specific to myelin or axons. Therefore, in this study we compared DTI metrics to a proposed proxy of myelin content, the T1-weighted image (T1-WI) to T2-weighted image (T2-WI) ratio with respect to their ability to: detect time-by-intervention interactions, predict processing speed ability, and their correlations with each other and age. We used longitudinal data from 169 cognitively healthy older adults (60-79yrs). MRI imaging (3T Siemens Trio) included 0.9mm3 MPRAGE, 1.7×1.7x3mm3 T2w and DTI (30 diff. dir., bval= 0 and 1000s/mm2, 1.7×1.7x3mm3). T1w/T2w was calculated using internal intensity calibration. We used FSL-FDT to extract DTI metrics, focused on major WM tracts using tract-based spatial statistics in FSL. From the WM skeleton, we calculated mean values for 12 regions-of-interest. Processing speed was assessed using the Virginia Cognitive Aging Battery. Results showed that the T1w/T2w produced greater time-by-intervention interactions than DTI-FA, especially in the posterior (β=0.27, p=0.01) and anterior (β=0.33, p=0.01) limb of the internal capsule. The T1w/T2w (in the whole WM) correlated with processing speed (β=-0.13, p=0.02). T1w/T2w correlated with DTI in regions with high fiber coherence/high myelin content; and with age in regions with high myelin content. Results suggest that the T1w/T2w offers greater ability than DTI to detect short-term longitudinal changes in WM, but they seem to reflect different microstructural properties in the WM. Further research is needed to gain a better understanding of its biological underpinnings and significance.
Open Access
Characterizing the role of the Hec1 tail domain at the kinetochore-microtubule interface in human cells
(Colorado State University. Libraries, 2020) Wimbish, Robert T., author; DeLuca, Jennifer, advisor; Markus, Steven, committee member; Reddy, Anireddy, committee member; Ross, Eric, committee member
Chromosome segregation is powered by interactions between the mitotic spindle and kinetochores. Kinetochores – large, protein-rich machines built on the centromere of each sister chromatid – must bind to spindle microtubules and harness the forces from their dynamic instability to drive chromosome movement. This interaction must be robust enough to ensure chromosomes remain bound to the growing and shrinking microtubule polymers, yet must also be reversible: incorrectly oriented kinetochore-microtubule attachments can cause chromosome mis-segregation leading to aneuploidy, which can be catastrophic for the newly formed cell. Thus, cells must be able to actively regulate the strength with which kinetochores bind to spindle microtubules – such a regulatory scheme ensures that incorrect attachments can be released, and correct attachments can be preferentially stabilized. The direct linkage between kinetochores and microtubules is the highly conserved, kinetochore-anchored NDC80 complex. This complex is also an effector of attachment strength regulation; specifically, the N-terminal "tail" region of the NDC80 complex subunit Highly expressed in cancer 1 (Hec1) is a target for phosphorylation by the Aurora family of kinases, which ultimately weakens kinetochore-microtubule attachments. Here, we investigate the molecular basis for kinetochore-microtubule attachment regulation in human cells. We find that Hec1 tail phosphorylation regulates kinetochore-microtubule attachments independently of the spindle and kinetochore associated (Ska) complex, a critical factor for attachment stability, contrary to previous reports that the two pathways are functionally coupled. We additionally map the domains of the NDC80 complex required for its coordination with Ska complexes to strengthen attachments. We also find that the Hec1 tail domain is dispensable for the initial formation of kinetochore-microtubule attachments, but provide evidence it plays a role in force generation. We further interrogate this role and how phosphorylation of the tail regulates attachment formation and force generation, and find that the length requirements for these functions of the tail are different. Moreover, we demonstrate that the phospho-regulatory pathway for attachment regulation is deficient for short tails, suggesting a new model for the means by which attachments are regulated. Together these results provide novel insight into how attachments between chromosomes and the spindle are formed and regulated, and how errors in this process can lead to chromosome mis-segregation.
Open Access
Allostery of the flavivirus NS3 helicase and bacterial IGPS studied with molecular dynamics simulations
(Colorado State University. Libraries, 2020) Davidson, Russell Bruce, author; McCullagh, Martin, advisor; Bernstein, Elliot, committee member; Barisas, George, committee member; Geiss, Brian, committee member
Allostery is a biochemical phenomenon where the binding of a molecule at one site in a biological macromolecule (e.g. a protein) results in a perturbation of activity or function at another distinct active site in the macromolecule's structure. Allosteric mechanisms are seen throughout biology and play important functions during cell signaling, enzyme activation, and metabolism regulation as well as genome transcription and replication processes. Biochemical studies have identified allosteric effects for numerous proteins, yet our understanding of the molecular mechanisms underlying allostery is still lacking. Molecular-level insights obtained from all-atom molecular dynamics simulations can drive our understanding and further experimentation on the allosteric mechanisms at play in a protein. This dissertation reports three such studies of allostery using molecular dynamics simulations in conjunction with other methods. Specifically, the first chapter introduces allostery and how computational simulation of proteins can provide insight into the mechanisms of allosteric enzymes. The second and third chapters are foundational studies of the flavivirus non-structural 3 (NS3) helicase. This enzyme hydrolyzes nucleoside triphosphate molecules to power the translocation of the enzyme along single-stranded RNA as well as the unwinding of double-stranded RNA; both the hydrolysis and helicase functions (translocation and unwinding) have allosteric mechanisms where the hydrolysis active site's ligand affects the protein-RNA interactions and bound RNA enhances the hydrolysis activity. Specifically, a bound RNA oligomer is seen to affect the behavior and positioning of waters within the hydrolysis active site, which is hypothesized to originate, in part, from the RNA-dependent conformational states of the RNA-binding loop. Additionally, the substrate states of the NTP hydrolysis reaction cycle are seen to affect protein-RNA interactions, which is hypothesized to drive unidirectional translocation of the enzyme along the RNA polymer. Finally, chapter four introduces a novel method to study the biophysical coupling between two active sites in a protein. The short-ranged residue-residue interactions within the protein's three dimensional structure are used to identify paths that connect the two active sites. This method is used to highlight the paths and residue-residue interactions that are important to the allosteric enhancement observed for the Thermatoga maritima imidazole glycerol phosphate synthase (IGPS) protein. Results from this new quantitative analysis have provided novel insights into the allosteric paths of IGPS. For both the NS3 and IGPS proteins, results presented in this dissertation have highlighted structural regions that may be targeted for small-molecule inhibition or mutagenesis studies. Towards this end, the future studies of both allosteric proteins as well as broader impacts of the presented research are discussed in the final chapter.
Open Access
Modeling the evolution of SIV progenitor viruses towards HIV-1 and HIV-2 in a humanized mouse surrogate model
(Colorado State University. Libraries, 2020) Curlin, James Zachary, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Stenglein, Mark, committee member; Wiese, Claudia, committee member
Human Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2), the causative agents of Acquired Immunodeficiency Syndrome (AIDS) first emerged in humans over the past century. Despite significant advances in treatment options, the pandemics continue with millions of new infections every year. Both HIV-1 and HIV-2 likely emerged through the cross-species transmission of primate lentiviruses originating from nonhuman primates (NHPs) including chimpanzees (SIVcpz), gorillas (SIVgor), and sooty mangabeys (SIVsm). SIVsm shares a remarkable degree of homology with HIV-2, while SIVcpz and SIVgor are most closely related to HIV-1. Nonhuman primates infected with these lentiviruses frequently come into contact with humans due to the prevalence of bushmeat hunting practices in various African countries. Other lentiviruses such as SIVmac239 represent independent instances of primate lentiviruses crossing into novel host species. The repeated exposure of primate lentiviruses to a human immune environment allowed the accumulation of adaptive genetic changes uniquely suited to overcoming the evolutionary pressures of a new host. Host-restriction factors such as tetherin, SAMHD1, APOBEC3G and SERINC3/5 exert species-specific antiviral activity and must be overcome for a virus to adapt to a new host cell. These evolutionary pressures could be a guiding force in the direction that these viruses adapt. In order to recapitulate these genomic cross-species adaptations, we used humanized mice engrafted with human hematopoietic stem cells (hu-HSC mice). These mice produce a full spectrum of human immune cells such as B cells, T cells, macrophages, monocytes, and dendritic cells, and are susceptible to HIV infection. Representative progenitor viruses of both HIV-1 (SIVcpzEK505, SIVcpzMB897, and SIVcpzLB715) and HIV-2 (SIVsmE041) as well as other viruses of interest, namely, SIVmac239, SIVhu and SIVB670 lineages were intraperitoneally injected into hu-HSC mice. Following successful infections, the derivative viruses were subsequently passaged serially through multiple generations to simulate the repeated exposures that originally produced HIV-1 and HIV-2. Viral adaptation was assessed primarily through three different criteria. Plasma viral RNA levels were measured on a weekly basis using qRT-PCR to determine changes in viral replication kinetics over time. We found that the plasma viral loads of the viruses tested varied during serial passages, and mostly increased over time in many cases. Human CD4+ T cell engraftment decline as assessed by flow cytometry biweekly acts as a measure of AIDS progression in cases of human infection. CD4+ T cell levels declined over time with increasing rapidity upon further passaging in many cases. Additionally, viral RNA collected from the infected mice at multiple timepoints in each generation was used to generate overlapping amplicons spanning the length of the viral genome in order to be used with Illumina-based deep sequencing. Numerous nonsynonymous mutations arose in the first generation of passaging and were maintained across multiple sequential passages. While the mutations occurred throughout the viral genome, the bulk of the mutations were found in env and nef. Many of these mutations were present in known CD4+ binding sites, motifs involved in protein interactions, and other areas involved in host-restriction factor antagonism. While these results are revealing, further inquiry is needed to determine the true functionality of these genetic changes. These data showcase the value of using humanized mice to model lentiviral evolution and provide important insights into understanding the origin of HIVs.
Open Access
The temporal elements of emotional identification with film characters
(Colorado State University. Libraries, 2020) McCormick, Stephanie, author; Romagni, Domenica, advisor; MacKenzie, Matt, committee member; Snodgrass, Jeffrey, committee member
I argue that the subjective experience of time passing, felt duration, is a crucial element in the emotional and immersive experience of narrative films. First, I review multiple theories of emotional identification to conclude that the most accurate and thorough account is provided by the simulation theory of emotions. Growing from this account, I establish a framework of emotional engagement (general and emotional identification) and immersion (emotional and temporal). Film theories about emotional engagement often overlook felt duration despite it being a feature of emotional experiences. A film's depiction of a character's felt duration facilitates the audience's emotional engagement and immersion. Additionally, the audience's felt duration can be manipulated by the film's pacing techniques to further engagement and immersion. There are two main upshots of my thesis I will briefly outline in the last chapter: aesthetic value and ethical value. The emotional and temporal experiences of the audience are vital to the understanding of narratives and the experience of films. Emotional identification exercises our capacity to relate to other people. This affects our ability to empathize and treat other people. In this thesis, I draw attention to felt duration as an element of emotional engagement and immersion that often goes unacknowledged.
Open Access
Transfer learning with weather radar
(Colorado State University. Libraries, 2020) Gooch, S. Ryan, author; Chandrasekar, V., advisor; Cheney, Margaret, committee member; Chavéz, José, committee member; Suryanarayanan, Sid, committee member
This work presents the culmination of the doctoral research by the author in exploring modern methods of Data Discovery in weather radar data, improvements in the cyberinfrastructure concerning multi-dimensional gridded data, with a concentration on real-time data streaming, and experimental use cases involving real world datasets. Included in this work is a successful method for the classification of weather radar image data using convolutional neural networks, with inspiration drawn from the subfield of Transfer Learning in the Computer Vision community. Once this model was developed, it was deployed on single radar data from each of the radars in the CASA DFW network to assign labels to support a human-in-the-loop semi-supervised method for data discovery in the weather radar scans. This model has been furthermore applied to the WSR-88D network of dual-polarimetric weather radars in the United States to demonstrate the model's generalizability, and its utility in discovering phenomena of interest in vast datasets. This work discusses the end-to-end development of the data discovery system, with special focus on initial data labeling, choices and tradeos in model architecture, and training concerns in the machine learning model. This represents the rst published research known to the authors on utilizing the power of transfer learning to transfer the learning of high quality convolutional neural networks trained on photographic images to the weather radar image domain.
Open Access
RNA sequencing identifies genes putatively involved at the aphid-Buchnera symbiotic interface
(Colorado State University. Libraries, 2020) Acharya, Shailesh Raj, author; Nalam, Vamsi J., advisor; Leach, Jan, committee member; Sloan, Daniel, committee member
In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium, Buchnera aphidicola. The endosymbiont converts abundant non-essential amino acids into essential amino acids that are supplied to the aphid. The long-term goal of the proposed work is to exploit the biochemical interdependence that exists between soybean aphid (Aphis glycines) and its primary endosymbiont to develop effective resistance in soybean (Glycine max). Little is known of the A. glycines and soybean amino acid transporters (AATs) that facilitate this exchange. The soybean aphid is the most important arthropod pest on soybean in North America and aphid outbreaks in major soybean growing regions of the country in the past has resulted in yield losses of up to 40%. In the current study, we used RNA-seq to identify amino acid transporters involved in the exchange of amino acids between the aphid and its endosymbiont. A total of 2121 genes were differentially expressed between the aphid and bacteriocytes with 516 genes showing up-regulation, while 1605 genes were down-regulated in the bacteriocytes. Analysis of GO terms revealed enrichment in membrane and transport associated processes. Our RNA-seq analysis of differentially expressed genes showed that one putative amino acid transporter: 72-RA, is up-regulated in the bacteriocytes. This work represents a first step towards understanding aphid dependency on its endosymbiotic bacteria and target them as a means of a novel aphid control strategy.
Open Access
Characterizing the pine wilt disease pathosystem in the Front Range Region of Colorado
(Colorado State University. Libraries, 2020) Atkins, David, author; Davis, Seth, advisor; Stewart, Jane, advisor; Wall, Diana, committee member; Tinkham, Wade, committee member
Pine wilt disease, caused by the pinewood nematode (Bursaphelenchus xylophilus; PWN), is one of the most damaging invasive species in Asia. Tree mortality associated with PWN has recently been reported in Colorado and surrounding states. There remains little documentation on PWN incidence in native pine species or the biology of potential vectors in the Rocky Mountain region. Here we regionally surveyed for PWN in host trees and two putative insect vectors (Monochamus clamator & M. scutellatus) for two years to develop vector flight phenology models and test the hypothesis that disturbance factors predict vector abundance across the landscape. Flight phenology was similar between vectors: flight initiated in mid-July and continued into October for both species. PWN was distributed throughout the Front Range at rates lower than those reported in the putative native range (Host: 3.6%; Vector 4.2%). Infection rate in hosts varied among sites (0-89%), and four 'epicenters' of vector infectivity were identified. We also report the first incidence of PWN-M. clamator association in the U.S. The four identified epicenter sites varied in the timing of anomalous infection frequency, and flight phenology of infective vectors differed between epicenter and peripheral sites. Monochamus populations were found primarily in natural forest areas and seasonally migrate in small numbers to urban areas. Landscape factors such as proximity to burned area were positively correlated with Monochamus abundance. Synthesis and applications: Our study describes PWN infection frequency to be greater than that expected of a newly introduced pathogen, but lesser than the eastern United States and Canada where PWN is known to be established. Our findings provide tools that can predict exposure windows of disease exposure, which were observed to be highest in the early season in Colorado. We also describe the threat that populations of PWN in wildland forests pose to urban landscapes, and how this risk varies seasonally. These findings collectively serve characterize PWN distribution in the native ecosystem and provide tools that can be used by decision-makers and managers to proactively manage the spread of pine wilt disease.
Open Access
The effect of resins on the aggregation behavior of asphaltenes
(Colorado State University. Libraries, 2020) Derakhshani Molayousefi, Mortaza, author; McCullagh, Martin, advisor; Szamel, Grzegorz, committee member; Van Orden, Alan, committee member; Ettema, Robert, committee member
Millions of barrels of crude oil are extracted on a daily basis. Crude oil has four main components separated by the SARA fractionation method.1 Asphaltenes are the heaviest component of the cured oil. They are known to be responsible for clogging oil wellbores and pipelines, which bedevils the oil industry financially. Additionally, the cleaning chemicals and the clogging waste has a huge negative impact on our environment. The majority of the research on understanding the clogging problem is focused on the asphaltenes as a fraction of crude oil without much consideration for the effects of specific chemical structure. Moreover, the role of other components of the crude oil such as resins is not clear. Here, we have performed structure specific studies of asphaltenes by performing all-atom molecular dynamics (MD) simulations to quantify the aggregation behavior of asphaltenes in the absence and presence of resins. In this research, we have studied the aggregation tendency of asphaltenes in connection with their molecular properties. Systems with 20 counts of model asphaltene molecules were studied for nanoaggregation behavior of eight model asphaltenes in their neat state. We have quantified the aggregation tendency of asphaltene molecules in n-heptane with isodesmic free energy of aggregation, ∆Giso, as well as a quantity called aggregation propensity (AP). Using ∆Giso and AP value, we have classified model asphaltene molecules to three main category of non-aggregating, mildly-aggregating, and readily-aggregating asphaltenes. Each category of asphaltene have different aggregation behavior. They differ in their molecular features that ultimately is related to their aggregation propensity. Subsequently, we have studied the aggregation tendency of asphaltene in the presence of resin with total of 48 systems comprising 8 model asphaltene molecules in the presence of 6 model resins. We wanted to determine the role of resins in the aggregation behavior of asphaltenes by observing the effect of presence of resin on the ∆Giso and AP values. Additional to ∆Giso, we have defined a normalized quantity called aggregation propensity ratio (APR) to compare the effect of resin on the aggregation of asphaltenes. Resins studied in this work had no promoting effect on the aggregation tendency of asphaltenes. In general, both ∆Giso and APR metrics suggest that aggregation of asphaltene in presence of resin is either not affected or is prevented to different degrees. We have studied the aggregation behavior of asphaltenes in nanoaggregation, clustering and flocculation stages proposed by Yen-Mullins model. Resins have from minimal disruptive to highly disruptive effect on the nanoaggregation of asphaltenes. We investigated the further aggregation of stable nanoaggregates into clustering and flocculation with 500 counts of mildly-aggregating and readily-aggregating asphaltene molecules. We found that both clustering and flocculation stages occur for the readily-aggregating asphaltenes and do not occur for the mildly-aggregating asphaltenes. Readily-aggregating asphaltene molecules with large negative ∆Giso and large AP values lead to clustering and flocculation whereas the mildly-aggregating asphaltenes stay in the form of nanoaggregates. Our results show that in order for asphaltenes to flocculate, there is a threshold for existence of adequate favorable molecular features. Asphaltenes containing large enough aromatic cores and/or heteroatom reach clustering and flocculation stages. Furthermore, we found that in the presence of a highly disruptive resin, clustering and flocculation does not occur. For the readily-aggregating asphaltenes the aggregation stops in the nanoaggregation stage and for the mildly-aggregating asphaltenes the size of the nanoaggregates decreases. Our results explain what kind of resins are capable of potentially solving the deposition problem with providing insight on the molecular features of both asphaltene and resin molecules. Such molecular insights paves the road to explore more natural based solutions in preventing the clogging problem in the oil industry by informed characterization of each oil reservoir and its capability to form aggregate or prevent aggregates within itself and in another reservoir.
Open Access
Electrophysiological analysis of Kv2 channel regulation by non-canonical and canonical mechanisms
(Colorado State University. Libraries, 2020) Maverick, Emily E., author; Tamkun, Michael, advisor; Amberg, Gregory, committee member; Krapf, Diego, committee member; Tsunoda, Susan, committee member; Vigh, Jozsef, committee member
Kv2 channels are the most abundant voltage-gated potassium channels in the mammalian nervous system and entire body. These channels regulate action potential ﬁring and apoptosis via their canonical conducting functions. However, Kv2 channels also play a non-conducting role in the cells in which they are expressed. Speciﬁcally, they form junctions between the endoplasmic reticulum and plasma membranes, and these junctions regulate a myriad of cellular process. Several studies have now shown that many Kv2.1 channels expressed on the plasma membranes of mammalian cells do not respond canonically to changes in membrane voltage. Instead of opening to allow potassium efﬂux, the pores of these non-canonical channels are locked in a non-conducting state. This state has likely evolved to prevent electrical paralysis that would otherwise be conferred upon cells expressing high levels of completely functional Kv2 channels. The mechanism bringing about the non-conducting state of Kv2.1 channels is unknown. The work described in the ﬁrst part of this dissertation was carried out with the ultimate goal of revealing the mechanism of the Kv2.1 channel non-conducting state. I describe an improved, all-electrophysiological method to quantify the numbers of nonconducting Kv channels expressed in heterologous systems. I validate this approach by measuring the fraction of non-conducting Kv2.1 channels that arise when expressed in HEK293 cells. I go on to use this approach to show evidence for a non-conducting state in the second Kv2 isoform, Kv2.2, for the ﬁrst time. I ﬁnd that like Kv2.1, the Kv2.2 nonconducting state is dependent on the density of channels in the membrane. Surprisingly, I also ﬁnd that two Shaker-related channels, Kv1.4 and Kv1.5 also show density dependence in the fraction of channels that conduct. These results suggest that the mechanism underlying the non-conducting state is more common than we thought, and I discuss hypotheses that should be tested in the future. In the last part of this dissertation I describe the effects of the assembly of Kv2 channels with a newly discovered family of Kv β subunits, the AMIGOs. The experiments in this portion of the dissertation focus on each AMIGO's ability to modulate canonical, conducting Kv2 channels, as well as Kv2's ability to alter AMIGO trafﬁcking and localization. I ﬁnd that both Kv2.1 and Kv2.2 promote AMIGO trafﬁcking to the plasma membrane and alter their localization there. I also ﬁnd that while all three AMIGO isoforms promote Kv2 channel opening, AMIGO2 confers an additional stabilizing effect on the open state by slowing inactivation and deactivation. In all, the work in this dissertation expands on our current understanding of Kv channel function. These ﬁndings should guide future experiments to probe both canonical and non-canonical functions of Kv channels.