2020-

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https://hdl.handle.net/10217/182111

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Open Access
Design and quantification of a tissue type specific genetic circuit in plants
(Colorado State University. Libraries, 2020) Oehmke, Sara, author; Medford, June, advisor; Argueso, Cris, committee member; Nishimura, Marc, committee member
Synthetic biologists aim to rationally design genetic circuits and utilize plant platforms to photosynthetically drive, self-sustainable circuits. Although plants are excellent platforms, issues and unpredictability arise from the innate complexity of multicellularity. The ability to quantitatively control gene expression within specific cell types can address some issues arising from multicellularity. In my research, I developed a genetic circuit with the ability to induce and quantitatively control expression in Arabidopsis thaliana root epidermal cells. The circuit design uses an externally applied ligand that activates a computationally designed transcriptional response driven by a tissue specific promoter to control output (GFP expression). In addition, I engineered a circuit that adds a positive feedback motif. To quantify the behaviors of these circuits I developed a MATLAB program to remove background signals from confocal microscopy images and quantify GFP signal in a high-throughput manner. The genetic circuit is highly specific for root epidermal cells, controllable with external ligand, and has increased sensitivity and memory with positive feedback. The concepts and components of these circuits can be implemented in future designs to engineer and produce plants with more predictable and diverse behaviors affording the operator greater control.
Open Access
Mechanisms and management for soil carbon sequestration
(Colorado State University. Libraries, 2020) Mosier, Samantha, author; Cotrufo, M. Francesca, advisor; Paustian, Keith, advisor; Davies, Christian, committee member; Denef, Karolien, committee member
Soil organic matter (SOM) holds more carbon (C) than the atmosphere and terrestrial aboveground biomass combined. SOM also provides many other co-benefits in the form of ecosystem services. The rate at which we lose or sequester more C in soils is of great importance for mitigating the rising atmospheric greenhouse gas concentrations as well as for maintaining the fundamental services that soils provide to humanity. Many of the mechanisms involved in accruing and storing soil C are not entirely clear, and factors like litter chemistry and minerology can all come into play when determining the sequestration potential of a specific ecosystem. Additionally, not all soil C is equal in its turnover time or in its ability to resist disturbance. Therefore it is crucial that we better understand how functionally different soil C pools form and persist in the soil environment. Several "climate smart" soil management practices have been analyzed for their potential to sequester more C. However there are still gaps in our knowledge regarding soil C sequestration and how it can be impacted by land use management. The southeast US is a region with particularly severe soil degradation from poor agricultural management, but also has a high potential for increased soil C sequestration and overall soil health. This dissertation looks at some potential mechanisms and management practices involved with storing more stable soil C in the southeastern US. Mechanisms include how litter chemistry and soil C saturation can enhance or inhibit soil C sequestration. Then, we evaluated management practices from pine plantations and grassland grazing in the southeastern US to evaluate if improved management could increase soil C stocks, their distribution, and overall soil health.
Open Access
From fields to genomes: towards a comprehensive understanding of the lifestyle and evolution of Claviceps purpurea the ergot fungus
(Colorado State University. Libraries, 2020) Wyka, Stephen Andrew, author; Nalam, Vamsi, advisor; Broders, Kirk, advisor; Charkowski, Amy, committee member; Pearce, Stephen, committee member; Jahn, Courtney, committee member
Claviceps purpurea (ergot), an ascomycete and member of the family Clavicipitacea, is considered a pathogen of all grass species (family Poaecea) including economically important cereal crops which infects ovaries resulting in the development of a fungal sclerotium rather than a plant seed. Ergot infections poses significant impacts to agriculture and livestock due to various toxic alkaloids present in the sclerotia. Severe ergot poisoning in humans and livestock, ergotism, can cause corrosion/loss of extremities from gangrene, internal bleeding, diarrhea, and reduced pregnancy and abortion. Due to these serious health concerns, strict restrictions are placed on the amount of ergot contaminated grain that can be accepted for food and livestock feed. However, these toxic alkaloids are also heavily researched in the field of pharmacology and have been shown to provide some beneficial aspects in human medicine. Despite the abundance of pharmacological and agricultural research on C. purpurea researchers have been unsuccessful in identifying crop or wild grass varieties that have resistance to ergot infection, leading to critical challenges in the control of ergot disease outbreaks. Recent studies have also suggested that C. purpurea is more of a conditional defensive mutualist as opposed to a plant pathogen. Taken together, these factors demonstrate that there are still gaps of knowledge surrounding the epidemiology, lifestyle, evolution, and adaptability of this species. We implemented a comprehensive analysis into the life history of C. purpurea through a combination of field surveys, greenhouse inoculations, and deep genomic data mining to help elucidate these gaps. Field surveys were conducted to investigate the role wild grass populations surrounding cereal crop fields play in epidemiology of ergot outbreaks. Results revealed that unmanaged grasses along ditch banks, even in drought years, represent significant inoculum reservoirs of ergot, particularly when Bromus spp. are present, and should be a focal point in future research for better disease control. Greenhouse inoculations were conducted to elucidate the effects of C. purpurea infections on hosts through inoculations of a single isolate on two commercial cereal crops in a controlled setting. Our results show that the effect of C. purpurea infections can range from negative to positive, depending on infection rate, plant species, and plant tissue, but overall showed a general trend of neutral effects. However, we did observe a potential for increased root growth as infection rates increased, which could signify an interesting plant-microbe interaction that imparts a benefit, of infection, on highly rhizomatous grass hosts such as Bromus spp.. Lastly, through a collaborative effort we sequenced, assembled, and annotated 50 Claviceps genomes, representing 21 species, for a comprehensive comparison of genome architecture, plasticity, and evolution within the genera. We also conducted a detailed analysis of C. purpurea through construction of a pangenome and investigations of the recombination and positive selection landscape across the genome. Our genus-wide comparison revealed that despite having nearly identical life-strategies, these closely related species have substantially altered genomic architectures and plasticity that are likely driving genome adaptation. One key difference we observed was a shift from characteristic one-speed genomes in narrow host-range Claviceps species of sections Citrinae and Paspalorum to two-speed genomes in broader host-range lineages of sections Pusillae and Claviceps. Claviceps purpurea was observed to have a large accessory genome that is likely influenced by a large effective population size, high recombination rates, and transposable element (TE) mediated gene duplication. Due to a lack of repeat-point induced (RIP) mutation, prolific TE expansion is likely controlled by high recombination rates, which subsequently may be influencing the overall trend of purifying selection observed within the species. However, secondary metabolites genes were found to have the highest rates of positive selection on codons within genes, indicating that these genes are a primary factor affecting the diversification of the species into new ecological niches and to potentially help maintain its global distribution and broad host range.
Open Access
The flavivirus NS3 helicase Motif V controls unwinding function and alters viral pathogenesis in mosquitoes
(Colorado State University. Libraries, 2020) Du Pont, Kelly Elizabeth, author; McCullagh, Martin, advisor; Geiss, Brian J., advisor; Szamel, Grzegorz, committee member; Snow, Christopher, committee member; Krummel, Amber, committee member; Ho, Shing, committee member
Over half of the world's population is at risk of flavivirus (e.g. dengue virus, West Nile virus, Japanese Encephalitis virus, and Zika virus) infection making it a global health concern. These specific mosquito-borne flaviviruses are responsible for causing a variety of symptoms and outcomes including flu-like fevers, encephalitis, hemorrhagic fevers, microcephaly, Guillain-Barré syndrome, and death. Unfortunately, vaccines and anti-viral therapeutics are not always effective in protecting against and treating viral infections. Sometimes these therapies cause more severe symptoms through an antibody dependent enhancement. Therefore, there is a pressing need for the development of effective anti-viral therapies against each flavivirus. For the advancement of these interventional strategies, a fundamental understanding of how flaviviruses replicate within hosts, including the mosquito vector, is required. This dissertation investigates how flaviviruses regulate viral replication, pathogenesis and mosquito transmission through the nonstructural protein 3 (NS3) helicase structure and function. A combination of virology, biochemistry, and computational simulations will be utilized to address how NS3 plays a role in viral infection, viral replication, and viral protein structure. An essential aspect of flaviviral genome replication is the unwinding of the double-stranded RNA intermediate via the C-terminal helicase domain of NS3. NS3 helicase translocates along and unwinds the double-stranded nucleic acids in an ATP-dependent manner. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well understood. Previous simulations in the group led us to hypothesize that Motif V is a critical component of the transduction mechanism. Here, we tested Motif V mutations in both sub-genomic replicon and recombinant protein systems to examine viral genome replication, helicase unwinding activity, ATP hydrolysis activity, and RNA binding affinity activity. NS3 helicase mutants, T407A and S411A, indicated reduced viral genome replication and increased turnover rates of helicase unwinding activity by a factor of 1.7 and 3.5 respectively. Additionally, we simulated Motif V mutants to probe the structural changes within NS3 helicase caused by the mutations. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. Motif V mutations T407A and S411A exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during viral genome replication. Next, we utilized T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo to probe the how these mutations play a role in pathogenesis and transmission of flaviviruses. Of the two Kunjin virus mutants, only S411A Kunjin virus was recovered. In cell culture, S411A Kunjin decreased viral infection and increased cytopathogenicity as compared to WT Kunjin. Similarly, in surviving Culex quinquefasciatus mosquitoes, S411A Kunjin decreased infection rates as compared to WT Kunjin, but S411A Kunjin infection increased mortality compared with that of WT Kunjin infection. Additionally, S411A Kunjin increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin. These data suggest that S411A Kunjin increases pathogenesis in mosquitoes. Overall, these computational simulation, biochemical assay, and virology data indicate that flavivirus NS3 helicase Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus, not just regulation of translocation and unwinding during viral genome replication. The molecular level insights presented in this dissertation provide the fundamental research for understanding how to target specific regions of NS3 helicase for the advancement of anti-viral therapeutics.
Open Access
The integration of yoga into occupational therapy practice for people with multiple sclerosis
(Colorado State University. Libraries, 2020) Candray, Haylee A., author; Schmid, Arlene A., advisor; Atler, Karen A., committee member; Fling, Brett W., committee member
The purpose of this study was to fill a research gap, by providing initial insight into the use of yoga in occupational therapy practice (OT) for people with multiple sclerosis (PwMS). More specifically, this study aimed to answer how and why occupational therapists (OTs) integrate yoga into clinical practice for PwMS. Eight OTs, residing across the United States, completed an online survey and semi-structured telephone interview. Telephone interviews were transcribed verbatim and inductively open coded. Themes, answering the questions of how and why OTs use yoga for PwMS, emerged through thematic data analysis including: (1) OT and yoga are a natural and complementary fit; (2) holistic benefits for clients beyond therapy; (3) leveraging personal ties to yoga; and (4) use of yoga is dependent on client factors and clinical environment. Since OTs use activities to promote health and well-being, yoga may be appropriate for PwMS because its use is context- and client-centered and allows for shared engagement in a meaningful activity for clients and therapists. Furthermore, as yoga and OT together are a natural and complementary fit, OTs use of yoga may be holistically beneficial to PwMS during and after being discharged from occupational therapy. Future research needs to establish the efficacy of integrating yoga into clinical OT practice as well as qualitatively assess PwMS' experience of engaging in yoga during occupational therapy.
Open Access
Subalpine forest ecosystem responses to long-term nitrogen loading at Loch Vale Watershed, Colorado, USA
(Colorado State University. Libraries, 2020) Weinmann, Tim, author; Boot, Claudia, advisor; Baron, Jill, advisor; Rhoades, Charles, committee member; Covino, Tim, committee member
This thesis presents the results of a long-term N fertilization experiment in the Loch Vale watershed of Rocky Mountain National Park and presents a conceptual model hypothesizing environmental controls on ecosystem responses to N fertilization. The experiment consisted of annual 25 kg N ha-1 application of NH4NO3 fertilizer to three 30 x 30 m subalpine forest plots from 1996 to 2017. Soil, plant, and lysimeter samples were collected over the course of the experiment to address three research goals. First, determine whether increased N input leads simply to increased N output, or whether observable ecosystem effects result. Second, describe changes in the ecosystem responses over time and determine the controls that govern those changes. Third, identify results of the experiment that may generalize to other coniferous forests around the world. The results show that N fertilization causes ecosystem effects in Loch Vale which vary over time and space due to the influence of hypothesized controlling factors such as soil moisture, pH, and plant uptake of N. The results of the Loch Vale experiment suggest that impacts of excess N on boreal forests may include increased plant growth where soil moisture is adequate, but when coupled with drought may lead to increased rates of nitrification, N mineralization, NO3- leaching, and soil acidification.
Open Access
Soil phosphorus availability and transformations following biosolids applications
(Colorado State University. Libraries, 2020) McDaniel, Jacob P., author; Butters, Gregory, advisor; Barbarick, Kenneth, advisor; Davis, Jessica, committee member; Frasier, Marshall, committee member
Land application of biosolids has many benefits such as providing nutrients for plant growth as well as adding organic matter to soil. However, there is the potential for negative impacts due to the high concentration of phosphorus (P) relative to nitrogen (N). Much work has been done looking at the effects of over application of P and best management practices for the application of biosolids in the eastern United States. However, little work has been done in the western United States where the environment is very different due to lower rainfall and higher soil pH and calcium (Ca) concentrations. This study was composed of four parts to help understand how P reacts in the western United States and to look at the implications for plant growth. These parts included: (1) fractionation of P in biosolids, (2) vertical distribution of P following long-term biosolids applications, (3) seasonal soil P cycling, and (4) plant uptake of P. The fractionation of P was designed to look at several biosolids and determine a method to be able to characterize the P that is present. This information would have the potential to aid in decision making about best managing the P from various sources. The vertical distribution of P following long-term application was designed to characterize the P in the soil profile. It is usually assumed that P is not mobile, but over long periods of time and with high application rates it can move. Seasonal changes of the different pools of P were studied to better understand how time of year affects the amount of P available in soil samples, and potentially to plants and the environment. The last study was designed to show how different sources of organic amendments supply different amount of P to a plant. The objective was to be able to better determine the amount of plant available P from an amendment. The results of the different studies begin to show the similarities and differences in the behavior of P in the western United States and in the east. The fractionation study found that the assumptions that are used for manure cannot be applied to all factions of P in biosolids and that a longer shaking time is needed. This study also found that there are differences among wastewater treatment plants that is mostly related to differences in treatment methods in an individual wastewater treatment plant. The vertical distribution of P study found that over time with repeated applications of P in dryland wheat (Triticum aestivum L.)-fallow rotations there is a significant effect on occluded P. Even with repeated applications most of the accumulations of P were limited to the plow layer and in a system where P chemistry is dominated by Ca, Fe still plays an important role. The seasonal change in the fractions of soil P study showed the changes in soil P were greatly affected by soil properties, water availability, climate, and application rates of biosolids. The high concentration of Ca in the soils favored the formation of Ca bound P, and saturated soils affected the forms of Fe bound P. The addition of organic matter and Fe with biosolids applications increased the concentration of soluble P, microbial biomass P, and Fe bound P. The plant uptake study found that there is a much larger effect of the soil on plant uptake than the source of the amendment. There is an inverse relationship between a soil's ability to adsorb P and the concentrations of plant tissue P when the plants are young. As the plants age and develop large root masses they can increase their tissue P concentrations. This work shows that in the western United States there is more of a need to focus on the soils than the biosolids being applied to be able to make the best management decision. All the studies that looked at the P after it had been added to soil found that Ca affects the transformations and uptake of P by plants. In the western United States, there are areas that have large amount of Ca in the soil and areas that do not. As a result, interactions of Ca and P need to be focused on and further studied to ensure adequate plant nutrition as well as being a steward of the environment.
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
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.