Theses and Dissertations

Permanent URI for this collectionhttps://hdl.handle.net/10217/100474

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You will know them by their movement: evaluating pain and mobility behavior in preclinical models of post-traumatic and infection-associated osteoarthritis
(Colorado State University. Libraries, 2025) Kloser, Heidi, author; Santangelo, Kelly, advisor; Henao-Tamayo, Marcela, advisor; Dobos, Karen, committee member; Goodrich, Laurie, committee member
Osteoarthritis (OA) is a progressive, multifactorial joint disease affecting an estimated 595 million people globally and is the leading cause of disability in the United States. Characterized by cartilage degradation, subchondral bone remodeling, and synovial inflammation, OA leads to pain, stiffness, and loss of mobility. These factors further contribute to comorbidities such as cardiovascular disease, metabolic dysfunction, and early mortality. While aging and female sex are primary risk factors, OA can also be triggered or exacerbated by joint trauma, repetitive stress, obesity, and systemic inflammation. Despite current therapies, an estimated 20-50% of joint injuries will typically progress to clinical post-traumatic osteoarthritis (PTOA) within 10-20 years, underscoring the urgent need for more effective therapies. Emerging evidence also implicates infectious diseases, including tuberculosis (TB), as potential facilitators of joint degeneration. To better understand these complex interactions, this work employed longitudinal assessments of pain and mobility behaviors to evaluate disease onset and progression across preclinical OA models. This dissertation aims to enhance the translational utility of rodent post-traumatic and infection-associated OA models by refining behavioral and mobility assessments, testing protocols, evaluating the therapeutic efficacy of stromal cell therapy and PTOA, and investigating the potential for infectious diseases to exacerbate joint degeneration. Chapter 1 presents a comprehensive review of rodent mobility and pain-related behaviors following destabilization of the medial meniscus (DMM) injury. This chapter highlights significant behavioral differences between injured and control animals in these PTOA models, identifies timelines for when changes are expected to occur, and provides a comprehensive summary of current efforts to understand behavioral and pain responses post-injury. Additionally, this review emphasizes the importance of standardizing and improving communication within the field to enhance reproducibility and translational relevance. Chapter 2 builds upon the behavioral insights presented in Chapter 1 by providing an in-depth analysis of short-term open-field testing in mice. This work establishes foundational behavioral profiles for naïve juvenile and adult male and female mice, offering evidence-based recommendations for optimal test durations and parameter selection. These findings support the refinement of behavioral assessments in preclinical research. Chapters 3 and 4 apply behavioral and pathological outcome measures to evaluate the effects of therapeutic and comorbid modifiers of OA. Chapter 3 investigates the potential of stromal cell therapies to modulate inflammation and improve clinical indicators of PTOA. Chapter 4 examines how chronic pulmonary TB infection affects joint degeneration and mobility-related behaviors in animal models, providing new insights into the pathogenesis of infection-associated OA. Chapter 5 concludes the dissertation by integrating the findings across studies, underscoring how behavioral outcomes are interconnected across different models and interventions. This work discusses the broader implications for the field and proposes future directions, including the development of composite behavioral scoring systems to enhance consistency and interpretability in OA research.
Open Access
Expanding mosquito control tools: novel approaches for surveillance and control
(Colorado State University. Libraries, 2025) Pugh, Gregory Scott, author; Foy, Brian D., advisor; Kading, Rebekah, committee member; Hemming-Schroeder, Elizabeth, committee member; Nachappa, Punya, committee member
Approximately 80% of the world's population is at risk of at least one vector-borne disease. Most of these diseases are preventable through protective measures such as insecticide treated bed nets or indoor residual spraying of insecticides along the walls of a domicile. However, these measures can be cost prohibitive and due to these costs, disproportionate infection rates are found within the communities which have lower incomes. Additionally, invasive species are overtaking ecological niches where endemic vectors once thrived. Because of this, novel diseases are being introduced into naïve human populations, which could have deleterious results. While disease prevention methods, such as vaccinations, are becoming more common, these methods often leave out the populations which need them the most, due to vaccinations typically require a solid cold-chain infrastructure. Furthermore, these prevention methods do not exist for some of these diseases, therefore controlling the mosquito populations with insecticides are the "gold standard" for preventing disease outbreak. However, mosquito vectors are exhibiting resistance, both biological and behavioral, to commonly used insecticide classes, resulting in cross-resistance to new products, before they come to market. Novel approaches to mosquito control are necessary to circumvent the rising insecticide resistance issues, such as the development of new control methods and identifying mosquito population dynamics. Endectocides, such as ivermectin and isoxazolines, could be of assistance in regions where resistant mosquitoes are feeding on human and livestock hosts when insecticides are commonly used. Ivermectin is a gluatamate-gate chloride channel agonist causing death by flaccid paralysis in arthropods which feed on treated individuals and is extremely efficacious on anopheline mosquitoes. Additionally, ivermectin is known to cause a reduction in fecundity and refeeding frequency of mosquitoes which imbibe a sublethal dose. This class of drug has been used for the last 30 years as an antihelminth drug in rural Africa and is accepted as a beneficial drug for community health. While ivermectin has a short half-life in human plasma, new models suggest that a change in dosing regimen may retain the mosquitocidal effects up to 28 days post-intervention. Prior studies have conducted clinical trials using this dosing regimen, observing the effects on the mosquito populations. However, these studies have paired ivermectin with malaria treatment methods, which could alter the mosquitocidal effects of ivermectin. The Repeat Ivermectin Mass Drug Administration for the control of MALaria II (RIMDAMAL II) clinical trial sought to control this by only administering ivermectin once a month to select villages in mass drug administration campaigns in Burkina Faso. In addition to observing the mosquitocidal effects of ivermectin on wild mosquitoes, plasma was obtained from participants to elucidate the pharmacokinetics and pharmacodynamics of human processed ivermectin. The goal of this clinical trial was to reduce the malaria burden within children, by controlling the older, possibly infectious mosquitoes. The extrinsic incubation period (EIP), or the time it takes a mosquito to become infectious after consuming an infected blood meal, is dependent on both the mosquito species, the pathogen, and external factures such as climate. Understanding the EIP is essential to determine if a mosquito population has higher risk of transmitting a pathogen to the human population, the older a mosquito population is, the greater the risk. For instance, the EIP for Plasmodium development for mosquitoes in the Anopheles gambiae complex is between 9 and 16 days before the parasite can disseminate throughout the mosquitoes, eventually arriving to the salivary glands, making her infectious when she imbibes her next bloodmeal. While the EIP of West Nile virus in Culex tarsalis is between 8 and 18 days, there are some instances where the virus can escape the midgut and reach the salivary glands within a few days, causing mosquitoes to become infectious before the established EIP. This makes time crucial when assessing the age of the mosquito population. However, the current methods to age mosquito populations are slow, expensive, and require a high degree of technical knowledge. Additionally, the current "gold standard" of mosquito age-grading is qualitative, observing the populations parity rate, which requires an exorbitant amount of time, especially when mosquito control experts are in the conducting control operations. Furthermore, these parity methods are difficult to conduct on certain species of mosquitoes due to their ability to be autogenous, or lay eggs without the need for a prior blood meal, causing an inappropriate classification of population age. Thus, a new age-grading method is needed for mosquito control experts and researchers to identify mosquito populations which contain the greatest risk for pathogen transmission. However, a novel technique is being researched. As a mosquito ages, she loses her wing scales, eventually becoming threadbare. Qualitative methods in the past placed mosquitoes into prescribed bins based on the look of their wings. We have previously identified a novel, high-throughput, and quantitative method to age mosquito populations using machine vison algorithms to determine the pixel intensity of wing photos. This dissertation has investigated the pharmacodynamics of human processed ivermectin on Anopheles gambiae mosquitoes and observing a possible feeding aversion to participants which contain a high venous concentration of ivermectin. Furthermore, we have further refined our pixel intensity method and developed a known age model of An. gambiae G3 strain. We then applied the known age model to the pixel intensities of wing photos collected from wild An. gambiae sensu lato (s.l.) mosquitoes captured during the RIMDAMAL II clinical trial, interpolating their ages. Additionally, using this novel method, we observed a significant decrease in pixel intensities once new dual-chemistry bed nets were issued halfway through our clinical trial, suggesting the pixel intensity method was sensitive enough to examine mosquito control interventions. We have also applied the pixel intensity method to wild Cx. tarsalis around the Greater Salt Lake and Northern Colorado during mosquito control interventions to elucidate if the observations in An. gambiae pixel intensity could be seen Cx. tarsalis. These age-grading techniques lay the foundation for an inexpensive and high throughput measurement for public health, mosquito control experts, and researchers to identify if a mosquito vector population has the capacity to transmit pathogens to the community.
Embargo
From mycobacterial native protein method development to graduate student professional development
(Colorado State University. Libraries, 2025) Lucas, Megan, author; Dobos, Karen, advisor; Henao-Tamayo, Marcela, committee member; McLean, Jennifer, committee member; Mehaffy, Carolina, committee member; Prenni, Jessica, committee member
Mycobacterium tuberculosis (Mtb) is a major global health concern, and is currently the leading cause of death due to an infectious disease. Among the challenges impeding the control of tuberculosis (TB) disease are the need for improved diagnostics, vaccines, as well as therapeutics. Research aimed at gaining a better understanding of the basic biology of the organism can aid in the development of these critical control mechanisms. Over the years, a wide array of methods have been developed for the isolation of individual bacterial components, including subcellular fractions, lipids, and native proteins. These isolated biomolecules have contributed to our overall understanding of the bacteria and its interactions with the host. These discoveries have brought the research community that much closer to reaching the goal of ending the TB pandemic. As part of our long-standing collaboration with BEI Resources, our lab has a history of isolating and distributing mycobacterial reagents for the research community at large. In chapter 1 of this dissertation, I will explore the isolation of biomolecules from mycobacterial cells and their utilization for global research, and discuss the management and scientific impact of this reagent program. I will also describe how the isolation of biomolecules such as native proteins has contributed to the understanding of the basic biology of mycobacteria, with examples pulled from my own publication history. Chapter 2 explores various novel methods for the screening and purification of native proteins. We hypothesize that we can exploit a priori knowledge of antigen targets, combined with targeted mass spectrometry, to inform and accelerate the development of purification methods for native proteins. Methods explored in this chapter include the use of multiple reaction monitoring mass spectrometry (MRM-MS) for the screening of protein fractions. It also describes a purification method which exploits protein-protein interaction for the isolation of native proteins, here used for purification of ESAT6 (Rv3875), CFP10 (Rv3874), and EspC (Rv3615c). Chapter 3 of this dissertation will explore characterization of the heparin binding hemagglutinin protein (HBHA) of Mtb. HBHA is a post-translationally modified, multi-functional bacterial adhesin, found to have roles both on the mycobacterial cell surface, as well as intracellularly, and it has exciting potential as a biomarker of latent infection when used in an interferon gamma release assay. In our lab, we observe that Mtb has as many as four bands identified by western blot analysis when probed with anti-HBHA antibody. A comparison of subcellular fractions and HBHA-enriched material used for native protein purification shows that the banding pattern varies among different fractions, and that these banding patterns are stable and reproducible. Some, but not all, of these protein bands exhibit evidence of methylation when probed using an anti-methylated lysine antibody. Therefore, we hypothesize that there are four distinct proteoforms of HBHA with differences in their hemagglutination activity, a measure of the protein's ability to function as an adhesin. To further explore these observations, efforts are underway to chromatographically separate the four proteoforms in order to look at differences in their biological activity as measured by hemagglutination assay, and analyze them by mass spectrometry to identify potential differences in post-translational modification. Finally, Chapter 4 of this dissertation chronicles a very different endeavor undertaken during my PhD, which was the creation of a course-based professional development class for graduate students in the Department of Microbiology, Immunology, and Pathology. Scientific training and expertise are only part of the skill set needed for a career in science. This is particularly true post-graduate school, where PhD level scientists are expected to be leaders in their respective fields. However, the demands of graduate school make it challenging for students to seek out and engage in extracurricular professional development opportunities to learn about other aspects of scientific careers, such as laboratory funding and time management. To address this need, we've created an experimental professional development course where we've brought together resources and expertise from across CSU's campus into a modular series, consisting of three one-credit modules, each of which focuses on the needs of students at different stages of their graduate education. The goal is to provide a setting where students can learn about, discuss, and practice relevant professional skills, and to encourage participation in these activities by making it a credit-bearing course that will contribute to their degree.
Embargo
Evaluating the biodistribution of osteosarcoma extracellular vesicles and their impact on lung macrophages
(Colorado State University. Libraries, 2025) Haines, Laurel Anne, author; Regan, Daniel, advisor; Avery, Anne, committee member; Thamm, Douglas, committee member; Yang, Ivana, committee member; Henao Tamayo, Marcela, committee member
Patients with osteosarcoma (OS), the most common primary cancer of the bone, often experience metastatic spread of their disease following their initial diagnosis. OS metastasis has a striking tropism for the lungs and is associated with extremely poor survival outcomes, with under 30% of these patients surviving an additional 5 years. Therapeutic options for patients with metastatic OS have not improved in over four decades, emphasizing the pressing need to better understand this disease process. Metastatic disease is preceded by a "pre-metastatic" phase in which tumors release factors into systemic circulation that can prime distant organs to create a favorable site for eventual circulating tumor cell seeding. One of the tumor-derived factors known to elicit the formation of these hospitable pre-metastatic niches in distant organs are extracellular vesicles (EVs), which are nano-sized membrane bound particles that carry biological information between cells. However, our understanding of how EVs released by primary OS tumor cells alter the lungs prior to metastasis is limited. We aimed to improve our scientific understanding of the impacts of OS EVs on the lungs. Our initial goal was to identify which cell types within the lungs are the primary targets of OS EVs. In order to accomplish this, we first optimized and validated a technique to fluorescently label small EVs (sEVs) in order to detect them within cells of the lungs. One of the widely used approaches for EV labeling employs lipophilic membrane dyes, which have been recently shown to have serious technical limitations due to the formation of non-EV fluorescent particles that can confound results. We hypothesized that by optimizing the sEV starting material to ensure high purity and concentration and by altering the buffer conditions to eliminate protein from the system, we could more efficiently label sEVs with lipophilic membrane dyes. We show that by doing so, we could achieve sEV membrane labeling while limiting false positive signal formation and detect these fluorescently-labeled sEVs within cells both in vitro and in vivo. This work improves upon a widely used technique to label EVs, revealing its limitations and how to overcome them to avoid confounding results. These results are described in Chapter 2 of this dissertation. After developing a highly validated technique to efficiently label sEVs, we determined the cellular targets of OS sEVs in the lungs. To do this, we designed a multi-parameter, spectral flow cytometry panel that could delineate many of the cell types that comprise the lungs including stromal and immune populations. Using multiple different OS sEV sources from three different species, we evaluated the biodistribution of OS sEVs in immunocompetent, syngeneic mouse models as well as immunocompromised models. We hypothesized that lung interstitial macrophages may be a target of OS sEVs as they are professional phagocytes that act as a gateway between the vasculature and lung interstitium. Interestingly, although we observed that all OS sEVs, regardless of cell source, were taken up by interstitial macrophages, there were additional cellular targets that appeared to be dependent on the OS EV source. OS sEVs sourced from the murine OS cell line K7M2 also uniquely targeted alveolar macrophages present within the airways whereas canine and human OS sEVs did not. These findings suggest an ability of K7M2 sEVs to access the airway spaces that could be in part related to the species-matched nature of these sEVs. Additionally, K7M2 OS sEVs led to subtle, but significant, alterations to both inflammatory monocyte and fibroblast populations in the lungs of immunocompetent mice. Because OS impacts both humans and dogs, we also leveraged dogs as a translationally relevant surrogate to study alterations to the pre-metastatic lung microenvironment. Using cytology, flow cytometry, and single cell RNA sequencing we documented alterations in specific canine alveolar macrophage populations across different stages of naturally occurring OS using bronchoalveolar lavage techniques. In summary, these results suggest unique, non-random, and potentially species-specific patterns of OS EV biodistribution within the lungs prior to tumor cell arrival. OS sEV priming additionally alters key lung cell populations that may influence metastatic progression. Finally, we show that interrogating the airways spaces of canine OS patients reveals shifts in alveolar macrophage populations that could inform mechanisms of metastasis or identify biomarkers of disease progression. These results are reflected in Chapter 3. Our results from Chapter 3 revealed that the cell types capable of taking up OS sEVs in the lungs were primarily macrophages. Thus, in Chapter 4 we aimed to directly evaluate the impact of OS sEVs on primary human lung resident and recruited macrophages. The existing literature evaluating OS sEV impact on macrophages has been performed in mouse cells or in immortalized, cultured human macrophages. We aimed to build on previous work by investigating the impact of OS sEVs on lung macrophages in a translationally relevant setting utilizing primary human donor-derived macrophages. We were able to analyze alveolar macrophages isolated from airway spaces as well as monocyte-derived macrophages from human blood (a proxy for interstitial or recruited lung macrophages) using RNA sequencing and cytokine analysis. Following treatment with OS sEVs we observed a strong inflammatory response in monocyte-derived macrophages, with canonical anti-viral pathway upregulation. On the other hand, alveolar macrophages displayed a more metered inflammatory response, with upregulation of wound healing pathways. The response of each cell type was largely unique, but there were several shared upregulated pathways related to innate immune stimulation. These data provide key directions for future investigation of the functional consequences of sEV-primed lung macrophages in the pre-metastatic lungs of OS patients. Collectively, this work has identified the cellular targets of OS sEVs and characterized their impact on these resident lung cells. With this, we have developed a strong foundation for future investigation of biomarkers and mechanisms of metastatic progression. Moving forward, we hope that our findings lead will help inform new therapeutic targets for the treatment of metastatic OS to improve the lives of both human and canine OS patients.
Embargo
Primary viral-secondary bacterial pneumonia: a novel Syrian hamster infection model for investigating modulation of host response pathways as therapeutic targets
(Colorado State University. Libraries, 2025) Romano, Ashley, author; Slayden, Richard, advisor; Abdo, Zaid, committee member; Bosco-Lauth, Angela, committee member; Podell, Brendan, committee member; Tjalkens, Ron, committee member
Acute respiratory tract infections remain a significant public health concern. Despite numerous clinical retrospective studies on primary viral-secondary bacterial pneumonia (VBP), there are no experimental reports addressing the influence of SARS-CoV-2 on susceptibility to secondary bacterial infection, VBP studies exploring the effects of biological variables, or assessment of host-directed therapeutics (HDTs) as a treatment strategy for VBP using immunocompetent lab animal model species. As such, I designed a three-pronged research project to address these critical knowledge gaps to create a novel small animal infection model with high translational impact for studying human VBP pathogenesis and treatment options. I first established an infection model characterizing VBP in Syrian hamsters, which is an immunocompetent lab animal species that is naturally susceptible to SARS-CoV-2 and extracellular bacterial species. I found that the ideal infection scenario comprised of: male hamsters older than 39 weeks of age; intranasal SARS-CoV-2 administered on Day 0; non-surgical intratracheal Haemophilus influenzae serotype B administered on Day 2.5 post-viral infection (PVI); study endpoints of Day 7 and Day 14 PVI. I then repeated this experiment and included virus-only, bacteria-only, and bacteria followed by virus infection groups to confirm that the pathogen infection sequence impacts disease phenotype. This study demonstrated that the virus followed by bacteria infection group resulted in the greatest amount of weight loss, most severe histopathology, and positive microbial titers on Day 7 post-primary infection (PPI). Moreover, to assess which pathways were most affected on Day 7 PPI, I performed qRT-PCR relative gene expression on lung tissue homogenates that revealed significant pro- and anti-inflammatory gene dysregulation in a subset of virus followed by bacteria infected hamsters that was not detected in any other infection group. These findings informed the choice of HDTs selected to modulate host pathways implicated in host-pathogen interactions that can lead to severe disease states. I discovered that either modulating expression of host receptors utilized by SARS-CoV-2 for cell entry or modulating host cyclooxygenase-2 function may be viable treatment targets for subsets of VBP patients. Together, this work establishes a new model for assessing VBP in an underutilized laboratory animal species and highlights the utility of HDTs as a treatment option for VBP.
Embargo
Boosting and modeling immunity: integrated approaches in tuberculosis vaccine development
(Colorado State University. Libraries, 2025) Maldonado, Pablo, author; Henao-Tamayo, Marcela, advisor; Anderson, G. Brooke, advisor; Dow, Steven, committee member; Elf, Jessica, committee member; Wilusz, Carol, committee member
Tuberculosis (TB) disease caused by Mycobacterium tuberculosis (Mtb) has returned as the leading cause of death by an infectious agent in 2023, and approximately 25% of the global population is infected with Mtb. There is only one licensed vaccine, Bacillus Calmette–Guérin (BCG), a live attenuated strain of Mycobacterium bovis. While the World Health Organization estimates that global BCG vaccination coverage was 87% as of 2023, TB remains a top 10 cause of mortality overall, claiming a life every 30s. It is without doubt that BCG alone is not enough to protect against TB and any newly developed vaccines must complement the baseline protection already offered by BCG. To address these gaps, we established two complementary approaches. This dissertation outlines the development of a vaccine booster to BCG using a novel inactivated Mtb (SolaVAX-Mtb) and the establishment of a Generalized Linear Model (GLM) statistical framework to evaluate high-dimensional flow cytometry data and the multivariable influences on immune responses using a clinical trial booster. We find that BCG prime and boost with SolaVAX-Mtb significantly reduces lung colony forming units and lesion burden, while increasing correlates of mucosal immunity. In our GLM analysis, mice showed increased probabilities of T cell activation in the lung following BCG + ID93-GLA-SE compared to BCG or ID93-GLA-SE alone, with females showing more robust B cell responses. This dissertation underscores the importance of multivariable influences in TB immunology and the benefits of boosting mucosal immunity to improve baseline BCG protection—ultimately paving the way for more effective TB prevention strategies.
Embargo
Advancing the understanding of osteosarcoma metastasis: characterizing the impact of exosomes on lung fibroblasts and metastatic colonization
(Colorado State University. Libraries, 2025) Palmer, Eric Patrick, author; Regan, Daniel P., advisor; Thamm, Doug, committee member; Podell, Brendan, committee member; Redente, Elizabeth, committee member
Osteosarcoma (OS) is the most common malignant bone cancer, typically diagnosed in children and young adults. 30-40% of all OS patients develop tumor recurrence that occurs almost exclusively in the form of lung metastasis, which is associated with a dismal 20% 5-year survival rate. Recent evidence suggests organ specific metastasis, organotropism, may be driven by tumor secreted extracellular vesicles called exosomes. Exosomes have been shown to traffic to specific organs where they are taken up by target cells, releasing their biological cargos, and reprogramming those cells towards tumor-promoting phenotypes that alter the microenvironment in advance of circulating tumor cells, thus creating a pre-metastatic niche. Cancer-associated fibroblasts are a critical cell type within the lung TME, which promote immune suppression, drug resistance, and tumor cell survival. Prior work shows tumor cells can co-opt fibroblasts to a pro-tumorigenic phenotype via exosome mediated intercellular communication. Currently, the mechanisms by which OS exosomes modulate resident lung fibroblast function and promote enhanced metastatic colonization of the lung has not been evaluated. To investigate this, we isolated exosomes from a panel of 6 OS cell lines. We assessed the uptake and response of human donor-derived primary lung fibroblasts (LF's; n=4) to OS exosome treatment in vitro via flow cytometry, confocal fluorescent microscopy, proliferation assays, phospho-kinase array, multiplex cytokine analysis and RNA-sequencing. We observed that LFs efficiently take up OS exosomes, which is associated with induction of MAPK pathway activation, fibroblast proliferation and significantly enhanced secretion of IL-6, CXCL8 and CCL2 compared to untreated LFs. RNA-seq of exosome treated LFs confirmed these responses and revealed significant enrichment of pathways related to cytokine secretion, proliferation, immune cell chemotaxis, migration, proinflammatory and profibrotic mediators. Finally, in an exosome-educated lung fibroblast-OS co-culture model, exosome educated LFs conferred significantly increased OS cell survival and proliferation as compared to untreated fibroblasts. To evaluate the impact of OS-derived exosomes on metastatic colonization in vivo, we sought to develop a highly sensitive mouse model of OS metastasis for detection of OS cells by bioluminescent IVIS imaging and spectral flow cytometry. To overcome the lack of universally expressed and distinguishing cell surface markers on OS cells, we evaluated using fluorescent cell labeling dyes, Cell Trace Yellow and DIR. By using a mouse lung resident cell type antibody panel, we determined these dyes are not restricted to the tumor compartment and therefore are likely not useful for tracking OS cells in vivo. Next, using a lentiviral transduction system, we generated a 143b, OS cell line that expressed nuclear red fluorescent protein (RFP) and Luciferase. Here, we demonstrate the use of this reporter system allows for the rapid and highly sensitive detection of OS cells in the lung by bioluminescent imaging and spectral flow cytometry within 24-hours of tumor cell, tail vein injections. Next, we evaluated the impact of repeated intravenous exosome educations on metastatic colonization of the lung using both human OS cell line, 143b in an immunocompromised xenograft mouse model and mouse OS cell line, K7M2, in an immunocompetent syngeneic mouse model. Bioluminescent intravital imaging 4 days post-tumor cell tail vain injections reveal exosomes derived from 143b OS cells but not K7M2 OS cells promoted enhanced metastatic lung colonization efficiency. Based on these observations, we sought to determine if changes in resident mouse lung cell populations and intracellular cell signaling were associated with increased metastatic lung colonization resulting from exosome treatments. By employing a 9-marker, anti-mouse panel, we show no significant changes to resident mouse lung CD45+ or stromal cell populations. Additionally, we observed no changes to lung concentrations of previously identified IL-6 and CCL2 upon exosome education. However, follow up studies would show tumor cell involvement may be required to stimulate significant changes to cell populations and cell signaling within this model. Finally, despite observations that OS exosomes can induce striking changes to lung fibroblasts in vitro and promote metastatic colonization in vivo, we did not observe any significant differences in overall survival among mice exposed to OS exosomes prior to tumor cell injection. Taken together, we show OS-derived exosomes can induce lung fibroblasts to shift towards a tumor-promoting phenotype in vitro. Additionally, chronic exosome treatments promote metastatic colonization in an immunocompromised xenograft mouse model. However, these findings were not correlated to alterations among resident lung cell populations, tumor-promoting intracellular signaling molecules, or overall survival.
Open Access
Injectable anesthesia and effects of analgesics on the immune response in Jamaican fruit bats
(Colorado State University. Libraries, 2025) Pollak, Marissa, author; Kendall, Lon, advisor; Schountz, Tony, committee member; Boscan, Pedro, committee member
Injectable anesthesia and immunomodulatory effects of buprenorphine and meloxicam in Jamaican fruit bats (Artibeus jamaicensis) (JFBs) have not been studied to date. Testing injectable anesthetic protocols in JFBs would allow for potential alternatives to isoflurane anesthesia which is relied upon in the field and in laboratory settings, but requires special equipment and can be difficult to manage in the field. Injectable anesthetic protocols in bats have been studied in limited capacity yet are valuable due to minimal equipment needs and the ability to induce multiple animals in succession. Four injectable anesthetic protocols (n=6) were compared with isoflurane inhalant anesthesia (control, n=5) in JFBs. Ketamine (K), alfaxalone (A), butorphanol (B), and nalbuphine (N), were each combined with dexmedetomidine (DX) and delivered intraperitoneally, followed by subsequent reversal with atipamezole. KDX and ADX induced anesthesia in 5 out of 6 bats, while NDX and BDX only induced mild to moderate sedation in all bats. All groups except for KDX resulted in return to flight within 60 minutes of injection. In summary, KDX and ADX induced anesthesia in the majority of bats tested and are viable alternatives compared to isoflurane alone. As KDX resulted in a prolonged return to flight time, it is not ideal for use in the field where timely recovery is needed to ensure the safety of bats. Bats are frequently used as a model to study emerging infectious diseases and bat immune responses. These studies may result in clinical signs, such as flu-like symptoms, in the bats. Minimizing pain and distress is a priority in laboratory animal medicine and is necessary to remain in compliance with regulations including the Guide for Care and Use of Laboratory Animals and the Animal Welfare Act. Opioids and non-steroidal anti-inflammatory drugs (NSAIDs) can be used to mitigate the potential pain and distress associated with infectious agents. Here we aim to understand how buprenorphine HCl (BUP), an opioid and meloxicam (MEL), an NSAID, affect the immune response in JFBs. Three male and three female bats in each group were immunized subcutaneously with keyhole limpet hemocyanin (KLH) with complete Freund's adjuvant on day 1. Immediately prior to immunization, bats were bled for baseline IgG determination. Subcutaneous doses of meloxicam (MEL) at 5 mg/kg, buprenorphine (BUP) at 1 mg/kg, or 0.15 mL of saline (SAL) were given daily for 21 days. On day 21, bats were given a subsequent immunization with KLH and incomplete Freund's adjuvant. On day 28 bats were euthanized and blood was collected to assess IgG responses in the serum and spleens were isolated to assess gene expression responses in KLH stimulated splenocytes. Antibody response to KLH was measured by ELISA and gene expression profiling was evaluated by RT-qPCR. There were no antibodies detected prior to immunization in any group. After immunization and treatments, all groups had elevated antibody responses. There were no statistical differences in the antibody responses of bats treated with either BUP or MEL compared to SAL treated bats. The only statistically significant difference in gene expression was decreased IL-13 expression in BUP treated bats compared to SAL treated bats. Overall, these results suggest that the use of BUP or MEL will have minimal impact on the bat immune response.
Open Access
Histological and behavioral characterization of a murine model of Parkinson's disease using systemic inoculation with Western equine encephalitis virus
(Colorado State University. Libraries, 2025) Thomas, Samantha Monica, author; Kendall, Lon V., advisor; Tjalkens, Ronald, committee member; Kading, Rebekah, committee member
Parkinson's disease (PD) is the second-most common neurodegenerative disease after Alzheimer's disease, constituting over 10 million ongoing cases in humans worldwide. Disability associated with deprecations in motor function in PD is substantial, highlighting the need for better animal models to study interventions. Although the causes of PD are largely unknown, infection with mosquito-borne alphaviruses, such as Western equine encephalitis virus (WEEV), induces symptoms in humans that closely resemble those of PD. This suggests that viral inoculation may be an appropriate model to study the disease in vivo. Intranasal inoculation of mice with WEEV induced dopaminergic neuronal loss in the striatum (ST) and substantia nigra pars compacta (SNpc), a key pathologic feature of PD in humans. However, significant mortality in animals results from this potent, direct impact on brain tissues. This requires adjunctive immunotherapy to improve survivability. Footpad inoculation, however, may prove to be a better model due to an indirect, gradual hematologic spread to the brain. Additionally, footpad inoculation may more closely replicate central nervous system (CNS) pathology, as it mimics the natural route of infection with this mosquito-borne virus. We hypothesize that systemic WEEV infection will induce behavioral and histopathologic changes resembling PD without requiring adjunctive immunotherapy. First, a dose escalation study was performed to evaluate the tolerability of mice to increasing doses (0.5 x 104, 1 x 104, 2 x 104, 0.5 x 105, 1 x 105, and 2 x 105 PFU) of WEEV inoculated subcutaneously into the hind footpads. Clinical observations, body weights, bioluminescent imaging, and behavioral testing, including a pole test and a grid hang test, were performed at various time points prior to brain tissue collection to evaluate the loss of dopaminergic neurons in the ST and SNpc. It was determined that while mice displayed difficulty recovering from anesthesia associated with imaging, the highest viral dose displayed the most consistent pathologic findings. However, behavioral testing was inconsistent as it was determined that the hang test was not suitable, and the pole test needed modifications to improve grip. Finally, C57BL/6 mice were inoculated with 2 x 105 PFU of systemic WEEV to characterize the behavioral and histopathologic traits of the model fully. Observations, weights, and behavioral testing were performed at various time points before brain tissue collection at 14- and 42-days post-infection (dpi) for histopathology and plaque assays. No mortality was seen in this study, indicating that the mortality seen in the dose escalation study mice was likely related to sensitivity to anesthesia induced by systemic WEEV infection. A grip strength meter replaced the hang test, and the pole test was modified for better traction. Mice displayed significant deficiencies in both grip strength and motor coordination by 42 dpi. Plaque assays indicated increasing viral CNS titers from 14 dpi to 42 dpi. Thus, we identify key characteristics of a PD phenotype in a suitable systemic WEEV model of PD. Future studies can utilize this mouse model to investigate novel therapeutic and neurodegenerative-reducing strategies for viral Parkinsonism. This mouse model will also be used in combination with other PD risk factors, such as manganese, to create a dual-hit model to more completely recapitulate the disease process.
Embargo
Insights into the cell of origin, pathogenesis, and translational potential of canine peripheral T-cell lymphoma
(Colorado State University. Libraries, 2025) Owens, Eileen, author; Avery, Anne, advisor; Basaraba, Randall, committee member; Duval, Dawn, committee member; MacNeill, Amy, committee member; Regan, Daniel, committee member
Peripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms in humans and dogs with short survival times and poor treatment responses. The clinical features and immunophenotype of PTCL in dogs closely resemble PTCL, not otherwise specified (PTCL-NOS), the most common and most poorly understood subtype of PTCL in humans. This has led to interest in their potential as a preclinical model for this disease. Because these are naturally occurring tumors in dogs, they offer some unique advantages as a preclinical model compared to traditional xenograft and genetically engineered mouse models, including the ability to study these cancers in an immunocompetent host with a more comparable tumor microenvironment, the potential to investigate the genetic origins of these tumors in hosts with similar genetic diversity to humans, and the consideration of environmental risk factors, since humans and dogs largely share the same environments. One remaining barrier to this proposed canine model is our limited understanding of how the molecular features of canine PTCL compare to human PTCL-NOS. In humans, gene expression and mutational profiling studies have unveiled prognostically significant molecular subtypes of PTCL-NOS, identified enriched oncogenic signaling pathways, recognized mutations in key tumor suppressor genes and oncogenes, and discovered large structural variants such as fusion genes that contribute to the pathogenesis of PTCL-NOS and represent opportunities for novel targeted therapies. However, similar gene expression and mutational profiling studies in canine PTCL are lacking. We hypothesized that canine PTCL would exhibit a similar global gene expression profile to PTCL-NOS, would be enriched for the same oncogenic signaling pathways, and would share some of the most common gene mutations of human PTCL-NOS. We tested this hypothesis by performing bulk RNA-sequencing, differential gene expression analysis, and variant analysis on canine PTCL, normal canine nodal lymphocytes, and normal canine thymocytes. We discovered that the gene expression profile of the most common CD3+CD5+/-CD4+CD8– immunophenotype of canine PTCL closely resembles human PTCL-NOS—particularly the GATA3-expressing subtype associated with an inferior prognosis—and canine PTCL often exhibits mutations in the same genes commonly mutated in human PTCL-NOS, including DNMT3A, TP53, TET2, and PTEN. The cell of origin of human PTCL-NOS remains a subject of ongoing speculation. The observation that subsets of human PTCL-NOS upregulate either TBX21 or GATA3—transcription factors controlling the differentiation of naïve T cells to mature T helper 1 (Th1) or T helper 2 (Th2) cells, respectively—led to suggestions that these tumors are derived from their respective normal mature T-helper cell counterparts. However, more recent studies have demonstrated that GATA3 is an oncogene upregulated by a variety of neoplasms, including immature precursor T-cell neoplasms and even non-hematopoietic neoplasms, and its expression in GATA3-PTCL is associated with promotion of general T-cell survival and proliferation rather than Th2 differentiation. Additionally, mature T cells have been shown to be highly resistant to oncogenic transformation, suggesting that even seemingly mature T-cell neoplasms may actually arise from earlier precursors. Given the similarities between human PTCL-NOS and canine CD4+ PTCL, investigations into the cell of origin in dogs may offer additional insight into the cell of origin in human PTCL-NOS. One previous study identified decreased surface expression of CD25 and MHC class II in canine PTCL, molecules normally associated with T-cell maturity and activation, suggesting that canine PTCL may arise from an earlier T-cell precursor. Recently, an RNA-sequencing study comparing the gene expression profile of canine T-zone lymphoma, a more indolent form of T-cell lymphoma in dogs, to gene signatures of human and murine naïve and activated T-cell subsets successfully identified the cell of origin for this disease as a mature, previously activated αβ T cell. Therefore, we sought to employ similar methods to test our hypothesis that the cell of origin of canine CD4+ PTCL is a thymic precursor cell. Additionally, to help overcome some of the limitations of using publicly available data in other species, we sought to develop a single-cell transcriptomic atlas of normal canine thymic and lymph node tissue to identify key changing genes throughout T-cell development and differentiation in dogs and evaluate those expression signatures in canine CD4+ PTCL. We found that canine CD4+ PTCL upregulated several markers of immaturity, including CD34, KIT, DNTT, and CCR9. Canine CD4+ PTCL was enriched for gene signatures associated with immature murine and human thymocytes compared to more mature T-cell subsets. Finally, based on data derived from our single-cell atlas of normal thymus and lymph node, canine CD4+ PTCL tended to upregulate genes expressed early in canine thymocytes and downregulate genes expressed as canine T cells progressed to more mature stages of development. Taken together, these findings support an immature thymic precursor cell of origin for canine CD4+ PTCL. For a subset of human PTCL-NOS, a type of mutation known as fusion genes play a role in tumorigenesis and offer an opportunity for targeted therapies. These mutations are characterized by the juxtaposition of two previously independent genes, which can then be transcribed and potentially translated together to oncogenic effect. In small subsets of human PTCL-NOS, the presence of a fusion gene drives malignant transformation through upregulation of specific oncogenic signaling pathways, and inhibitors of these pathways have shown promise as an alternative treatment option in these tumors. Despite their potential as actionable drivers in many hematologic cancers, fusion genes have not been previously investigated or described as a feature of canine PTCL. We hypothesized that recurrent fusion genes could be identified in canine CD4+ PTCL, and that some would correlate with enrichment for particular oncogenic pathways. To test this hypothesis, we utilized fusion calling algorithms for bulk RNA-sequencing data and long-read DNA sequencing techniques to investigate expression of fusion mRNA transcripts and the presence of large chromosomal aberrations, respectively, in canine CD4+ PTCL. We identified 13 recurrent fusion genes at the mRNA level, 3 of which were tumor-specific and not detected in dogs without lymphoma. 2 of these 3 PTCL-specific fusions, TOX2-LMO4 and PER1-EIF5A, were associated with enrichment for TNF and NF-ĸB signaling and greater enrichment for elements of the tumor microenvironment, respectively, suggesting possible mechanisms by which these fusions could contribute to CD4+ PTCL. Long-read sequencing additionally revealed 3 fusion genes in CD4+ PTCL DNA that were also predicted in bulk RNA-seq data: STAG2-SH2D1A, PLEKHA5-AEBP2, and SERPINB5-ENSCAFG00000031329. The evidence of concurrent chromosomal aberrations and fusion transcript expression for these candidates make them stronger contenders for potential driver mutations that should be explored in future studies. Taken together, the work in this dissertation supports that canine CD4+ PTCL is a naturally occurring neoplasm in dogs that arises from a thymic precursor cell of origin and closely resembles the GATA3-expressing subtype of human PTCL-NOS. Additionally, similar to human PTCL-NOS, recurrent fusion genes are a feature of canine CD4+ PTCL that may arise from a combination of structural chromosomal variants and alternative RNA splicing events, and these fusions may contribute to the pathogenesis of CD4+ PTCL through activation of certain oncogenic signaling pathways. The shared features between canine CD4+ PTCL and human PTCL-NOS represent potential opportunities to explore novel therapies in the dog that may be translated to the human disease.
Embargo
The role of galactosyleceramides in flavivirus infection
(Colorado State University. Libraries, 2025) Laurence, Hannah, author; Perera, Rushika, advisor; Ebel, Greg, committee member; Nachappa, Punya, committee member; Belisle, John, committee member
Mosquito-borne flaviviruses are an intensifying threat to global health without specific antiviral therapies or a widely available vaccine. Between 2000 and 2019, there was a 10-fold surge in dengue virus cases reported globally [1]. 2024 was a record year with more than 14 million dengue cases reported worldwide [2]. Flaviviruses are reliant upon host cell membranes to complete their life cycle. Preliminary data for this work included an siRNA loss of function screen of each enzyme in the sphingolipid metabolic pathway performed in liver cells (Huh7) infected with dengue virus 2. This screen identified numerous enzymes of interest in the metabolic pathway. This dissertation investigates the roles of two key enzymes in the sphingolipid metabolic pathway that affect synthesis and degradation of galactosylceramides, UDP-galactosyltransferase 8 (UGT8) and galactocerebrosidase (GALC), and how they affect flavivirus infections in vitro. We studied how modulating the expression of UGT8 and GALC during infection with dengue virus, serotype 2 (DENV2) and Zika virus (ZIKV) impact the virus life cycle in human hepatoma cells (Huh7). We additionally studied how UGT8 knockdown affected ZIKV titer in neuroblastoma cells (SH-SY5Y). Chapter 2 focuses on the effect of UGT8 during DENV2 infection of Huh7 cells. We showed that with UGT8 knockdown, there was an increase in titer, while overexpression caused a decrease in titer. Mechanistic studies demonstrated that modulating UGT8 expression affected entry and release of DENV2 particles. We did not observe any effect on DENV2 genome replication or the specific infectivity of the virus particles. We hypothesized that modulation of UGT8 expression alters the cell and viral membranes to influence virus release and virus entry. Chapter 3 focuses on GALC, which performs the reciprocal reaction as UGT8 and degrades galactosylceramides in the lysosome. We discovered that GALC knockdown increased both extracellular and intracellular DENV2 titers, but did not affect genome replication or specific infectivity. This suggested that GALC knockdown caused an increase in the efficiency of viral assembly. We proposed that the oxidative stress resulting from loss of function of the GALC enzyme upregulated sphingomyelinases to increase cellular pools of ceramide, resulting in an increase in efficiency of viral assembly. Chapter 4 explored the effects of UGT8 in ZIKV infection of both Huh7 cells and SH-SY5Y cells. Modulation of UGT8 expression in these cells showed a similar phenotype to DENV2. Together, the findings in this dissertation highlight the importance of sphingolipid metabolism in flavivirus infections and provides new insights into the roles of these specialized lipids during infection. Hypothesis of the study: The flavivirus life cycle is critically influenced by the balance of galactosylceramide synthesis and degradation, processes regulated by UDP-galactosyltransferase 8 (UGT8) and galactocerebrosidase (GALC) respectively.
Embargo
Non-tuberculous mycobacterium pulmonary disease: challenges and strategies for the preclinical modeling of M. abscessus and M. avium complex
(Colorado State University. Libraries, 2024) Pearce, Camron, author; Gonzalez-Juarrero, Mercedes, advisor; Jackson, Mary, committee member; Henao-Tamayo, Marcela, committee member; Amberg, Gregory, committee member
Mycobacterium avium complex (MAC) and Mycobacterium abscessus (Mab) each present significant clinical challenges. Both mycobacterial complexes are notorious for their ability to cause chronic and severe pulmonary infections, resistance to standard antibiotics, and intricate host-pathogen interactions that complicate disease management. Although in vitro characterization and preclinical mouse models are important for developing novel therapies, they often fail to replicate the full complexity of human disease. This dissertation presents work evaluating the efficacy of inhaled antibiotics delivered via liquid aerosol in treating MAC pulmonary infections in mice and explores a cystic fibrosis-like mouse strain as a potential preclinical model for Mab pulmonary infection. Building on the Mab studies, whole-body plethysmography (WBP) was established as a robust tool for longitudinally studying the effects of Mab pulmonary infection. This technique enabled monitoring of respiratory parameters and provided a detailed assessment of mouse respiratory function over time. Subsequently, a machine learning (ML) pipeline was developed to classify infection status based on WBP data, which demonstrated the potential of WBP-coupled infection studies to monitor disease progression. By identifying the respiratory parameters most predictive of infection, this work showed the potential for WBP modelling to not only track disease progression, but also better align preclinical mouse models with clinically relevant patient-reported outcomes.
Open Access
Dodging wrenches in the time of COVID: exploring flavivirus replication mechanisms and SARS-CoV-2 antibody development
(Colorado State University. Libraries, 2024) Terry, James Steven, author; Geiss, Brian J., advisor; Wilusz, Jeffrey, committee member; Ebel, Gregory, committee member; Snow, Christopher, committee member
Flaviviruses pose a significant threat to global health, threatening hundreds of millions of people who live in endemic areas. Infection with flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV) can trigger symptoms ranging from a mild cold-like illness to microcephaly, encephalitis, hemorrhagic fever, and death. As climate change alters global temperature ranges, habitable environments for the flavivirus arthropod vectors are expanding into previously unexposed regions. Due to a lack of flavivirus vaccines and antivirals, most efforts to combat infection fall within vector population control and palliative care for infected individuals. To develop antivirals and vaccines against flaviviruses, we need to better understand the fundamental mechanisms through with the viruses replicate. By investigating incompletely understood processes in the replication cycle, new antiviral targets can be identified and pursued. This dissertation investigates two components of the flavivirus replication cycle to better understand the key processes necessary for successful flavivirus infection. Additionally, this dissertation reports on efforts during the SARS-CoV-2 COVID-19 pandemic to develop novel reagents to assist in research and diagnostic development. An important determinant of successful flavivirus infection is the generation of subgenomic flavivirus RNA (sfRNA). This RNA is composed of exoribonuclease resistant RNA (xrRNA) structures in the flavivirus 3' untranslated region (3'UTR). These structures allow the flavivirus 3'UTR to withstand degradation by stalling the host Xrn1 exoribonuclease, halting viral RNA degradation and creating sfRNA. The production of sfRNA is critical for flavivirus replication success as the new RNA entity actively suppresses the host cell immune response to viral infection. There are blind spots in our understanding of the key stages of sfRNA generation, namely how the Xrn1 substrate is produced from the flavivirus genome. It has previously been postulated that host decapping enzymes remove the flavivirus Type 1 cap structure, allowing Xrn1 to bind to the 5' monophosphate and degrade the viral RNA. The enzyme responsible for decapping has not yet been identified. Following preliminary evidence from the Geiss Lab, we investigated the host decapping enzyme Dcp2 as the protein responsible for priming flavivirus RNA for Xrn1 degradation and sfRNA production. We developed a pipeline using splint-ligation to specifically label monophosphorylated WNV RNA with an RNA adapter at the 5' end. Following this the ratio proportion of viral RNA that is monophosphorylated is revealed using a qRT-PCR reporting system. With this pipeline, it was determined that suppressing Dcp2 expression increased the proportion of monophosphorylated WNV RNA in infected cells while having no significant effect on monophosphorylated RNA in newly produced virions. Additionally, northern blot analysis revealed that sfRNA generation was not reduced by Dcp2 knockdown. From this study we determined that Dcp2 is not necessary for sfRNA generation, and thus other processes are responsible for the generation of monophosphorylated viral genomes for Xrn1 degradation. One hole in our understanding of flavivirus replication concerns the viral replication compartment. The compartment is an invagination in the endoplasmic reticulum membrane that is formed through viral protein manipulation. This environment then hosts the viral replication machinery, protecting the vulnerable viral RNA from host cell immune detection as new viral genomes are produced. Proper viral protein-protein interactions are critical for the successful formation of this viral RNA factory. While studies have been conducted to determine the replication compartment location and some interactions between nonstructural proteins, our understanding of how these proteins interact with each other in situ is limited. To address this, we employed crosslinking mass spectrometry. First, a flavivirus replication compartment purification and crosslinking pipeline underwent a series of evolutions and significant optimizations followed mass spectrometry data acquisition. Then, a crosslinked protein analysis pipeline using the Bonvin Lab programs DisVis/HADDOCK was validated with crosslinked bovine serum to ensure its utility with crosslinked viral compartment samples. MaxQuant analysis revealed some viral protein crosslinks while highlighting areas for improvement in our methodology. Nevertheless, the identified intramolecular crosslinks within NS1, NS3, and NS5 hint at potential dimer interfaces. An intermolecular crosslink between NS3 and NS4b was identified that supports the observations of previous studies while establishing in situ evidence for interactions along an NS3 N-terminus and NS4b residue K172 interface. The results provided intriguing preliminary evidence for future investigations into the replication compartment protein-protein interactions and established a protocol for analyzing viral proteins with crosslinking mass spectrometry. In addition to chronicling on flavivirus replication cycle studies this dissertation includes a chapter chronicling work during the COVID-19 pandemic on SARS-CoV-2. Monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid protein were generated, characterized, and sequenced during the height of the pandemic. These antibodies were the first of their kind to be published and were made available for use during the global SARS-CoV-2 research effort. This chapter also reports on collaborative efforts surrounding the use of antibodies for diagnostics and predictive computational pipelines. Work was done to assist the Henry Lab in developing inexpensive electrochemical and colorimetric ELISA devices targeting SARS-CoV-2 NP for bedside diagnostic use. Lastly, wet lab verification was performed to validate Jacob Deroo's epitope-predicting PAbFold AlphaFold2 pipeline. The work covered in this dissertation spans five years, two viruses, and three separate target areas. These projects, while varied, are all bound together by the common goal of contributing to the advancement of knowledge and techniques for stopping viral threats to global health. Knowing how a virus creates a safe environment for genome replication or identifying which host proteins help create an immune-modulating viral RNA molecule is important for identifying new paths towards intelligently designed antivirals. Similarly, developing and characterizing antibodies to supply a global research effort and validate cutting-edge computational tools is necessary for actively combatting a global pandemic and preparing for the next one. With this work, scientific inquiry ranging from foundational knowledge to translational science is explored.
Open Access
From the ovine to human rotator cuff; tenocyte as to MSC derived exosomes for tendon healing
(Colorado State University. Libraries, 2024) von Stade, Devin P., author; Regan, Daniel, advisor; McGilvray, Kirk, advisor; Santangelo, Kelly, committee member; Hollinshead, Fiona, committee member
Tendinopathies comprise one of the most widespread and economically significant diseases in developed nations. The societal value of rotator cuff tear surgical intervention alone has been estimated at greater than 3.4 billion US dollars despite frequent repair failures (30-79%). This drives great interest in adjunct therapies; however, research is complicated by a limited understanding of the underlying pathogenesis. Recent data suggests that the primary driver is cell-to-cell communication during the acute and chronic stages of rotator cuff tears. Most notably, the paracrine signaling of macrophages, which are preferentially recruited earlier and persist longer than other immune cells, may direct the structural function of injured tendons. Extracellular vesicles (EVs) are the primary contributors to the paracrine signaling responsible for many successful cell therapy studies. Investigations into mesenchymal stromal cell (MSC) derived EVs have served as a launching point toward this end, however, cell origin can dramatically change the effect of EVs on target cells. To explore the effects of exosomes as a function of cell source on tendon healing, we have developed in vitro models in human and ovine cell lines to test the effects of tissue native, tenocyte derived EVs as they compare to MSC derived EVs on key effectors of rotator cuff tears, tenocytes and macrophages. The goal of this work is to (a) describe the direct effect of EV education, as a function of cell source, MSC vs tenocyte, on macrophage gene regulation and cytokine production and tenocyte bioactivity; (b) to then assess the indirect effects of such EV educated macrophages on tenocyte bioactivity. (c) Additionally, the underlying pathogenesis of tendinopathy and the animal models of rotator cuff tears we use will be explored and further defined in the context of contemporary histologic and biomechanical methods.
Embargo
SARS-CoV-2 evolution and within-host variation in nonhuman animals
(Colorado State University. Libraries, 2024) Bashor, Laura, author; VandeWoude, Sue, advisor; Stenglein, Mark, committee member; Bosco-Lauth, Angela, committee member; Sloan, Dan, committee member; Gagne, Roderick B., committee member
The COVID-19 pandemic originated following spillover of SARS-CoV-2 from non-human animals into humans. Despite concentrated efforts before and after the pandemic, current research is constrained by the impracticality of witnessing initial host shift events and transmission dynamics that shape infectious disease emergence. SARS-CoV-2 transmission from humans to a range of domestic and wild species has been well documented; furthermore, spillback into humans from white-tailed deer, mink, hamsters, domestic cats, and lions has also been reported. SARS-CoV-2, like other RNA viruses, has the ability to adapt rapidly following host shifts. These cross-species transmission events can accelerate novel variant emergence through selection for genetic variation that improves virus fitness in a novel host environment. To evaluate the possibility that cross-species transmission accelerates SARS-CoV-2 evolution and variant emergence, we employed next-generation sequencing of viral genomes recovered from experimentally and naturally infected animals to characterize within-host virus populations. We demonstrated the use of experimental exposure studies as a controlled system to test hypotheses surrounding SARS-CoV-2 adaptation in cats (Felis catus), dogs (Canis lupus familiaris), hamsters (Mesocricetus auratus), ferrets (Mustela putorius furo), deer mice (Peromyscus maniculatus), bushy-tailed woodrats (Neotoma cinerea), Brazilian free-tailed bats (Tadarida brasiliensis), striped skunks (Mephitis mephitis), red foxes (Vulpes vulpes) and mule deer (Odocoileus hemionus). We also evaluated publicly available sequencing data from infected felids, and investigated within-host dynamics in natural infections of Amur tigers (Panthera tigris altaica), African lions (Panthera leo), and spotted hyenas (Crocuta crocuta) in a zoo environment. Our initial work investigated SARS-CoV-2 evolution across three passages in Vero cells and experimentally infected cats (n = 6), dogs (n = 3), hamsters (n = 3), and a ferret (n = 1). We observed the rapid selection and fixation of five SARS-CoV-2 mutations in Vero cells, followed by their reversion in dogs, cats and hamsters 1-3 days post-infection. We noted 14 emergent variants across the SARS-CoV-2 genome, including increased variation in the SARS-CoV-2 spike protein. Emergent variants included mutations not detected in the original virus stocks used for inoculation, and several defining mutations of variant lineages of concern in humans. Finally, we noted increased signs of adaptation in dogs, which did not shed infectious virus, including six nonsynonymous mutations in the SARS-CoV-2 open-reading frames (ORFs) encoding proteins for virus replication. In particular, this work underscored the potential for accelerated viral evolution in cell culture systems used commonly in virological research. This work has been published and represents Chapter 2 of this dissertation. Our next study built upon this work by investigating SARS-CoV-2 evolution in three experimental cohorts of domestic cats (n=23) infected through direct inoculation and cat-to-cat contact transmission. We observed high numbers of within-host variants in SARS-CoV-2 genomes recovered from cats compared to what is documented in humans, over half of which were nonsynonymous changes. The number of variants detected was positively correlated with the experimental dose of virus inoculum, and fewer variants were observed in contact cats. Similar to the previous study, mutations occurring at the same positions as defining VOC mutations, and signatures of positive selection in the viral spike (S) gene were observed. Our concurrent analysis of publicly available SARS-CoV-2 sequences showed no evidence for independent evolutionary trajectories associated with natural infections of domestic cats or other felids, and confirmed susceptibility of felids to the breadth of variants circulating in human populations. This work has also been published and represents Chapter 3 of this dissertation. We subsequently investigated SARS-CoV-2 evolution in longitudinal samples collected from Amur tigers (n=2), African lions (n=11), and spotted hyenas (n=4) infected during an outbreak at the Denver Zoo. Longitudinal nasal swabs were collected from infected individuals over an approximately three-month sampling period. We determined that the outbreak was caused by a single introduction of the Delta sublineage AY.20, which was a rare variant circulating in human populations at the time. We inferred a transmission chain from tigers to lions to hyenas, which was consistent with the appearance of clinical signs in infected animals. We observed expansion and diversification of within-host virus populations, and signatures of both purifying and positive selection. The strongest signs of positive selection were evident in the viral nucleocapsid (N) gene, and in viruses recovered from hyenas. Four candidate species-specific adaptive mutations, two of which are in the N gene, were identified in lions and hyenas (N A254V) and hyenas alone (ORF1ab E1724D, S T274I, and N P326). This work is presented in Chapter 4 of this dissertation. In Chapter 5, we evaluated a large dataset of peridomestic wildlife species experimentally infected with two SARS-CoV-2 variants, WA01 and Delta. Study species included deer mice (n=3), bushy-tailed woodrats (n=3), Brazilian free-tailed bats (n=4), striped skunks (n=5), red foxes (n=9), and mule deer (n=6). Distinct dynamics were observed in within-host virus populations recovered from WA01- and Delta- infected animals. This included increased within-host variation, relative effective population size, and genomic signatures of positive selection in WA01 animals. In contrast to our first study in domestic dogs, Brazilian free-tailed bats, which also did not shed infectious virus, did not show increased signs of adaptation. We also observed a potential host barrier to infection in skunks and one fox, followed by the emergence of potential de novo mutations. Six novel mutations were also detected in contact-exposed mule deer. Our findings suggest that mule deer populations, similar to what has been documented in closely related white-tailed deer, should be investigated for accelerated SARS-CoV-2 evolution. Collectively, our work reveals the unique dynamics of SARS-CoV-2 evolution and transmission in both naturally- and experimentally- infected felids. We observed rapid viral adaptation both in vitro and in vivo, highlighting advantages and limitations of experimental animal infections for studies of viral evolution. In each study, we used publicly available data to contextualize our experimental data and identify broader patterns. Furthermore, we identified specific SARS-CoV-2 mutations and genomic regions under selective pressures across a range of animal species, setting the groundwork for future mechanistic studies. Our findings underscore the importance of a One Health approach to understanding SARS-CoV-2 evolution, and the need for surveillance in animal populations.
Open Access
From computation to communication: unveiling Salmonella metabolic plasticity and public perceptions of the microbial world using multi-omics and thematic analysis
(Colorado State University. Libraries, 2024) Kokkinias, Katherine, author; Wrighton, Kelly C., advisor; Kelp, Nicole, committee member; Borlee, Brad, committee member; Weir, Tiffany, committee member
Research and communication on microorganisms and microbiomes has become increasingly important in recent decades due to evolving threats posed by infectious diseases and microbial contributions to ecological systems. Antibiotic resistance presents a significant challenge to global health equity, with nontyphoidal Salmonella infections being a prominent concern. Despite its prevalence and impact, Salmonella infections lack effective vaccines, posing a serious threat to vulnerable populations. Concurrently, misconceptions and misinformation about microorganisms and microbiomes can arise given the dynamic nature of scientific research which can hinder effective science communication and health outcomes. Despite this, little is known about public perceptions of microorganisms and microbiomes, impeding our ability to create effective, tailored science messaging. Both basic pathogen research and science communication research are essential to identify targeted prevention strategies and to understand public perceptions of microorganism and microbiomes. This dissertation spans microbiome and science communication research, employing both qualitative and quantitative methods. The overarching research goals of this dissertation are to 1) lay the groundwork for therapeutics by studying Salmonella metabolism and metabolic plasticity, 2) develop a multi-omics repository to expand the usability of our omics datasets, and 3) understand public perceptions of microorganisms and microbiomes to improve future microbial science communication efforts. Chapter 1 as the introductory chapter reviews the current state of Salmonella and science communication research, providing a context for the new research presented in this dissertation. Through a multi-omics approach, Chapter 2 explores the metabolic strategies of Salmonella under different diet backgrounds and over time, offering insights into potential therapeutic targets. Chapter 3 introduces the CBA_DREAMM database, facilitating centralized storage and sharing of multi-omics datasets to enhance communication of our research and collaboration in microbiome research. Chapter 4 investigates public perceptions of microbes and microbiomes in the United States, revealing a need for tailored science communication efforts. Additionally, the study emphasizes the importance of clear communication, trust, and emotions, like apathy, in science communication. Chapter 5 is the conclusion, summarizing findings from Chapter 2, 3, and 4 and describing future directions. By bridging natural and social sciences, this dissertation aims to inform strategies for tackling global issues by advancing microbiome and science communication research.
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The adventures of Lactobacillus acidophilus: evaluating a recombinant probiotic rotavirus vaccine from host and microbial perspectives
(Colorado State University. Libraries, 2024) Gilfillan, Darby L., author; Vilander, Allison, advisor; Dean, Gregg, advisor; Abdo, Zaid, committee member; Wilkins, Mike, committee member
Rotavirus is an enteric infection of global importance causing diarrheal-associated illness that can be fatal in young children and the elderly. There is a gap in vaccine efficacy between high- and lower-middle-income countries (LMIC) with LMIC often experiencing diminished vaccine-conferred protection. Rotaviruses, whether attenuated vaccine strains or primary pathogens, do not exist in isolation within the host's gastrointestinal tract. Other actors present within the microbiome can inhibit or augment vaccine efficacy by influencing the vaccine itself or the mucosal immune response. Understanding and exploiting interactions between host and microbe is a promising frontier for mucosal vaccinology. This dissertation will explore the probiotic Lactobacillus acidophilus (LA) as a vaccine platform for a microbiome-minded, next-generation approach to rotavirus immunization. We developed and confirmed a novel recombinant LA (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results demonstrate the potential of rLA platforms to generate protective mucosal immunity. Additionally, metagenomic and metatranscriptomic analyses of exogenous probiotic administration within the murine small intestine revealed differences between LA genome expression and the whole metatranscriptome in recombinant- versus wild-type LA-vaccinated mice. LA genome expression in rLA-vaccinated mice had decreased carbohydrate metabolism and increased stress responses. We also detected antigen and adjuvant transcript expression only in mice exposed to the rLA platform. There was relative enrichment of probiotic species in the wild-type group with overall increased α- and β-diversity in the buffer compared to probiotic groups. These results highlight the interactions between an exogenous probiotic and the host microbiome at an immune inductive site. Finally, we used an in vitro model to evaluate modulation of polyunsaturated fatty acid (PUFA) metabolism on host cell and (r)LA interactions. Both (r)LA and PUFA treatments significantly changed pathogen recognition receptor expression. (r)LA treatment mainly altered inflammatory cytokine expression while PUFA supplementation primarily influenced mucin expression. rLA strains adhered more to host cells than wild-type LA while the rLA strain expressing both antigens and adjuvants may better prevent E. coli adhesion. These results and methodologies provide a starting point for further investigation into PUFA metabolism as a mechanism for improving rLA immunogenicity and competition against other enteric pathogens.
Open Access
Design and application of a droplet-digital PCR assay for detection of the STAT5BN642H mutation in feline T cell neoplasia
(Colorado State University. Libraries, 2024) Bork, Sydney Bonnie, author; Avery, Anne, advisor; Olver, Christine, committee member; Webb, Craig, committee member
Lymphoma is a commonly diagnosed hematopoietic neoplasm in cats. Small Cell T-cell Epitheliotropic Intestinal Lymphoma (SCL) is the most reported subtype of lymphoma in cats. Cats with SCL are presented with non-specific clinical signs such as chronic vomiting, diarrhea, and weight loss. Diagnostic work-up often includes collection of intestinal biopsies with histopathology for diagnosis. SCL is characterized by infiltration of neoplastic lymphocytes into the intestinal epithelium and lamina propria of the small intestines. Neoplastic cells are small to intermediate in size and of T-cell origin. Diagnosing SCL can be challenging for pathologists because cats also commonly develop a condition called inflammatory bowel disease (IBD), which has an almost identical clinical presentation and similar histopathologic patterns. However, in IBD, the lymphocytic infiltration is often heterogeneous (termed "lymphoplasmacytic enteritis"). When histopathology results are inconclusive, assessment of expression with immunohistochemistry markers can help further characterize the cell population. Additionally, advancements have been made with lymphocyte clonality testing by PARR (PCR for Antigen Receptor Rearrangement), a DNA-based assay that evaluates T-cell receptor (TCR) and Immunoglobulin (Ig) gene rearrangements. Cats diagnosed with SCL demonstrate a clonal TCR result, while cats with IBD demonstrate a polyclonal TCR result. Unfortunately, there are still cases where histopathology and PARR results are equivocal. Recent work in feline medicine has demonstrated that cats with SCL exhibit high expression of phosphorylated STAT5B with immunohistochemical staining on small intestinal biopsy samples compared to cats with IBD. Importantly, one group detected a STAT5BN642H mutation in cats diagnosed with SCL. In this study, 40% (17/42) of cats with intestinal lymphoma were classified as SCL by histopathology. A combination of Sanger sequencing and ARMS qPCR detected the STAT5BN642H mutation in 29.4% (5/17) of cats with SCL. This work correlates to a comparable disease entity in people, monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), which has reported the prevalence of the STAT5BN642H mutation to be 22-57%. Our group aimed to develop a droplet digital PCR (ddPCR) assay to detect wild-type and mutated STAT5B in cats. Our first aim was to design specific primers and locked-nucleic acid hydrolysis probes to detect and discriminate between wild-type and mutated STAT5B. The first step included analyzing data from control samples using wild-type DNA from cats without neoplasia and a positive control gene fragment block ("gBlock"). The second step included analyzing two cohorts of young cats (<6 years of age) without a diagnosis of lymphoid neoplasia to assess assay performance and determine if the mutated STAT5B could be considered a germ line polymorphism. The fractional abundance was calculated from the ddPCR data, estimating the percentage of mutated copies within a positive sample. The results of the first aim demonstrated that our ddPCR assay can distinguish between wild-type and mutated STAT5B with high sensitivity. Most young cats without a diagnosis of lymphoid neoplasia do not carry the STAT5BN642H mutation. Two cats with marked lymphoplasmacytic enteritis had detectable mutated STAT5BN642H. The second aim was to evaluate the prevalence of the STAT5BN642H mutation in cats with confirmed SCL. A sub-aim was to evaluate cats with CD4 T-cell leukemia to determine if this mutation could be found in other forms of T cell lymphoid neoplasia. The results from this aim demonstrate that cats with SCL frequently carry the STAT5B mutation (66.7%). We also discovered that this mutation is not exclusive to cats with SCL, as almost half of the cats with CD4 T-cell leukemia also carry this mutation (47.7%). These findings shed light on the prevalence of the STAT5BN642H mutation in cats with SCL and CD4 T-cell leukemia. This data suggests potential implications for ddPCR mutation detection to help further differentiate and diagnose cats with T cell neoplasia versus those with inflammatory conditions (such as SCL versus IBD), and investigate novel therapies (i.e., JAK/STAT inhibitors). Further research is warranted to investigate other JAK/STAT pathway mutations, particularly in cats where the STAT5BN642H mutation was not detected. Larger outcome studies should investigate the correlation of STAT5BN642H mutation status and the fractional abundance to evaluate disease risk, treatment response, and survival.
Embargo
Assessing antimicrobial mechanisms in Langerhans cells during a Mycobacterium leprae infection
(Colorado State University. Libraries, 2024) Fischbacher, Linda, author; Belisle, John, advisor; Gries, Casey, committee member; Tesfaye, Dawit, committee member
Langerhans cells are essential immune cells in the skin that maintain homeostasis and clear pathogens. Despite their importance, much is unknown about Langerhans cells, including their innate antimicrobial mechanism. Single-cell sequencing of leprosy skin lesions identified genes upregulated in Langerhans cells of reversal reaction lesions that may be associated with antimicrobial activity. CCL22, MPEG1, and IDO1 were selected to study further as potential effectors in Langerhans cells for killing Mycobacterium leprae. We hypothesized that altered metabolic processes in Langerhans cells impact antimicrobial effects on M. leprae. An in vitro model was developed to induce antimicrobial gene expression in Langerhans cell-like dendritic cells (LCDCs). IL-1β was identified as the best inducer of CCL22 and MPEG1, and IFN-γ as the best inducer of IDO1. Induction was measured by gene expression and protein production, as well as enzyme activity for IDO1 by measuring metabolites. The antimicrobial effect of IDO1 on intracellular M. leprae in LCDCs was assessed by inducing IDO1 with IFN-γ or inhibiting IDO1 activity with 1-methyltryptophan. Stimulation by this agonist or this antagonist modulated IDO1 expression and activity but did not affect M. leprae viability. The changes of intracellular tryptophan catabolites in non-stimulated and M. leprae-infected LCDCs were measured. The M. leprae infection increased two kynurenine pathway catabolites after 24 and 48 hours, 3-hydroxyanthranilic acid and quinolinic acid. These data indicate that while M. leprae did not induce IDO1 expression, it did increase IDO1 and kynurenine pathway activity. Neither metabolite has reported antimicrobial properties, but quinolinic acid may benefit M. leprae for synthesizing nicotinamide adenine dinucleotide. A different tryptophan catabolite pathway leads to serotonin production. In M. leprae-infected LCDCs, serotonin was decreased, but 5-hydroxyindoleacetic acid, a breakdown product of serotonin, was increased. The implication of these changes for an M. leprae infection of LCDCs is unknown. 5-hydroxyindoleacetic acid is also increased in Mycobacterium tuberculosis patients. This metabolite may benefit these mycobacteria as it is reported to increase PPARγ activity, which is known to support M. leprae and M. tuberculosis in macrophages. The main antimicrobial mechanism of IDO1 is depleting tryptophan from tryptophan auxotrophic pathogens. Whether the tryptophan biosynthesis pathway in M. leprae is functional was assessed using 13C-tracing, to determine if tryptophan depletion by IDO1 could kill M. leprae. In axenic media, M. leprae did not synthesize tryptophan from 13C-glucose and 13C-palmitic acid nor synthesize tryptophan from intracellular 13C-glucose. In vitro, M. leprae only synthesized tryptophan from an intermediate, anthranilic acid. Using the same method, M. tuberculosis synthesis of tryptophan from 13C-glucose was confirmed as a control. The functionality of the tryptophan biosynthesis pathway in M. leprae could not be confirmed. However, because of the homology between the M. leprae and M. tuberculosis genes for tryptophan biosynthesis, this pathway likely is functional, and M. leprae would not be killed by IDO1-mediated tryptophan depletion. These findings indicate that IDO1 is not associated with antimicrobial activity towards M. leprae in LCDCs. Instead, increased IDO1 activity induced by M. leprae infection resulted in increased tryptophan catabolites likely to benefit rather than kill M. leprae in LCDCs. M. leprae likely evades the primary killing mechanism of IDO1, tryptophan depletion, by possessing an intact pathway for tryptophan biosynthesis. Further studies to elucidate the importance of quinolinic acid and 5-hydroxyindoleactic acid for M. leprae and validate that the M. leprae tryptophan biosynthesis pathway is functional will aid in identifying essential pathways for M. leprae that can be targeted with therapeutics. Other potential antimicrobial effectors in LCDCs, including CCL22 and MPEG1, will need to be assessed to study this innate mechanism in Langerhans cells further.
Open Access
Linking mosquito midgut and virus population biology at the molecular and cellular level
(Colorado State University. Libraries, 2024) Fitzmeyer, Emily Anne, author; Ebel, Gregory D., advisor; Stenglein, Mark, committee member; Kading, Rebekah, committee member; Anderson, Brooke, committee member
Vector competence (VC) refers to the efficiency of pathogen transmission by vectors. Each step in infection of a mosquito vector constitutes a barrier to transmission that may impose bottlenecks on virus populations. West Nile virus (WNV) is maintained by multiple mosquito species with varying VC. However, the extent that bottlenecks and VC are linked is poorly understood. Similarly, quantitative analyses of mosquito-imposed bottlenecks on virus populations are limited. We used molecularly barcoded WNV to quantify tissue-associated population bottlenecks in three variably competent WNV vectors. Our results confirm strong population bottlenecks during mosquito infection that are capable of dramatically reshaping virus population structure in a nonselective manner. In addition, we found that mosquitoes with differing VC uniquely shape WNV population structure: highly competent vectors are more likely to contribute to the maintenance of rare viral genotypes. These findings have important implications for arbovirus emergence and evolution. The mosquito midgut functions as a key interface between virus and vector. However, studies of midgut physiology and associated virus infection dynamics are scarce, and in Culex tarsalis - the primary vector of West Nile virus (WNV) in the contiguous United States - nonexistent. We performed single-cell RNA sequencing on dissociated, WNV-infected Cx. tarsalis midguts. We identified populations of distinct midgut cell-types consistent with existing descriptions of insect midgut physiology and found that all midgut cell populations were permissive to WNV infection. However, we observed high levels of viral RNA suggesting enhanced replication in enteroendocrine cells and cells enriched for mitochondrial genes. In addition, we found no significant upregulation of mosquito immune genes associated with WNV infection at the whole-midgut level, rather, a significant positive correlation between immune gene expression and WNV viral RNA load at the individual cell level. These findings illuminate the midgut infection dynamics of WNV, providing insight into cell-type specific enhancement of, and immune response to, WNV infection in a primary vector.

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