Theses and Dissertations

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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.
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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.
Embargo
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.
Embargo
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.
Open Access
A QuPath workflow utilizing machine learning to analyze homing protein specificity and penetration into lung granulomas of Mycobacterium tuberculosis infected mice
(Colorado State University. Libraries, 2024) Patterson, John, author; Gonzalez-Juarrero, Mercedes, advisor; Lyons, Mike, committee member; Ghosh, Soham, committee member
Targeted delivery of drugs to the lungs can improve TB chemotherapy and thus our goal is to develop TB-drug loaded nanoparticles tagged to pulmonary homing peptides. In a previous study, homing peptides to the lungs of TB diseased animals were identified using preclinical TB models (Balb/c and C3HeB/FeJ mice). The selection of homing peptides was carried out using a phage library containing peptides with known homing affinity in other diseases (e.g. cancer). Having identified and selected the homing peptide PL1 (PPRRGLIKLKTS) to granulomas present in the lungs of murine TB models, the PL1 peptide and a negative control (scrambled LinnTT peptide) were tagged to Fluorescein Amidites (FAM). To facilitate tracking in vivo of the nanoparticles to be loaded with TB drugs, silver nanoparticles (SNP) were conjugated to Cy3 fluorochrome, a fluorescent marker used in in vivo tracking studies, followed by functionalization with the PL1 homing peptide (PL1-SNP) or biotin as negative control (Ctrl-SNP). Tracking and homing of the PL1 peptide to granulomas was possible after in vivo administration via intraperitoneal (IP) or intravenous (IV) route of either the FAM tagged synthetic peptides or Cy3-SNPs to Mycobacterium tuberculosis (Mtb) infected C3HeB/FeJ mice. Visualization of the fluorescence-tagged carriers within the lungs was performed using microscopic slides affixed with lung sections from each mouse followed by whole slide imaging. The semi-quantitative analysis of the fluorescence whole slide images performed using the QuPath workflow confirmed that PL1-FAM, or PL1-SNP homed to the granulomas. Thereafter, a QuPath workflow was developed that uses machine learning approaches (MLP) for unbiased identification of tissue types. Other tools were used for characterization and quantification of FAM (synthetic peptides) and Cy3 (SNP) positive cells within granulomatous lesions of the C3HeB/FeJ TB mouse model. Moreover, it was important to quantify the penetration capacity of the FAM tagged peptide as well as the peptide coated SNP into granulomas. QuPath also includes a built in MLP pixel classifier for unbiased segmentation of the whole slide. In addition, a modified QuPath script was developed to segment the granulomas into concentric regions (outer, inner and center) followed by detection and quantification of positive cells for either fluorochrome within each region. Specific colocalization of PL1 with its known receptor (FN-EDB), either as a synthetic peptide or coupled to the SNP, was also studied using lung sections from mice treated with PL1-FAM or PL1-SNP and counter stained with Alexa 647 conjugated anti-FN-EDB monoclonal antibodies. The modified QuPath script was trained to quantify fluorescence from Alexa 647 in cells within granulomas and the Pearson coefficient and QuPath script was used to assess PL1 and FN-EDB colocalization within each region of the granuloma. The results demonstrated that when compared to their respective control samples, the IP route of administration provides equal or better homing of PL1 peptide to the granulomas than the IV route. Both the PL1-FAM and PL1-SNP home to the granulomas and specifically colocalize with its receptor FN-EDB. The FAM tagged peptide and SNP penetrate to the inner and center regions of the granuloma whereas the control SNP were unable to penetrate the barrier in the outer region of the granulomas. The QuPath workflow developed here can be used for tracking and quantification of other homing peptides and nanoparticles for development of new TB therapeutics.
Open Access
Spatial analyses of vector-borne disease risk for allocation of disease prevention and control resources
(Colorado State University. Libraries, 2009) Winters, Anna M., author; Eisen, Lars, advisor; Moore, Chester G., advisor
Vector-borne diseases are major threats to human health and well-being, inflicting enormous health and economic burden around the world. Meeting the challenge of emerging and resurging vector-borne diseases is a difficult undertaking as vaccines are limited, and future vaccine prospects are slow in coming. This underscores the importance of appropriately applied vector control and disease prevention efforts to decrease morbidity and mortality caused by vector-borne diseases. The objective of this dissertation was, therefore, to investigate spatially and temporally-explicit methods to enhance targeting of vector control and disease prevention efforts. Three diseases were investigated: West Nile virus disease in Colorado, USA; human plague in the West Nile region of Uganda; and dengue in Merida, Mexico. Various methods were explored to present spatial information representing risk of vector-borne diseases. Spatial models were developed indicating the risk of human exposure to WNV in the state of Colorado; and Yersinia pestis, the causative agent of plague, in Uganda. The resulting models provided robust validations indicating that the spatial risk maps contained useful information for disease prevention and control efforts. The final chapter expanded from spatially-explicit methods to also account for the temporal aspect of disease outbreaks or epidemics. This chapter describes the evaluation of the feasibility of an early detection system to identify outbreaks in a timely fashion in Merida, Mexico. The early detection system has the potential to provide Merida public health authorities with a resource to clearly recognize when the current dengue burden is exceeding historical norms and may be applicable and useful in other dengue endemic areas. The chapters composing this dissertation describe the application of cross-cutting methods used to determine spatial risk in different vector-borne disease systems. Furthermore, the methods used are applicable to the current public health situation where new vector-borne diseases are emerging and "old" diseases are resurging, further underscoring the importance of targeting limited public health resources.
Open Access
Drug efflux systems and antibiotic resistance in Burkholderia pseudomallei
(Colorado State University. Libraries, 2009) Trunck, Lily, author; Schweizer, Herbert P., advisor
Burkholderia pseudomallei, the etiologic agent of melioidosis, is intrinsically resistant to most antibiotics. A predicted 10 RND efflux transporters are encoded by the B. pseudomallei genome; 3 have been characterized (AmrAB-OprA, BpeAB-OprB, and BpeEF-OprC) as major contributors to the intrinsic aminoglycoside, macrolide, chloramphenicol, and trimethoprim resistance of this organism. AmrAB-OprA is constitutively expressed in most strains and confers resistance to aminoglycosides. Gentamicin susceptible clinical isolates have been identified and work in this dissertation demonstrates that such susceptibility occurs as a result of either insufficient expression or deletion of amrAB-oprA. The mechanisms regulating expression in these strains are unclear, but are not related to mutations in this operon's putative repressor (AmrR) or mutations in the regulatory regions of amrAB-oprA. Expression analysis of seven B. pseudomallei RND efflux pumps (amrB, bpeB, bpeF, bpeH, BPSL0309, BPSL1267, and BPSL1567) in 60 clinical and environmental B. pseudomallei isolates from Thailand demonstrated (i) efflux pump expression is prevalent in both clinical and environmental strains (ii) bpeH is expressed at a higher level in clinical isolates as compared to environmental isolates and (iii) efflux pump expression correlates with resistance/susceptibility to several antibiotics. These data suggest possible substrates for uncharacterized efflux pumps or, alternatively, co-regulation of resistance determinants. Since a deficit in efflux resulted in susceptibility to otherwise clinically useful drugs and that expression of efflux pumps was prevalent in B. pseudomallei, we suggest that efflux pump inhibitors would broaden the spectrum of antibiotics useful for treatment of melioidosis. To facilitate discovery and characterization of such compounds, we have developed a panel of efflux deficient B. thailandensis strains that express the B. pseudomallei efflux pumps, amrAB-oprA and bpeAB-oprB, which can be handled under BSL2 conditions. When expressed in the surrogate background, these pumps have the same substrate profile and can be inhibited by clinically insignificant efflux pump inhibitors to a degree similar to that observed in the native background. In summary, we have assessed the contribution of efflux to antibiotic resistance in B. pseudomallei and described a tool for discovery and characterization of efflux pump inhibitors for pumps expressed in this organism.
Open Access
Tumor-associated macrophage recruitment and regulation of angiogenesis
(Colorado State University. Libraries, 2008) U'Ren, Lance W., author; Dow, Steve W., advisor
Tumors are no longer thought of as purely a mass of transformed cells. A major component of the cellular composition of a tumor is infiltrating immune cells. Macrophages can constitute a large proportion of infiltrating immune cells. In many cases, increased numbers of Tumor-Associated Macrophages (TAMs) can be associated with a poorer prognosis. Utilizing mice which lack a functional type I interferon receptor (IFN-α/βR-/-), we found that endogenous levels of type I IFNs control tumor growth and angiogenesis. We also determined that tumors grown in IFN-α/βR-/- mice have an increase in macrophage infiltrate. In vitro assays suggest that suppression of macrophage responsiveness to CSF-1 by type I IFNs was responsible for the increased macrophage accumulation in tumors of mice unable to respond to type I IFNs. These results indicate that endogenous production of type I IFNs by tumor cells or inflammatory cells may be an important means of suppressing the accumulation of TAMs and their subsequent induction of angiogenesis. The ability of TAMs to produce VEGF is one of the major means by which TAMs are known to induce tumor angiogenesis. Since VEGF expression is in part induced by hypoxia, it has been speculated that the hypoxic tumor environment is responsible for driving TAM VEGF production. As an alternative possibility we suggest that the engulfment of apoptotic tumor cells can stimulate TAM production of VEGF. We determined that the use of Liposome DNA-complex (LDC) therapy can induce anti-tumor immunity through the combined activation of systemic innate and adaptive immune responses. We show that LDC can traffic into macrophages and induce expression of activation markers. In vitro results show that LDC therapy can inhibit the production of VEGF by macrophages after their consumption of apoptotic cells, suggesting that LDC may be an effective way to circumvent the pro-tumor function of TAMs. Additionally, we determined that LDC combined with chemotherapy can be used as a safe and effective immunotherapy for the treatment of canine hemangiosarcoma. Taken together, these findings could uncover new avenues in which TAMs can be targeted and identified a novel immunotherapy as a potential candidate.
Open Access
Investigating molecular determinants of FIV pathogenesis
(Colorado State University. Libraries, 2009) Thompson, Jesse Alan, author; VandeWoude, Susan, advisor
Five FIV clades have been identified and are distinguished by envelope sequence. Two isolates, FIV-PPR and FIV-CPG (molecular clone FIV-C36), belonging to clades A and C, respectively, are variable with regard to disease potential. Chimeric viruses constructed between phenotypically distinct strains of FIV are potentially useful tools to identify molecular determinants of virulence. Several chimeric constructs were therefore developed by exchanging elements between FIV-C36 and FIV-PPR. FIV-PCenv and FIV-PC3'LTR were two resulting chimeras that were capable of competent in vitro replication. Studies in Chapter One aimed to characterize these viruses in the domestic cat model and to test the hypothesis that elements surrounding the env region contribute to in vivo pathology. FIV-PC3'LTR, containing FIV-C36 rev2 and 3' LTR was infectious, although attenuated compared to parental constructs. FIV-PCenv, containing FIV-C36 vif, otfA, env, and the first exon of rev, displayed a phenotype intermediate to parental viruses with regard to replication kinetics, and CD4+ T-cell and neutrophil declines, but peak viral load and development of clinical disease was delayed by three weeks compared to FIV-PPR, FIV-PC3'LTR, and FIV-C36. Studies in Chapter Two evaluate potential mechanisms for the delayed phenotype of FIV-PCenv. To test the hypothesis that delayed kinetics observed during FIV-PCenv infections was due to adaptation of the construct during first round infections, pooled plasma from the in vivo study described in Chapter One was used to inoculate a second cohort of cats. Passaged FIV-PCenv again displayed intermediate phenotype in terms of viral replication and immunopathology, but onset of acute viremia was no longer delayed. To further pinpoint particular genes that contribute to FIV pathogenesis, three additional chimeras were generated using PCR-driven overlap extension as described in Chapter Three. Overlapping-PCR was utilized to produce chimeras with specifically substituted ORF genes encoding the FIV-C36 regulatory proteins Vif and OrfA for those from FIV-PPR; chimeras FIV-PCvif, FIV-PCvif/orfA, and FIV-PCorfA were successfully constructed. Upon infection of the feline MYA-1 T-cell line, all three chimeras produced measurable RT activity. Further, in vitro analysis of FIV-PCvif/orfA demonstrated that this construct had replication properties equal to those of FIV-C36 as measured by viral capsid and RNA genome production.
Open Access
Host cell antigen and T-lymphocyte subset contribution to simian immunodeficiency virus pathogenicity
(Colorado State University. Libraries, 2008) Stump, Debora Shawn, author; VandeWoude, Sue, advisor
The continuity of the host cell plasma membrane and the simian immunodeficiency virus (SIV) envelope at the time of budding results in the incorporation of host membrane antigens. Of these host antigens, major histocompatibility complex class II (MHCII), is abundantly represented on the virion surface. In Chapter 1, the investigation the potential of antibodies specific for MHCII to block viral infection by binding viral envelope MHCII in vitro is presented. Our results did not demonstrate viral neutralization associated with anti-MHCII antibodies but illustrate that viral infectivity is influenced by target cell membrane and immunological signaling characteristics. In Chapter 2 we investigated the utility of alloimmunization of genetically divergent rhesus macaques in eliciting immune responses specific for host cell antigens capable of limiting SIV infectivity in vivo. Our results suggest that alloimmune responses are not sufficient to protect animals from SIV challenge. We were also able to assess differences in response to pathogenic SIV infection in rhesus macaques of Chinese origin (ChRh) compared to Indian origin (InRh) in Chapter 3. ChRh in our study were better able to control viral replication and resist disease progression compared to InRh. Peripheral immunocyte kinetics were evaluated using four color flow cytometry in order to define parameters of the differential immune response. No consistent differences were evident, demonstrating that peripheral immune correlates of viral control and disease progression remain unknown. Natural SIV infection has been identified exclusively in primate species inhabiting continental Africa. Serological evidence of exogenous lentiviral infection has been noted in wild lemurs in Madagascar. In Chapter 4, we investigated evidence of a naturally occurring lentivirus, possibly related to African SIVs, in samples from a captive population of L. catta at the Indianapolis Zoo. We show confirmatory serological reactivity to diverse lentiviral antigens but failed to amplify lentiviral specific sequences using established degenerate primer sets. In total, this work represents investigations that interrogate important aspects of nonhuman primate lentiviral pathogenicity. While results were primarily negative in nature, these studies provide important new information and point to additional studies required that will continue investigations into the complex nature of lentiviral host: virus relationships.
Open Access
Vector and virus interactions: La Crosse encephalitis virus and the mosquito vector Aedes (stegomyia) albopictus
(Colorado State University. Libraries, 2008) Sutherland, Ian W., author; Beaty, Barry J., advisor
La Crosse encephalitis continues to be an important cause of pediatric arboviral encephalitis in the United States. Since 1985, the invasive mosquito vector, Aedes albopictus, has spread across the country and into La Crosse virus endemic regions. As an aggressive, daytime feeder, this vector has the potential to change the epidemiology of La Crosse encephalitis. This study investigated 4 components of the La Crosse virus- Ae. albopictus system: (1) time course of disseminated and filial infection rates (FIR) among recently colonized field strains, (2) anatomic basis of ovarian infection during the 1st gonotrophic cycle, (3) mitochondrial DNA (mtDNA) variation among geographically dispersed populations in the U.S., and (4) development of transovarially susceptible and refractory strains of Ae. albopictus. All geographic strains tested are susceptible to La Crosse virus oral infection and capable of transovarial transmission (TOT). No regional or geographic patterns emerged with respect to dissemination or TOT. 1st gonotrophic cycle vertical transmission was observed at low levels, with an FIR averaging 1%. 2nd gonotrophic cycle FIR averaged approximately 10% and was significantly lower than that of the natural vector, Ae. triseriatus. La Crosse virus antigen (Ag) was detected in ovaries by Day 2 after oral infection and prior to detection in head tissues. Ag was not detected in follicles through Day 7, suggesting vertical transmission. Examination of variation in the ND5 mtDNA marker revealed high levels of homogeneity among U.S. Ae. albopictus populations. Only 2 haplotypes were observed from 16 geographically dispersed states, including Hawaii. Such broad homogeneity could be due to multiple factors, including founder effects and cytoplasmic incompatibility. Ae. albopictus responded poorly to selection based on FIR for the development of susceptible and refractory strains. This study supports prior literature suggesting Ae. Albopictus may be an important secondary vector of La Crosse virus.
Open Access
Characterization of the mannosyltransferase RV3779, which forms polyprenyl phosphomannose for the biosynthesis of phosphatidylinositol mannoside and lipoarabinomannan, and elucidation of the polymerization stages of galactan biosynthesis in mycobacteria
(Colorado State University. Libraries, 2008) Scherman, Hataichanok, author; Brennan, Patrick J., advisor
The cell wall complex of mycobacteria, dominated by highly unique structural polysaccharides and a rather impermeable layer of lipids, can give these bacteria the ability to resist the immune response and cause a prolonged and deadly illness. Various lipoglycans and glycolipids in the cell wall complex of mycobacteria, such as lipoarabinomannan (LAM), as well as the synthetic precursors of LAM; lipomannan, and the phosphatidyl myo-inositol mannosides, are essential for the normal growth and viability of mycobacteria, although the specifics of their synthesis are still not fully defined. Their synthesis are governed by a myriad of enzymes, including a class of enzymes called glycosyltransferases, which are one of the most diverse and important groups of enzymes in nature. A particular glycosyltransferase, Rv3779, is the primary focus of this study, and from sequence and bioinformatic analysis, we identified it as a putative mannosyltransferase belonging to the GT-C superfamily found to be involved in various aspects of synthesis of the higher forms of the phosphatidyl myo-inositol mannosides and the subsequent lipoglycans. Rv3779 is present in a prominent gene cluster involved in cell wall biosynthesis. From the experimental evidence gathered from construction and analysis of a Rv3779 knockout mutant of M. tuberculosis, the in vitro assay of the membrane fractions of an Rv3779-overexpressed strain of M. smegmatis, and analysis of the reaction products by thin layer chromatography and mass spectrometry, we have concluded that Rv3779 possesses polyprenyl phosphomannose synthase activity in a homologous manner to Ppm1. Polyprenyl phosphomannose is the key mannosyl donor that is utilized heavily by later enzymes in the extracytoplasmic hypermannosylation of the higher forms of these lipoglycans. The galactan chain serves as the covalent attachment point for mycolated arabinan and is a central structural polymer in the cell wall complex that is the second focus of this study. The exact stages of the buildup galactan are not fully elucidated, and involve unique polymerization steps. We have utilized various organic extraction and analytical techniques from a cell-free assay utilizing UDP-D-galactofuranose as a donor and the results of these studies suggest that the galactan chain is polymerized one galactofuranosyl residue at a time.
Open Access
Molecular epidemiology of leprosy in Cebu, Philippines and essentiality and characterization of an arabinosyltransferase involved in the cell wall synthesis of mycobacteria
(Colorado State University. Libraries, 2009) Sakamuri, Rama Murthy, author; Vissa, Varalakshmi D., advisor
Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae, which affects the skin, peripheral nerves and mucous membranes. Approximately a quarter million new cases are detected annually throughout the world. Since M. leprae is an obligate intracellular bacterium, not cultivable under in vitro conditions, and with a long incubation time, the causes for continued prevalence in endemic countries are still unknown. In this regard, molecular tools for differentiation of isolates of M. leprae are needed to track and control transmission of leprosy. Genome sequencing of M. leprae facilitated the identification of genetic markers such as variable number of tandem repeat (VNTR) and single nucleotide polymorphic (SNP) loci. We standardized methods for DNA extraction from the clinical samples and also developed rapid, high throughput and inexpensive methods like multiplex-PCR and fragment length analysis (FLA) for VNTR analysis; polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for SNPs to facilitate molecular strain typing of clinical samples. We have applied these methods on the DNA extracts of stored and new samples obtained from leprosy patients from Leonard Wood memorial (LWM), Cebu, Philippines. We compiled a database containing epidemiological information from over 200 leprosy patients. VNTR alleles were found to be highly concordant when obtained from biopsies and slit skin smears (SSS) from the same patients indicating that either can be used for strain typing. Based on parsimony cluster analysis of 207 clinical isolates, five major groups were observed. VNTR markers alone were able to differentiate the SNP type 3 isolates from the majority of the SNP type 1 isolates. VNTR profiles of the M. leprae isolates within the multicase families (MCFs) were similar indicating common source of transmission. In a continuation study, VNTR data was obtained for another seventy samples from newly diagnosed leprosy patients from LWM, Cebu, Philippines. Multiplex PCR and FLA methods were found to be more efficient and accurate compared to short tandem repeat (STR) amplification and DNA sequencing. Intra-patient VNTR variability using different SSS was found to be minimal. Population structure of M. leprae isolates from Cebu remained similar with stable clusters in the phylogentic tree, even with the inclusion of 70 additional samples. Drug resistance is a cause for concern in the treatment and control of any infectious disease. Drug resistance in leprosy was known to be caused during the dapsone monotherapy control program and patients' irregular treatment. WHO MDT which includes anti-leprosy drugs such as dapsone, rifampicin and clofazimine was introduced in 1982. However drug resistance was reported even in the areas where the MDT was implemented successfully. So to evaluate the primary drug resistance in newly diagnosed leprosy patients who are being treated with the standardized MDT regimen, we developed a multiplex PCR for amplifying the drug resistance determining regions (DRDR) in genes rpoB, folP1, gyrA and gyrB, which are targets of the drugs rifampicin, dapsone and oflaxacin, respectively. No mutations were detected in rpoB and folP1 DRDRs for 77 samples and in the gyrA DRDR for 15 samples tested thus far. A separate chapter describes the studies on the cell wall biosynthesis. The cell envelope of mycobacteria are comprised of a core macromolecule known as mycolyl arabinogalactan (mAG) which is covalently attached to the peptidoglycan layer forming the mAGP complex and other macromolecules such as lipomannan and lipoarabinomannan. The mAGP complex is essential for survival of the bacteria and is a permeability barrier. The synthesis and assembly of this mAGP complex is still not fully understood. In this regard, through comparative genome approaches we identified Rv3805c as a putative glycosyltransferase located in the cell wall synthesis gene cluster. Rv3805c predicted to have ten transmembrane domains, contains a conserved glycosyltransferase and proline rich motif also present in Emb proteins which are involved in cell wall biosynthesis. In this study we identified that, Rv3805c encodes an arabinosyltransferase through an in vitro arabinosyltransferase assay. Furthermore, a knock out mutant of an ortholog gene NCgl2780 was generated in C. glutamicum. The structural analysis of AG of NCgl2780 mutant in C. glutamicum revealed the loss of β(1→2) Araf residues at the non-reducing ends. We also indentified that the ortholog MSMEG_6400 is essential for M. smegmatis, as we were unable to obtain a genetic knock out mutant in the absence of an additional functional copy of the gene.
Open Access
Investigations of the evolutionary, epidemic, and maintenance potential of La Crosse virus
(Colorado State University. Libraries, 2008) Reese, Sara M., author; Beaty, Barry J., advisor
Arthropod-borne viruses are resurging and emerging worldwide, and La Crosse virus (LACV) is a prototypical emergent virus in the United States. In this dissertation, the evolutionary, epidemic, and maintenance potential of LACV is investigated. In laboratory and field studies, LACV has shown significant evolutionary and epidemic potential through point mutations and segment reassortment. Through sensitive molecular epidemiological techniques, significant genetic variation was observed in LACV RNA amplified from field-infected Aedes triseriatus mosquitoes, suggesting the potential for frequent segment reassortment of LACV in nature. Maximum parsimony phylogenetic analysis and linkage disequilibrium analysis revealed that 25-38.6% of the mosquito samples contained reassortant viruses. The geographical, environmental and temporal factors that condition the genetic structure of LACV were also investigated. The analysis revealed that there are no physical barriers to viral flow in the study site, indicating that the more virulent LACV strains could traffic and be transmitted throughout the entire 15,360 km2 study range (southeastern Wisconsin, southwestern Minnesota and northeastern Iowa). Although there were no barriers to viral gene flow and no isolation by distance, a significant temporal association with viral genotype was revealed. The maintenance of LACV in nature is not well understood. Mathematical models have revealed that field infection rates are well below those required to maintain the virus in nature. However, the mathematical models have not considered the possibilities of stably-infected Ae. triseriatus mosquitoes or a LACV induced mating advantage for infected females. Super-infected Ae. triseriatus mosquitoes in nature were identified in southeastern Wisconsin and southwestern Minnesota in these studies (0.011% prevalence rate) suggesting that LACV could be maintained in nature through a stabilized infection in a small number of females. LACV maintenance in nature may also be assisted by a mating advantage for Ae. triseriatus females infected with LACV. In this study, LACV transovarially-infected female mosquitoes become inseminated faster than uninfected mosquitoes, and this could increase the chance for transovarial transmission as well as venereal transmission of the virus. The evolutionary and maintenance potential of LACV was investigated in this dissertation and the results provide insight into the determinants of arbovirus emergence and epidemic potential in nature.
Open Access
Of mice, genes, and radiation: the genetics of non-hereditary breast cancer explored using the common laboratory BALB/c mouse
(Colorado State University. Libraries, 2008) Ramaiah, Lila, author; Ullrich, Robert L., advisor; Benjamin, Stephen A., advisor
In this dissertation we describe the generation and characterization of two novel strains of mice carrying alternate genetic variants of the DNA repair gene Prkdc (DNA-PKcs). Strains congenic for the common (PrkdcB6) and variant (Prkdc BALB) alleles of Prkdc are developed, genotypically validated, and used to examine the functional consequences of Prkdc BALB and its linkage with radiation susceptibility. DNA-PKcs protein expression, post-irradiation double strand break repair, post-irradiation cell survival, breeding depression, and constitutive and radiation-induced gene expression are examined. By western blot we demonstrate that PrkdcBALB is required and sufficient to decrease DNA-PKcs protein expression. Using three different DSB repair quantification methods we show that PrkdcBALB is required for reduced radiation-induced DSB rejoining in BALB/c. We also show that Prkdc BALB is both sufficient and required for decreased cell survival after exposure to ionizing radiation. Thus we demonstrate that Prkdc BALB modulates and even diminishes the ability of cells to maintain genomic homeostasis. Using our newly developed congenic mice, we present the first evidence that PrkdcBALB has a significant effect on gene expression in unirradiated as well as irradiated mice. Microarray analysis of gene expression reveals that PrkdcBALB may have a greater impact on overall gene expression than does radiation, and that Prkdc may play a role in constitutive and DNA damage-induced apoptotic and transcriptional responses. The results presented within this dissertation support the hypothesis that the main role of PrkdcBALB in radiation-induced breast cancer is the initiation of mammary epithelial cells. Our data show that PrkdcBALB is strongly associated with diminished DNA-PKcs expression and function, diminished survival, and altered transcriptional regulation. The congenic strains developed and characterized in this proposal will be instrumental in ongoing studies aiming to clarify the role of Prkdc and genomic instability in radiation-induced mammary carcinogenesis in the BALB/c mouse. Future studies should endeavor to quantify DNA-PKcs specific kinase activity and protein metabolism and to evaluate cytogenetic instability, with particular emphasis on telomeres. The congenic strains developed and characterized in this work serve as compelling rodent models of sporadic and radiation-induced human breast cancer, and provide proof of principle for the role of genetic polymorphisms and genomic instability in breast cancer susceptibility.
Open Access
The acquisition of dendritic cell tolerance during malaria infection results in differential T-cell activation
(Colorado State University. Libraries, 2008) Perry, James A., author; Avery, Anne, advisor
Malaria is caused by intracellular protozoan parasites belonging to the genus Plasmodia. These single cell eukaryotes have a complex life cycle requiring both mammalian (and in certain Plasmodium species, avian) and mosquito hosts. Clinical malaria in humans and other animals is the result of red blood cell (RBC) infection. Although infection direcdy destroys erythrocytes, causing anemia, a significant degree of anemia and morbidity is the result of the host immune response. Inflammatory cytokines have been implicated in the pathogenesis of severe malaria anemia (SMA) and cerebral malaria (CM), two diseases that are responsible for most malaria-related morbidity. Therefore, understanding the regulation of host immunity and inflammatory cytokine production during malaria infection will improve our understanding of malaria related illness.
Open Access
Avian immunity to West Nile virus
(Colorado State University. Libraries, 2008) Nemeth, Nicole M., author; Bowen, R. A., advisor; Spraker, Terry R., advisor
As West Nile virus (WNV) becomes endemic throughout much of North America, it continues to have detrimental effects on countless birds of various taxonomic groups. However, many birds survive infection, mounting an effective immune response. This dissertation focuses on the avian immune response to WNV, including naturally and experimentally-induced antibody duration and passive transfer of immunity. In addition, persistent WNV infection is a potential factor in altering pathogenesis if immunity were to wane. The duration and protection provided by anti-WNV antibodies was documented in house sparrows (Passer domesticus) and raptors for 3-4 years. Antibody levels were relatively stable over time, and protected against viremia in the former and recurrence of clinical disease in the latter. Passive transfer of WNV immunity from hen to eggs and chicks was characterized in domestic chickens (Gallus gallus domesticus). Eggs from both seropositive and seronegative hens were either sacrificed to test for WNV antibody in yolks or chicks artificially inoculated to examine viremic and serologic responses. Concurrently, age-associated differences in response to WNV infection were documented. The passive transfer experiment was repeated in house sparrows to explore this phenomenon in a passerine species; passive transfer was less prevalent in sparrow versus chicken chicks, was of shorter duration, and was less protective. Persistent WNV shedding, viremia, and tissue infection was examined in house sparrows, with juveniles sampled more intensively on a shorter time scale (30-65 days) and adults sampled at 1, 6, 12, 18, and 24 months post-infection. Infectious WNV was isolated from an oral swab, spleen, and kidney of several individuals at 30 DPI, but not from sera after 6 DPI or swabs after 15 DPI. However, WNV was detected in an oral swab by RT-PCR at 44 DPI and was in multiple tissues from most sparrows at 30 DPI, and from kidney and spleen of two individuals at 65 DPI. These findings suggest that WNV infection in tissues may persist beyond the acute stage of infection, while implications for natural transmission and avian health remain unknown.