Browsing by Author "Dow, Steven, committee member"
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Item Open Access Complex regulation of BpeEF-OprC mediated drug efflux in Burkholderia pseudomallei(Colorado State University. Libraries, 2016) Rhodes, Katherine, author; Belisle, John, advisor; Schweizer, Herbert, advisor; Dow, Steven, committee member; Stargell, Laurie, committee memberBurkholderia pseudomallei (Bp) is a Gram-negative bacillus and the etiologic agent of melioidosis, a multifaceted syndrome causing high mortality in tropical regions of the world. The bacteria is classified as a Tier-1 Select Agent due to the seriousness of infection, low infectious dose, lack of effective vaccine, and difficulty of treatment. Bp’s many acquired and intrinsic antimicrobial resistance determinants make the study of these factors vital to improving the efficacy of bi-phasic treatment currently used to treat melioidosis. This study examines one factor in particular: the BpeEF-OprC efflux pump, a member of the resistance-nodulation and cell division family of efflux proteins, and capable of extruding both trimethoprim and sulfamethoxazole. A combination of these compounds (co-trimoxazole) is the first line of eradication phase therapy, making BpeEF-OprC the most clinically important efflux pump encoded by Bp. In spite of this, little is understood of the regulation of bpeEF-oprC, other than it is controlled in part by two LysR family proteins, BpeS, and BpeT. We hypothesized that these regulatory proteins 1) exert their action(s) by interacting with bpeEF-oprC at a specific site within the bpeT-llpE-bpeEF-oprC intergenic region, 2) are capable of influencing transcription of additional operons, and 3) that mutations to these proteins altered ability to form multimers, thereby influencing their function as observed by increased co-trimoxazole resistance and bpeF transcript levels. In Aim I of the study, we identified the cis regulatory regions by which these proteins interact within the bpeT-llpE-bpeE intergenic region using a combination of 5’ deletion assays, S1 nuclease protection, fluorescent-linked oligo extension and electrophoretic mobility assays. With this information we were able to locate bpeT transcriptional start sites and promoter regions as well as binding sites for both BpeT and BpeS. In Aim II, we examined the function of BpeT and BpeS as trans regulatory factors of BpeEF-OprC through mutation and deletion of both genes in part I, and as global regulatory factors in part II. Through overexpression and qRT-PCR or MIC analysis of wild type and mutant forms of both genes, we observed that while BpeT is a direct transcriptional activator of bpeEF-oprC, BpeS is not. Additionally, mutation position in BpeS seems to play a role in the expression phenotype of bpeEF-oprC. However, these mutations do not influence the ability of BpeS or BpeT to form multimers, as we observed no change between wild type and mutant protein oligomer formation through low-pressure gel chromatography and native gel electrophoresis. These same mutations also appeared to have no deleterious effect on the ability of the protein to bind their consensus region within the IR. Additionally, the loss of both genes did not interrupt the ability of bpeEF-oprC to be induced by substrates of BpeEF-OprC, suggesting an additional regulatory factor is at play. In Part II, RNA sequencing analysis and confirmation of select transcriptionally altered operons by RT-qPCR revealed that BpeS might influence expression of the Bsa Type 3 Secretion System (T3SS), while BpeT seems only to target bpeEF-oprC. This may have implication in the pathogenesis of Bp, and must be confirmed in in-vivo cell models using Select Agent excluded strain Bp82 in order to solidify the link between efflux and T3SS during infection. Ultimately, more work is needed to identify the missing regulatory factors in play during expression of bpeEF-oprC, understand how mutations to BpeT and BpeS alter their function, and confirm the relevance of a putative link between co-regulation of efflux and virulence during Bp infection.Item Open Access Development and application of an improved in vitro model for aerosol toxicology(Colorado State University. Libraries, 2014) Hawley, Brie, author; Volckens, John, advisor; Dow, Steven, committee member; Ryan, Elizabeth, committee member; Reynolds, Stephen, committee memberIn vitro cellular studies offer an economical and rapid screening tool for assessing aerosol toxicity. Traditional submerged in vitro cell models and exposure techniques are often criticized for their inability to (1) simulate in vivo cellular morphology (2) maintain the chemical and physical characteristics of sampled aerosol and (3) estimate 'delivered' exposure levels. Further, the exposure levels applied in traditional submerged in vitro systems are often orders of magnitude above inhalational exposures that occur in vivo. Improved airway cell culture models and direct air-to-cell exposure systems have been developed over the last few decades; these improvements offer greater 'real-world' significance to in vitro aerosol toxicology. Air-liquid interfaced airway cell cultures offer greater physiological relevance than previous, submerged cell cultures. Further, direct air-to-cell exposure systems offer the ability to (1) better maintain the chemical and physical characteristics of test aerosols and (2) more closely control and approximate exposure levels. Presented here, are two improved direct air-to-cell aerosol exposure systems that rely upon electrostatic deposition or gravitational settling to directly expose well-differentiated airway cell cultures to three different aerosols of interest, with regard to occupational and environmental health. The first and second study presented here used electrostatic deposition to expose well-differentiated normal human bronchial epithelial cells to diesel particulate matter and complete diesel exhaust. Cells were exposed to either (1) diesel particulate matter or (2) complete diesel exhaust from an engine run on either petro- or biodiesel, and with and without a diesel particulate filter. Cellular response was assessed by measuring transcripts associated with inflammation, oxidative stress, aromatic hydrocarbon response and overall cellular dysfunction at 1, 3, 6, 9, and 24 hours after exposure to diesel particulate matter. Cellular response to complete diesel exhaust was assessed by measuring transcripts associated with oxidative stress and aromatic hydrocarbon response at two hours after exposure. The main aims of these two studies were to (1) characterize the time course of the proinflammatory response of normal human bronchial epithelial cells after exposure to diesel particulate matter and (2) screen for the effects of exposure to petro- and biodiesel exhaust, with and without a diesel particulate filter. The third study presented here used gravitational settling to expose well-differentiated human bronchial or nasal epithelial cells to two different particle size fractions from inhalable dust collected at a local dairy parlor. Cellular response was assessed by measuring transcripts associated with inflammation at two hours after exposure. Cell compromise was also measured in all three studies by measuring percent lactate dehydrogenase release. Significant airway cellular responses were observed in all three studies, at levels of exposure far lower than reported in previous traditional in vitro studies. Results from the work presented here strongly support the use of improved airway cell models and direct air-to-cell exposure systems in future in vitro studies in aerosol toxicology.Item Open Access Development of Lactobacillus acidophilus as an oral vaccine vector and effects of rice bran ingestion on the mucosal health of Malian infants(Colorado State University. Libraries, 2020) Vilander, Allison C., author; Dean, Gregg, advisor; Abdo, Zaid, committee member; Dow, Steven, committee member; MacNeill, Amy, committee member; Ryan, Elizabeth, committee memberMost pathogens enter the body at the mucosa and induce innate and adaptive immune responses at these surfaces essential for protection against infection and disease. Induction of mucosal immune responses is best achieved locally but mucosal vaccines have been difficult to develop with few currently approved for use. Almost all are attenuated live vaccines which limits their use and efficacy in some populations. Strategies to enhance the mucosal immune response to vaccination and move away from attenuated live vaccines are needed. Prebiotics (nondigestible food ingredients that promotes growth of beneficial microorganisms) and probiotics (live microorganisms that are beneficial when ingested) are an active area of interest for improving mucosal health and increasing oral vaccine performance. Here we present the development of the probiotic Gram-positive lactic acid bacteria Lactobacillus acidophilus (LA) as a novel oral subunit vaccine. LA has many advantages as an oral vaccine vector including endogenous acid and bile resistance, heat tolerance, and numerous proteins that interact with the mucosal immune system. We show that LA can induce immune responses to weakly immunogenic neutralizing peptides from HIV-1 and rotavirus. To enhance the immune response, we developed the E. coli type I pilus protein, FimH, as a LA vaccine adjuvant. FimH increased the immune response to vaccination and increased LA trafficking by antigen presenting cells to the mesenteric lymph node, an important site of mucosal immune induction. We also evaluate the effects of ingestion of the nutrient dense prebiotic rice bran on mucosal health in a cohort of healthy Malian infants at risk for malnutrition and the subclinical condition environmental enteric dysfunction. Rice bran ingestion was found to decrease episodes of diarrhea, decrease the age to elevated fecal microbiome α-diversity, and stabilize total fecal secretory IgA concentrations over time. These results indicate that rice bran protects from diarrhea and improves the mucosal environment.Item Open Access Influence of adipose-derived mesenchymal stromal cells on osteosarcoma minimal residual disease(Colorado State University. Libraries, 2015) Aanstoos-Ewen, Megan, author; Ehrhart, Nicole, advisor; Kipper, Matthew, committee member; Dow, Steven, committee member; Custis, James, committee memberIntroduction: Mesenchymal stromal cells (MSCs) have been shown to improve bone integration and healing in several preclinical studies and have therapeutic potential in limb salvage following massive bone loss due to tumor resection. However, MSCs have also been shown to promote primary and pulmonary metastatic tumor growth when injected in the presence of gross tumor or when co-injected with tumor cells in rodent models. While these results raise concerns about the safety of using MSCs in sarcoma patients, MSCs are unlikely to be utilized in a clinical setting when gross tumor is present. The objective of this dissertation project was to develop murine models of minimal residual osteosarcoma following primary tumor removal then to utilize these models to determine whether the administration of adipose-derived MSCs with or without chemotherapy treatment in a minimal residual disease setting would promote either pulmonary metastatic osteosarcoma progression or local disease recurrence. We hypothesized that surgical site or intravenous administration of MSCs will influence either osteosarcoma pulmonary metastatic burden or local disease recurrence in a minimal residual disease setting. Materials & Methods: Two syngeneic, orthotopic models of luciferase-expressing osteosarcoma were developed. In the first model, tumor-bearing mice underwent a coxofemoral amputation and were followed to assess development of pulmonary metastases. In the second model, a femorotibial amputation was performed in order to develop a model of consistent local tumor recurrence. In this model, all gross tumor was removed, however, microscopic tumor remained at the surgical margin. In this dissertation project, three principle projects were completed to test our hypothesis. The first project explored the use of MSCs delivered either to the surgical site or intravenously to ascertain their influence on pulmonary disease burden. A follow-on pilot explored concurrent MSC and chemotherapy treatment on development of pulmonary disease. The second project evaluated the use of MSCs delivered either to the surgical site or intravenously on local recurrence of osteosarcoma at the surgical site. Gross recurrent tumor size was measured for comparison between treatment groups. The third project examined the use of cisplatin and MSCs on survival of mice following removal of primary osteosarcoma. Data were expressed in mean +/- SD or median with 95% CI. ANOVA test, Kruskal-Wallis test, Fisher’s Exact test, Welch’s test, t-test, and Mann Whitney test were used for statistical analysis. Significance was set at p<0.05. Results: Mice treated with intravenous MSCs had a faster time to first pulmonary metastatic disease detection than mice treated with MSCs injected into the surgical site or control mice (no MSCs) (p=0.022). No treatment effect was seen between groups with respect to time to tumor recurrence or size of recurrent tumor in the second study. Survival curves were significantly different when comparing cisplatin, cisplatin and MSC treatment, MSC alone treatment and untreated mice (p<0.001) as well as in pairwise comparisons. Mice treated with MSCs had a 73% chance of earlier death than untreated controls. Discussion/Conclusion: Intravenous administration of MSCs in a minimal residual osteosarcoma environment resulted in a faster time to first detection of pulmonary disease and in a higher chance of earlier death compared to untreated mice. However, administration of MSCs locally in a surgical site following sarcoma excision appears to be safe, even in the setting of known residual microscopic disease. Further, the use of cisplatin treatment appeared to ameliorate the effects of intravenous MSCs on survival. Based on these results, further study is warranted to evaluate the influence of intravenously administered MSCs on minimal residual pulmonary metastatic disease.Item Open Access Investigation of dietary rice bran for protection against Salmonella enterica Typhimurium infection in mice(Colorado State University. Libraries, 2014) Kumar, Ajay, author; Ryan, Elizabeth P., advisor; Dow, Steven, committee member; Leach, Jan, committee member; Schenkel, Alan, committee member; Weir, Tiffany, committee memberRice bran is a byproduct of rice milling for white rice. Rice bran is a rich source of nutrients such as vitamins, minerals, soluble and insoluble fibers, fatty acids, polyphenols and proteins. Research has shown the beneficial health effects of rice bran in hyperlipidemia, diabetes, immune modulation, allergies and cancer. This dissertation focuses on evaluation of rice bran for protection against Salmonella using a mouse model of oral infection. Salmonella is a food and water borne pathogen that affects a variety of hosts including plants, animals and humans. Salmonella infections are a major public health challenge around the globe. Currently, salmonellosis is treated using high doses of synthetic antimicrobials and the problem of drug resistance has increased. In this scenario, alternative and sustainable interventions are needed to control Salmonella infections. Several dietary agents have been studied for protective effects in Salmonella infection models. We tested the prophylactic effects of dietary rice bran in a Salmonella model of infection using female 129S6/SvEvTac mouse model with infection of Salmonella enterica Typhimurium 14028s strain. Feeding of 10% dietary rice bran for one week prior to infection significantly (p<0.05) reduced fecal excretion of Salmonella in orally infected mice. Salmonella-infected, rice bran fed mice also showed a significant decrease in systemic inflammatory cytokines such as TNF-α, IFN-γ and IL-12 as compared to control diet fed animals. The colonization resistance against enteric pathogens is highly influenced by composition of gut microflora. Supplementation of dietary rice bran increased the number of Lactobacillus spp. in feces of mice as compared to mice that were fed control diet. Research has shown that oral administration of some species of Lactobacillus reduces the colonization of Salmonella. We hypothesized that rice bran components also enhance mucosal protection by preventing Salmonella entry into the epithelial cells. Methanolic rice bran extracts were assessed in mouse small intestinal epithelial (MSIE) cells for blocking Salmonella entry and intracellular replication. Rice bran extract significantly reduced Salmonella entry and intracellular replication into MSIE cells. These results suggest the potential mechanisms for dietary rice bran induced improvement of colonization resistance against Salmonella. Given that rice crops have a large variation in genotype and phenotype such as in yield, disease and pest resistance, drought resistance, and nutrient quality, we hypothesized that variation in rice bran across cultivars induces differential protection against Salmonella infection due to differences in their phytochemical profile. A panel of six varieties namely IAC 600, Jasmine 85, IL 121-1-1, Wells, Red Wells and SHU 121 were tested in the in vitro and in vivo model of Salmonella infection. We found that rice bran extracts across varieties inhibited Salmonella entry into the MSIE and Caco-2 cells to different extents. IAC 600 fed animals significantly (p<0.05) reduced Salmonella fecal excretion as compared to the control diet fed animals. IAC 600 fed animals also reduced Salmonella fecal shedding significantly (p<0.05) as compared to SHU 121 diet fed animals at 2 and 6 days post Salmonella infection. Histopathological analysis revealed that IAC 600 diet fed animals had better ileal pathological scores as compared to SHU 121 and the control diet fed animals post Salmonella infection. SHU 121 and the control diet fed groups showed higher ulceration and inflammatory changes in ileum as compared to IAC 600 fed animals. Next we analyzed the fatty acid profile, mineral profile and total phenolic contents of rice bran. Stearic acid, lignoceric acid, boron and total phenol content were significantly correlated with Salmonella fecal shedding in mice across varieties. However, further studies are required to confirm the role of these nutrients from rice bran in protection against Salmonella. These results suggest that the variety of rice plays an important role in bran-induced protection against Salmonella infection and this difference in protection across the varieties could be attributed to a combination of bioactive components. Our studies suggest that dietary rice bran improves colonization resistance against Salmonella in mice. Rice bran could have important role in prevention of enteric infections in resource scarce populations and further human clinical studies are required. Rice bran may also be evaluated for supplementing diets of food animals to prevent Salmonella infections and therefore could have a potential role in food safety.Item Embargo Investigation of environmental factors on the intranuclear landscape of mesenchymal stromal cells(Colorado State University. Libraries, 2022) Kaonis, Samantha, author; Ghosh, Soham, advisor; Johnstone, Brian, committee member; Popat, Ketul, committee member; Dow, Steven, committee memberMesenchymal stromal cells (MSC), also known as mesenchymal stem cells, are popular candidates for tissue engineering and regenerative medicine. They can differentiate into many tissue types, and they can also help in regeneration through their trophic and immunomodulatory properties. Despite being investigated thoroughly for the last four decades and being under clinical trial in more than a thousand FDA approved studies, their application in clinics is very limited. One of the most important challenges in using MSC is that after harvesting from the patient, they need to be expanded to millions of cells for successful clinical outcomes. During this process, MSCs lose their differentiation potential, and trophic and immunomodulatory properties. In this thesis, I investigated the potential mechanisms of how environmental factors cause the MSC to divert from their phenotype during the expansion process. Subsequently, I intervened these mechanisms to achieve high quality MSCs without compromising the number of cells, i.e., their proliferation potential. Specifically, I investigated how two critical biophysical factors - mechanical stiffness and oxygen concentration of the MSC environment affects the cell phenotype and function through mechanisms involving epigenetic modifiers, transcription factors, and the chromatin architecture. First, the regulation of mechanics-induced population heterogeneity in MSCs was examined. Plastic culture and fibrotic conditions post-transplantation experienced by the MSC is completely different from the natural biomechanical niche of the MSC. Accordingly, the role of the mechanical environment has been shown to be a critical determinant of MSC gene expression and function. In this study, we report that human bone marrow-derived primary MSC population becomes phenotypically heterogenous when they experience an abnormal mechanical environment, compared to their native environment. Using a newly developed technique to quantify the heterogeneity, we provide evidence of phenotypical heterogeneity of MSC through high-resolution imaging and image analysis. Additionally, we provide mechanistic insight into the origin of such substrate mechanics-driven heterogeneity, which is further determined by the cell-cell mechanical communication through the substrate. In the second study, we investigated how the chromatin architecture and epigenetic landscape changes in MSCs by the substrate mechanical stiffness, thus causing a shift from the MSC phenotype. Using high-resolution confocal microscopy and advanced image analysis we identified the key epigenetic drivers in the mechanical stiffness mediated chromatin organization changes. Subsequently, we targeted several components of a proposed mechanobiological pathway to achieve MSCs with higher growth factor secretion without compromising their proliferation. The outcome of these studies might provide mechanism-driven design principles to the molecular, cellular and tissue engineering researchers for the rational design of MSC culture conditions and scaffolds, thus improving their functional outcome. Finally, the effect of oxygen concentration on MSC proliferation and performance wereexplored. Culture under physiological oxygen concentration (physioxia) can increase the proliferation of MSCs through a pathway initiated by the stabilization of the hypoxia-inducible factor-1 (HIF-1). Stabilized HIF-1α translocates into the nucleus, triggering the transcription of target genes conducive to MSC activity and proliferation. However, stabilized HIF-1α also triggers the p21 pathway causing cell cycle arrest, decreasing the MSC proliferation thereby limiting the beneficial effect of physioxia. Maintaining low oxygen conditions can be challenging, especially at a large scale, so rational exploitation and selective manipulation of such pathways through biochemical means has the potential to culture MSCs easily at scale. In this work, we created a mathematical model to predict optimal physioxic culture parameters to achieve the highest MSC proliferation. Through analysis of a gene downstream of the HIF-1 pathway, we also compared standard physioxic culture (2% O2) to treatment with deferoxamine mesylate (DFO), a physioxia-mimicking drug. The outcomes of this study might provide the rationale for MSC culture under standard hyperoxic conditions with only a simple addition of a combination of drugs to the culture medium to improve the scalability of MSC culture. Together, the results of the work will identify the mechanistic details of culture environment factors that play a role in determining the phenotype of MSCs during in vitro expansion process. The combination of these techniques to optimize MSC culture in vitro has the potential to resolve the current impediment to the clinical success of MSC therapies.Item Open Access Mechanisms of pulmonary fibrosis in PECAM-1 deficient FVB/n mice(Colorado State University. Libraries, 2014) Lishnevsky, Marta, author; Schenkel, Alan R., advisor; Dow, Steven, committee member; Slayden, Richard, committee member; Orton, Christopher, committee memberIdiopathic Pulmonary Fibrosis (IPF) is a fatal disease of the aging population that affects nearly 100,000 Americans, and its incidence has been steadily on the increase. Patients typically present in late-stage disease, and effective early stage diagnosis and treatment methods are thoroughly lacking. The cellular and molecular events involved in disease initiation are still unknown. There is increasing evidence that alveolar bleeding and coagulation play an important role in the initiation and progression of IPF, and anticoagulant therapies have been shown to exacerbate the process of the disease. Most current mouse models have difficulty in reproducing the spontaneous occurrence of the disease. Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) deficient mice on the FVB/n background spontaneously develop a fatal chronic pulmonary disease in the absence of a detectable acute inflammatory event. The studies here found that the disease observed in these animals is characterized by alveolar collapse frequently accompanied by extravasated red blood cells in the alveolar space, presence of hemosiderin-positive alveolar macrophages, fibrin deposition, and myofibroblasts in areas of developing collagen deposition. The early events hallmarked by alveolar collapse and extravasated red blood cells in alveoli are of particular importance, as identifying early pathogenic events that lead to the development of fibrotic disease can contribute to the development of preventive, diagnostic, and treatment options for the patient population with IPF.Item Open Access Mining the Mycobacterium tuberculosis cellular envelope for diagnostic and drug targets(Colorado State University. Libraries, 2013) Wolfe, Lisa Marie, author; Dobos, Karen M., advisor; Belisle, John T., committee member; Dow, Steven, committee member; Lewinsohn, David, committee member; Prenni, Jessica, committee memberThe cellular envelope of Mycobacterium tuberculosis is a highly complex structure containing many lipids, carbohydrates and proteins. Together these components maintain cellular homeostasis and play an active role in the establishment and maintenance of intracellular infection. The World Health Organization estimates that upon exposure, 90% of infected persons will not succumb to active disease and likely remain a potential source of M. tuberculosis transmission upon the emergence of clinical symptoms. Throughout this complex disease course, physiological and structural changes occur within the cell envelope of the bacillus and facilitate its survival within an infected host. These physiological changes also influence the immunological interplay between bacteria and host cell, further contributing to the success of the pathogen. To understand these changes we must have a comprehensive knowledge of the cell wall proteins that contribute to the overall makeup of the mycobacterial envelope, understand the unique antigens that reside in or that are secreted from the bacillus and monitor these proteins under different physiological disease profiles. In the last decade many large-scale descriptive studies focusing on gene transcript profiles and proteomic composition of subcellular fractions have pioneered efforts in the understanding of M. tuberculosis physiology, immunology and pathogenesis at the level of systems biology. These studies have shown that M. tuberculosis is capable of retaining essential gene products for respiration, nutrient uptake and energy metabolism both in vitro and in vivo and that the proteins within the cell wall are highly immunogenic for M. tuberculosisspecific T lymphocytes. Our first objective established a comprehensive description of the cell wall proteome of M. tuberculosisusing traditional two-dimensional gel-based techniques and liquid-chromatogoraphy mass spectrometry (LC-MS). From this work, over 500 proteins were identified using a combination of differential detergent extraction and multi-dimensional-LC. A highly lipoprotein enriched fraction revealed that the majority of cell wall associated proteins were functionally annotated to mechanisms of intermediary metabolism (35%) and macromolecular synthesis and degradation (25%) building upon evidence that the M. tuberculosis cell wall is actively engaged in cellular homeostasis and remodeling events. Secondly, we investigated the role of the cell envelope proteins in the search for novel immunodiagnostic epitopes. It is well known that the cell wall of M. tuberculosis is highly immunogenic and contains both non-protein and protein antigens. Specifically, the proteins associated with the cell wall were shown to be uniquely responsible for the activation of human CD8 T cell clones generated from both actively and latently infected individuals. The immunological response to CD8 T cell antigens may be an effective means of distinguishing between latent TB infection (LTBI) and active disease. To broadly define the repertoire of CD8 T cell antigens, 56 proteins from the cell wall proteome study were included in the design of a synthetic peptide library. Exhaustive screening of the peptide library for novel antigens and epitopes that elicit an immunological response in TB patients, resulted in the identification of eight cell wall antigens that are currently being investigated for their clinical utility. In addition, the cell wall proteome was also mined in the identification of an HLA-E restricted CD8 T cell epitope. HLA-E has low polymorphism in the human population and seems to be enriched in M. tuberculosis - containing phagosomes, therefore identification of this antigen could be used as a novel diagnostic or vaccine candidate. Using a MS-based proteomics approach, we discovered the HLA-E antigen to be the post-translationally modified glycoprotein Mpt32 (45kDa/Apa). Glycosylated proteins and lipids within the mycobacterial cell envelope are dominant and the role of this modification in the host immune response can now be elucidated. Lastly, the composition and integrity of the M. tuberculosiscell envelope facilitates its adaptation and survival within various microenvironments. These physiological functions are influenced by the presence or absence of functionally linked genes and proteins whose relative abundance may change over time or within altered metabolic states. Our final efforts used nucleotide analog probes, to specifically bind and enrich proteins with an ATP-binding function and measure their relative abundance between altered states of growth (i.e. between active disease and hypoxia-induced dormancy). With these efforts we classified 122 ATP-binding proteins in either metabolic state and demonstrated differential abundance patterns between actively growing and hypoxic cells within the functionally linked protein networks of energy metabolism, cell wall and lipid biosynthesis. These protein families represented in the M. tuberculosis ATPome are a subset of essential (60% of the Mtb-ATPome) gene products and may be relevant therapeutic targets for the future development of novel small molecule inhibitors against M. tuberculosis. The spectrum of studies undertaken to mine the cellular envelope for diagnostic and drug targets demonstrates a natural evolution of MS-based proteomics in the study of biologically relevant questions. From a purely descriptive characterization of the cell wall proteome, this data was utilized in a practical approach in the design of a high-throughput antigen/epitope-screening library and finally these studies culminate in a functionally relevant profile of the ATP-binding proteins of M. tuberculosis. Future work will continue to focus on developing hypothesis-driven proteomic studies for the identification of novel diagnostic antigens and drug targets.Item Open Access Minipigs as a neonatal animal model for TB vaccine efficacy(Colorado State University. Libraries, 2016) Ramos Arriaza, Laylaa, author; Gonzalez-Juarrero, Mercedes, advisor; Bowen, Richard, committee member; Izzo, Angelo, committee member; Guth, Amanda, committee member; Dow, Steven, committee memberCurrently, the only vaccine available to prevent tuberculosis (TB) is Bacillus Calmette-Guerin (BCG). The vaccine lacks efficacy against pulmonary disease or reactivation of latent TB but prevents disseminated TB in children and is thus widely used in countries with endemic TB as part of the neonatal vaccine regimen. There are several new vaccines that have shown efficacy against TB in adult animal models yet fail to protect infants from TB disease in clinical trials. Failure in the development of new pediatric vaccines may be due to incomplete knowledge in the elicited immune response to BCG vaccination and testing of vaccine efficacy in adult rather than neonatal animal models. In this novel approach, we used the mini-pig as a neonatal animal model for evaluation of immune responses to BCG vaccine. We demonstrate young mini-pigs are susceptible hosts to the highly virulent Mycobacterium tuberculosis (Mtb) strain, HN878 and that the pathological course of infection resembles that seen in human TB. In this study we longitudinally monitored the immune response of neonatal mini-piglets vaccinated with BCG until adulthood, with the same monitoring applied to a group of unvaccinated mini-piglets. Further, we challenged both vaccinated and non-vaccinated animals via the aerosol route with HN878 and we characterized important changes between the two groups in the course of immune responses following challenge. Based on comparison of immune responses to BCG in mini-pigs and infants, our findings suggest that mini-pigs have the potential to serve as an effective neonatal animal model for TB vaccine development.Item Open Access T cell independent mechanisms for protection against Mycobacterium tuberculosis infection(Colorado State University. Libraries, 2019) Bickett, Thomas, author; Izzo, Angelo, advisor; Dow, Steven, committee member; McLean, Jennifer, committee member; Bowen, Richard, committee member; Argueso, Lucas, committee memberThe live attenuated Mycobacterium bovis strain Bacille Calmette Guérin (BCG) is a potent innate immune stimulator. Innate Immunity provides the host with the ability to immediately respond to invasion by pathogens and can be utilized through the use of molecular adjuvants to trigger specific innate mechanisms leading to adaptive immunity. In the C57BL/6 mouse model of tuberculosis, BCG stimulated immunity causes a significant reduction of M. tuberculosis burden after pulmonary infection. Our studies indicate that BCG induced protection against pulmonary M. tuberculosis through early monocyte recruitment is present as early as 7 days after vaccination. This protection showed longevity, as it did not wane when mice were infected 30 days post vaccination. As BCG induced mycobacterial killing after 7 days, we sought to identify the contribution of different innate immune components to better understand mechanisms required for mycobacterial killing. When BCG was administered through subcutaneous inoculation, we found that there was significant monocyte recruitment in the lungs within 7 days after vaccination. Further studies revealed that killing of mycobacterium is dependent on BCG being viable and is monocyte derived, independent of trained innate immunity, highlighting a novel mechanism for killing M. tuberculosis. With the rise of drug resistant strains of Mycobacterium tuberculosis, new vaccine development is paramount. A better understanding of the BCG vaccine will hopefully lead to the development of a more effective alternative.Item Open Access Telomere length, telomerase activity, and structural variants as biomarkers of extreme environments(Colorado State University. Libraries, 2017) McKenna, Miles J., author; Bailey, Susan M., advisor; Dow, Steven, committee member; Kato, Takamitsu, committee member; Simon, Steven, committee member; Thamm, Douglas, committee memberMammals, and in particular humans, are masterful at overcoming and adapting to extreme environments. Whether astronauts in low earth orbit aboard the International Space Station (ISS) or U.S. military veterans exposed to nuclear fallout from atomic weapons testing, humans can persist through a wide range of physical, psychological, and environmental stressors. The overall goal of the studies presented here was to evaluate the biological influences of extreme environments not commonly experienced by the general population. Whether spaceflight or exposure to nuclear fallout, results improve our understanding of short- and long-term effects of low gravity environments, exposure to ionizing radiation (IR) of mixed qualities, as well as low dose and low dose effects of IR. We explored these scenarios by evaluating biomarkers of stress, specifically telomere length dynamics, and biomarkers of DNA damage, specifically transmissible structural rearrangements. Telomeres are not only regarded as valuable biomarkers of aging and age-related degenerative pathologies like cardiovascular disease and cancer, and so are reflective of overall health status, they also serve as "hallmarks" of radiosensitivity. Stable chromosomal structural rearrangements (translocations and inversions) persist with time and so provide informative signatures of IR exposure as well. During the 1950's United States military personnel and weathermen, collectively known as the atomic veterans, were unintentionally exposed to nuclear fallout during atomic bomb testing following WWII. Here, directional Genomic Hybridization (dGH) for high-resolution detection of IR-induced chromosomal inversions and translocations was assessed as a more sensitive, quantitative retrospective biodosimetry approach. The influence of IR exposure on telomere length dynamics was also evaluated to determine the long-term influence of such exposures. Our results illustrate that even for nuclear events that occurred six decades in the past, evidence of exposure is still present. We find that although translocations and inversions are reliable biodosimeters independently, a combined approach provides a more sensitive measurement of past radiation exposure. We also report, for the first time, the influence of age and smoking on background inversion frequencies. Furthermore, telomere length was inversely related to IR dose, suggesting that a single acute exposure to nuclear fallout may lead to persistent long-term effects on overall health. Telomere length dynamics and structural rearrangements were also monitored longitudinally in monozygotic twin and unrelated astronauts. NASA astronauts are a unique group of individuals who experience an extreme environment that the human body is not adapted for. Little is known about the biological health effects of a low gravity environment with increased IR exposure including galactic cosmic rays (GCRs), solar particle events, and secondary neutrons. Not only do astronauts have shorter telomeres than age-/gender-matched controls at baseline (pre-flight), but a transient increase in telomere length during space flight was also observed. Results suggest this unexpected finding may be due to an upregulation of telomerase, the enzyme responsible for maintaining telomere length. Moreover, telomerase activity also increased post-flight in both twins, Scott and Mark Kelly. Although not spaceflight specific, this is the first report of telomerase upregulation in humans due to a psychologically traumatic event. A dose dependent increase in inversions, and to a lesser extent, translocations, as a consequence of IR exposure on the ISS was also seen in Scott Kelly. Collectively, the studies presented here demonstrate a profound influence of extreme environments, particularly those involving low dose IR, on human biological responses. Telomere length dynamics and chromosome aberration frequencies (e.g. translocations and inversions) provide insight into the long-term health effects and implications of spaceflight and exposure to nuclear events.Item Open Access The pathogenesis of diabetes-tuberculosis comorbidity(Colorado State University. Libraries, 2014) Podell, Brendan K., author; Basaraba, Randall J., advisor; Dow, Steven, committee member; Rovnak, Joel, committee member; Jackson, Mary, committee member; Ishii, Douglas, committee memberExposure to the bacterium, Mycobacterium tuberculosis, only leads to the active form of tuberculosis disease (TB) in 5-10% of infected individuals. The development of active TB, at any stage of infection, is often the result of a known TB risk factor, either intrinsic to the individual or acquired as a communicable or non-communicable disease. An association between diabetes and TB has long been recognized, but only recently was diabetes confirmed to increase the risk of developing active TB disease. The convergence of a growing diabetes epidemic on regions with endemic TB has positioned diabetes as an emerging global threat to TB control. Of particular importance is the rapidly growing incidence of type 2 diabetes, which accounts for up to 95% of the global diabetic population. Since the potential impact of this growing comorbidity has only been recently emphasized, little is known regarding the mechanisms of dysregulated immune function and metabolism by which diabetes predisposes to active TB disease. The current understanding of this comorbidity is further limited by the lack of appropriate animal models that replicate the pathogenesis of both human type 2 diabetes and TB. The guinea pig is a well-established model of TB that replicates human pathology and disease progression. This species was emphasized in this series of studies with the goal of better understanding the impact of type 2 diabetes on TB progression and the mechanisms that may change the host response to M. tuberculosis infection. In Chapter 2, we investigated the impact of hyperglycemia alone, induced as post-prandial hyperglycemia through daily administration of sucrose, on TB disease progression in non-diabetic guinea pigs. Guinea pigs receiving daily sucrose developed both higher bacterial burdens in pulmonary and extrapulmonary tissue and also more severe pathology by day 60 of infection. This exacerbated disease manifestation was accompanied by the accumulation of advanced glycation end-products, which are inflammatory by-products of chronic hyperglycemia with known involvement in the development of diabetes-related complications. Interestingly, by monitoring glucose and lipid metabolism in these guinea pigs, we learned that TB alone leads to severe metabolic disturbances, manifesting as hyperglycemia and accumulation of circulating total free fatty acids. From this study, we were able to conclude that not only does mild post-prandial hyperglycemia worsen the course of TB disease in guinea pigs, but also, infection with M. tuberculosis alone induces metabolic disease resembling diabetes, similar to what has been previously reported in human TB. These conclusions rationalize the investigation of novel adjunctive therapies to restore metabolic homeostasis, which may improve the host response to infection, limit bacterial growth, and increase the efficacy of frontline antimicrobial drugs. In Chapter 3, we developed a novel model of type 2 diabetes in the guinea pig to be used in future investigations of type 2 diabetes-TB comorbidity. Previously, the guinea pig as a diabetic model has been described only in the context of β-cell cytotoxicity with the drug, streptozotocin (STZ), but with variable efficacy. In this study, we initially optimized the dose response and STZ preparation to achieve an induction of hyperglycemia that was uniform with limited mortality. This hyperglycemic response was transient but could be stabilized through continued β-cell stress, in the form of a high fat, high sugar diet. Feeding of this modified diet led to impaired glucose tolerance and a compensatory β-cell response that could be abrogated with the use of a single optimized dose of STZ. This novel model of type 2 diabetes develops both insulin resistance and β-cell failure, which replicate the typical progression of type 2 diabetes in humans, all within a reasonable experimental timeframe. From this study, two models emerged, a type 2 diabetic guinea pig as well as a model of impaired glucose tolerance, or prediabetes, that would be used to investigate the mechanisms of diabetes-TB comorbidity. In Chapter 4, the newly developed guinea pig models were used to investigate the overall impact of type 2 diabetes and impaired glucose tolerance on TB progression and the host immune response to M. tuberculosis infection. Although impaired glucose tolerance alone had limited impact on TB progression with exacerbation of disease only at chronic end points, M. tuberculosis infected type 2 diabetic guinea pigs closely resembled the reported manifestations of human diabetes-TB comorbidity including more severe TB disease, higher bacterial burdens, and a robust innate and cell-mediated immune response. Despite evidence of strong Th1 cell-mediated immunity, which is known to be critical for limiting bacterial growth and disease progression, diabetic guinea pigs were unable to control bacterial growth and developed damaging neutrophilic inflammation. To better understand the immune mechanisms leading to uncontrolled bacterial growth and severe disease, in Chapter 5, we investigated the innate and adaptive immune response over the course of early infection in type 2 diabetic guinea pigs. Diabetic guinea pigs were slow to develop early lesions with delayed bacterial transport to the lung draining lymph node, and a corresponding delay in antigen-specific Th1 immunity. Early alterations in cytokine expression were identified that may explain the delayed development of cell-mediated immunity and allow for substantial growth of M. tuberculosis in the lung of infected diabetic guinea pigs. These data indicate that not only does type 2 diabetes increase the severity of TB but also that the chronic inflammatory process associated with TB itself may worsen diabetes. This has important implications worthy of further investigation revolving around the diagnostic criteria for diabetes when associated with TB, the impact of active TB on medical management of diabetes, and the investigation of novel therapeutic targets, both metabolic and immunological, to enhance the host immune response to infection and limit TB disease severity in diabetics.Item Open Access Titania nanotube arrays: interfaces for implantable devices(Colorado State University. Libraries, 2012) Smith, Barbara Symie, author; Popat, Ketul, advisor; Gonzalez-Juarrero, Mercedes, committee member; Prasad, Ashok, committee member; Dasi, Lakshmi Prasad, committee member; Dow, Steven, committee memberFor the 8-10% of Americans (20-25 million people) that have implanted biomedical devices, biomaterial failure and the need for revision surgery are critical concerns. The major causes for failure in implantable biomedical devices promoting a need for re- implantation and revision surgery include thrombosis, post-operative infection, immune driven fibrosis and biomechanical failure. The successful integration of long-term implantable devices is highly dependent on the early events of tissue/biomaterial interaction, promoting either implant rejection or a wound healing response (extracellular matrix production and vasculature). Favorable interactions between the implant surface and the respective tissue are critical for the long-term success of any implantable device. Recent studies have shown that material surfaces which mimic the natural physiological hierarchy of in vivo tissue may provide a possible solution for enhancing biomaterial integration, thus preventing infection and biomaterial rejection. Titania nanotube arrays, fabricated using a simple anodization technique, provide a template capable of promoting altered cellular functionality at a hierarchy similar to that of natural tissue. This work focuses on the fabrication of immobilized, vertically oriented and highly uniform titania nanotube arrays to determine how this specific nano-architecture affects skin cell functionality, hemocompatibility, thrombogenicity and the immune response. The results in this work identify enhanced dermal matrix production, altered hemocompatibility, reduced thrombogenicity and a deterred immune response on titania nanotube arrays. This evidences promising implications with respect to the use of titania nanotube arrays as beneficial interfaces for the successful implantation of biomedical devices.Item Embargo Using gene expression and mutational profiling to characterize canine acute myeloid leukemia and assess their comparative features with human acute myeloid leukemia(Colorado State University. Libraries, 2023) Harris, Adam, author; Avery, Anne, advisor; Avery, Paul, committee member; Dow, Steven, committee member; Duval, Dawn, committee memberAcute myeloid leukemia (AML) is an aggressive heterogenous hematopoietic neoplasm that afflicts both dogs and people. Over 10,000 individuals (about the seating capacity of Cameron basketball stadium at Duke University) in the United States succumb to AML-related deaths every year. Treatment options for AML have made little progress in the past few decades and prognosis for both human and canine AML (cAML) remains dismal. However, there are large ongoing multi-institutional studies devoted to advancing medical management for human AML (hAML) by providing targeted therapeutics to patients based on their molecular characteristics. A preclinical model for testing novel therapies could accelerate the development of better treatments in people. We hypothesize that cAML will have similar underlying molecular features as human AML and dogs could be a translational model for developing therapeutics focused on treating AML. The goals of this thesis were to assess the gene expression programs and mutational profiles of cAML and compare our findings with available human AML data. First, we established diagnostic criteria for defining cAML using flow cytometry. Next, we globally assessed normal hematopoiesis in dogs using single cell transcriptomics to generate a hematopoietic tree for defining the cellular composition of cAML. Additionally, we investigated the mRNA expression and genetic variants in cAML to ultimately compare the molecular features with pediatric and adult AML subtypes. We hope this work advances our knowledge of cAML molecular characteristics and adds further credentials to the dog as spontaneous model for human AML.