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Browsing by Author "Dobos, Karen, advisor"

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    Characterizing the role of the HspX protein from Mycobacterium tuberculosis as a subunit vaccine candidate in the small animal model of tuberculosis
    (Colorado State University. Libraries, 2011) Wieczorek, Agatha E., author; Dobos, Karen, advisor; Taylor, Jennifer, committee member; Curthoys, Norman, committee member
    Tuberculosis (TB) is a bacterial disease of the lung, caused by Mycobacterium tuberculosis, and currently remains an important human pathogen. The only vaccine against tuberculosis licensed for human use is a live, attenuated strain of the closely related Mycobacterium bovis Bacille-Calmette Guerin (BCG), which offers little protection against pulmonary disease in adults, particularly against latent infection. Current vaccine strategies against TB include the development of subunit vaccines, which contain one to a few antigens. Subunit vaccines are delivered with an adjuvant formulation to mount an appropriate T cell response against the pathogen. One such antigen for vaccine design is the 16kDa small heat shock protein and molecular chaperone from Mtb, HspX. This protein has been implicated as a latency-associated antigen due its late expression and ability to re-stimulate T cells from latently infected patients. A previous study in our laboratory revealed that native HspX purified from Mtb was protective in mice against pulmonary TB when given as a vaccine. HspX expressed and purified from E coli in its recombinant form was not able to protect. We hypothesize that because HspX functions as a molecular chaperone, it requires its binding partners to remain biologically active as a vaccine. In this study, we test this hypothesis and we also tested the capacity of native HspX to protect guinea pigs experimentally infected with TB. Our results illustrate that, while native HspX protects in the mouse model, it does not confer protection in guinea pigs, suggesting differences in the establishment of Mtb physiologically in the lung.
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    Development of an ultrasensitive ELISA for the detection of Mycobacterium tuberculosis antigens: an impossible challenge or a promising feat?
    (Colorado State University. Libraries, 2022) Early, Kala, author; Dobos, Karen, advisor; Mehaffy, Carolina, advisor; Schenkel, Alan, committee member; Henry, Charles, committee member
    Tuberculosis (TB) has been classically characterized as a two-state disease with active and latent phases. Latent TB infection (LTBI) is diagnosed by either the tuberculin skin test (TST) or the Interferon Gamma Release Assay (IGRA) test. However, both diagnostic tests are unable to differentially diagnose active TB and LTBI and perform poorly in immunocompromised patients. The TST is further complicated by cross-reactivity with BCG vaccination. Therefore, further diagnostic discovery for LTBI is needed for differential diagnosis and to identify those at risk of progression to active TB for subsequent treatment. Extracellular vesicles (EVs) are nanovesicles released by eukaryotic cells. EVs from TB patients contain Mycobacterium tuberculosis (Mtb) proteins, and these protein biomarkers show promise for TB and LTBI diagnostics. Our lab previously identified 31 Mtb peptides in trypsin-treated serum EVs isolated from patients with LTBI using multiple reaction monitoring-mass spectrometry (MRM-MS) methods. MRM-MS is a highly sensitive technology but is not feasible for widespread use as a diagnostic. The goal of this study was to develop an ultrasensitive ELISA against Mtb proteins for potential use as a point-of-care diagnostic. A sandwich ELISA was initially developed against Mtb proteins DnaK, Mpt32, and GroES. Reagent development for the sandwich ELISA included polyclonal antibody production using a rabbit model, murine monoclonal antibody purification and biotinylation from an existing collection of hybridoma cell lines for each antigen, and detection using a streptavidin-HRP system with a chemiluminescent substrate for signal expansion. We observed that the sandwich ELISA was complicated by non-specific binding of the DnaK and GroES antigens to the BSA block. We hypothesized that the chaperone function of these two proteins influenced them to bind to BSA. This non-specific interaction was further characterized using SPR technology and demonstrated a concentration dependent binding of DnaK to BSA. A direct-biotinylated ELISA was subsequently developed and optimized. Limit of detection (LOD) and limit of quantification (LOQ) of the direct-biotinylated ELISA was determined for each antigen: 1) GroES had an LOD of 1.959 ng/mL and an LOQ of 6.531 ng/mL, 2) Mpt32 had an LOD of 1.884 ng/mL and an LOQ of 6.278 ng/mL, and 3) DnaK had an LOD of 6.310 ng/mL and an LOQ of 21.032 ng/mL. This direct-biotinylated ELISA platform demonstrated high sensitivity with low background for all three antigens. Thus, we successfully developed and optimized an ultrasensitive ELISA for the detection of Mtb antigens.
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    Establishment and systematic characterization of Mycobacterium tuberculosis in bioreactors
    (Colorado State University. Libraries, 2016) Knabenbauer, Phillip, author; Dobos, Karen, advisor; Slayden, Richard, committee member; McNeil, Michael, committee member; Peebles, Christie, committee member
    Mycobacterium tuberculosis infection is characterized by active and latent disease states. Granuloma-induced oxygen tension may shift bacteria into bacteriostatic persistence. Current models of hypoxia-induced mycobacteria have limitations, requiring establishment of novel culturing methods. Here, M. tuberculosis was propagated under defined oxygen concentration in bioreactors. Initial analyses confirmed mycobacterial non-replicating persistence. This study will provide insight into core physiological adaptations of M. tuberculosis while reducing bias from the contaminants during adaptation into dormancy. Here we describe a novel method of propagation using defined oxygen concentrations, then enrich the final culture for viability to remove transcriptional bias, and finally interrogate the presence of viable but non-culturable tubercle bacilli in order to obtain a greater sense of true viability. The current study will further contribute to our understanding of the physical adaptation of Mtb during growth and dormancy, by removing bias from the contaminating transcriptome gradient generated by the temporal adaptation of M. tuberculosis into dormancy. This will enhance the accuracy of downstream structural and transcriptomic analyses as well as give rise to a novel high throughput approach to M. tuberculosis propagation for research materials.
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    Inactivation of Mycobacterium tuberculosis for safe use outside of the BSL-3 laboratory
    (Colorado State University. Libraries, 2020) Watkins, Jackson, author; Dobos, Karen, advisor; Goodrich, Raymond, committee member; Slayden, Richard, committee member; Hall, Ed, committee member
    Techniques for pathogen inactivation have been long employed by research laboratories to help ease the financial, physical, and health strains associated with (A)BSL-3 work. While robust protocols exist for many of these techniques, there are many holes in research associated with characterization of damage to treated organisms, and standardized methods for comparative analysis of successful sterilization. The work in this thesis aimed to develop methods to better understand current techniques of inactivation. At the same time, it also aimed to characterize the limitations of both a novel approach to inactivation, as well as the pathogenic models we commonly use for infectious disease research. We accomplished this by first developing a D10 value and standardized curve to describe inactivation of Mycobacterium tuberculosis by traditional cesium irradiation techniques. D10 modeling is a stepping stone for comparative analysis of organisms to one another, and also maintains calibration potential to measure radioactive source decay over time. We then focused on the effect of riboflavin photochemistry on Mycobacterium smegmatis as a model for Mtb grown from large-scale culture. We demonstrated the potential for M. smegmatis to enter a viable but non-culturable state, as well as potential reluctance to readily uptake exogenous riboflavin, making it uniquely resistant to this specific method of photosensitizer damage. Characterization of damage to M. smegmatis through propidium iodide flow cytometry assays, and 8-oxoguanosine detection assays, also offered insight into the limitations of M. smegmatis as a model for Mtb.
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    Probing the metabolic secrets of Anopheles stephensi mosquitoes to enhance cryopreservation techniques
    (Colorado State University. Libraries, 2024) Ramirez, Gabriela, author; Dobos, Karen, advisor; Ebel, Greg, committee member; Barfield, Jennifer, committee member; Broeckling, Corey, committee member
    The lipid profile and cryopreservation methods of Anopheles stephensi mosquitoes have significant implications for mosquito management and research. The intricate life cycle of mosquitoes is governed by lipid metabolism, involving lipogenesis, lipolysis, and fatty acid metabolism, which are critical for transitioning between life stages. Despite the importance of these processes, mosquito cryopreservation has faced challenges, mainly due to the impenetrable nature of mosquito eggs to traditional cryoprotective agents. While other insects like honeybees and fruit flies have seen some success in cryopreservation, mosquitoes have posed unique difficulties. Initial attempts to cryopreserve mosquito eggs were unsuccessful, and there remains a need for effective cryopreservation protocols that maintain the viability and normal development of mosquitoes post-thaw. This study investigates lipid metabolism across mosquito life stages and advances cryopreservation techniques. The lipid profile analysis focused on major lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelins (SM), and triacylglycerides (TGs), as well as non-bilayer lipids like diacylglycerol (DG) and lysophospholipids (LysoPC, LysoPE). For cryopreservation, a new protocol using methylformamide (MF) with trehalose was developed for first-instar larvae (L1s) older than 1.5 hours post-hatching. The hypotheses were that 1) Lipid metabolism plays a crucial role in the successful development and reproduction of mosquitoes, and 2) Inducing diapause and using suitable cryoprotectants can improve the recovery rates of cryopreserved mosquitoes. The lipid profile analysis revealed that PE is vital for protein anchoring required for embryogenesis and immune responses and that steroids like 20-hydroxyecdysone (20E) are crucial for molting and development. The cryopreservation study showed that supercooling larvae for up to 60 minutes resulted in normal development to adulthood, although longer durations inhibited adult emergence. Supercooled larvae and their offspring exhibited typical sex ratios and developmental patterns, indicating genetic and phenotypic stability. This research underscores the well-orchestrated metabolic strategies in mosquitoes. It suggests that understanding these biochemical processes is essential for effective cryopreservation, potentially paralleling natural cold survival strategies seen in other insects.
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    Proteomic and biological diversity of closely related clinical isolates of Mycobacterium tuberculosis
    (Colorado State University. Libraries, 2009) Mehaffy, Martha Carolina, author; Dobos, Karen, advisor
    Recent studies have indicated that some Mycobacterium tuberculosis {Mtb) strains may be more easily transmitted and successful in causing disease. While this phenotypic diversity exits and is well documented, protein variation between Mtb strains appears to be small. Historically, the analysis of virulence determinants in Mtb has focused on the comparison of virulent to avirulent Mtb strains as well as on the comparison of single gene knock-out mutants to Mtb reference strains. Biological and biochemical comparison of Mtb clinical isolates with different degrees of virulence, although more complex than those described above, might provide additional information regarding the natural phenotypic variability that exists in Mtb. The focus of this study was to determine if a group of closely related clinical isolates of Mtb (BE, C28 and H6) as well as the more distantly related Mtb CDC 1551 present differences in both, virulence in the guinea pig model of TB infection and in the protein expression profiles of secreted and cytosolic fractions. The three closely related Mtb isolates comprise the S75 group, which has been reported to be very successful in causing disease. In contrast, Mtb CDC 1551, although reported to be highly transmissible, is associated with only a few number of active TB cases. To test the hypothesis that closely related Mtb strains would have similar virulence between each other, but higher virulence when compared to Mtb CDC 1551, guinea pigs were infected with each Mtb strain and time-to-death analysis was performed. Proteomes of secreted and cytosolic fractions of these strains were also analyzed and compared by 2D-GE and isobaric tag labeling (iTRAQ) followed by tandem mass spectrometry in order to test whether protein levels of these strains would present statistically significant differences when compared to each other and when compared to Mtb CDC1551.