Theses and Dissertations

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Embargo
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.
Embargo
Of microbes and mothers: evaluating the complex maternal-neonatal interaction and microbiome-immunity development with novel Lactobacillus vaccination
(Colorado State University. Libraries, 2024) Ecton, Kayl E., author; Abdo, Zaid, advisor; Dean, Gregg, advisor; Wrighton, Kelly, committee member; Vilander, Allison, committee member; Argueso, Lucas, committee member
The task of identifying an optimal vaccination strategy for neonates has been challenging scientists and physicians alike. Multiple factors contribute to the difficulty in establishing an optimal platform including the complexity of the maternal-fetal dyad, a neonatal Th2 skewed profile and the role of the parallel development of the immune system and the gut microbiome (8). Disease remains a main cause of infant morbidity and mortality, encouraging the discovery of novel infant vaccinations to be delivered during the first 28 days of life to provide protection (41). Passive protection from the maternal transfer of transplacental IgG and both IgG and IgA in breastmilk has a limited window of operation, leaving the maturing neonate at risk (128). Although exact mechanisms remain to be elucidated, here we examine the complex crosstalk between mother-fetus and maternal-neonate dyads, neonatal microbiome-immunity development, and optimal delivery strategies for neonatal vaccine development. In this dissertation we investigated the role of maternal infection prior to gestation, neonatal challenge after vaccination, and vaccine effectiveness after exposure to virus. We evaluated the use of a novel vaccine platform developed previously in the lab as an orally delivered mucosal targeting subunit vaccine in Lactobacillus acidophilus. We investigated the effectiveness of the recombinant vaccine with and without adjuvants in a neonatal experimental design model and discovered increased virus specific responses in neonates vaccinated with adjuvants when challenged with rotavirus. We show a significant impact of maternal influence on neonatal outcomes. Beyond the immunogenic strength of the novel Lactobacillus acidophilus vaccine platform in neonates, we identified induced shifts to the gut microbial communities that occurred with vaccination or infection. We saw a shift in the gut microbiome over the course of a 7-day rotavirus challenge in neonates that did not return to baseline during the observation period, even after no virus shedding was detected in fecal samples. We also evaluated the impact of different doses, 1x106 CFU/dose and 1x109 CFU/dose, on the immune response and the gut microbiome. We confirmed the role of fecal microbiome transplants in breeding does to normalize for the maternal microbiome prior to gestation. Our results indicate that there are modifications to the gut microbiome and changes in immune antibodies during vaccination and infection. While we did not pursue a specific mechanism crosslinking the maternal-neonatal interaction and the gut-immunity relationship, we do consider the presence of such a connection.
Embargo
Mapping the metabolic protein interactome that supports energy conservation at the limits of life
(Colorado State University. Libraries, 2024) Williams, Seré Anne, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Pilon, Marinus, committee member; Anderson, G. Brooke, committee member; Snow, Christopher, committee member
Distinct metabolic strategies yield energetic gains from a wide variety of substrates, yet only three overarching methods of energy conservation have been defined: substrate level phosphorylation, the generation of a charged membrane, and electron bifurcation. The dominant theme of known energy conservation mechanisms suggests that energy is conserved through the selective movement and management of electrons, thus essentially all life relies on redox (reduction and oxidation) reactions. Small molecule redox cofactors (such as NAD(P)+) and proteinaceous electron carriers (such as ferredoxins) are employed as electron carriers throughout the biosphere. Proteinaceous electron carriers offer the potential for selective protein-protein interactions to bridge reductive flow from catabolic reactions to the membrane, providing a "proteinaceous electron highway" for efficient electron shuttling. Specific redox protein partnerships have been shown to adapt to changing physiological conditions, suggesting that proteinaceous electron flux is tunable and provides a level of selectivity not possible with small molecule electron transport. While electron flux through a tunable and regulated system of protein interactions can offer exceptional energy conservation strategies, large gaps remain in our knowledge of how electron flux is regulated in vivo. Identification of bona fide in vivo protein assemblies – and how such assemblies dictate the totality of electron flow and thus cellular metabolism – is an important milestone to understand the regulation imposed on metabolism, energy-production, and energy conservation. Resolving the dynamic nature of nanoscale interactions in living systems is arguably the current frontier of molecular biology, and combinatorial methods – which layer multiple in vitro and in vivo techniques with large data analysis – have come to the forefront. This dissertation addresses energy conservation strategies of in vivo protein associations in a model, genetically accessible, hyperthermophilic archaeon (Thermococcus kodakarensis) by mapping the metabolic protein interactome using affinity purification mass spectrometry (AP-MS) and generating engineered strains where fusion proteins selectively redirect electron flux in vivo. Twenty-five proteins involved in distinct metabolic functions were tagged to reveal that each tagged-protein interacts with ~ thirty proteins on average. These interactions connected disparate functions suggesting catabolic and anabolic activities may occur in concert -- in temporal and spatial proximity in vivo. The AP-MS method also refined our understanding of previously determined stable complexes suggesting that protein complexes in vivo likely adapt to redox conditions. Engineered strains linking a proteinaceous electron donor to a proposed electron acceptor were viable and impacted electron flux in vivo. Fusion strains linking a ferredoxin to the hydrogen-generating respiratory system increased hydrogen gas output ~8% on average with one strain showing a ~45% increase over wild type. Fusion strains impacting lipid saturation were shown to inhibit saturation, and future studies aim to determine if electrons can be redirected from the vast reductant sink of lipids to the generation of hydrogen gas, a valuable biofuel.
Embargo
Characterization of modes and kinetics of mutation accumulation in Saccharomyces cerevisiae through the analysis of defined cellular lineages
(Colorado State University. Libraries, 2024) Stewart, Joseph, author; Argueso, Juan Lucas, advisor; Moreno, Julie, committee member; Regan, Daniel, committee member; Wiese, Claudia, committee member
In the field of evolution, gradualism is the process of incremental adaptation supported by a slow and random accumulation of mutations that, over time, lead to genetic diversification and fitness gains. Although this Darwinian model is well supported and widely accepted, it cannot always explain the rapid changes seen in some instances such as tumors with extremely high and complex mutation loads. Recent reports in various organisms, including from our group using Saccharomyces cerevisiae, provide evidence for an additional mode of rapid and non-independent accumulation of chromosomal rearrangements. We have used a yeast model to follow the accumulation of structural genomic rearrangements such as loss of heterozygosity (LOH). We found that while chances of a single LOH event happening are very low, two or more LOH tracts co-occurred at rates 25- to 200-fold higher than expected if these events were independent of each other; therefore, the conventional process of slow and independent accumulation of mutations are not sufficient to account for every change in the genome. In the present study, we focused on temporal kinetics of bursts of LOH accumulation in yeast. We developed a hybrid diploid yeast experimental strain that enables identification of LOH event both through counter-selection and visual screening for colony color. This hybrid strain, made from the S288c and SK1 genetic backgrounds, possesses ~55,000 heterozygous SNPs distributed throughout the genome and allows for ease of tracking LOH events through sequencing. The screening approach was used in combination with microcultures (one cell grown for 5 or 6 divisions) in phylogenetic analyses that unambiguously revealed 18 cases where multiple LOH events co-occurred in the same cell division cycle. Collectively, these studies offer support for punctuated bursts of mutation accumulation caused by systemic genomic instability (SGI). Additionally, we have investigated a potential mechanism that influences SGI, namely global noise in gene expression.
Open Access
HopBA1, a pathogen virulence factor, reveals tissue-specific immune responses within the Pseudomonas syringae pv. tomato–Nicotiana benthamiana pathosystem
(Colorado State University. Libraries, 2024) Todd, Tyler Scott, author; Nishimura, Marc, advisor; Sloan, Dan, committee member; Roberts, Robyn, committee member
Plant pathogens represent a major threat to food security as they dramatically reduce crop yield, impact the expression of desirable traits, and reduce post-harvest longevity. To infect host plants, bacteria like Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) utilize a type III secretion system to deliver virulence proteins (also known as effectors) into the host cytoplasm to suppress immunity. In response, plants have evolved intracellular immune receptors that perceive immunosuppression and reactivate immunity. Thus, a given pathogen effector can both suppress and activate immunity depending on the host genome. Dissecting the molecular mechanisms of plant pathogen effectors helps inform our understanding of both disease and resistance. The present work reveals an uncharacterized role for Pst DC3000 as an aggressive vascular pathogen, causing systemic infection in the model plant Nicotiana benthamiana (Nb). Further, it establishes that the bacterial effector HopBA1 inhibits movement through the vascular system, despite increasing pathogen persistence within the primary infection site in leaves. Simultaneously, HopBA1 was found to induce irreversible upward vertical bending (i.e., hyponasty) in the petioles of infiltrated leaves, a novel phenotype for a bacterial effector. LC-MS/MS and RNA-Seq revealed phytohormone alteration (notably, a reduction in auxin and jasmonic acid-related metabolites) and transcriptional reprogramming of both developmental and defense genes. HopBA1-dependent growth restriction was suppressed in Nb eds1 (ENHANCED DISEASE SUSCEPTIBILITY 1) mutant plants, which still undergo HopBA1-induced hyponasty. Together, these results suggest that (1) HopBA1 triggers tissue-specific immune responses and (2) hyponasty is due to HopBA1's virulence activity, rather than host immune activation. Thus, HopBA1 in combination with the model pathogen Pst DC3000 becomes an important tool to dissect the poorly understood area of vascular-specific immunity. Vascular pathogens are particularly devastating and difficult to manage in crop species owing to the pathogen's internal location and systemic route of infection, making this research useful in crop improvement.
Open Access
Detection of bovine respiratory pathogens using real-time PCR and bead-based technologies
(Colorado State University. Libraries, 2024) Holmes, Joey, author; Pabilonia, Kristy, advisor; Mayo, Christie, advisor; Ehrhart, Nicole, committee member
The global cattle industry suffers financial losses of $900 million USD annually from infections caused by respiratory pathogens in the bovine respiratory disease complex (BRD). Accurate and timely detection of BRD pathogens provides cattle producers with a diagnosis so they can institute patient care and prevent pathogen spread. We sought to implement Luminex xTAG technology to detect four pathogens that cause BRD - bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), bovine herpes virus-1 (BHV-1), and Mycoplasma bovis (M. bovis). We compared singleplex real-time polymerase chain reaction (real-time PCR) to a newly developed xTAG testing protocol. Nucleic acids were extracted from 28 bovine lung samples that previously tested positive on PCR for each of the viral pathogens: BRSV (5), BVDV (5), BHV-1 (5), and M. bovis (5). All samples for BRSV and BHV-1 were detected on xTAG with a mean fluorescent index (MFI) well above 10,000 while detection of BVDV is limited to an MFI of 10,000 and M. bovis is detected inconsistently by xTAG. Lungs from six co-infected animals that tested positive for two BRD pathogens were tested on xTAG and real-time PCR side-by-side, revealing similar findings to the single positive lungs where BHV-1 and BRSV targets are more detectable than BVDV and M. bovis. Spiked pools of all pathogens resulted in MFI decreases as the number of pathogens per sample increases. With proper optimization, Luminex xTAG may be utilized in the veterinary diagnostic setting to circumvent issues with multiplex real-time PCR while maintaining high standards of diagnostic testing.
Open Access
Novel therapeutics, associated adverse effects, and changes in immune responses during pulmonary infection with Mycobacterium tuberculosis
(Colorado State University. Libraries, 2024) Ali, Malik Zohaib, author; Gonzalez-Juarrero, Mercedes, advisor; Basaraba, Randall J., committee member; Moreno, Julie A., committee member; Chen, Chaoping, committee member
Patients diagnosed with multidrug resistant (MDR) or extensively drug resistant (XDR) tuberculosis (TB) have limited treatment options. The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) collectively termed as BPaL for the treatment of TB. This regimen achieved remarkable results as almost 90% of the participants suffering from MDR- or XDR-TB had favorable outcomes. Despite the extraordinary outcomes, many patients also developed severe adverse effects (AEs) which were associated with the long-term administration of the oxazolidinone protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a potent protein synthesis inhibitor of Mycobacterium tuberculosis (Mtb) with an excellent safety profile, but which lacks oral bioavailability. In chapter 2, we hypothesized that inhaled spectinamide 1599, combined with BPa ––BPaS regimen ––, has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the BALB/c (permissive resistant) and C3HeB/FeJ (permissive susceptible) murine chronic TB efficacy models. Both regimens promoted similar bactericidal effects in lung and spleen of both models after 4 weeks. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cell counts while increased the number of B and helper and regulatory T cells. This combined data suggests that inhaled spectinamide 1599 combined with BPa is an effective TB therapy that avoids L-associated AEs. The granuloma formation is the pathological hallmark of TB, and several studies suggest that there are temporal and spatial changes in their distinct immune responses. These changes differ not only from one granuloma to another in a single individual but also depend on the severity of the disease. In chapter 3, we attempted to understand longitudinal changes in immune cells, their relationships, and their spatial distribution in granulomas of Mtb infected BALB/c and C3HeB/FeJ mouse models using a novel technique of multispectral imaging microscopy. Multiplex fluorescence immunohistochemistry (mfIHC) is unique in its ability to provide both expression and location of several immune cells along with their co-localization in a single tissue section while preserving tissue architecture and spatial context. The results showed that as the infection progresses, there are also dynamic changes in the immune phenotypes forming the granulomas and those located within the parenchymal tissue. Moreover, the histologically similar granulomas manifested complexity in their immune cell composition mainly due to the presence of adaptive immune responses. The advanced cellular granulomas in BALB/c TB model were mainly predominated by CD4 and CD8 T cells, Ly6G stained neutrophils, B220 B cells and all these were surrounded by F4/80 macrophages. With time post infection, there was an increased uniform recruitment of CD4 and Foxp3 T cells, F4/80 macrophages and Ly6G neutrophils within granulomas compared to parenchymal tissue where IFNγ and IL-10 secreting cells were in abundance. Moreover, B220 B cells and CD8 T cells also showed increased but heterogeneous distribution among the advancing granulomas especially B220 B cells formed clusters. The spatial analysis showed an increased median distance for Ly6G neutrophils, whereas this distance was decreased for B220 B cells when measured from CD4 and CD8 cells. In summary, combining the spatial and temporal data in addition to the mere cell counts helps to uncover interactions and relationships between different immune cells within the granuloma.
Open Access
Comparison of radiobiological endpoints in cells from CXB RI mice
(Colorado State University. Libraries, 2008) Xiao, Guanxiong, author; Weil, Michael M., advisor
Recombinant inbred (RI) mouse strains have been used both for trait cosegregation studies and genetic linkage analysis. They are created by using a breeding scheme that consists of a cross between two inbred mouse strains (progenitor strains) followed by at least 20 generations of brother-sister inbreeding. Thus, RI strains are inbred (homozygous at every locus) and derive roughly half their genome from each of the two progenitor strains. The CXB RI strain set consists of 13 RI strains derived from matings of BALB/c (C) and C57BL/6(B) mice. The CXB progenitor strains, BALB/c and C57BL/6, differ in their susceptibility to radiation-induced mammary tumors with BALB/c being susceptible and C57BL/6 being resistant. In part, the susceptibility difference can be explained by a polymorphism in the Prkdc gene which encodes the catalytic subunit of DNA-dependent protein kinase. However, other, as yet unknown, loci may be involved. The CXB RI strain set provides a useful tool to unravel the events that lead to radiation-induced mammary tumorigenesis and to understand the interrelationships of cellular radiobiological endpoints to one-another. We have generated fibroblast strains from each of the CXB RI strains and from the progenitor strains. The fibroblast strains were assayed for a number of radiobiological endpoints including clonogenic survival following acute and low dose-rate exposures, γ-H2AX focus formation and clearance following acute and low dose-rate exposures, and G2 chromosomal aberrations. In addition, we genotyped the strains for a polymorphism in the gene encoding the catalytic subunit of the DNA-dependent protein kinase, Prkdc. We then determined the correlations of different endpoints between the RI strains. As expected, clonogenic survival at low dose rates and following acute exposures were positively correlated. γ-H2AX focus formation at low dose rate correlated well with survival endpoints, particularly clonogenic survival under low dose rate irradiation and the surviving fraction at 2 Gy acute exposures. These three endpoints are all significantly associated with the Prkdc genotype with radiosensitive strains having the BALB/c genotype. The data we have collected provides a baseline description of cellular radio sensitivity in CXB fibroblasts. The approach used in this dissertation can be used to correlate these cellular radiobiological endpoints with susceptibility to clinically significant adverse outcomes from cancer radiotherapy, such as normal tissue injury and radiation-induced second cancers.
Open Access
Examination of molecular genetic factors involved in sensitivity to breast cancer following radiation exposure
(Colorado State University. Libraries, 2008) Williams, Abby J., author; Ullrich, Robert L., advisor; Bailey, Susan M., advisor
Understanding DNA repair is not only an important aspect of cell biology, but also has important implications for the field of carcinogenesis since cancer most likely occurs from genetic damage that occurs over one's lifetime. DNA repair needs to be accurate and efficient in order for a cell to maintain genomic stability, and defects in repair systems can result in radiosensitivity. Because radiation exposure, DNA repair deficiency and telomere malfunction are associated with cancer risk, we investigated Lymphoblastoid Cell Lines (LCLs) from breast cancer patients and controls for chromosomal radiosensitivity, relative telomere length, and gene expression changes. The importance of studying peripheral blood lymphocytes from cancer patients lies in the fact that minimally invasive techniques are lacking for the detection of individuals with high risk for cancer, and that telomere length has been proposed to be useful in this regard. Identification of radiosensitivity markers would be a valuable contribution for clinicians in hopes of avoiding excessive radiation or chemotherapy treatment given to patients. Failure to adequately repair DNA damage can result in cell suicide or halting of cell cycle progression in an attempt to allow repair mechanisms to operate. If damage persists, a cell can be pushed toward transformation and the pathway of carcinogenesis. A second aspect of the current work was to study the Homologous Recombination double-strand break repair protein, Rad51D. The emerging interrelations between DNA repair and telomere maintenance also prompted us to evaluate Rad51D's role in telomere function. The final aspect of this research involved examination of how DNA repair related proteins are linked to the indirect effect of ionizing radiation exposure known as the bystander effect (BSE). We are the first to demonstrate that DNA-PKcs and ATM are required to generate, but not receive, a bystander signal. We also show that mouse embryonic fibroblasts do not generate bystander signals to neighboring cells, while their adult cell counterparts do. Taken together, this work makes important contributions to our appreciation of the many and varied roles DNA repair related proteins play in maintenance of chromosomal integrity, proper telomere function, inhibition of carcinogenesis and now, regulation of the BSE.
Open Access
Interactions between plants and an opportunistic human pathogen, Pseudomonas aeruginosa
(Colorado State University. Libraries, 2008) Weir, Tiffany L., author; Schweizer, Herbert P., advisor; Vivanco, Jorge M., advisor
Pseudomonas aeruginosa is an opportunistic human pathogen that can be found living in soil, water, or saprophytically on plant tissues. It is important to understand the pathology of this organism under variable conditions because of its ability to survive in diverse environments, its role in human disease, and its use as a model organism in studies on biofilm formation, quorum-sensing, and pathogenicity. To this end, a number of unconventional model systems, including plants, nematodes, and fruit flies, have been developed to study the pathology of P. aeruginosa. In the present study, the interactions between P. aeruginosa and plants, with respect to pathogenicity, quorum-sensing, and microbial ecology are further explored. To examine what factors are important in the pathogenicity of P. aeruginosa in a plant system, compatible and incompatible cultivars of Nicotiana tabacum were infiltrated with the pathogen. Bacterial growth in planta was monitored and P. aeruginosa PAO1 gene expression was examined 24 hours after infiltration into the hosts. The data suggests that, in addition to known virulence factors, the acquisition of micronutrients such as sulfate and inorganic phosphate are also important in disease development. The results of this study also suggest that type III secretion systems may be important in P. aeruginosa's ability to infect plants, and that differences in host response, i.e. salicylic acid signaling, are determining factors in host compatibility. Another aspect of this study was to utilize the natural interactions between plant roots and soil-borne bacteria to identify root exudates that interfere with bacterial quorum sensing (QS), particularly in P. aeruginosa. Quorum sensing in P. aeruginosa controls the expression of several secreted factors that are important in virulence of the pathogen, and preventing infections by inhibition of quorum sensing is a current therapeutic target. Unfortunately, while many of the exudates appeared to have some affect on QS in general, none had strong activity against P. aeruginosa QS systems. However, one class of chemicals, triterpene saponins, was shown to be active in a lux-based QS reporter. Finally, preliminary data suggesting that root exudates can influence competitive outcomes between two soil-borne bacterial species are also presented. The interactions between bacteria are typically studied in nutrient rich medium under defined laboratory conditions. Under these conditions, P. aeruginosa outcompetes Agrobacterium tumefaciens, two bacteria that potentially compete for the same niche in the soil. However, when Arabidopsis thaliana is factored into this equation, growth of A. tumefaciens is favored. Furthermore, the negative effects of P. aeruginosa on the growth of A. thaliana were reduced.
Open Access
Isolation and characterization of proteins that interact with a pollen-specific calmodulin-binding protein
(Colorado State University. Libraries, 2008) Shin, Sung-Bong, author; Reddy, A. S. N., advisor
Calcium and calmodulin, a calcium sensor, are implicated in pollen germination and tube growth. However, the mechanisms by which calcium and calmodulin regulate these processes are largely unknown. Calcium bound calmodulin regulates diverse cellular processes by modulating the activity of other proteins called calmodulin-binding proteins. Maize pollen-specific calmodulin-binding protein (MPCBP) and its homolog (NPG1, no pollen germination) from Arabidopsis were isolated previously. Studies with a knockout mutant have shown that AtNPG1 is not necessary for pollen development but is essential for pollen germination. Analysis of the Arabidopsis genome sequence with AtNPG1 revealed the presence of two other proteins (AtNPGR1, NPG-Related1; AtNPGR2, NPG-Related 2) that are closely related to AtNPG1. To gain insights into the function of AtNPG1 and AtNPGRs, I focused my research on characterization of these proteins. Specifically, my research focused on in vivo localization of AtNPG1 in pollen grain and tube, interaction between AtNPGs, isolation and characterization of AtNPG1 interacting proteins, and functional analysis of AtNPGR1 in plant development. Transgenic plants containing GFP fused to AtNPG1 promoter showed GFP expression only in mature and germinating pollen, suggesting that the promoter is active only in pollen. Localization of GFP-AtNPG1, driven by AtNPG1 promoter, during different stages of pollen germination revealed uniform cytosolic distribution of GFP-AtNPG1 in the growing pollen tube that was similar to GFP alone. However, the observed uniform localization of GFP-AtNPG1 is not due to degraded fusion protein. AtNPGRs, like AtNPG1, bind calmodulin in a calcium-dependent manner. The calmodulin-binding domain in AtNPGs was mapped to a short region. AtNPG1 and AtNPGRs have several tetratricopeptide repeats (TPRs) that are known to be involved in protein-protein interaction. I tested the interaction among AtNPGs using the yeast two-hybrid analysis. AtNPG1-BD interacted with itself-AD and AtNPGR1-AD and AtNPGR2-AD. AtNPGR1-BD interacted with itself-AD, AtNPG1-AD and AtNPGR2-AD. However, AtNPGR2-BD did not interact with AtNPG1-AD or AtNPGR1-AD and showed a very weak interaction with itself-AD. To study the role of AtNPG1, AtNPG1 interacting proteins from a petunia pollen library were isolated in a yeast two-hybrid screen and identified as pectate lyase-like proteins. Using in vivo and in vitro protein-protein interaction assays, I show that AtNPGs interacts with four Arabidopsis pectate lyase-like (PLL) proteins with the highest similarity to petunia PLLs. Truncated AtNPG1 lacking the TPR 1 did not interact with most of partners or showed drastically decreased interaction with some proteins, suggesting that the TPR 1 domain is essential for this interaction. To understand the role of Arabidopsis PLL proteins, we characterized these using molecular and biochemical tools. Of the 26 Arabidopsis PLLs, fourteen were expressed in pollen and four AtPLLs were highly expressed. These four AtPLLs showed expression in other tissues also. Analysis of pectate lyase activity in Arabidopsis tissues (flower, root, stem, and leaf) revealed enzyme activity in all four tissues and the activity varied depending on the buffer pH. To see if AtNPG1 interacting AtPLLs have enzyme activity, four AtPLLs were expressed in bacteria or yeast and assayed for their enzyme activity under different conditions with different substrates. None of the AtPLLs expressed by bacterial or yeast showed pectate lyase activity. To discover the role of AtPLL in Arabidopsis development, one AtPLL mutant, atpll8, was isolated. Phenotypic analysis of atpll8 under different growth condition showed no significant differences as compared to wild type. AtNPGR1, unlike AtNPG1, is expressed in tissues other than pollen. To understand the role of AtNPGR1 in plant development, I isolated an atnpgr1 knockout mutant and characterized its phenotype under different growth conditions. The atnpgr1 showed a sugar resistance phenotype, suggesting that it might be involved in sugar sensing and/or signaling pathway. Expression of hexokinase (Hxk), an important component in sugar signaling in plants, and other genes in the Hxk pathway, revealed that NPGR1 might be involved in an Hxk independent pathway.
Open Access
Serial protein misfolding cyclic amplification (sPMCA) to detect surrogate markers for chronic wasting disease in surface water, municipal water and soil
(Colorado State University. Libraries, 2008) Nichols, Tracy A., author; Zabel, Mark, advisor
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy of deer and elk. Research has indicated that CWD is transmitted horizontally, and that both blood and saliva can transmit disease. Environmental exposure to pens where infected animals have been kept has resulted in disease transmission to deer. However, examination of environmental components such as soil and water for prions has been hampered by sensitivity limitations of conventional western blotting and inoculation limitations of bioassays. In this study we evaluated the ability of protein misfolding cyclic amplification (PMCA) to detect protease-resistant prion protein (PrPres) in environmental samples such as water and soil. Serial protein misfolding cyclic amplification (sPMCA) of PrPres, the misfolded proteinase-resistant protein associated with prion disease, was used to detect prion-infected brain homogenate spiked into soil and water to determine detection limits of this assay in environmental samples. The PrPres detection limit for water after 6 rounds of PMCA was 1:26 x 106. Detection of a CWD spike in soil with our current methodology was not possible. We next evaluated surface and drinking water from a CWD endemic region of Colorado for PrPres by sPMCA. PrPres was detected in Cache la Poudre River and flocculant samples at a time of high snowmelt runoff, suggesting that sPMCA can be a useful tool in evaluating water for PrP res in CWD-endemic areas.
Open Access
Actin dynamics in silico, in waves, and in rods
(Colorado State University. Libraries, 2009) Pak, Chi W., author; Bamburg, James, advisor
The current paradigm of actin dynamics and superorganization has advanced in the past decade from emerging technologies and perspectives, which include the discovery of actin nucleators, real time imaging of the dynamics of single filaments in vitro, and single molecule imaging of actin superstructures in vivo. These advances have influenced each of our studies on multiple levels, sometimes directly. A novel analysis of single actin filament dynamics revealed faster than expected dynamics during treadmilling but not during bulk polymerization. Using an exact stochastic simulation, we investigated whether filament-annealing and -fragmentation might account for faster than expected dynamics; their influence on actin dynamics had not been investigated before in a comprehensive model. Results from our work demonstrated that filament-annealing and -fragmentation alone cannot account for faster than expected dynamics during treadmilling. Thus, strictly through computational modeling, we are able to investigate various hypothetical models and offer insights into a process that cannot be achieved by experimentation. A concept that has also gained support during the past decade has been the self-organizing nature of actin, which was demonstrated by the Listeria actin-comet-tail reconstitution assay. We have proposed that this is a fundamental property of all actin superstructures, whether they are assembled in vitro or in vivo or whether they are involved in development or disease. The concept of actin's self-organization has influenced our study of neuronal waves, which are growth cone-like structures that travel along neurites and which were hypothesized to transport actin to growth cones and support neuritogenesis. Using diffusional analysis, we were able to demonstrate that neuronal waves transport actin. Neuronal waves provide a unique mechanism for transporting actin in that the delivery of actin is dependent upon actin itself and its dynamics. In disease states, the self-organization of actin is often changed but not disrupted, sometimes resulting in the formation of orderly-structured aggregates of cofilin and actin known as cofilin-actin rods (or rods). Using glutamate excitotoxicity as a model system for the cofilin pathology observed in Alzheimer disease (AD), we have determined signaling mechanisms for cofilin-actin rod induction, which in young rat hippocampal neurons require AMPA receptors and are calcium-independent. In addition, cofilin-actin rod interactions with microtubule associated proteins, and associated changes to the microtubule cytoskeleton were studied for its potential relevance to the pathology of AD. Our results suggest that disruptions to the normal organization of actin and microtubules might underlie several pathological hallmarks of early AD.
Open Access
Glial inflammatory signaling in manganese neurotoxicity
(Colorado State University. Libraries, 2009) Moreno, Julie Ann, author; Tjalkens, Ronald B., advisor
Degenerative movement disorders affecting the basal ganglia, including, Parkinson's (PD) and Huntington's diseases, are debilitating and currently incurable. Increased inflammatory gene expression in astrocytes promotes neuronal loss in these disorders, but the signaling mechanisms underlying this phenotype are not fully understood. In order to enhance understanding of this phenotype, the degenerative movement disorder, manganism, is a useful model, because patients suffering from excessive exposure to manganese (Mn) develop a neurodegenerative condition affecting the same brain region and with clinical features resembling PD. Recently, the potential effects of Mn on the developing brain have gained attention due to an increase in cognitive deficits with overexposure to Mn. Moreover, astrocytes are a known target of Mn, and reactive gliosis seems to precede neuronal injury. Mn toxicity enhances production of the inflammatory mediator nitric oxide (NO) in astrocytes by a mechanism involving NF-κB, the principal transcription factor responsible for expression of inducible nitric oxide synthase (NOS2). However, the role Mn toxicity plays in the developing brain along with the signaling mechanism(s) by which Mn enhances activation of NF-κB remains poorly understood in astrocytes. Therefore, in order to address the gap in knowledge I have characterized the role of glial cells in the promotion of neuronal damage in the developing and adult brain in a mouse model of Mn neurotoxicity, as well as the mechanism by which Mn enhances inflammatory activation of NF-κB dependent genes in astrocytes. First, it was identified that sGC relays signals to ERK and NF-κB, initiating NO signaling in astrocytes. Also it was determined that the glial inflammatory response leads to an age- and sex-dependent vulnerability of the basal ganglia which can be modulated by E2. This indicates that Mn toxicity in the developing brain results in locomotor deficits, reduction in normal dopaminergic neurotransmitter release, increased NOS2 expression in glial cells and neuronal injury. These findings are significant because once the mechanism of Mn-induced inflammatory activation of glial cells is understood, it will promote a better understanding of manganism and potentially other disorders of the basal ganglia.
Open Access
Role of gamma-aminobutyric acid (GABA) in hypothalamic nuclear development
(Colorado State University. Libraries, 2008) McClellan, Kristy Michelle, author; Tobet, Stuart A., advisor
The hypothalamus is involved in energy balance, appetite regulation, stress and anxiety, reproduction including sex behavior, and aggression. It is divided into cell groups based on cell staining, functional similarities and projections. This dissertation looks at the development of three of these cell groups, the ventromedial nucleus (VMN), the paraventricular nucleus (PVN), and the arcuate nucleus (ARC). The VMN and PVN have a unique pattern of GABA expression in elements surrounding the nuclei, with a void of GABA within the region of the developing nuclei. The ARC, on the other hand, has dense GABAergic immunoreactive elements throughout the nucleus. Because of the expression pattern of GABA immunoreactivity surrounding the VMN and PVN, it is likely that GABA is acting as a boundary cue to influence migrating neurons. Chapter 2 reviews the development of the VMN, including a discussion of the heterogeneity of the nucleus, a description of what cues are involved in cell migration, and descriptive information on the directions of cell movement during development. Chapter 3 examines the role of GABA on cell migration within the VMN and ARC. A live slice culture system allowed visual tracking of cell movement in the VMN and ARC. There was a difference between the average movement speeds of cells in the VMN vs. cells in the ARC, and upon addition of GABA receptor antagonists to the slices, VMN cells increased in speed while ARC cells did not change. In mice lacking functional GABAB receptors there was a change in cell positions of neurons containing immunoreactive estrogen receptor (ER)α, which may be due to changes in cell movements and migration. There was no change in amount of cell positions of immunoreactive ERα cells in the ARC. Chapter 4 looks at the role of GABA and brain derived neurotrophic factor (BDNF) in PVN development. There was a sex-selective effect in GABAB R1 subunit knockout mice of positions of cells containing immunoreactive ERÎα, indicating that females may be particularly more susceptible to changes in GABA signaling as it may be influencing the final positions of cells. There was also a decrease in BDNF expression in GABABR1 subunit knockout mice, suggesting that GABA may play a role in cell differentiation. In conclusion, several lines of evidence indicate roles for GABA in the development of the hypothalamus, in particular, within the VMN and PVN.
Open Access
Dengue viral pathogenesis and immune response in humanized Rag2-/-gamma c-/- (RAG-hu) mice
(Colorado State University. Libraries, 2009) Kuruvilla, Jes G., author; Akkina, Ramesh K., advisor
Dengue viral pathogenesis and vaccine studies are hampered by the lack of an ideal animal model mimicking human disease and eliciting an adaptive immune response. We utilized a novel mouse model that permits multi-lineage human hematopoiesis and immune response following transplantation with human hematopoietic stem cells. To generate immuno-competent humanized mice, we xenografted neonatal Rag2-/-γc-/- mice with human CD34+ hematopoietic stem cells, resulting in the de novo development of functional human adaptive immune system. To evaluate susceptibility to dengue viral infection, humanized mice were challenged with dengue virus (DENV). We observed viremia that lasted three weeks post infection with all four serotypes of dengue virus. We also demonstrated the presence of human anti-dengue antibodies. Anti-dengue IgM was first detected at 2 weeks post infection followed by IgG at 6 weeks. Our results demonstrated for the first time that humanized mice were capable of dengue viral primary immune responses, thus paving the way for further dengue immunopathogenesis studies. We challenged the mice that survived a primary infection with a secondary infection of dengue virus. We report increased viremia, severe dehydration, hemorrhage and morbidity in several mice during a secondary infection. We demonstrate the first instance of dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS) like phenomenon reported in any humanized mouse model for dengue infections. We also report the identification of cells that are actively infected with DENV in the spleen and lymph nodes of these mice using in situ hybridization and immunofluoresence staining methods. We used a SCID-hu model with an engrafted human thymus to study the direct infection of T-cells by dengue virus. We demonstrate that T-cells and CD34+ hematopoietic stem cells do not support a dengue viral infection. We were also able to infect monocytes and dendritic cells that were isolated and developed from human peripheral blood in vitro. In order to develop a consistent assay for antibody dependent enhancement (ADE) of dengue infection we were able to infect macrophages that were differentiated in vitro from CD34+ hematopoietic stem cells. In summary, this model provides a new tool to further our understanding of a complex disease like dengue hemorrhagic fever and dengue shock syndrome as well as provide a new system to test new vaccines as well as therapeutics against dengue viruses in the human context.
Open Access
Characterization of the flow cytometry mutation assay and its use in novel genotoxicity studies
(Colorado State University. Libraries, 2009) Keysar, Stephen Berkeley, author; Fox, Michael H., advisor
The flow cytometry mutation assay (FCMA) has been previously demonstrated to be a rapid and sensitive assay for measuring mutations induced by a wide variety of genotoxic agents. After treatment with a mutagen, the mutant fraction measured by the FCMA increased to a peak over time and subsequently decreased to a stable plateau. Using ethyl methanesulfonate (EMS), ionizing radiation (IR) and asbestos, I determined that the return to normal cell survival is an indicator of peak mutant expression. Decreased survival significantly contributes to the decline in the mutant fraction and confirms that mutations that are not clonogenically viable are measured by the FCMA on the peak day of expression. Also, I analyzed clones isolated from several CD59- regions and generated mutant spectra for EMS using flow cytometry. I then investigated hypoxia induced mutagenesis. It has been previously shown that hypoxic stress can generate DNA damage and mutations which are likely caused by reactive oxygen species (ROS). Here I demonstrate that the oxygen radical scavenger dimethyl sulfoxide significantly decreased cell killing and mutagenesis after hypoxia treatment, supporting the concept that ROS are responsible for hypoxia induced mutations. I also investigated the effects of silencing of DNA repair proteins on cell survival, cell cycle and mutagenesis. The knockdown of homologous recombination repair protein Rad51C slightly increased sensitivity to IR and drastically increased killing by EMS treatment. Rad51C knockdown also caused a significant G2 phase buildup after EMS treatment. Silencing of the non-homologous end joining (NHEJ) protein Ku80 increased cell sensitivity to IR and decreased the mutant yield after EMS treatment. This implies that EMS generates significant double strand breaks (DSBs) during S phase that are possibly repaired by NHEJ. In summary, I have demonstrated that the FCMA is a fast and reliable method to measure mutagenesis induced by an agent and to quantify the degree of damage by obtaining a mutant spectrum. I have used this assay to investigate mechanisms of mutagenesis by EMS, IR, asbestos and hypoxia and evaluated the effects of DNA repair pathways on cell cycle, cell killing and mutant induction.
Open Access
Long chain polyunsaturated fatty acids and oxytocin signaling in pregnant human myometrial smooth muscle cells
(Colorado State University. Libraries, 2009) Kim, Paul Y., author; Allen, Kenneth G. D., advisor
Since the suggestion by Olsen and colleagues that the pattern of exceptionally high birth weight in the Faroe Islands is related to a high intake of seafood, a number of epidemiological studies and interventional clinical trials have explored the possibility that consumption of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) abundant in fish and fish oil, such as 20:5 eicosapentaenoic acid (EPA) and 22:6 docosahexaenoic acid (DHA), may lengthen gestational duration. A dose response relation between dietary LC n-3 PUFA and gestational duration has emerged wherein expectant women who consume small amounts of fish stand to benefit the most from LC n-3 PUFA supplementation. The mechanisms are not well understood. Although prostaglandins (PG) of the 2-series are known to play a role in the initiation and progress of labor, an interpretation based on modulation of PG biosynthesis appears unlikely since idiopathic preterm labor is accompanied by low PG concentrations. Consequently, we investigated one potential PG-independent mechanism of LC n-3 PUFA action using a pregnant human myometrial smooth muscle cell line, PHM1-41, as a model. Our primary goal was to characterize the effect of DHA treatment on the signaling pathway of oxytocin, a potent uterotonic hormone involved in labor. The addition of 10 µM to 100 µM DHA to the culture media for 48 hrs resulted in incorporation and dose dependent enrichment of DHA in membrane lipid. DHA significantly inhibited IP3 elaboration (at 30 µM and 100 µM) and [Ca2+]i mobilization (at 100 µM) in response to oxytocin stimulation compared with bovine serum albumin (BSA) control and equimolar 18:1 oleic acid (OA). DHA at 30 µM and 100 µM significantly reduced receptor density in the membrane (Bmax) without altering the binding affinity (Kd) or rate of receptor internalization. These findings may be relevant to the reports of dietary fish and fish oil consumption prolonging gestation.
Open Access
Progress towards an understanding of radiation-induced mammary cancer using a murine model
(Colorado State University. Libraries, 2009) Klingler, Rebekah Henderson, author; Ray, F. Andrew, advisor
Theoretically, any exposure to ionizing radiation (IR) results in an increased risk of developing breast cancer. We have used a mouse model of radiation-induced breast cancer to study the effects of genetic background and molecular mechanisms of carcinogenesis. The BALB/c mouse strain is susceptible to radiation-induced mammary cancer while other laboratory strains are not. In this dissertation telomere-specific FISH was used to show that mammary epithelial cells derived from BALB/cByJ mice develop significantly more telomere-DSB fusions after IR exposure compared to those derived from C57BL/6J mice. The kinetics of telomere instability follow the same kinetics as the strain-specific genomic instability observed in earlier studies. An experimental system involving transplantation of cells from the breast of one mouse and regenerated in another mouse has been used extensively to demonstrate the genetic susceptibility of the BALB/c mouse to radiation-induced mammary cancer. The numbers of cells necessary for successful transplantation suggests that the cell capable of regenerating a mammary gland is a rare cell, perhaps a pluripotent stem cell. In this dissertation detailed protocols were created for isolation and tissue culture of murine mammary stem cells as mammospheres grown at high density, clonal density, and grown in basement membrane extract. A great deal of size variation was found in each culture of mammospheres. To test the hypothesis that only large mammospheres contained true stem cells the self-renewal capacity of specific sizes of mammospheres was tested using serial passaging. The data suggest that cells derived from larger mammospheres are capable of more passages than small mammospheres. Additionally, mammospheres were dissociated and tested for the presence of multiple cell lineages, as expected for pluripotent cells. Finally, we developed an assay to assess the radiation response of mammospheres derived from five strains of inbred mouse related to the BALB/c model of radiation-induced mammary cancer. These data show that mammary stem cells are more resistant to the killing effects of IR than fibroblasts derived from the same strains of mice. The data also show varying radiation sensitivities between genetically distinct mouse strains.
Open Access
Ovine pulmonary adenocarcinoma as an animal model for human lung adenocarcinoma
(Colorado State University. Libraries, 2008) Hudachek, Susan, author; Dernell, William S., advisor
Appropriate animal models of disease allow defined and controlled investigations that can ultimately be applied to the management of human disease. Based on symptomatic, histopathologic, and possible molecular signaling similarities, we hypothesized that sheep experimentally affected by OPA are a relevant animal model for the study of human lung adenocarcinoma and, in particular, for the evaluation of lung cancer therapeutics. The value of this model is dependent upon its predictability, reproducibility, amenability, and validity. The former two features have been previously reported; OPA induction in sheep is both predictable and reproducible following JSRV inoculation of neonatal lambs. The overall objective of this body of work was to assess the amenability of this animal model for therapeutic research and to assess the validity of OPA-affected sheep as an animal model for human lung adenocarcinoma in terms of genetic similarities. We determined that this animal model is amenable for therapeutic studies because, using CT, OPA can be detected early, before the onset of clinical signs, and cancer development can be monitored noninvasively. However, not only did we observe OPA disease progression during this study, but surprisingly, we also witnessed spontaneous regression of OPA. In fact, the latter was the more common outcome seen in our research after JSRV inoculation of neonatal lambs. We propose that the immune system, particularly CD3+ T-cells, is an important mediator of the spontaneous regression of JSRV-induced OPA seen in our work. Regardless of the cause, the mere occurrence of spontaneous regression of cancer in OPA-affected sheep severely restricts the use of this animal model for therapeutic research. In addition to assessing the amenability of OPA-affected sheep for therapeutic research, we also found that OPA tumors do not harbor genetic mutations in the TK domain of the EGFR, KRAS codons 12 and 13, or the DNA-binding domain of P53 and therefore, are not genetically similar to human lung adenocarcinomas that contain these mutations. Based on these genetic disparities, OPA-affected sheep are not an ideal animal model for human lung adenocarcinoma. Overall, the genetic profile combined with the disease development data provided further characterization of OPA and facilitated an assessment of the utility and relevance of this animal model for human lung cancer studies.