Browsing by Author "Tjalkens, Ronald, committee member"
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Item Open Access Behavioral effects of estrogen receptor beta acting locally to regulate the expression of tryptophan hydroxylase 2 (THP2) in serotonergic neurons of the dorsal raphe nuclei(Colorado State University. Libraries, 2008) Donner, Nina Caroline, author; Handa, Robert J., advisor; Tjalkens, Ronald, committee member; Clay, Colin McKeown, committee member; Tobet, Stuart, committee memberAffective disorders often involve serotonin (5-HT)-related dysfunctions and are twice as common in women than men. Interactions between estrogen and the brain 5-HT system have long been proposed to contribute to sex differences in mood and anxiety disorders, but the mechanisms underlying this phenomenon have yet to be revealed. Estrogen signaling is mediated by two different receptors termed estrogen receptor alpha and estrogen receptor beta. While estrogen receptor alpha (ERalpha) has mainly reproductive responsibilities, in brain, estrogen receptor beta (ERbeta) has been shown to attenuate anxiety- and despair-like behaviors in rodent models. However, little is known about ERbeta regulation of function in the brainstem raphe nuclei. The raphe nuclei are the main 5-HT system of the brain, and projections from the dorsal raphe nuclei (DRN) innervate many important forebrain and limbic areas. The work presented in this thesis addressed the possibility that ERbeta may be involved in the regulation of 5-HT gene expression specifically in DRN neurons. My studies examined the effects of systemic versus local, intracerebral application of the selective ERbeta agonist diarylpropionitrile (DPN) and the nonselective ERligandestradiol (E) on tryptophan hydroxylase 2 (TPH2) mRNA expression within the DRN of female rats. TPH2 is the brain-specific, rate-limiting enzyme catalyzing 5-HT synthesis, and is expressed in every 5-HT neuron. Thus, it provides an excellent tool to assess the capacity for 5-HT production with the DRN. In these studies, TPH2 mRNA expression was assessed via in situ hybridization. In addition, relevant behavioral parameters were tested in all animals to evaluate each compound’s effect on two closely related, but yet different mental states, anxiety-like and despair-like behavior. Both, chronic systemic and chronic local DPN administration to ovariectomized (OVX) female rats significantly enhanced TPH2 mRNA expression in mid- and caudal subregions of the DRN after 8 days of treatment. Respective controls received systemic vehicle (27% hydroxypropyl-beta-cyclodextrin) or blank control pellets. Local application of DPN caused a stronger effect than systemic drug delivery. Chronic local delivery of E (0.5 μM) increased TPH2 mRNA expression in the same subregions of the DRN as did DPN, but its overall effect was weaker compared to the selective ERbeta agonist. Interestingly, while systemic DPN-administration confirmed the anxiolytic nature of ERbeta in two separate anxiety tests (elevated plus maze and open field test), the effect was lost when DPN was delivered locally. However, local DPN- as well as E-treatment both resulted in attenuated despair-like behavior, as measured in the forced-swim test. Chapter 3 describes the experimental design, results and interpretation of these studies in depth. Taken together, my data indicate that local actions of ERbeta agonist onto DRN neurons are sufficient to decrease despair-like behavior, whereas ERbeta stimulation of other brain regions is necessary to alter anxiety-like behaviors. Correspondingly, ERbeta acts locally to control TPH2 mRNA expression and presumably 5-HT synthesis in the certain subregions of the rat DRN. These results suggest an important role of ERbeta for regulating cellular events in the female DRN, and offer new opportunities for therapeutic treatments of depressive disorders.Item Open Access Biologic and biochemical features of prion pathogenesis(Colorado State University. Libraries, 2016) Hoover, Clare Elizabeth, author; Hoover, Edward A., advisor; Zabel, Mark D., advisor; Avery, Anne, committee member; Tjalkens, Ronald, committee memberPrions are the causative agents of a group of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies. Prions are unique in that disease is initiated when the normal prion protein (PrPC) undergoes a conformational change and propagates through a process of templated conversion to an infectious, misfolded, isoform (PrPRES, PrPCWD, or PrPSc) which can assemble into oligomers and amyloid fibrils. Disease is associated with prion accumulation in the central nervous system, causing the pathologic lesions of neurodegeneration, white matter spongiosis, and a reactive astrogliosis. Previous work has demonstrated the process of prion propagation and disease pathogenesis can be influenced by conversion cofactors, inhibitors, and biologic systems. Heat shock proteins have been shown to protect against the toxic disease effects of denatured and aggregated proteins in several models of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and spinocerebellar ataxia. In this dissertation, I investigated if heat shock protein 72 (HSP72) expression in neurons could protect against prion disease-associated pathology through a cell culture and mouse model of murine-adapted scrapie strain RML. In contrast to the role in other neurodegenerative diseases, HSP72 did not alter the prion disease course or amount of prion conversion in either disease model. Chronic wasting disease (CWD) is a naturally occurring, horizontally transmitted prion disease affecting wild and captive cervid populations that is rapidly expanding into new states and countries. Studies investigating the distribution of PrPCWD during early subclinical CWD infection have detected prions in the oropharyngeal lymphoid tissues as early as 1.5 months; however, the complete tissue distribution of PrPCWD immediately following prion exposure and the chronological progression of prion tissue accumulation remains unknown. Here, I show prions initially accumulate in the oropharyngeal lymphoid tissues following mucosal exposure and rapidly disseminate to all systemic lymphoid tissues prior to neuroinvasion. These findings will help better understand the early pathogenesis of CWD prior to clinical disease and potentially identify therapeutic targets. Prion disease diagnosis relies on demonstration of the misfolded isoform by immunodetection, amyloid seeding assays, or animal bioassays, all assays which may require separate sample preparations precluding examination by multiple tests. To address this limitation, I developed a new technique to detect PrPCWD amyloid seeding in fixed paraffin-embedded (FPE) tissues by real-time quaking induced conversion (RT-QuIC). FPE RT-QuIC proved to be more sensitive than IHC for prion detection and the use of RT-QuIC amyloid formation kinetics yielded a semi-quantitative estimate of the prion burden in samples without the cost and time of animal bioassays. The normal cellular prion protein resides in cell membrane lipid rafts, which has been shown to be a site of pathogenic conversion. Previous in vitro assays have highlighted the ability of lipids to promote prion formation but knowledge is limited regarding the capacity of lipids to inhibit prion formation. Here, I show endogenous polar brain lipids directly inhibit prion amyloid formation in RT-QuIC in a dose-dependent manner. This work is the first to identify an inhibitory role of lipids and suggests the prion conversion process is influenced by a balance of pro-conversion and inhibitory molecules.Item Open Access Design of a gait acquisition and analysis system for assessing the recovery in a classical murine model of Parkinson's disease(Colorado State University. Libraries, 2015) Damale, Pranav, author; Chong, Edwin K. P., advisor; Tjalkens, Ronald, committee member; Ebert-Uphoff, Imme, committee memberGait deficits are important clinical symptoms of Parkinson's disease (PD). Data focusing on gait can be used to measure recovery of motor impairments in rodents with systemic dopamine depletion. This thesis presents a design for a gait acquisition and analysis system able to capture paw strikes of a mouse, extract their positions and timing data, and report quantitative gait metrics to the operator. These metrics can then be used to evaluate the gait changes in mice. This work presents the design evaluation of the system, from initial cellphone captured video concepts through prototyping and testing to the final implementation. The system utilizes a GoPro camera, optimally lit walkway design, image processing techniques to capture footfalls, and algorithms for their quantitative assessment. The results gained from live animal study with methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced murine model of PD and treated with 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) are presented, and it is shown how the quantitative measurements can be used to determine healthy, injured, and recovering gait.Item Open Access Glial inflammation as a key regulator and therapeutic target for prion disease(Colorado State University. Libraries, 2023) Hay, Arielle, author; Zabel, Mark, advisor; Moreno, Julie, advisor; Tjalkens, Ronald, committee member; Chanda, Soham, committee member; Santangelo, Kelly, committee memberPrion diseases are lethal neurodegenerative diseases characterized by the misfolding of the cellular prion protein, PrPC, into the infectious PrPSc. PrPSc accumulation in the brain contributes to the activation of microglia and the subsequent increase in reactive astrocytes, which together contribute to neuroinflammation. PrPSc aggregation triggers and leads to the dysregulation of a variety of cellular stress pathways, including the oxidative stress response, unfolded protein response, ubiquitin-proteosome system, autophagy and lysosomal degradation. Most critically, PrPSc contributes to neuronal toxicity and death, but the mechanism behind this is poorly understood. Prion diseases affect humans and a variety of mammalian species, with no available treatments. The majority of therapeutics developed to combat these diseases have targeted the prion protein itself. As these have been unsuccessful, it is time to turn our attention to treatments that target the cellular pathways and neuroinflammation caused by PrPSc accumulation in the brain. The overarching goal of this work is to identify glial-induced inflammation as a candidate for therapeutic intervention of prion diseases. We assessed the use of mesenchymal stromal cells (MSCs), which are potent regulators of inflammatory signaling and glial polarization, in cell culture and animal models of prion disease. Additionally, we investigate the role of a key inflammatory signaling pathway, Nuclear Factor-Kappa B (NF-κB) in microglial response to prion infection. Our findings both characterize contributions of specific glial cells to prion-induced inflammation, as well as uncovering novel targets for the treatment of prion diseases. First, we assessed the therapeutic potential of adipose-derived mesenchymal stromal cells (AdMSCs) in a cell culture model of glial prion infection. MSCs are known for their ability to migrate to sites of inflammation and produce immunomodulators. We evaluated the ability of cultured AdMSCs to respond to molecular factors present in brain homogenates from prion-infected animals. We found that these cells upregulate anti-inflammatory genes in response to both specific inflammatory cytokines and crude prion brain homogenates. Moreover, AdMSCs migrate towards prion brain homogenates in an in vitro model. Co-culturing AdMSCs with prion-treated BV2 cells or infected primary mixed glial cultures resulted in a significant decrease in markers of inflammation and disease-associated microglia and reactive astrocyte markers. These findings were independent of PrPSc, as AdMSCs had no effect on prion accumulation in mixed glial cultures. Collectively, these findings highlight AdMSCs as an intriguing candidate for modulating glial-induced inflammation in prion disease. Next, we evaluated AdMSCs in a mouse model of prion disease. Prior to delivery into prion-infected mice, AdMSCs were stimulated with TNFα, which we show increases their upregulation of anti-inflammatory molecules and growth factors. Stimulated AdMSCs were delivered intranasally to prion-infected mice every two weeks beginning from early in infection (10 weeks post-infection (wpi)) and ending late in infection (20 wpi). A cohort of mice was euthanized at various stages in infection, at 14 wpi, 16 wpi and 18 wpi. We show that AdMSCs are able to migrate throughout the brain when delivered intranasally, with the most cells being found in the hippocampus and thalamus. Although AdMSCs were not successful in improving behavior or increasing survival in prion-infected mice, they did induce changes in prion pathology at early time points in disease. A decrease was seen in inflammatory cytokines and markers of glial activation. No changes were seen in PrPSc accumulation or neuronal loss compared to untreated controls. However, at both 16- and 18 wpi, we identified significant changes in both glial numbers as well as morphology, indicating that AdMSCs attenuate reactivity in microglia and astrocytes. Together, these findings highlight AdMSCs as potent regulators of prion-induced glial inflammation, and warrants further investigation to optimize these cells as a treatment for prion disease. In addition to assessing therapeutics that decrease inflammation and reprogram glial cells to a homeostatic phenotype, we wanted to better characterize specific inflammatory pathways and understand how these were being regulated in glial cells in response to prion infection. NF-κB-related genes have long been identified in the brains of animal models with prion disease, but studies that have investigated its role in prion pathogenesis have focused on neurons and astrocytes. Microglia are critical innate immune regulators in the brain, and interact closely with both neurons and astrocytes to regulate inflammation and cell survival. Therefore, we saw an immediate need to characterize NF-κB signaling in microglia, and its contribution to prion-induced neuroinflammation. IKβ kinase (IKK) is a complex that responds to cell stressors and is critical for NF-κB signaling to occur. We utilized a primary mixed glial model containing wild-type (WT) astrocytes and IKK KO microglia. Upon infecting these mixed glial cultures with prions, we saw a drastic decrease in NF-κB-related genes compared to cultures containing WT astrocytes and WT microglia. Despite this, cultures containing IKK KO microglia still contribute neurotoxic signals that induce neuronal cell death. Moreover, we found that cultures with IKK KO microglia showed significantly more PrPSc accumulation, suggesting that these cells may have impaired autophagy. This work implicates microglial NF-κB-signaling and IKK as a potent inducer of inflammation and regulator of autophagy in prion disease.Item Open Access Immune modulatory and antimicrobial properties of canine and human mesenchymal stem cells(Colorado State University. Libraries, 2018) Chow, Lyndah, author; Dow, Steven, advisor; Koch, Peter, committee member; Zabel, Mark, committee member; Tjalkens, Ronald, committee memberTo view the abstract, please see the full text of the document.Item Open Access Immune response modulation using Tolfenamic acid as an effective therapeutic for Burkholderia pseudomallei infection(Colorado State University. Libraries, 2016) Wilson, William J., author; Hanneman, William, advisor; Slayden, Richard, committee member; Legare, Marie, committee member; Tjalkens, Ronald, committee memberMelioidosis is a tropical, often fatal, disease caused by the aerobic, Gram-negative facultative intracellular bacterium Burkholderia pseudomallei. Despite its growing global burden and high fatality rate, little is known about the disease. Recent studies demonstrate that Cyclooxygenase-2 (COX-2) inhibition is an effective post-exposure therapeutic to pulmonary melioidosis by inhibiting the production of Prostaglandin E2 (PGE2). However, this effective treatment was conducted using an experimental COX-2 inhibitor which is not approved for human or animal use, therefore, an alternative COX-2 inhibitor needs to be identified for further studies. Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) developed for COX-2 inhibition and is marketed outside of the United States for the treatment of migraines. While this drug was developed for COX-2 inhibition, it has been found to modulate other aspects of inflammation. In this study, we analyzed the effect of TA on cell survival, regulation of COX-2 and Nuclear factor- kappaB (NF-ĸB) protein expression as well as PGE2 production using RAW 264.7 cells infected with B. pseudomallei. We used this information to develop a protocol to evaluate the effectiveness of post-exposure treatment with TA as a therapeutic and compared these results to effective Ceftazidime treatments and the co-treatment of TA with a sub-therapeutic treatment of Ceftazidime in BALB/c mice. TA effectively increased cell viability in vitro and was able to reduce COX-2 expression and PGE2 production while also decreasing NF-ĸB activation during infection. Oral administration of TA to BALB/c mice infected with B. pseudomallei was able to significantly increase survival outcome, however, this did not alter bacterial load or dissemination within organ tissues. The co-treatment of TA with a sub-therapeutic treatment of Ceftazidime was able to substantially increase survival outcome and clear the bacterial load within organ tissue. We demonstrate that post-exposure treatment with TA and sub-therapeutic Ceftazidime is effective to treat melioidosis in BALB/c mice. Additionally, we further elucidate the inflammatory response to B. pseudomallei.Item Open Access Inflammatory modulation of synovial fibroblasts in rheumatoid arthritis(Colorado State University. Libraries, 2017) Afzali, Maryam F., author; Legare, Marie E., advisor; Allen, Christopher, committee member; Goodrich, Laurie, committee member; Hanneman, William, committee member; Tjalkens, Ronald, committee memberRheumatoid arthritis (RA) has a prevalence of 1-2% and is one of the more common causes of chronic morbidity among people over 65 years of age. It is characterized by hyperplasia of fibroblast-like synoviocytes (FLS) within the synovium and recruitment of multiple leukocyte populations that drive the inflammatory process. Although disease etiology is unknown, it is thought that both genetic and environmental factors trigger the onset of RA. A key mediator of cellular inflammation and joint destruction in rheumatoid arthritis (RA) is the presence of fibroblast-like synoviocytes (FLS), a unique cell type that distinguishes RA from other inflammatory conditions of the joint. Due to their presence within the disease, FLS represent a possible target for next generation RA therapeutics, used in conjunction with immunomodulators, to control disease pathology without augmenting immunosuppression. The complex network of signal transduction pathways controlling FLS include inflammatory proteins such as cytokines, matrix metalloproteinases, cyclooxygenase (COX-2), mitogen-activated protein kinase (MAPK), nuclear factor kappa-B (NF-kB), and janus kinase (JAK-STAT), all of which have been implicated in the pathogenesis of RA. Under rheumatoid conditions, FLS express the tumor necrosis factor (TNF)-recognition complex (TNFR1, TNFR2, VCAM-1 and ICAM-1), which induces local macrophage activation and leads to downstream NF-kB signaling, that is partially responsible for propagating inflammatory damage within the joint. It is postulated that specific inhibition of NF-kB signaling in this system could mitigate FLS-driven inflammation without the negative off-target effects of global immune suppression. We investigated into C-DIM mechanism of action, DIM-C-pPhCl was examined in the RAW264.7 macrophage cell line treated with LPS to stimulate cytokine production. DIM-C-pPhCl treatment reduced the expression of inflammatory proteins such as NF-κB, iNOS, COX-2, and prostaglandin production following LPS stimulation. In addition, DIM-C-pPhCl treatment increased the amount of nuclear p65 and Nurr1 protein. As a final investigation into the mechanism of action of C-DIM12, was examined in primary murine synovial fibroblasts treated with TNF- to stimulate adhesion molecules and cytokine productions. These experiments provide evidence that NF-B directly mediates the induction of VCAM-1 in synovial fibroblasts by TNF- and furthermore C-DIM12 suppresses this activation. Demonstrating that C-DIM12 induces activity in synovial fibroblasts through a Nurr1 dependent mechanism. Providing a novel mechanism to decrease expression of NF-B regulated inflammatory genes in synovial fibroblast cells relevant to degenerative joint diseases.Item Open Access Metronidazole neurotoxicity(Colorado State University. Libraries, 2021) Vick, Zaria Denise, author; Moreno, Julie, advisor; Legare, Marie, advisor; Bouma, Jerry, committee member; Tjalkens, Ronald, committee memberMetronidazole is a broad-spectrum antibiotic approved for clinical therapeutic use in veterinary and human medicine. Although the literature has reported neurotoxic unintended side effects with the use of this drug, these incidences occur in less than 1% of human cases making this instance rare. The mechanism of this neurotoxicity has not been fully elucidated, nor the susceptible population identified. We explore in this work that these susceptible populations are humans and animals with concurrent localized and/or systemic inflammation. Some proposed mechanisms are axonal swelling with increased water content due to toxic injury, vascular spasm with mild reversible localized ischemia, modulation of the gamma-aminobutyric acid (GABA) receptors within cerebellar and vestibular systems, RNA binding with inhibition of protein synthesis, and axonal degeneration. While these mechanisms offer some insights into the neurotoxicity, we propose a novel connection between cholesterol inhibition and the reductive activation of metronidazole resulting in poor glial myelination that explicates low dose neurotoxic clinical outcomes in vulnerable humans and animals with the use of this drug. In order to investigate this, we have implemented physiologically based pharmacokinetic computational models of a human, equine, murine, and rabbit with metronidazole exposure. Furthermore, in combination with computational techniques, we assess cellular and molecular analyses to address this neurotoxicity in a primary murine glial cell model. Additionally, we use liquid chromatography and mass spectrometry work in order to address the reductive activation of metronidazole. We then ask if inflamed glia are more susceptible to metronidazole-dependent neurotoxic outcomes. With these data, we offer insight into this elusive mechanism and will aid human and veterinary literature in a way that improves the quality of life of affected patients and better predicts populations vulnerable to this neurotoxicity.Item Open Access Molecular characterization of novel transcription of antisense toxin-antitoxin RNA in regulating Mycobacterium tuberculosis(Colorado State University. Libraries, 2020) Dawson, Clinton C., author; Slayden, Richard A., advisor; Basaraba, Randall, committee member; Belisle, John, committee member; Karkhoff-Schweizer, RoxAnn, committee member; Tjalkens, Ronald, committee memberDespite more than seventy years of available anti-tuberculosis (TB) treatments, Mycobacterium tuberculosis (Mtb) remains the deadliest human pathogen. Novel short-course therapies are needed that effectively treat latent TB infection (LTBI), which is like a major source for new infections. However, the molecular determinants of LTBI, including a large repertoire of regulators encoded by Mtb that mediate survival, are largely uncharacterized. Gene expression studies have implicated numerous regulators and particularly toxin-antitoxin (TA) systems in Mtb pathogenesis. Whole genome sequencing (i.e. WGS) studies have linked the massive genomic expansion of TA systems along with other pathogen-specific gene families to the emergence of TB-causing mycobacteria. In addition, a multitude of TA systems show genotypic differences that distinguish between ancient and modern lineages of Mtb. These predominantly include lineage-specific changes in amino acids, altering antitoxin DNA-binding, and nucleotides, generating new promoters. These mutations have led to an overrepresentation of differentially expressed Mtb TA genes responsible for mediating epigenetic changes that are associated with gains in virulence of modern lineages. Thus, the work presented in this dissertation begins to define the novel co-regulation of TA systems that underlie Mtb pathogenesis. Unraveling of more complex regulation of Mtb TA systems will provide keen insights into the phenotypic changes responsible for Mtb survival and persistence in vivo. This will ultimately help to streamline research and development of novel antibiotics as well as host directed immunotherapies against hard-to-treat tubercle bacilli, effectively shortening the duration of TB treatment. TA systems are ubiquitous among bacteria, especially pathogens, and increasingly found to be essential for adaptation to host immune defenses and in vivo drug pressures, resulting in the development of persistent or chronic infections. Phylogenomics comparisons have revealed that Mtb encodes a significantly expanded repertoire of TA systems that are solely conserved by tubercle bacilli, including homologous ParDE/RelBE systems like RelBE2 (i.e. Rv2865-Rv2866). Herein, we report a novel antisense (as)RNA, we call asRelE2, which is uniquely encoded by Mtb and involved in differentially post-transcriptionally regulating relE2 mRNA levels as part of the response to host-associated stress such as low pH in a cAMP-dependent manner. This dynamic regulation of the tripartite relBE2/asrelE2 TA locus appear to be essential for long-term survival under acidic stress in vitro. In addition, the overexpression of relE2 is found to mediate phenotypic development of a persistent state in Mtb associated with increasing tolerance towards frontline anti-TB drugs isoniazid (Inh) and rifampicin (Rif). In mice, asRelE2 acts in differentially regulating bi-cistronic relB2 and relE2 mRNA levels in a host tissue-specific manner dependent upon the downstream effector functions of interferon gamma (i.e. IFN-γ) in murine TB. Specifically, relE2 and relB2 mRNA levels are found to steadily increase in lungs and in spleens, respectively, in the development of the chronic phase of Mtb infection. To our knowledge, this is the first time a Mtb TA system has been shown to be co-regulated by an asRNA antitoxin. Furthermore, this is linked with the development of the adaptive host immune response to Mtb, demonstrating that the post-transcriptional regulation of TA systems is an important mechanism, coordinating the epigenetic changes that are a hallmark of Mtb persistence and pathogenesis.Item Open Access The development of microfluidic devices for environmental and food quality analysis(Colorado State University. Libraries, 2012) Jokerst, Jana Catherine, author; Henry, Charles, advisor; Barisas, George, committee member; Strauss, Steven, committee member; Borch, Thomas, committee member; Tjalkens, Ronald, committee memberWhether termed micro-total analysis systems, lab-on-a-chip, or microfluidic devices, the technologies that define the field of microfluidics have shown great promise for overcoming many challenges in environmental, clinical, and biological analyses. The numerous advantages these devices bring to analysis, such as cost, reduced analysis time, minimal sample and reagent requirements, integration of multiple processing steps in a single device, and the possibility for automation and on-site analysis, make them attractive alternatives to conventional instrumentation. The work comprising this dissertation focuses on novel, miniaturized platforms for both food and water quality analysis. The development of two devices for these applications will be presented: a microchip capillary electrophoresis (MCE) method for the determination of perchlorate in drinking water and a paper-based analytical device (μPAD) for the detection of foodborne, pathogenic bacteria. Recent advances in microfluidics have had a significant focus in environmental analysis. The portability and rapid analysis these devices provide has brought us closer to on-site monitoring of environmental matrices and real-time measurements in these systems. Perchlorate has gained considerable attention over the past decade as a water contaminant. Prolonged consumption of contaminated drinking water has been linked to impaired thyroid function, leading to a number of adverse health effects. Current detection methods rely on expensive bench-top instruments, housed in a centralized laboratory. While these techniques offer high sensitivity and low detection limits, they are costly, time-consuming, and do not provide the ability to monitor perchlorate on-site. This dissertation describes the development of a microchip capillary electrophoresis device capable of rapid analysis of ppb levels of perchlorate in drinking water. Unique separation chemistry in which zwitterionic surfactant micelles are incorporated into the running buffer allows for selective analysis of perchlorate. The device performance was tested via analysis of spiked drinking water samples with detection limits of the system below the U.S. Environmental Protection Agency requirement. Further advancement of the device for analysis of more complex environmental samples is also discussed herein. The remaining chapters present a novel, paper-based device for bacterial detection. Faster, simpler methods of detecting foodborne pathogens are highly desired, particularly in the food industry. Existing methodologies are time-consuming, cumbersome, and require highly trained personnel. Often, several days are required for confirmation of food contamination, and in an industry with time-sensitive products, this delay is a major hindrance. Presented here is a paper-based analytical device (μPAD) developed for more rapid and simple detection of three foodborne pathogens: Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica. The μPAD provides a fast, easy-to-use technology for first-level screening that compliments existing methods. The device comprises a simple spot test on filter paper and utilizes species-specific enzymatic assays for colorimetric bacteria detection. In this work, the device is used to detect the three pathogens in spiked ready-to-eat meat samples. Concentrations as low as 101 cfu/cm2 were detected within an 8-12 h of enrichment. While this first phase of development shows great promise, work is ongoing to enhance assay selectivity and reduce overall analysis time.Item Open Access The physiological function and pathological significance of transient receptor potential ankyrin 1 channels in the cerebral artery endothelium(Colorado State University. Libraries, 2015) Sullivan, Michelle Nicole, author; Earley, Scott, advisor; Feng, Yumei, advisor; Dinenno, Frank, committee member; Tjalkens, Ronald, committee member; Amberg, Gregory, committee memberEndothelial cell Ca2+ dynamics have a significant influence on cerebrovascular tone. Several transient receptor potential (TRP) channels have been shown to mediate Ca2+ influx in the endothelium, including TRP vanilloid 4 (TRPV4), TRPV3, and TRP ankyrin 1 (TRPA1), which activates endothelium-dependent vasodilatory pathways. High resolution Ca2+ techniques have allowed for the recording of unitary TRP channel Ca2+ influx events, called TRP sparklets, in endothelial cells where they have been found to underlie vascular function. The following studies first characterize the biophysical properties of TRPV4 and TRPA1 sparklets in endothelial cells. TRPA1 channels are present in the endothelium of cerebral arteries and absent from other vascular beds, suggesting a critical, yet previously unknown function for the channel in this tissue. Research here describes the physiological function of TRPA1 channels as sensors of oxidative membrane degradation in cerebral artery endothelial cells. Further, the involvement of TRPA1 channels in delaying the onset of hypertension-associated spontaneous hemorrhagic stroke is examined.Item Open Access The role of cell-secreted vesicles in equine ovarian follicle development(Colorado State University. Libraries, 2013) da Silveira, Juliano Coelho, author; Bouma, Gerrit, advisor; Carnevale, Elaine, advisor; Winger, Quinton, committee member; Veeramacheneni, DN Rao, committee member; Tjalkens, Ronald, committee memberOvarian follicular development is a process responsible for generating a gamete and steroid hormones, which are important for reproduction and general health. Failure of intrafollicular cell communication is one of the causes behind infertility. Recently, cell-secreted vesicles (microvesicles and exosomes) were described as mediators of cell communication through the transfer of bioactive material such as protein, mRNA and miRNA. Cell-secreted vesicles are present in different body fluids. The overall hypothesis is that cell-secreted vesicles are present in ovarian follicular fluid and are involved in regulating TGF-β signaling members during follicular development. In order to test this hypothesis we utilized the mare as an animal model due to the well described follicular dynamics and the easy access to sufficient experimental material. Firstly, we described the presence of microvesicles and exosomes in ovarian follicular fluid from pre-ovulatory follicles. Further we demonstrated the presence of cell-secreted vesicles markers such as miRNA and proteins. We also demonstrated that microvesicles are taken up by granulosa cell in vitro and in vivo. Secondly, we demonstrated the role of exosomes mediating regulation of TGF-β signaling members during follicular development at mid-estrous and pre-ovulatory stages. Thirdly, we demonstrated association between relative levels of TGF-β signaling members and exosomal miRNAs during follicular development in young and old mares. The data indicates that cell-secreted vesicles play an important role mediating cell proliferation and differentiation through the regulation of TGF-β signaling members during follicular development.Item Open Access Unraveling prions: the complexities between the prion protein, complement, and B cells in diverse pathogenic settings(Colorado State University. Libraries, 2017) Kane, Sarah, author; Zabel, Mark, advisor; Bamburg, James, committee member; Tjalkens, Ronald, committee member; Avery, Anne, committee memberPrions diseases affect numerous mammalian species and may arise spontaneously, from genetic predisposition of the prion protein PrPC to misfold and aggregate, or from contacted with prion-contaminated materials. The first described prion disease, Scrapie, manifests in sheep, and records date back to the 18th century. Other mammalian species susceptible to prion diseases include humans, cats, mink, cervids (deer, elk, and moose), and cattle. The term Transmissible Spongiform Encephalopathy (TSE) arose to describe this new class of infectious diseases which exhibit spongiform degeneration in the central nervous system (CNS). TSEs are invariably fatal diseases, and only herd culling or breeding resistance mitigate disease spreading. However, chronic wasting disease (CWD) in cervids represents the first known TSE to occur in free-ranging wildlife, and the apparent facile spread demands strategies to halt its spread and prevent species eradication. Human prion disease characterization dates back to the 1920s. However, the bovine spongiform encephalopathy (BSE or mad cow disease) outbreak and subsequent transmission into a small number of humans in the 1980s and 90s pressed the need to understand the TSE agent. Many researchers since the 1960s postulated protein at least partially comprised the agent, but Stanley Prusiner provided the first scientific evidence of protein composition correlating with infectivity. Further, he coined the term proteinaceous particle, or prion. Follow-up research elegantly highlighted a host protein requisite to cause disease. Researchers now broadly accept the disease mechanism involves prions perverting the cellular prion protein to alter its conformation and join the highly stable growing prion aggregate. Upon peripheral exposure, most prion strains propagate in the lymphoreticular system prior to invading the CNS. Many elegant studies reveal the Complement system promotes initial prion trafficking and propagation in spleen and lymph nodes because mice deficient in various Complement proteins or receptors exhibit delayed or no disease. Once in the LRS, many postulate prions retrogradely infect the brain via sympathetic nerve fibers and the spinal cord. Once in the brain, prions provoke astrogliosis, neurodegeneration, and invariable death. While prion researchers made great strides in characterizing TSEs within a short few decades, many fundamental questions remain unaddressed. For example: what additional host factors foster prion pathogenesis? What is the normal function of the properly-folded, cellular prion protein? Lastly, do prion binding partners provide therapeutic targets? Data presented in this dissertation highlight crucial roles for Complement regulatory protein Factor H and Complement receptor CD21 in Scrapie pathogenesis, suggest C1q may strain-specifically impact prion disease, highlight PrPC as a crucial mediator in the adaptive immune system, and provide potential therapeutic tools and targets to combat prion disease.Item Open Access Western equine encephalitis virus: neuroinvasion, pathogenesis, and immunomodulatory treatment strategies(Colorado State University. Libraries, 2013) Phillips, Aaron Timothy, author; Olson, Kenneth, advisor; Brennan, Carol Blair, committee member; Aboellail, Tawfik, committee member; Tjalkens, Ronald, committee memberWestern equine encephalitis virus (WEEV; Alphavirus) is a mosquito-borne virus that can cause severe encephalitis in humans and equids. WEEV is closely-related to eastern equine encephalitis virus (EEEV) and may model similar pathogenesis in a mouse model. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan (McM) strain result in high mortality within 4 days of infection, thus providing a model of exposure to airborne encephalitic alphavirus. In addition, WEEV McM causes high mortality within 5-7 days following peripheral inoculation of mice. Therefore, WEEV McM may be used to model infection following exposure to infected mosquitos. The route of WEEV entry into the central nervous system (CNS) is not well-understood. In the studies presented here, bioluminescence (BLM) imaging and recombinant reporter viruses based on WEEV McM were applied to detect and track virus in mice following intranasal or subcutaneous inoculation, and used to determine correlation between BLM and viral titer. Additionally, histopathology analysis was guided by corresponding BLM images and used to identify specific CNS regions affected during infection. The major findings from these studies indicate that WEEV McM uses a different route for entry into the CNS for each of the two inoculation methods (intranasal or footpad). Intranasal challenge resulted in neuroinvasion occurring primarily through cranial nerves, mainly in the olfactory tract. Olfactory bulb neurons were initially infected followed by spread of the infection into different regions of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few infected non-neuronal glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem. Neuroinvasion from footpad inoculation demonstrated a consistent pattern in the spatiotemporal distribution of virus among the imaged brains, none of which involved the olfactory bulb. These studies support the hypothesis that neuroinvasion of WEEV likely occurs in areas of the CNS where the blood-brain barrier is naturally absent. These areas include the median eminence of hypothalamus (hypothalamic output), posterior pituitary, pineal body, and the area postrema. There are no antiviral therapies against alphaviral disease and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, a novel alphavirus vaccine comprised of cationic-lipid-nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto) was designed and tested. Interestingly, the CLNC component alone had therapeutic efficacy as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes; LANACs) using a prime/boost regimen provided strong protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. In addition, the LANAC immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV, indicating potential as a 'pan-alphavirus' vaccine candidate. Mice immunized with LANAC mounted a strong humoral immune response, but did not produce neutralizing antibodies.