Browsing by Author "Moreno, Julie, committee member"
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Item Open Access Adult onset male hypogonadism: diagnosis and treatment(Colorado State University. Libraries, 2022) Akagi Bath, Natalie, author; Legare, Marie, advisor; Dooley, Greg, committee member; Moreno, Julie, committee member; Nickoloff, Jac, committee member; Popichak, Katriana, committee memberHypogonadism affects an estimated 2–4 million American men with increasing prevalence seen in older men. Normal aging processes lead to decreased biological production of testosterone. However, levels below physiologic function can decrease the quality of life and the life expectancy of men. A clinical diagnosis of hypogonadism, results from this failure to produce testosterone and or normal amounts of sperm and is more commonly referred to as low-Testosterone (low-T). Pharmacologic intervention with exogenous testosterone, hormone replacement therapy, can improve quality of life. However, this intervention is not without risks and should only be done when serum testosterone is below 300 ng/ml and is accompanied with symptoms associated with low testosterone. The aims of this work are to report the most commonly-used clinical symptoms associated with low-T for diagnosis, to provide a list of risks associated with hormone replacement therapy, and to analyze the different forms of pharmacologic intervention known commonly as Testosterone Replacement Therapy (TRT).Item Open Access Brain protein synthesis rates and energy sensing in sulfur-amino acid restricted mice(Colorado State University. Libraries, 2022) Martinez, Wenceslao, author; Hamilton, Karyn, advisor; LaRocca, Thomas, committee member; Moreno, Julie, committee memberProtein homeostasis (proteostasis) is the maintenance of the cellular proteome through protein synthesis, folding, trafficking, and degradation. Loss of proteostasis is considered one of the hallmarks of aging and is a driver for age-related neurodegenerative diseases. Dietary sulfur amino acid restriction (SAAR), a life-/healthspan extending treatment, activates mechanisms that maintain proteostasis in the liver. However, it is unknown if dietary SAAR activates mechanisms promoting proteostatic maintenance in the brain. To address this knowledge gap, wild-type male C57Bl/B6 mice were fed one of two levels of SAAR (expressed as % kcal); 0.18% methionine and 0% cysteine (Low SAAR) or 0.12% methionine and 0% cysteine (High SAAR), or a matched control diet sufficient (0.85-0.88%) in methionine. Deuterium enriched water was used to measure rates of newly synthesized proteins and DNA (as a marker of cellular proliferation). Brains were collected at days 1, 3, 7, 14, 21, and 35 of treatment. Mitochondrial, cytosolic, and mixed fractions of frontal cortex were analyzed for rates of protein synthesis and cell proliferation using GC/MS. A one phase association was used to determine the rate of the rise of newly synthesized protein and DNA to capture the kinetic parameter k (1/d). Phosphorylated and total protein content for AMP-protein kinase (AMPK), ribosomal Protein S6 (RPS6) and eukaryotic Initiation Factor 2 (eIF2) were measured at day 1 (acute) and day 35 (long-term) via western blot. Mitochondrial protein synthesis rates were significantly greater in the Low SAAR diet compared to the matched control diet, but did not differ in the cytosolic and mixed fractions. Protein synthesis rates in all fractions of the High SAAR diet were not different from control. There was no significant difference in cell proliferation rates between the SAAR diets and their control matched diets. However, in the High SAAR diet, as reflected by greater protein synthesis to DNA synthesis ratios, more newly synthesized proteins were allocated toward mitochondrial proteome maintenance rather than cell proliferation compared to control. At day 1, eIF2 activation tended to greater (p=0.0922) in the Low SAAR diet compared to control, but was not different at day 35 in the Low SAAR or High SAAR diets. AMPK activation did not differ in the Low SAAR or High SAAR diet compared to their controls at day 1 and day 35. RPS6 activation was not significantly different at day 1 or day 35 in either SAAR diet compared to their controls. This is the first study to simultaneously assess rates of protein synthesis and cell proliferation in the frontal cortex during dietary SAAR, as well as assess activation of key energy sensing proteins. The results from this study show that despite restriction of the sulfur amino acids, rates of protein synthesis were maintained in the cytosolic and mixed fractions with Low SAAR, while the rate of mitochondrial protein synthesis was greater than the control group with Low SAAR but not High SAAR. Dietary SAAR also promoted allocation of more newly synthesized proteins towards maintenance of the existing proteome, with less for new cell proliferation.Item 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 memberIn 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.Item Open Access Chronic wasting disease strain diversity, distribution and transmission(Colorado State University. Libraries, 2021) Wagner, Kaitlyn, author; Zabel, Mark, advisor; Avery, Anne, committee member; MacNeill, Amy, committee member; Moreno, Julie, committee memberChronic wasting disease (CWD) is an invariably fatal prion disease affecting captive and free-ranging cervids, including white-tailed deer, mule deer, moose, elk and reindeer. Since the initial discovery of the disease in the 1960's, CWD has spread across the US and Canada, South Korea, and, most recently, Europe. While some outbreaks of CWD were caused by transport of infected animals from endemic regions, the origin of CWD in other epizootics is unclear and not all outbreaks have been characterized. Previous studies have shown that there are multiple strains of CWD; however, the continuous spread and the unclear origin of several outbreaks warrant continued surveillance and further characterization of strain diversity. Moreover, studies implicating extraneural prions as more zoonotic motivated us to examine within-host prion strain diversity. The overarching goal of the work presented here was threefold: 1) address CWD strain differences between lymphoid and brain tissue from the same animal, 2) assess if there are any differences in CWD from either within or between contiguous and non-contiguous outbreaks and 3) address aspects of plant-vectored CWD transmission. The work presented here has important implications for understanding strain diversity within and between deer, as well as identifies samples that appear to be novel strains that warrant follow up assessment. Finally, we show how plants may be playing a role in vectoring infectious prions shortly after exposure. This research has important implications for our understanding of prion strain diversity and distribution as well as adds insight to plant-vectored prion transmission. First, we assessed differences between lymph node-derived and brain-derived prions from within the same animal to characterize strain differences within a single animal. To do this, we assessed isolates using biochemical techniques including electrophoretic mobility, glycoform ratio and conformational stability. Interestingly, we found that there were biochemical differences between lymph node and brain isolates, novel intermediate conformations of the prions in the brain (but not the lymph node) and increased variability in the lymph node-derived prions. Collectively, these results suggest that there are more diverse prion strains in the periphery and are distinct from neurological prions. The research discussed here advances our understanding of the differences between lymph node-derived and brain-derived prions. In addition to within-host strain comparisons, we also wanted to assess biochemical strain differences from naturally infected cervid species. Numerous studies have examined CWD strains upon passage into transgenic mouse models. For the purposes of our research, we wanted to examine CWD strains from the natural host for a number of reasons: 1) bioassay is expensive and time consuming, making strain characterization challenging, 2) research indicating that host factors other than PrPC may be influencing strain characteristics and 3) to determine if we could detect dramatic biochemical differences in strains, thereby providing an easier method to determine CWD strain prevalence in cervid populations without bioassay. Because the origin of CWD is unknown and some outbreaks of CWD have no clear exposure/connection to ongoing CWD outbreaks, this research would provide insight into the evolution and origin of CWD. Here, we show that there are some cases of CWD that present with novel biochemical characteristics that distinguish them from other CWD isolates. These instances suggest a new strain has emerged or that there is differential evolution in these subpopulations. Importantly, this work highlights that there is a lot more variability CWD biochemical characteristics than previously described. As a part of the strain typing project, two samples were received from captive white-tailed deer in Texas. These samples immediately proved to be a challenge to work with because they were behaving in an unusual way in our biochemical strain typing assays. In short, these isolates behaved in strange ways depending on the detergent class with which they were being digested. Because there was no known introduction of CWD to this captive herd, we were suspicious that we were seeing a novel strain of CWD. Isolates were passaged into cervid and human PrP mice. Upon passage, these isolates looked like classical CWD in Tg33 mice and, fortunately, don't appear to have any zoonotic transmission potential into human PrP mice. Importantly, this work highlights that CWD can present in a unique way in a cervid host but cause a classical-type disease in transgenic animals. Finally, we examined the role of plants to transmit CWD. Previous research implicated plants as having a possible role as a vector in prion transmission. We built upon this previous research by using CWD prions rather than hamster prions and a different plant model. The research presented here will show that plants are able to uptake prions shortly after exposure, but that these prions are no longer detected by 72 h. The work presented here implicates plants as potential CWD vectors in the short term.Item Embargo Evidence for acute inflammation in the pathogenesis of different mouse models of post-traumatic osteoarthritis(Colorado State University. Libraries, 2022) Timkovich, Ariel E., author; Santangelo, Kelly, advisor; Olver, Christine, committee member; Moreno, Julie, committee member; Goodrich, Laurie, committee memberPost-traumatic osteoarthritis (PTOA) is a debilitating, degenerative condition affecting more than 5.6 million people in the United States. Following injury to the knee, a person is 4.2 times more likely to develop PTOA in that joint, compared to an uninjured joint. PTOA is characterized by clinical symptoms such as pain, swelling, and decreased range of motion of the knee or affected joint. As a leading cause of pain and disability, PTOA is a major contributor to a decreased quality of life. Notably, loss of mobility associated with PTOA can increase the risk of a multitude of other ailments including heart disease and diabetes. PTOA due to traumatic joint injury can result in damage to intra-articular structures, such as menisci, ligaments, cartilage, and subchondral bone. Despite current treatment options, many of these joint injuries progress to end-stage PTOA within 10 to 20 years. Rupture of the most commonly injured knee ligament, the anterior cruciate ligament (ACL), typically occurs in athletes and people who are physically active, with the majority of ACL tears occurring in patients who are less than 30 years old. The detrimental pathways driving PTOA remain loosely defined and little progress has been made in predicting, preventing, and treating the disease. The goal of this work was to further our understanding of PTOA mouse models through both development and characterization of models, as well as through the testing of therapeutics in pre-established PTOA mouse models. In the first part of this work (Chapter 2), we created and characterized a novel full and partial ACL rupture model. This work is the first published partial ACL rupture model to date and provides a novel platform to use as a preclinical model for testing of potential therapeutics and to further our understanding of PTOA following ACL rupture. Chapter 3 evaluates the therapeutic potential of a toll-like receptor 4 (TLR4) antagonist on a full depth PTOA model. We found that short term treatment with a systemic TLR4 antagonist significantly improved relevant gait parameters, and improved cartilage structural metrics and modified Mankin PTOA scores, implying improvement of clinical signs. Chapter 4 addresses the therapeutic potential of intra-articular bone marrow aspirate concentrate (BMAC), for treatment of PTOA in a well characterized meniscus degeneration model, compared to red blood cell (RBC) depleted BMAC or no injection controls. These results suggest that RBC-depleted BMAC improved treatment of PTOA in an animal model and may have translational therapeutic potential for humans suffering from similar disease processes. Finally, Chapter 5 sought to: (1) Identify transcriptional sex differences in knee joint tissue from naïve animals; and (2) Identify compensatory gene expression changes related to sex that are specific to animals receiving DMM surgery as compared to a naïve animal when taking into account surgery changes (i.e., sham surgery). The work described in this dissertation has improved our understanding of the mouse knee joint during PTOA progression and potential therapeutics and targets for clinical treatment of PTOA.Item Open Access From Retroviridae to Flaviviridae: adventures in molecular virology(Colorado State University. Libraries, 2021) Butler, Molly, author; Rovnak, Joel, advisor; Quackenbush, Sandra, committee member; Stenglein, Mark, committee member; Moreno, Julie, committee memberThe work presented here encompasses two avenues of investigation: the first, regarding the identification of a novel retrovirus in Gunnison's prairie dogs, and the second regarding the role of cyclin-dependent kinases 8 and 19 (CDK8 and CDK19) as transcriptional regulators during infection with dengue virus serotype 2 (DENV2) and during the innate immune response. Part I: During the course of research and wildlife disease surveillance efforts, we identified three cases of thymic lymphoma in free-ranging Gunnison's prairie dogs (Cynomys gunnisoni). As Gunnison's prairie dogs are keystone species, that is, critical for the maintenance of their ecosystems, we investigated the potential for an association between the observed thymic lymphomas and retroviral infection. We identified a novel retroviral sequence which exhibits genetic organization consistent with a type D betaretrovirus and which was highly associated with thymic lymphoma in Gunnison's prairie dogs. The proposed name of this virus is Gunnison's prairie dog retrovirus (GPDRV). Part II: CDK8 and CDK19 are transcriptional regulators which are critical for modulating gene expression changes during induced states such as hypoxia and starvation. We investigated the role of CDK8 and CDK19 in two distinct but related induced states: infection with DENV2 and the type I interferon response. We found that in the context of DENV2 infection, CDK8/19 regulate metabolic gene expression changes, the result of which is a reshaping of the host cell metabolic environment which is ultimately beneficial to viral replication. Therefore, chemical inhibition or reduced expression of CDK8 or CDK19 significantly restricted viral replication. Both within the context of DENV2 infection and with non-viral stimulation of innate immunity, we identified a role for CDK8 and CDK19 as regulators of the type I interferon response. CDK8 and CDK19 have distinct and overlapping functions as regulators of IFN-β expression dependent on the nature of the stimulus. This work not only furthers our understanding of host transcriptional regulation during DENV2 infection and within innate immunity, but also the diverse and complex functions of CDK8 and CDK19 as key modulators of cellular stress responses.Item Open Access Mediators of mucosal integrity in the context of agriculture dust exposure(Colorado State University. Libraries, 2023) Chesterman, Carly Sue, author; Nordgren, Tara, advisor; Moreno, Julie, committee member; Podell, Brendan, committee memberInhalation of particulate matter, such as agricultural dust, can lead to lung inflammation and increased risk for chronic respiratory diseases. Agricultural workers, such as hog farmers, are constantly exposed to organic dusts from the workplace. Chronic pulmonary obstructive disease (COPD) is a type of lower respiratory disease, which results in an excessive inflammation cycle that leads to disease progression. Currently, available treatments merely treat the patient's symptoms with no effect on the prevention of disease progression. Metabolites of omega-3 fatty acids, called specialized pro-resolving mediators (SPM), can aid in inflammation resolution and promote immunity in the context of respiratory infection. IL-22 promotes mucosal immunity by regulating inflammation, inducing the production of antimicrobial peptides (AMP), and promoting epithelial repair. Mucosal surfaces in the airways are lined with epithelial cells, a mucus layer, and immune cells that act as the first line of defense against inhaled pathogens. The respiratory epithelium cells express antimicrobial peptides like beta-defensin-2 and Regenerating islet-derived protein 3 gamma (Reg3g); this expression can be stimulated by IL-22. In this study, we explored the effects of omega-3 fatty acids after organic dust exposure using a fat-1 transgenic mouse model, which represents the ideal ratio between omega-3 and omega-6 fatty acids. To further explore the impacts of IL-22 on AMP expression after organic dust exposure, a whole-body IL-22 knockout mouse model also was used in this study. Since omega-3 fatty acids and IL-22 promote inflammation resolution, we studied their impact on mucosal immunity and epithelial repair following repetitive challenges with extracts of organic dust in vivo. Wildtype, IL-22 knockout, and fat-1 mice were exposed by intranasal installations five times a week for 3 weeks. Lung tissue from mice exposed to either organic dust or saline were obtained and evaluated for AMP and wound repair markers. To evaluate the impact of IL-22 on AMP expression in the context of organic dust exposure, immunofluorescence (IF) staining, enzyme-linked immunosorbent assays (ELISAs), and RT-qPCR arrays were used. IF staining was done to assess beta-defensin-2 expression within the bronchial epithelial cells in IL-22 knockout and wildtype mice. While trends of positive staining for beta-defensin-2 were observed, no statistical significance was found. ELISAs were performed to assess for Reg3g expression in mice lung tissue; concentrations were found to be present in both the wildtype and IL-22 knockout models after saline and dust exposure. To assess for markers of wound repair in IL-22 knockout and wildtype mice models, a custom RT2 profiler PCR array was ordered to detect gene expression of different antimicrobial peptides, anti- and pro-inflammatory markers, and wound repair markers. The findings were as follows. Expression of AMPs—specifically S100A8 and S100A9—indicates a decrease among organic dust-exposed groups. The expression of wound repair markers CCL7 and ITGA3 exhibited decreases in the context of organic dust exposure. Expression of the anti-inflammatory marker MIF also exhibited a decrease among dust-exposed groups. To evaluate the impact of omega-3 fatty acids on AMP expression in the context of organic dust exposure, lung tissue from fat-1 and wildtype mice were stained for Reg3g expression. While trends of positive staining for Reg3g were observed, no statistical significance was found. IL-22 signaling exhibited trends of increased expression of AMP and pro-resolution mediators of mucosal integrity in the context of chronic dust exposure. Further studies should be conducted to determine the effects of omega-3 fatty acids on AMP expression in the context of organic dust exposure. These findings can be utilized to develop new treatment strategies for lung disease that focus on pro-resolution rather than solely anti-inflammatory methods.Item Open Access Microglial innate and adaptive immune function modulates disease pathology in and environmental pesticide model of Parkinson's disease(Colorado State University. Libraries, 2022) Rocha, Savannah M., author; Zabel, Mark, advisor; Tjalkens, Ronald B., advisor; Bouma, Jerry, committee member; Kading, Rebekah, committee member; Moreno, Julie, committee memberParkinson's Disease (PD) is the world's foremost movement disorder with pathological features including loss of dopaminergic neurons (DAn) within the substantia nigra pars compacta (SNpc), chronic activation of glial cells, and the misfolding and aggregation of a-synuclein (a-syn). Compounding evidence gathered over the past two centuries suggests environmental exposures, genetics, and aging can induce complicated cell-to-cell interactions that evoke and facilitate chronic inflammatory states; but the role that individual glial cells, in particular microglia, have in the progression of disease remains unknown. Difficulties in recapitulating the three pathological hallmarks of PD underscore the need for better animal models. To address this gap in functional investigation, the studies herein provide, for the first time, an optimized environmental exposure model with the pesticide rotenone (2.5mg/kg/day) in murine, which has proven effective at mirroring DAn degeneration, gliosis and misfolded a-syn accumulation. The pathology observed was region-, time- and dose-dependent, emphasizing the importance of environmental exposure and associated PD diagnosis. The successful optimization of this exposure model has allowed for its implementation in transgenic mice, which was previously unfeasible. To determine microglial specific innate inflammatory reactions in the progression of PD, we targeted the inflammatory transcriptional regulator NF-kB by use of transgenic CX3CR1-CreItem Open Access Neuroinflammation and the two-hit hypothesis of Parkinson's disease(Colorado State University. Libraries, 2019) Bantle, Collin M., author; Tjalkens, Ronald B., advisor; Zabel, Mark, committee member; Moreno, Julie, committee member; Kato, Takamitsu, committee member; Randall, Elissa K., committee memberThe ever-increasing prevalence of neurodegenerative diseases, Alzheimer's Disease (AD) and Parkinson's disease (PD), impose one the most significant medical and public health threats throughout the world. Characteristic PD symptoms include loss of voluntary motor control due to α-synuclein protein-aggregation, neuroinflammatory glial activation, mitochondrial dysfunction, oxidative stress, and progressive neuronal loss. There are currently no disease-modifying therapies for the disease nor has the etiology of PD been elucidated. Epidemiologic and experimental evidence suggests that genetic susceptibility, environmental pesticide exposure, and viral infections are possible risk-factors for PD, but a clear understanding of the environmental links to PD and how these factors can act in concert remains extremely limited. Research is beginning to shed light on neuroinflammation as a converging and coalescing pathway in the pathogenesis and pathophysiology of genetic, sporadic, and postencephalitic PD. While it has been appreciated since the late 1980s that brain inflammation is a hallmark of PD and other age-related neurodegenerative diseases, the immunological role of glia and the key trophic and inflammatory factors and pathways responsible for neurotoxicity and neuronal death in PD have not been clearly elucidated. Understanding how these pathways are regulated in glia during genetic, sporadic, and postencephalitic PD, and how they can directly or secondarily affect the onset and progression of PD is of keen interest. Therefore, the subject of this work will be to explore mechanisms by which glial cells modulate neuronal injury in genetic, sporadic, and postencephalitic cases of PD, with an emphasis on the role of neuroinflammatory activation of glia in single and two-hit models of PD.