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Browsing by Author "Winger, Quinton, committee member"

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    Elucidating mother to offspring transmission of chronic wasting disease using a transgenic mouse model
    (Colorado State University. Libraries, 2016) Willingham, Kassandra, author; Mathiason, Candace, advisor; Zabel, Mark, committee member; Suchman, Erica, committee member; Winger, Quinton, committee member
    Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE), or prion disease, of free-ranging and farmed cervids (deer, elk and moose). CWD is the only TSE in a wildlife population, which was initially discovered in a captive mule deer herd in a study shared between Colorado State University and University of Wyoming in 1967. CWD is the most readily transmitted of all the prion diseases and since its discovery has been identified in cervid populations in 24 states, 2 Canadian provinces, and the Republic of Korea. Horizontal transmission of prion diseases is thought to account for its exceptional transmission efficiency [2-10]. Recent studies published by our group provide evidence that transmission from mother to offspring may also be a contributing factor. In the work of this thesis, we employed a transgenic mouse system that expresses the cervid prion protein Tg(CerPrP-E226) to help elucidate the role of mother to offspring CWD transmission via hemochorial placentation. Females were inoculated with known CWD-positive material and subsequently bred with CWD-naïve males at various timepoints post inoculation to investigate if maternal/vertical transmission occurs in this host, as well as to further understand how this might occur. We examined the likelihood of prion trafficking in utero by analysis of mother: offspring pairs at different timepoints in CWD-infection and gestation, in addition to looking for infectious prions in milk collected from CWD-positive dams. We have demonstrated that CWD-infected Tg(CerPrP-E226) females successfully breed and bear offspring irrespective to TSE disease stage. Offspring born to CWD- infected females did not exhibit signs of TSE disease and lacked detectible PrPres via conventional methodologies. Interestingly, conversion competent prions were identified in the brains and spleens of offspring by highly sensitive amyloid seeding assays. The lack of symptoms in these offspring indicates covert prion transmission from mother to offspring, resulting in a potential silent-carrier status. As for our studies to further the understanding of the mechanisms behind this transmission, we identified CWD-prions in reproductive and mammary tissue, and spleen of Tg(CerPrP-E226) mouse mothers as early as 72 days post inoculation. In addition, we found minute quantities of amyloid conversion material in placenta and fetal tissues from mother:offspring pairs at varying timepoints in CWD-infection. We were unable to detect prions in milk collected from CWD-positive transgenic dams, leading us to hypothesize that the route of TSE transmission to offspring is likely a combination of environmental exposure, and/or very low concentrations of prions breaching the feto-maternal interface.
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    Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines
    (Colorado State University. Libraries, 2019) Salman, Saleh M., author; Bruemmer, Jason E., advisor; Bouma, Gerrit, advisor; Magee, Christianne, committee member; Pinedo, Pablo, committee member; Winger, Quinton, committee member
    Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. A human resistant telomerase reverse transcriptase (hTERT) lentivirus was utilized to establish stable equine embryonic cell lines. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast are the allantochorion (AC). Embryonic CG cells are unique to horses compared to other farm animals' embryos. The CG cells are the predecessor of endometrial cups (EC) that differentiate, proliferate, and invade the endometrium by day 38 of pregnancy, yet morphologically, both have similar characteristics supporting the fetal origin for EC. The CG cells have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study has three objectives: 1) establishing a stable cell line from day 30 CG cells and AC using lentivirus encoding hTERT; 2) Characterization of day 30 CG cells and AC cell morphology and expression of eCG alpha (eCGα) and beta (eCGβ) subunits, major histocompatibility complex class II (MHC II), and Kisspeptin receptor (KISS1R) in CG and AC cells; 3) investigating eCG protein production in vitro from day 30 CG and AC cells. Reverse transcriptase (RT) PCR was used to study gene expression in cells and radioimmunoassay (RIA) was used to investigate protein presence in the media. We established a hygromycin-resistant day 30 CG and AC cell lines that express eCGα, eCGβ, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages, 10 of which were cultured after the lentiviral infection steps. Also, we characterized CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGα and eCGβ subunits are expressed by both day 30 CG and AC cells, but MHC II and KISS1R genes were not expressed in either of cells. Moreover, RIA results showed that day 30 CG cells did produce eCG protein in vitro earlier than what previous literature has shown. However, day 30 AC cells did not produce eCG protein in vitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection.
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    Evaluating learning resource selection to support gross anatomy education
    (Colorado State University. Libraries, 2023) Martin, Jason, author; Magee, Christianne, advisor; West, Andrew, committee member; Meyer, Carolyn, committee member; Kaiser, Leann, committee member; Winger, Quinton, committee member
    Anatomy educators are tasked with developing, maintaining, and revising comprehensive curricula that strengthen the foundation of a growing body of scientific knowledge necessary to be successful medical professionals. This work sought to evaluate the impact of instructional timing, spatial ability, self-efficacy, and resource preference in the animal anatomy classroom for undergraduate and graduate students at Colorado State University. It identified factors relevant to optimizing student performance on animal gross anatomy examinations. The first chapter provides a background on anatomy learning resources in the context of self-efficacy and spatial ability research. Chapter two found that following a transition to remote instruction, students value resources that assist with navigating their learning ecology and assist with content mastery. Chapter three was a five-year retrospective study that identified a correlation between prior examination experience and dissection examination scores. Chapter four compared two measures of visuospatial ability with atlas- and specimen-based animal anatomy assessments following experimental single-resource and real-world multi-resource instruction. Chapter five was a mixed-methods analysis that developed an experimental framework used to describe the relationship between learner self-efficacy and animal gross anatomy assessment scores. Using this framework, increased time attending in-person didactic lectures and teaching assistants mediated open laboratories was found to be beneficial for low self-efficacy students while independent exploration of open-laboratory was beneficial for high self-efficacy students. An experimental study reported in chapter five failed to find a relationship between resource preference and performance on experimental anatomy assessments. When taken together, the research provides suggestions for anatomy educators seeking to use the selective distribution of learning resources to improve the instruction of animal anatomy students.
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    Gas6/AXL signaling contributes to GnRH-dependent activation of pituitary gonadotropes
    (Colorado State University. Libraries, 2023) Mohammad Zadeh, Pardis, author; Amberg, Gregory C., advisor; Hoerndli, Frederic, committee member; Stasevich, Timothy, committee member; Winger, Quinton, committee member
    Gonadotropin-releasing hormone (GnRH) receptor plays a fundamental role in reproduction and is prevalent in various urogenital, reproductive, and non-reproductive cancers. Beyond the conventional G protein-coupled receptor signaling, GnRH receptors interact functionally with multiple receptor tyrosine kinases. AXL, a receptor tyrosine kinase found in various tissues and numerous tumors, is the focus of this dissertation to discover its impact, along with its endogenous ligand Gas6, on GnRH receptor signaling. In this study, clonal murine pituitary αT3-1 and LβT2 gonadotrope cell lines were utilized to evaluate the effect of AXL activation on GnRH receptor-dependent signaling pathways. A combination of ELISA and immunofluorescence techniques was employed to analyze AXL and GnRH receptor expression in αT3-1 and LβT2 cells, as well as in murine and human pituitary sections. Additionally, ELISA was used to quantify alterations in ERK phosphorylation, pro-MMP9 production, and the release of LHβ. The abundance of Egr-1 transcripts was measured using digital droplet PCR. To assess αT3-1 and LβT2 cell migration responses to GnRH and AXL, trans-well migration assay was used. Results showed the presence of AXL, alongside GnRH receptors, in αT3-1 and LβT2 gonadotrope cell lines, as well as in murine and human pituitary sections. In line with AXL's potentiating role, Gas6 enhanced GnRH-dependent ERK phosphorylation in αT3-1 and LβT2 cells. Furthermore, Gas6 increased the abundance of Egr-1 transcripts, suggesting enhanced post-transcriptional GnRH receptor responses. Notably, in LβT2 cells, Gas6/AXL signaling not only stimulated LHβ production but also enhanced GnRH receptor dependent pro-MMP9 protein generation and promoted cell migration, underlining its functional significance. In summary, our findings unveil a hitherto undiscovered role for AXL as a modulator of GnRH receptor signaling.
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    Role of α/β hydroxylase domain containing protein 2 in stallion sperm activation
    (Colorado State University. Libraries, 2019) McQuagge, Matthew, author; Bruemmer, Jason, advisor; Pinedo, Pablo, committee member; Graham, James, committee member; Winger, Quinton, committee member
    Cell signaling pathways involved in stallion sperm activation are not completely understood. Furthermore, failure of equine in vitro fertilization is commonly attributed to an inability to successfully capacitate sperm. Sperm activation describes the process by which sperm undergo capacitation, hyperactivation, and acrosome reaction in preparation for interaction with an oocyte. 2-arachidonoylglycerol (2AG) is found in the human sperm membrane and prevents calcium influx through the CatSper channel. The α/β hydroxylase domain containing protein 2 (ABHD2) is also found in the human sperm membrane and functions as a progesterone receptor. When progesterone binds to ABHD2, it removes 2AG from the membrane allowing CatSper to open, which leads to calcium entrance into the cell, resulting in sperm activation. It is unclear if this mechanism holds true in stallion. Experiments were conducted to test the hypothesis that progesterone causes sperm activation through interaction with ABHD2 by 1) determining whether the ABHD2 receptor exists on stallion spermatozoa, 2) determining if progesterone binds to ABHD2 on stallion spermatozoa and 3) demonstrating the role of ABHD2 in sperm activation through correlations between ABHD2 and hyperactivation and/or acrosome reaction. Immunoblotting identified ABHD2 protein in stallion sperm and immunocytochemistry (ICC) localized the receptor to the tail region of stallion spermatozoa. Immunocytochemistry also demonstrated that ABHD2 binds progesterone by restricting fluorescence exhibited by ABHD2 when incubated with progesterone. Stallion sperm were evaluated for hyperactivation with computer assisted sperm analysis (CASA) following incubation in capacitation medium with either 1) an endogenous activator of sperm; 3 µM progesterone (P4), 2) a positive pharmacological stimulator of hyperactivation not associated with ABHD2; procaine or 3) a known ABHD2-action inhibitor methyl arachidonyl flourophosphatnate (MAFP). MAFP is a serine hydroxylase inhibitor and functions by preventing the removal of 2AG caused by exposure of ABHD2 to progesterone and, thus, limits hyperactivation. Flow cytometry was used to measure the acrosomal status of treated sperm as a subset of the hyperactivation measurements. When MAFP was administered prior to treatment with either P4 and/or procaine the hyperactive movement was inhibited (p < 0.05) in the presence of P4 but did not affect procaine induced activity. Results were similar for all ejaculates. The reduced hyperactivation of sperm when incubated with both progesterone and MAFP illustrates a potential connection between ABHD2 and CatSper. No change in acrosomal status was discovered through incubation with P4, procaine, or MAFP. These results indicate 1) that ABHD2 is present on stallion sperm, 2) that progesterone binds to ABHD2 and 3) that progesterone has the potential for causing hyperactivation but does not affect the acrosome reaction.
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    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 member
    Ovarian 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.