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Browsing by Author "Hanneman, William H., advisor"

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    A multimethod simulation paradigm for investigating complex cellular responses in biological systems of aging and disease
    (Colorado State University. Libraries, 2019) Hoffman, Timothy Edward, author; Hanneman, William H., advisor; Legare, Marie E., advisor; Wallis, Lyle E., committee member; Moreno, Julie A., committee member
    Classical studies in toxicology and disease research have relied on the use of high-dose experiments and often lacked quantitative and comprehensive components essential to understanding biological queries. These shortcomings in the research community have been the result of modern methodological limitations, however, more robust and expansive experimental and computational methods are emerging. In this dissertation, I present a novel multimethod computational simulation paradigm that adds value to new and existing studies of toxicological and pathological endeavors. First, I established the use of this approach for pharmacokinetic and pharmacodynamic applications, with published examples in regulatory exposure toxicology and contemporary dose-response nuances. Following establishing the success of this approach in toxicology, I then applied this methodology to the broader question of degenerative aging, as it has been arduous with conventional techniques to understand the various mechanisms that contribute to and protect against cellular aging. The foundational simulation created for general cellular aging was then expanded in the context of tauopathies and Alzheimer's disease to better quantify and understand the pathways involved in this age-dependent disorder. The final results presented here improve experimental translatability, robustness and descriptiveness in order to better understand age-related diseases. More broadly, this dissertation in totality attempts to minimize quantitative deficits in toxicological and pharmacological research.
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    Characterization of DJ-1 mutation in mouse astrocytes
    (Colorado State University. Libraries, 2008) Ashley, Amanda Kathleen, author; Legare, Marie E., advisor; Hanneman, William H., advisor
    Mutations in DJ-1 cause early-onset Parkinson's disease (PD), a progressive, irreversible neurodegenerative condition. Currently, the only known cause of PD is mutation of certain genes including DJ-1, however these mutations account for only 5-10% of overall PD cases. The initial studies attempt to discern if expression of VEGF and HIF1α, factors thought to contribute to both PD as well as carcinogenesis were altered as a result of DJ-1 mutation. In fact, VEGF expression decreased in the brain of DJ-1-/- mice, and increased in lung tissue. As PD is a complex, multi-factorial condition, our studies are designed to incorporate mutation of the PD gene DJ-1 in our target cell type, astrocytes, which are exposed to toxic agents. Overall our results indicate that DJ-1-/- astrocytes do not have an exaggerated phenotype compared to DJ-1+/+ counterparts, however subtle alterations in cell function are observed in mitochondrial membrane potential, expression of proinflammatory mediators, as well as intracellular calcium (Ca2+) dynamics. First, DJ-1-/- astrocytes' resting mitochondrial membrane potential is significantly lower than that of DJ-1+/+ cells. Following treatment with 10μg/mL lipopolysaccharide (LPS), expression of COX2, and NOS2 were similar in both genotypes, however expression of TNFα was significantly lower in DJ-1-/- astrocytes. Finally, a delay in return to baseline intracellular Ca2+ levels following treatment with 1μM ATP was observed in DJ-1-/- cells. Interestingly, expression and secretion of TNFα were decreased in our DJ-1-/- astrocytes following LPS exposure, while expression of COX2 and NOS2 were similar. In conclusion, these changes, though modest, indicate basal dysfunction in astrocyte homeostasis induced by mutation of DJ-1. Secretion of TNFα may be the most significant finding, as it may predispose neurons to degeneration due to lack of sufficient protection against early neurotoxic insults that secreted TNFα may provide. These specific indicators are significant because mitochondrial dysfunction, altered neuroinflammation, and reactive gliosis are all implicated in PD. While altering astrocyte cellular function may not be the primary cause of DJ-1-linked PD, it is possible that changes in this cell type may contribute the progression of parkinsonism.
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    Protein expression of HER-2, CAV-1 and ER alpha in canine mammary tumors and canine osteosarcoma
    (Colorado State University. Libraries, 2007) Flint, Alfred Frederick, author; Hanneman, William H., advisor
    Human Epidermal Growth Factor 2 (HER-2, neu or erbB-2) is a protein that influences cell proliferation, morphological differentiation, and cell motility. Additionally, over expression of HER-2 has been shown to promote growth and invasion of cells of mammary neoplasia in vivo. Over expression of HER-2 has been identified in 25-30% of human and canine mammary neoplasms and osterosarcomas though the prognostic significance remains unclear. HER-2 over expression in human breast cancer correlates with a more aggressive tumor type, poor prognosis and resistance to chemotherapeutic agents. However, patients with breast cancer over-expressing HER-2 have benefited from anti-HER-2 therapy. By targeting HER-2, cell proliferation is subsequently inhibited by blocking intracellular signaling with direct targets to the cell cycle machinery. The studies presented examine the complex protein interactions of HER-2 in mammary neoplasia and osteosarcoma in canine patients. Real-time RT-PCR was used to evaluate HER-2 expression in 7 canine OSA cell lines and 10 canine OSA tissue samples. HER-2 is significantly over expressed in 86% (6/7) of the cell lines and 40% (4/10) of the OSA tissues samples. Given the importance of HER-2 in human breast cancer, the finding of HER-2 over expression in canine OSA may be important in further understanding the pathogenesis and possible therapies of OSA. Histomorphologic characterization and immunohistochemical analysis of HER-2, caveolin 1 (CAV-1), and estrogen receptor alpha (ERα) was performed on 144 canine mammary tumors from 44 different breeds. HER-2 was over expressed (score 3) in 23.4% (85/137) of the lesions. Patients with lesions over-expressing HER-2 had a 109d decrease in the median time to reoccurrence and a 276d decrease in median survival time. CAV-1 showed little or no expression in 31% (45/139) of lesions. However when CAV-1 was over expressed (score 2-3) patients had a decrease in the median time to reoccurrence of 236d and a decrease in median survival time of 292d. Finally, ERα expression in the cytoplasm was correlated to lesions that were classified as benign. The immunohistochemical evaluation of HER-2, CAV-1, and ERα support their use in prognostic evaluation.
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    Significance of ERα, HER2, and CAV1 expression and molecular subtype classification to canine mammary gland tumor
    (Colorado State University. Libraries, 2013) Shinoda, Hitomi, author; Hanneman, William H., advisor; Legare, Marie E., committee member; Mason, Gary L., committee member
    Canine mammary gland tumor and human breast cancer share many similar features regarding their risk factors, histopathological features, and behavior. Despite the increasing evidence of molecular marker expression as a prognostic factor for human breast cancer, there are only little studies using this approach on canine mammary gland tumor. Our aim was to evaluate the significance of the expression of Estrogen Receptor-alpha, Human Epidermal Growth Factor-2, and Caveolin-1 to the behavior and the clinical outcome of canine mammary gland tumor by Immunohistochemistry. We also assessed the correlation between 5 subtype classification (Luminal A, Luminal B, HER2-overexpressing, Basal-like, and Normal-like) and tumor behavior and prognosis. Canine mammary gland tissues were stained for Estrogen Receptor-alpha, Human Epidermal Growth Factor-2, and Caveolin-1 and evaluated for the positivity, and classified into 5 subtypes according to the staining status. Although there was no statistical significance among the subtypes, the positivity of Nuclear Estrogen Receptor-alpha, Extranuclear Estrogen Receptor-alpha, Human Epidermal Growth Factor-2, and Caveolin-1 showed significant correlations (p<0.05) in the behavior and the prognosis of the tumor. This study indicates the prognostic value of immunohistochemistry staining status of Estrogen Receptor-alpha, Human Epidermal Growth Factor-2, and Caveolin-1 for canine mammary gland tumor. In addition, some trends were seen in 5 subtypes on the prognosis of the tumor, implying that although further analysis is needed, the potential application of 5 subtype classification to canine mammary gland tumor.