Repository logo

Browsing by Author "Legare, Marie E., advisor"

Now showing 1 - 3 of 3
  • Thumbnail Image
    ItemOpen Access
    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.
  • Thumbnail Image
    ItemOpen Access
    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.
  • Thumbnail Image
    ItemOpen 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 member
    Rheumatoid 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.