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Browsing by Author "Regan, Daniel, committee member"

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    Cannabidiol induces apoptosis and perturbs mitochondrial function in canine and human glioma cells
    (Colorado State University. Libraries, 2020) Gross, Chase, author; Gustafson, Daniel, advisor; McGrath, Stephanie, advisor; Regan, Daniel, committee member
    Cannabidiol (CBD), the major non-psychoactive compound found in cannabis, is frequently used both as a nutraceutical and therapeutic. Clinical studies suggest CBD may be beneficial for treating rare forms of epilepsy and inflammatory conditions but despite anecdotal evidence as an anticancer agent, little is known about the effect it has on cancer cells. Given the intractability and poor prognoses of brain cancers in both human and veterinary medicine, we characterized the in vitro cytotoxicity of CBD on human and canine gliomas. Glioma cells treated with CBD showed a range of cytotoxicity from 4.9-8.2 μg/mL; canine cells appeared to be more sensitive than human. Treatment with >5 μg/mL CBD invariably produced large cytosolic vesicles. Next, the mode of cell death was interrogated using pharmacologic inhibitors. Inhibition of apoptosis was sufficient to rescue CBD-mediated cytotoxicity. Inhibition of RIPK3, a classical necroptosis protein, rescued cells from death and prevented the formation of the large cytosolic vesicles. Analysis of cell death by resazurin reduction showed much higher sensitivity than other cytotoxicity methods and suggested disruption of the mitochondria. Cellular mitochondrial activity in the presence of CBD was assessed and within two hours of treatment CBD reduced oxygen consumption in a dose dependent manner with almost complete ablation of activity at 10 μg/mL CBD. Fluorescent imaging with a mitochondrial-specific dye revealed that the large cytosolic vesicles were, in fact, swollen mitochondria. Lastly, calcium channels were inhibited and the effect on cell death was measured. Inhibition of mitochondrial channel VDAC1, but not the TRPV1 channel, rescued cells from CBD-mediated cytotoxicity. These results demonstrate the cytotoxic nature of CBD in human and canine glioma cells and suggest a mechanism of action involving dysregulation of calcium homeostasis and mitochondrial activity.
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    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 member
    In 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.
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    Culture-expanded articular chondrocytes: a potential cellular therapeutic for osteoarthritis with MSC-like properties
    (Colorado State University. Libraries, 2022) Liebig, Bethany Ellen, author; Goodrich, Laurie, advisor; Kisiday, John, advisor; Regan, Daniel, committee member; Santangelo, Kelly, committee member; McGilvray, Kirk, committee member; Bahney, Chelsea, committee member
    Osteoarthritis (OA) is a highly prevalent and debilitating joint disease in horses, dogs, and humans. OA affects more than 303 million people globally with an annual economic loss to Americans approaching $200 billion. It has a considerable impact on the patient, resulting in pain and disability and more than 1 million people undergo knee arthroscopy or joint replacement surgery each year due to end-stage OA in the United States. Therefore, OA therapies that produce lasting symptom- and disease-modifying effects are a medical priority. Mesenchymal stromal cells (MSCs) are considered 'medicinal signaling cells' that have been postulated to treat OA by reducing inflammation and restoring joint function. However, IA injection of MSCs into diseased human or companion animal joints has demonstrated only a modest benefit to date, as symptom-modifying effects are often temporary, and evidence of disease-modification has been minimal. It has been reported that culture-expanded chondrocytes (CECs) can assume many of the hallmark properties of MSCs, such as immunomodulation and immunophenotype. However, unlike MSCs, chondrocytes are known to thrive in suspension, which is important as IA injections release cells into synovial fluid. The goal of this research aims to characterize the growth, immunomodulatory properties, and gene expression of equine CECs as a function of expansion in vitro as well as CEC persistence in the joint after intra-articular injection using a validated model of OA in rats. Additional goals of this research are to 1) determine how CECs may (persistence) or may not (immunomodulation and molecular fingerprint) differ from bone marrow derived MSCs, and 2) compare cellular properties of CECs across age to determine an ideal donor for generating allogeneic therapies. The results shown in chapters 2 and 3 indicate that chondrocytes retain a strong propensity for immunomodulation, that increases with expansion and dedifferentiation does not coincide with other temporal changes in gene expression. Further, these data do not indicate a benefit of neonatal donors. Future in vitro studies should further characterize the immunomodulatory, redifferentiation (chondrogenic) and angiogenic potential of CECs. The preliminary results described in chapter 4 indicate that CECs may have greater persistence than MSCs in the first 3 days post IA injection. Future in vivo studies should focus on determining the symptom- and disease-modifying effects following IA injection of CECs in relevant preclinical models, such as the rodent, horse, and dog.