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- All Subjects: Cellular Biology
Description
Cellular and molecular biologists often perform cellular assays to obtain a better understanding of how cells work. However, in order to obtain a measurable response by the end of an experiment, the cells must reach an ideal cell confluency. Prior to conducting the cellular assays, range-finding experiments need to be conducted to determine an initial plating density that will result in this ideal confluency, which can be costly. To help alleviate this common issue, a mathematical model was developed that describes the dynamics of the cell population used in these experiments. To develop the model, images of cells from different three-day experiments were analyzed in Photoshop®, giving a measure of cell count and confluency (the percentage of surface area covered by cells). The cell count data were then fitted into an exponential growth model and were correlated to the cell confluency to obtain a relationship between the two. The resulting mathematical model was then evaluated with data from an independent experiment. Overall, the exponential growth model provided a reasonable and robust prediction of the cell confluency, though improvements to the model can be made with a larger dataset. The approach used to develop this model can be adapted to generate similar models of different cell-lines, which will reduce the number of preliminary range-finding experiments. Reducing the number of these preliminary experiments can save valuable time and experimental resources needed to conduct studies using cellular assays.
ContributorsGuerrero, Victor Dominick (Co-author) / Guerrero, Victor (Co-author) / Watanabe, Karen (Thesis director) / Jurutka, Peter (Committee member) / School of Mathematical and Natural Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Bexarotene is a Food and Drug administration (FDA)-approved therapeutic used in the treatment of cutaneous T-cell lymphoma (CTCL). However, bexarotene therapy causes significant side effects like hyperlipidemia and hypothyroidism due to crossover activity with retinoic acid receptor (RAR), thyroid hormone receptor (TR), and liver X receptor (LXR) signaling, respectively. More recently bexarotene has shown promise to reverse neurodegeneration, improve cognition and decrease levels of amyloid- β in transgenic mice expressing familial Alzheimer’s disease (AD) mutations. Bexarotene is a high affinity ligand for the retinoid X receptor (RXR) that heterodimerizes with the liver- X- receptors (LXR) and with peroxisome proliferator-activated receptor-gamma (PPARϒ) to control cholesterol efflux, inflammation, and transcriptionally upregulates the production of apolipoprotein (ApoE) in the brain. Enhanced ApoE expression may promote clearance of soluble Aβ peptides from the brain and reduce Aβ plaques, thus resolving both amyloid pathology and cognitive deficits. The present study assessed the potential of bexarotene and a group of 62 novel rexinoids to bind and activate RXR using a series of biological assays and screening methods, including: 1) a mammalian two-hybrid system (M2H) and an 2) Retinoid X Receptor response element (RXRE)-mediated reporter assays in cultured human cells. Moreover, Liver X Receptor response element (LXRE)-mediated luciferase assays were performed to analyze the ability of the novel analogs to activate LXRE - directed transcription, and to induce ApoE messenger ribonucleic acid (mRNA) in U87 glial cells. Furthermore, the most potent analogs were analyzed via quantitative polymerase chain reaction (qPCR) to determine efficacy in modulating expression of two critical tumor suppressor genes, activating transcription factor 3 (ATF3) and early growth response 3 (EGR3). Results from these multiple assays indicate that the panel of RXR ligands contains compounds with a range of activities, with some analogs capable of binding to RXR with higher affinity than others, and in some cases upregulating ApoE expression to a greater extent than bexarotene. The data suggests that minor modifications to the bexarotene core chemical structure may yield novel analogs possessing an equal or greater capacity to activate RXR and may be useful as therapeutic agents against CTCL and Alzheimer’s disease.
ContributorsReshi, Sabeeha Mushtaq (Author) / Jurutka, Peter (Thesis advisor) / Wagner, Carl (Committee member) / Marshall, Pamela (Committee member) / Arizona State University (Publisher)
Created2023