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Phosphoinositol-Dependent Kinase 1 (PDK1) acts in conjunction with phosphorylated lipids such as Phosphoinositol-3,4,5-triphosphate (PIP3) to activate a variety of proteins that regulate mechanisms ranging from cell growth and survival to cytoskeletal rearrangement. In this investigation PDK1 was examined in the context of cellular division. The techniques of immunocytochemistry and live

Phosphoinositol-Dependent Kinase 1 (PDK1) acts in conjunction with phosphorylated lipids such as Phosphoinositol-3,4,5-triphosphate (PIP3) to activate a variety of proteins that regulate mechanisms ranging from cell growth and survival to cytoskeletal rearrangement. In this investigation PDK1 was examined in the context of cellular division. The techniques of immunocytochemistry and live cell imaging were used to visualize the effects of the inhibition of PDK1 on division in HeLa cells. Division was impaired at metaphase of mitosis. The inhibited cells were unable to initiate anaphase cell-elongation ultimately leading to the flattening of spherical, metaphase cells. Preliminary studies with imunocytochemistry and live cell imaging suggested that insulin treatment reversed PDK1 inhibition, but the results were not statistically significant. Therefore, the recovery of PDK1 inhibition by insulin treatment could not be confirmed. Based on these observations a possible reason for the inability of the treated cells to complete cytokinesis could be the role of PDK1 in the Rho-kinase pathway that is required for the processes cell-elongation necessary for anaphase of mitosis.
ContributorsMasserano, Benjamin Max (Author) / Capco, David (Thesis director) / Baluch, Debra (Committee member) / Chandler, Douglas (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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One of the largest problems facing modern medicine is drug resistance. Many classes of drugs can be rendered ineffective if their target is able to acquire beneficial mutations. While this is an excellent showcase of the power of evolution, it necessitates the development of increasingly stronger drugs to combat resistant

One of the largest problems facing modern medicine is drug resistance. Many classes of drugs can be rendered ineffective if their target is able to acquire beneficial mutations. While this is an excellent showcase of the power of evolution, it necessitates the development of increasingly stronger drugs to combat resistant pathogens. Not only is this strategy costly and time consuming, it is also unsustainable. To contend with this problem, many multi-drug treatment strategies are being explored. Previous studies have shown that resistance to some drug combinations is not possible, for example, resistance to a common antifungal drug, fluconazole, seems impossible in the presence of radicicol. We believe that in order to understand the viability of multi-drug strategies in combating drug resistance, we must understand the full spectrum of resistance mutations that an organism can develop, not just the most common ones. It is possible that rare mutations exist that are resistant to both drugs. Knowing the frequency of such mutations is important for making predictions about how problematic they will be when multi-drug strategies are used to treat human disease. This experiment aims to expand on previous research on the evolution of drug resistance in S. cerevisiae by using molecular barcodes to track ~100,000 evolving lineages simultaneously. The barcoded cells were evolved with serial transfers for seven weeks (200 generations) in three concentrations of the antifungal Fluconazole, three concentrations of the Hsp90 inhibitor Radicicol, and in four combinations of Fluconazole and Radicicol. Sequencing data was used to track barcode frequencies over the course of the evolution, allowing us to observe resistant lineages as they rise and quantify differences in resistance evolution across the different conditions. We were able to successfully observe over 100,000 replicates simultaneously, revealing many adaptive lineages in all conditions. Our results also show clear differences across drug concentrations and combinations, with the highest drug concentrations exhibiting distinct behaviors.

ContributorsApodaca, Samuel (Author) / Geiler-Samerotte, Kerry (Thesis director) / Schmidlin, Kara (Committee member) / Huijben, Silvie (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of Politics and Global Studies (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05