Biology Theses and Dissertations

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Browsing Biology Theses and Dissertations by Author "Barkan, Alice"

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    Characterization of Small Molecules that Reduce CUG Repeat RNA in Myotonic Dystrophy
    (University of Oregon, 2015-08-18) Siboni, Ruth; Barkan, Alice
    Myotonic dystrophy (DM) is an inherited disease characterized by myotonia, insulin resistance, cardiomyopathy, and cognitive deficiencies. DM is a triplet repeat disorder, meaning that affected individuals carry anywhere between 50 and thousands of CTG/CCTG repeats in their genetic makeup. When transcribed into RNA, these repeats become “toxic” in the sense that they serve to bind and sequester important RNA binding proteins. One such family of proteins, the Muscleblind-like (MBNL) family, is important in the regulation of alternative mRNA splicing, and thus the sequestration of MBNL proteins leads to a number of mis-splicing events. Many of these events are directly correlated to DM symptoms. While there is no known cure for DM, the use of small molecules to treat symptoms is a well-characterized therapeutic tactic with immense promise. Pentamidine is a small molecule that was found to reverse mis-splicing in both DM cell and mouse models. Mechanistically, this molecule is particularly unique because unlike many small molecules, which physically displace MBNL from the toxic CUG RNA, pentamidine reduces CUG RNA levels, possibly through inhibition of CTG transcription. Chapter I summarizes alternative splicing mechanisms and regulation, defines MBNL protein structure and function, describes DM pathophysiology and molecular mechanism, and finally provides an overview of pentamidine characterization as a small molecule therapeutic. Chapter II reports the development of an in vitro T7 transcription assay, which allowed us to compare the relative efficacy by which pentamidine is able to inhibit the transcription of various repeat and non-repeat DNA sequences. This chapter further reports the characterization of a series of methylene linker analogues of pentamidine, which were also characterized through the T7 transcription assay. Chapter III details our thorough structure-activity relationship investigation of bisbenzamidine analogues of pentamidine, both in in vivo and in vitro models. Chapter IV describes our characterization of actinomycin D, a known transcription inhibitor and chemotherapeutic, within the DM disease framework. Chapter V summarizes these data, which ultimately serve as a proof of concept for the potential of CTG transcription inhibition in therapeutic contexts and broadly describe their application in other repeat diseases. This dissertation contains previously published and unpublished co-authored material.
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    Investigating Light-Induced psbA Translation in Chloroplasts
    (University of Oregon, 2020-02-27) Ljungdahl, Sonja; Barkan, Alice
    Light, while necessary for plants, can cause photo-oxidative damage. Adaptations to fluctuating light conditions optimize photosynthetic yield and minimize light-induced damage. Light-regulated synthesis of the chloroplast gene psbA and its protein product D1 is at the core of these responses. Shifting light intensity regulates D1 synthesis at the level of translation. This thesis investigates specific proteins we hypothesized mediate the effects of light on D1 synthesis. Our experiments on TPJ1, a maize mutant lacking one of these proteins, showed that TPJ1 does not influence psbA translation. Instead, our results show it is required for the translation of the chloroplast psbJ mRNA. In addition, I elucidated the biochemical interactions between two known psbA translational activators, OHP2 and HCF244, by identifying a short segment of OHP2 that is sufficient for its interaction with HCF244. This thesis includes published, co-authored material.