Browsing by Subject "Molecular Biology"

Now showing 1 - 2 of 2
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    Methods of Enhancing Triterpenoid Production in Yeast
    (2023-07) Scott, Samuel
    Microbial cell factories, particularly those using eukaryotic yeasts, are ideal platforms for producing plant secondary metabolites, including flavonoids, alkaloids, and terpenoids. Accordingly, this study aimed to increase triterpenoid production in the Saccharomyces cerevisiae strain BY4743 through CRISPR/Cas9 and cultivation engineering. The ROX1, DGK1, and PAH1 genes were targeted for knockout experiments. Sanger sequencing showed all three targets were successfully mutated; however, only the DGK1 knockout strain had a significant change in triterpenoid production at 130% compared to the wild-type. Various cultivation strategies were also explored, but none increased triterpenoid production significantly. Additionally, to illustrate the potential applications of engineered yeast, five uncharacterized oxidosqualene cyclases (OSCs) from Erysimum cheiranthioides were tested in the ROX1 knockout strain, revealing one responsible for producing the steroid core of medicinal cardenolides. In summary, this thesis provides engineered yeast strains with improved MVA pathway derivative production potential and comprehensive CRISPR/Cas9 methodologies for S. cerevisiae.
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    Molecular mechanisms of inhibitory signaling in the heart and brain
    (2014-06) Wydeven, Nicole Marie
    G protein-gated inwardly-rectifying K+ (GIRK/Kir3) channels mediate the inhibitory effect of many neurotransmitters on excitable cells of the heart and brain. Dysregulation of GIRK signaling is known to underlie a number of disorders, including arrhythmia, epilepsy, depression, anxiety, schizophrenia, and drug addiction. GIRK channels are gated by inhibitory Gi/o proteins and temporally modulated by Regulators of G protein Signaling (RGS) proteins. GIRK channels are tetramers consisting of various combinations of four mammalian Girk subunits (GIRK1-4). This dissertation focuses on neuronal and cardiac GIRK signaling cascades as targets for new pharmacotherapies in the treatment of anxiety-related disorders and cardiac arrhythmias.Both robust GIRK channel activity and modulation by a new class of GIRK-specific drugs depend on the GIRK1 subunit. The presence of GIRK1 in channel complexes is necessary for robust channel activity. We first sought to better understand the potentiating influence of GIRK1, using the GABAB receptor and GIRK1/GIRK2 heteromer as a model system. We found residues in both the distal carboxyl-terminal domain and channel core that underlie the GIRK1-dependent potentiation of receptor-dependent and receptor-independent heteromeric channel activity. Further, ML297, the prototypical member of a new family of small molecule GIRK channel modulators, selectively activates GIRK1-containing channels. We found that ML297 activates GIRK channels via a unique mechanism that requires two amino acids specific to the GIRK1 subunit. In addition, ML297 reduces anxiety-related behavior in mice, in a GIRK1-dependent manner, without triggering addiction-related behavior. Thus, ML297 is a new tool for probing the therapeutic potential of GIRK channel modulation, which may benefit individuals with anxiety-related disorders. Cardiac GIRK signaling plays a role in the parasympathetic regulation of heart rate (HR). Parasympathetic activity decreases HR by inhibiting pacemaker cells in the sino-atrial node (SAN). RGS proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K+ channel IKACh (a GIRK1/GIRK4 tetramer). We first identified RGS6 as a temporal regulator of cardiac M2R-IKACh signaling in atrial myocytes and SAN cells. Both RGS4 and RGS6 have been implicated in the negative modulation of the parasympathetic regulation of HR and the M2R-IKACh signaling pathway. We next looked at the contribution of RGS4 and RGS6 to the modulation of M2R-IKACh signaling. Ablation of Rgs6, but not Rgs4, correlated with decreased resting HR and a significant delay of M2R-IKACh deactivation rate. Thus, RGS6, and not RGS4, is the primary RGS modulator of cardiac M2R-IKACh. Taken together, these findings suggest that RGS6 is a potential pharmacotherapeutic target as the dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia.