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  1. Modified nucleotides and nucleic acids as molecular probes [electronic resource]

    Ghosh, Samantak

    This thesis discusses a number of projects involving the use of modified nucleotides and oligonucleotides in addressing some basic science questions and some clinical and technological applications.The first chapter details our efforts at using telomere-encoding circular DNA in elongating zebrafish telomeres. We microinjected our synthetic circular DNA into zebrafish embryos and studied their telomere length 24 hrs later. Using Quantitative Fluorescence in situ Hybridization (Q-FISH) as the analytical tool to determine telomere length, we observed no significant difference in telomere length between the group injected with the synthetic DNA and the control group. In the second chapter we studied the potential of a non-polar shape mimic of iodo-uracil as an imager of tumors. We continued discussing our work with non-polar nucleotide isosteres in the third chapter where we used them in investigating a novel active site in polymerase from Pyrococcus furiosus (Pfu). From our studies we concluded that although shape was an important factor in distinguishing bases, this binding site also employed hydrogen bonding to identify nucleoabases. The lack of any recognition of the syn-oxidized bases suggested that the enzyme preferred to recognize bases in the anti conformation rather than syn. In the fourth chapter we were interested in understanding the factors determining the fidelity and selectivity observed in RNA Polymerase II mediated transcription. Once again we used non-polar shape mimics of thymidine (dF) and adenine (dQ) to study the importance of shape and hydrogen bonding. We observed that the thymidine mimic was recognized better by the RNA Polymerase II active site than the mis-match bases. The adenine isostere on the other hand, was poorly recognized. This preliminary study demonstrates the importance of both shape and hydrogen bonding. The last chapter discusses our studies using polyfluorophores on a DNA backbone to detect gases. Following a combinatorial method, a library of oligodeoxyfluorosides (ODFs) were synthesized from which sensors of gases were selected. Using this method we were able to select optical sensors for a diverse set of small molecules in vapor state.

  2. Studies toward the enantioselective synthesis of briarane diterpenes

    Farr, Joshua Daniel
    [Stanford, California] : [Stanford University], 2022.

    First isolated over fifty years ago, the briaranes are a diverse family of approximately 800 diterpenoid natural products that are unified by a unique carbon skeleton which houses a trans-[8.4.0]-tetradecane ring system. This natural product family collectively possesses a broad range of interesting bioactivity and several unusual structural features, such as halogen-bearing stereocenters. This, in combination with the presence of multiple highly conserved structural features has attracted the attention of synthetic chemists for the last three decades. Despite considerable effort, no successful synthesis of any member of this family has been reported in the literature. At the outset of our work, the reports toward these natural products focused largely on the synthesis of structural motifs, rather than targeting the synthesis of any specific member. A synthesis which succeeded in establishing these important motifs in a streamlined manner as well as incorporating the rest of the carbon skeleton was completely unknown. Our group targeted a representative member, (+)-briareolide J, for synthesis, with the goal that the successful development of a route toward this member would enable the synthesis of other structurally related members which differ only by oxidation state. We developed a first-generation approach toward these natural products which hinged on the convergent coupling of two fragments using an enantioselective aldol reaction. Further elaboration of this coupled product enabled the installation of important structural motifs while incorporating necessary functionality to complete the synthesis. Ultimately, we found that our final disconnection was not able to establish the challenging trans-[8.4.0]-tetradecane ring system, leading us to revise our synthetic strategy. Targeting a redesigned second-generation synthesis, we developed a radical-polar crossover reaction to couple two enantioenriched fragments, establishing an array of important structural motifs in a single step. Further elaboration of this intermediate afforded an advanced precursor containing all the carbon atoms needed to complete the synthesis. However, we once again found that this intermediate was incapable of forming the trans-[8.4.0]-tetradecane ring system through our planned disconnection. Lastly, model studies helped to establish possible root causes of these challenges, prompting us to once again redesign our synthetic strategy. Although ultimately unsuccessful, the insights from these studies pave the way for future synthetic endeavors.

  3. Identification of E. coli amyloid assembly inhibitors, modes of action, and influence on biofilm formation

    Visser, Joshua Alan
    [Stanford, California] : [Stanford University], 2021

    Biofilms are multicellular communities of cells surrounded in a self-produced extracellular matrix (ECM), a complex mixture of polymeric substances. This matrix is associated with several beneficial functions for bacteria, depending upon the type of biofilm. These can include resistance to antibiotics and host defenses. Biofilm formation in the human host can contribute to the persistence and recurrence of infection. Uropathogenic E. coli is the most common causative agent of urinary tract infection and can assemble unique biofilms through the production of functional amyloid fibers termed curli and a modified form of cellulose -- phosphoethanolamine (pEtN) cellulose. Curli play a vital role in bacterial adhesion to biotic and abiotic surfaces as well as promoting multicellular biofilm formation. Curli fibers are assembled from CsgA protein subunits that non-covalently polymerize into the fibril form through the nucleation-precipitation pathway. Herein, we describe the characterization of the natural product NDGA and the salicylanilide closantel as inhibitors of curli formation. We demonstrate the effects of inhibitors on bacterial curli production, bacterial adhesion, bacterial biofilm formation as well as protein polymerization in vitro. Molecular analyses are accompanied by the evaluation of curli gene transcription to consider compound effects on gene expression. NDGA exhibits curlicide activity, preventing CsgA polymerization, and does not bind to intact curli. These results are consistent with the inhibition of early steps in curli biogenesis, e.g. amyloid nucleation, transport through CsgG, or CsgA-CsgF interactions. Closantel exhibits curlicide activity at very low concentrations and also binds to intact curli fibers. Thus, closantel is able to bind CsgA in its polymerized amyloid form and may also recognize early amyloidogenic conformations of CsgA or additionally recognize the soluble and unstructured form of CsgA preceding nucleation and polymerization. This comprehensive and mechanistic approach in the full cellular context of bacteria provides an avenue to identify and develop curli-specific amyloid inhibitors and may hold promise in identifying the best inhibitors of amyloidogenesis and assembly of amyloid oligomers and fibers associated with human diseases. Finally, we expanded our understanding of the functions and contributions of both curli and cellulose in uropathogenic E. coli biofilm formation through comparisons of curli and pEtN cellulose gene mutants in the uropathogenic E. coli clinical isolate UTI89. We examined biofilm formation at the agar-air and air-liquid interfaces. We also examined mixed cultures of mutants lacking either curli or modified cellulose and discovered that they could recapitulate the formation of a biofilm similar to wild-type UTI89. This work breaks new ground in understanding the contributions of complex and insoluble biopolymers to the assembly of architectures that extend beyond the surface of single bacteria and that are built for community behaviors


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