p53 is a critical tumour suppressor which acts to repair or remove abnormal cells and thus prevent cancer. Aberrant function of p53 is therefore a critical step in tumourigenesis and p53 is mutated in half of all cancers. Mutation of p53 leads to both a loss of normal wildtype function as well as the gain of oncogenic function. p53 is considered to be a promising therapeutic target and therapeutic strategies for targeting of the p53 pathway include: 1. Activation of wild-type p53 (wtp53) protein function, 2. Refolding of mutant p53 (mtp53) into the wtp53 conformation, 3. Reduction of mtp53 protein levels. In this work a number of small molecule screening assays were used to identify potentially novel regulators of both wtp53 and mtp53. Screening of a protein kinase inhibitor library for small molecules which can stimulate wtp53 activity identified the GSK3 pathway and a CDK pathway as dominant suppressors of wtp53 function. Screening of the library for inhibitors which reduce mtp53 protein levels led to the identification of two IKKβ inhibitors. The work then focused on investigating the effects of one of these compounds, IMD0354, on the mutant p53 pathway; with a specific focus on MDM2 as the most rapidly responding biomarker. IMD0354 is a well characterised inhibitor which has been shown to specifically inhibit IKKβ leading to the repression of the Nf-κB pathway. This study shows that IKKβ inhibition leads to the loss of a number of oncogenic proteins including mtp53, MDM2 and cyclin D. Mass-spectrometry based (ITRAQ) proteomic analysis was then employed to identify potential mediators and/or co-regulated factors in response to IKKβ-inhibition via IMD0354 treatment. This led to the identification of RPS3 as a potential negative regulator of MDM2 protein expression; the reduction in MDM2 protein in response to IMD0354 treatment is shown to be partially dependent on RPS3. Together this data has identified, using small molecule kinase inhibitor libraries: (i) dominant kinase signalling pathways that suppress wt-p53 and (ii) a dominant kinase signalling pathway that sustains expression of mutant p53 and MDM2 in cancer cell lines. This latter data supports further investigation to aid understanding of how the IKK signalling pathway cross-talks to the p53-MDM2 axis.

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Thesis advisor: Udayan Mohanty / The stabilization of particular conformations of protein and nucleic acid structure is believed to play an important role in many important biological functions. In chapter one, the α -helical conformation and structural stability of single and double stapled all- hydrocarbon cross-linked p53 peptides when bound and unbound to MDM2 are investigated. Our study provides a comprehensive rationalization of the relationship between peptide stapling strategy, the secondary structural stability, and the binding affinity of p53-MDM2 complex. In chapter two, we study counterion-mediated collapse of a strongly charged model polyelectrolyte chain by Group-II divalent metal cations using coarse-grained Brownian dynamics simulations. Polyelectrolyte effects govern the association of counterions with the chain. Large ions are less effective in counterion condensation than small ions. However, upon counterion condensation, the reduction of the backbone charge is independent of size of the metal cations. Above a threshold value of Coulomb strength parameter, counterion release entropy drives the formation of counterion-induced compact states. In chapter three, the nature of surface tension in the random first order theory of supercooled liquid is analyzed within the framework of Landau-Lifshitz fluctuation theory. We show that the surface tension of a droplet satisfies the differential equation 4πr2(dσ)+ 8πrσ(r)− Br1/2 = 0 , where B/ T = 12πkBcv , T is temperature, kB is dr Boltzmann constant, and cv is heat capacity. A consequence is that the slope of the relaxation time at the glass transition temperature, i.e., the fragility index, is expressed as the square of the ratio of heat capacity and configurational entropy of the supercooled liquid. When backbone extended nucleosides are incorporated into a double helix, a unique helical structure is formed. In chapter four, we find that the predicted stability of modified backbone DNA strands in aqueous solution is in good agreement with experimental melting temperature data. The incorporation of extended backbone nucleosides into a duplex results in elongation of the end-to-end chain distance due to the distortion of the B-DNA conformation at the mutated base-pair insertion. We also find that the modified backbone helical twist is approximately 40 degrees, larger than B-DNA helical twist and closer to the twist angle predicted for D-form DNA. The folding of RNA tertiary structure has been described as an equilibrium between partially folded I (intermediate) states, and the fully folded native conformation, or N state. RNA is highly sensitive to the ionic environment due to its negative charge, and tertiary structures tend to be strongly stabilized by Mg2+. There is a need for models capable of describing the ion atmosphere surrounding RNA with quantitative accuracy. In chapter 5, we present a generalized Manning condensation model of RNA electrostatics for studying the Mg2+-induced RNA folding of the 58mer ribosomal fragment. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.