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Kynurenine 3-monooxygenase is a FAD-dependent aromatic hydroxylase (FAH) which is a widely suggested therapeutic target for controlling the balance of bioactive metabolite levels produced by the mammalian kynurenine pathway (KP). Prior to starting this work no structural information was known for the enzyme, with studies of the human form complicated by the presence of a C-terminal transmembrane helix. The bacterial Pseudomonas fluorescens enzyme (PfKMO) lacks the transmembrane region and has been previously characterised by Crozier-Reabe and Moran [1, 2]. Therefore PfKMO, which shares 32 % sequence identity with the human enzyme, was selected as a target for structure solution. Initial substrate bound PfKMO crystals showed poor X-ray diffraction. Subsequent growth optimisation and the generation of a C252S/C461S PfKMO mutant (dm2) yielded crystals suitable for structure solution. Selenomethioninelabelled substrate bound dm2 crystals were used to solve the first structure to a resolution of 3.40 Å. With just one protein molecule per asymmetric unit, a high solvent content was responsible for the poor diffraction properties of this crystal form. The overall fold resembled that of other FAH enzymes with a Rossmann-fold based FADbinding domain above a buried substrate binding pocket. Interestingly PfKMO possesses an additional, novel C-terminal domain that caps the back of the substrate-binding pocket on the opposite side to the flavin. Residues proposed to be involved in substrate binding were identified and shown to be highly conserved among mammalian KMO sequences. Subsequently single crystals of substrate-free dm2 PfKMO were obtained and showed significantly stronger diffraction due to new lattice packing in an orthorhombic space group bearing four molecules per asymmetric unit. The structure was solved to a resolution of 2.26 Å and revealed a clear conformational change of the novel C-terminal domain. This movement opens a potential route of substrate/product exchange between bulk solvent and the active site. The investigation of a set of C-terminal mutants further explored the relevance and mechanics of the conformational change. In addition the presence of chloride ions in the substrate-free crystal growth solution caused a small number of localised subtle alterations to the structure, with a potential chloride binding site identified adjacent to the flavin cofactor. This may have relevance for the observed inhibition of PfKMO activity by monovalent anions – a feature widely common to FAH enzymes [3]. The first discovered KMO inhibitors were analogues of the substrate L-Kyn, however one such compound (m-NBA) was recently shown to instigate uncoupled NADPH oxidation leading to the release of cytotoxic hydrogen peroxide [1]. A set of substrate analogues were tested and characterised for inhibition of PfKMO. The picture was shown to be complex as some substrate analogues completely inhibited the enzyme whilst the binding of some still stimulated low-levels of NADPH oxidation. Crystallographic studies with m-NBA and 3,4-dichlorobenzoylalanine (3,4-CBA) bound revealed indistinguishable structures from that of substrate-bound PfKMO. These studies suggest that the analogue 3,4CBA is a potent PfKMO inhibitor whose therapeutic potential may be re-visited. The previous most potent KMO inhibitor whose structure was not analogous to the substrate was Ro 61-8048 [4], which unfortunately did not pass pre-clinical safety tests. A novel series of 1,2,4-oxadiazole amides based on the structure of Ro 61-8048 was created by Gavin Milne (PhD, University of St Andrews) and tested on PfKMO. Rounds of refinement led to the discovery and refinement of low nanomolar competitive inhibitors of the bacterial enzyme. PfKMO was co-crystallised with each of the four most potent compounds forming a third different lattice arrangement, which yielded structures to resolutions of 2.15-2.40 Å. The structures displayed conformational changes resembling the substrate-free fold possibly caused by displacement of a crucial substrate-binding residue, R84. Overall the wealth of structural data obtained may be transferable to predictions about the structural features of human KMO and to the rational design of therapeutic inhibitors. The potent novel inhibitors tested may additionally present a new exciting development for the therapeutic inhibition of human KMO.

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We have hypothesized that dietary modulation of intestinal miRNA expression may contribute to the chemoprotective effects of nutritional bioactives (fish oil and pectin). Using a rat colon carcinogen model, we determined miRNAs-let-7d, miR-15b, miR-107, miR-191 and miR-324-5p were modulated by fish oil + pectin. We also demonstrated that BACE1 and PTEN are targets of miR-107 and miR-21, respectively. To further elucidate the biological effects of diet and carcinogen on miRNAs, we integrated global miRNAs, total and polysomal gene expression datasets obtained from the above mentioned study and used four computational approaches. We demonstrated that polysomal profiling is tightly related to microRNA changes when compared with total mRNA profiling. In addition, diet and carcinogen exposure modulated a number of microRNAs and complementary gene expression analyses showed that oncogenic PTK2B, PDE4B, and TCF4 were suppressed by the chemoprotective diet at both the mRNA and protein levels. To determine the function of select diet and colon carcinogen modulated miRNAs and to validate their targets, we carried out a series of loss and gain of function experiments along with luciferase reporter assays. We verified that PDE4B and TCF4 are direct targets of miR-26b and miR-203, respectively. PTK2B was determined to be an indirect target of miR-19b. In addition, microRNA physiological function was assessed by examining effects on apoptosis and cell proliferation. To better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we investigated the chemoprotective effects of dietary agents on miRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creERT2 knock in mice injected with AOM. We demonstrated that based on relative expression of miR-125a-5p, miR-190b and miR-191 in stem cells vs. daughter cells and differentiated cells, these miRNAs may be stem cell specific miRNAs. We also identified miR-21 to be significantly reduced in stem cells compared to differentiated cells and selectively modulated by these dietary agents in stem cells. In summary, our results indicate for the first time that fish oil plus pectin protect against colon tumorigenesis in part by modulating a subset of miRNAs and their target genes (mRNAs) implicated in the regulation of the colon stem cell niche and tumor development.

The aim of this bachelor's thesis has been to investigate surfaces that are the main contributions to scattering amplitudes in a type of string theory. These are constant mean curvature surfaces in hyperbolic 3-space. Classically the way to find such surfaces has been to solve a non-linear partial differential equation. In many spaces constant mean curvature surfaces are intimately connected to certain harmonic maps, known as the Gauss maps. In 1995 Dorfmeister, Pedit, and Wu established a method for constructing harmonic maps into so-called symmetric spaces. I investigate a generalization of this method that can be applied to find constant mean curvature surfaces in hyperbolic 3-space by using the intimate connection between these surfaces and harmonic maps. This method relies on a factorization of a Lie-group valued map. I show an explicit method for finding the factorization in terms of what is known as the Birkhoff factorization. Because approximation methods for the Birkhoff factorization are known, this allowed me to use the method constructively to find constant mean curvature surfaces in hyperbolic 3-space.