<p>Sol-gel derived silica provides a bio-compatible material for the solid-phase entrapment of viable cells. A selection of <em>E. coli</em> cells containing unique promoter-linked GFP expression vectors were applied to fluorescence microwell plate assays, plate counting and various microscopy methods to assess changes in the entrapped bacteria and compatibility towards compound screening. Materials screening showed that a fastgelation sol-gel composition from sodium silicate precursor and PBS buffer provided a consistently greater fluorescence signal than non-entrapped cells. It is shown for the first time that entrapped cells are capable of dividing within pockets of the silica gel, and can di vide at a comparable rate to free cells. The entrapment of cells within a silica matrix does not induce the basal expression level of promoters tested here. Silica entrapment provides improved storage capabilities over non-entrapped cells in solution. A set of 12 related GFP-linked promoters were induced in solution and within silica when screened by two DNA gyrase inhibitors, providing similar expression profiles but greater signal-tonoise ratios in silica. The sol-gel derived material is amenable in an array format, and is a prospective material for the fabrication of sol-gel cell microarrays.</p> / Master of Science (MSc)

<p>Immunodeficient-causing diseases such as HIV and leukemia have no cures, often require meticulous treatments and result in high morbidity or mortality. Although bone marrow transplants are an option for a subset of leukemia patients, the shortage of donors and the requirement for donor matching restricts the efficacy of this treatment option. Therefore there is a prominent clinical need for alternative sources of hematopoietic stem/progentior cells with lymphopoietic potential. Recently we described the direct conversion of human dermal fibroblasts to multilineage hematopoietic progenitors by ectopic expression of OCT4. This direct conversion method was used to assess whether OCT4-transduced fibroblasts had the capacity to derive cells of the lymphoid lineage. This work shows the transient co-expression of CD34 and CD45 of fibroblasts within 7 days of OCT4 transduction followed by stable expression of CD45 on fibroblasts by day 15. The acquisition of hematopoietic markers, however, did not coincide with colony formation as previously described. Furthermore, CD45+ cells that were enriched and cultured in hematopoietic conducive conditions did not acquire co-expression of CD34 as previously shown. Interestingly, CD34 expression was shown to be inversely correlated with OCT4 expression. Therefore the constitutive expression of OCT4 may have (1) inhibited the acquisition of CD34 expression on CD45+ cells (2) downregulated the expression of CD34 on the day 7 CD34+CD45+ fibroblasts, thereby resulting in the transient expression of these markers. Furthermore, this work shows that expression of CD45 on OCT4-transduced fibroblasts is required for survival on the MS5 stromal cell line used to support hematopoietic progenitors with lymphopoietic potential, while supplementation of CD45+ fibroblasts with hematopoietic progenitor supportive conditions resulting in co-expression of CD34 and CD45 is required for acquisition of CD19, a pan-B cell marker on CD45+ fibroblasts. These findings suggest OCT4-transduced fibroblasts have lymphopoietic potential.</p> / Master of Science (MSc)

<p>The process of in vitro selection has led to the isolation of many catalytic DNA molecules, called deoxyribozymes, which can catalyze a range of biologically-relevant reactions. Despite these advances, questions still remain as to why DNA, which seems more suited to information storage than catalysis can efficiently catalyze chemical reactions. In this thesis, a group of deoxyribozymes that can catalyze their own phosphorylation using NTP substrates are used a model system to study how DNA is able to fold into complex structures necessary for catalysis. Using a variety of structural probing techniques, these studies elucidated a common secondary structure shared by three deoxyribozymes, which do not appear to share a common ancestor sequence. This suggests that this motif may be most efficient motif to catalyze self-phosphorylation by DNA. It also more generally demonstrates that DNA can undergo convergent evolution to reach the same complex folding arrangement. A fourth deoxyribozyme was found to fold into a complex tertiary structure containing a novel quadruplex-helix pseudoknot motif. The finding of this pseudoknot and comparison with other quadruplexes found in other functional nucleic acids led us to investigate whether these stable motifs could be incorporated into nucleic acid libraries to improve the process of in vitro selection and give researchers a better chance of isolating functional nucleic acids. Design and characterization of structured libraries revealed that DNA libraries could be made in which the majority of sequences are folded into quadruplex arrangements. The incorporation of this quadruplex scaffold into DNA sequence libraries may ease the isolation of functional nucleic acids that contain this useful structural motif. In the final part of this thesis, a self-phosphorylating deoxyribozyme was converted from a tool for study of DNA structure to a sensor for GTP and Mn²⁺, demonstrating that deoxyribozyme substrates can be converted into targets for biosensors.</p> / Doctor of Philosophy (PhD)

<p>Antibiotic producing organisms often bear cognate export and/or resistance mechanism encoded in the same cluster of antibiotic biosynthetic genes, but how expression of these protective genes associated with the cognate biosynthetic machinery was poorly understood. This work focuses on elucidating the mechanism by which the antibiotic producing bacteria initiate and sustain self-resistance in individual cells and across the colonies. Actinorhodin is a blue pigmented antibiotic produced by <em>Streptomyces coelicolor</em>. Within its biosynthetic gene cluster, an <em>actAB</em> operon encodes two putative efflux pumps and is regulated by the transcriptional regulator ActR. This work (1) identified that normal yields of actinorhodin require <em>actAB</em> expression and both actinorhodin and its 3-ring pathway intermediate (e.g. DHK) could relieve <em>actAB</em> expression from ActR repression <em>in vivo</em>; (2) created an ActR mutant that interacts productively with a 3-ring intermediate (e.g. (<em>S</em>)-DNPA) but not actinorhodin and responds to actinorhodin biosynthetic pathway with induction of <em>actAB</em> and normal actinorhodin production; (3) demonstrated that actinorhodin producing cells could induce <em>actAB</em> expression in non-producing cells and actinorhodin is the intercellular signal in this case; (4) showed <em>actAB</em> expression in “intermediate only” ActR mutant is short lived and in the absence of the actinorhodin-mediated signaling step the culture experiences widespread cell death. Based on these results, we proposed a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance.</p> / Doctor of Philosophy (PhD)

<p>All living things must replicate their DNA. Despite being essential for life, this process is also inherently dangerous. Replication stress, which induces replication fork stalling, is an unavoidable risk that can trigger potentially harmful changes to the genome. Eukaryotes have a replication checkpoint pathway that stabilizes stalled replication forks to prevent damage. One of the critical protein interactions in this pathway, between Dbf4 and Rad53, pauses the cell cycle in budding yeast. This is important to give the cell time to recover from stress. The molecular details of this interaction were investigated to shed light on how this association is regulated by the cell. The structure of an N-terminal domain from Dbf4 was solved through X-ray crystallography and discovered to have a modified <em>BR</em>CA-1 <em>C</em>-<em>t</em>erminal (BRCT) fold, which included an additional N-terminal helix. This domain could interact with the <em>F</em>ork<em>H</em>ead <em>A</em>ssociated 1 (FHA1) domain from Rad53 <em>in vitro</em>, and the additional helix was necessary for complex formation. Although the FHA1 domain has a well-characterized binding site for phospho-epitopes, a combination of chemical cross-linking and NMR spectroscopy experiments demonstrated that the N-terminal domain from Dbf4 is contacting an alternative surface. However, the full-length Dbf4 protein <em>in vivo</em> may be contacting both this distal site and the phospho-epitope binding pocket. This bipartite interaction between Dbf4 and Rad53 would lend specificity to the complex and also suggests a kinase may be regulating the association. As FHA and BRCT domains are prevalent in eukaryotic nuclear proteins, these findings are instructive for how these domains mediate interactions in other signaling pathways.</p> / Doctor of Philosophy (PhD)

<p>KDM5b acts as a transcriptional repressor through its ability to demethylate tri-methylated lysine (K) 4 on histone H3 (H3K4me3). Demethylation of this histone modification leads to transcriptional repression and downstream biological effects on gene expression. KDM5b is involved in the regulation of differentiation and can exert an oncogenic and a tumour suppressive role depending on cellular context, making it an attractive future target for pharmaceutical intervention. Work from our group has shown that KDM5b expression is linked to differentiation, and that recruitment of the enzyme does not always result in an alteration of H3K4me3. Additionally, work from our group, as well as others, has failed to observe H3K4me3 demethylation by KDM5b in nucleosomal preparations. We therefore hypothesized that KDM5b may exert its demethylase potential on alternative histone targets and that KDM5b requires enzymatic co-factors to demethylate nucleosomes, similar to what is observed for other histone-modifying proteins. In this thesis, we describe KDM5b as having an alternate histone target, di-methylated histone H2B lysine 43 (H2BK43me2). We show that this methyl mark is the primary target for KDM5b, and that the expression level of H2BK43me2 is directly related to the process of differentiation. We additionally present a novel co-factor for KDM5b, the co-repressor TLE4 of the Groucho/TLE family. The presence of TLE4 is required and sufficient to confer nucleosomal demethylase activity to KDM5b, a novel discovery for any of the KDM5 family members. Overall, this work has described both an additional KDM5b target, and detailed requirements for KDM5b nucleosomal demethylation, advancing our understanding of how this enzyme is regulated <em>in vivo</em>. The novel aspects of KDM5b regulation presented within this thesis provide a framework from which future studies can be designed. This work contributes to our overall understanding of epigenetic regulation and will potentially aid in the development of novel anti-cancer therapeutic strategies.</p> / Doctor of Philosophy (PhD)

Master of Science (MSc)

GPCR accessory proteins regulate the strength, efficiency and specificity of signal transfer upon receptor activation. Due to the inherent difficulties of studying membrane proteins in vitro and in vivo, little is known about the structure and topology of these small accessory proteins. Two examples of GPCR accessory proteins are the Melanocortin-2 receptor accessory protein (MRAP) and the Receptor-activity-modifying protein (RAMP) family. MRAP and RAMP1 are the main focus of this thesis in which they are thoroughly characterised by solution-state NMR and further biophysical techniques. The single-pass transmembrane domain protein MRAP regulates the class A GPCR melanocortin receptors. It is specifically required for trafficking the melanocortin-2-receptor from the endoplasmic reticulum to the cell surface and subsequent receptor activation. A remarkable characteristic of MRAP is its proposed native dual-topology, which leads to an antiparallel homodimeric conformation. Investigation of the biochemical and biophysical properties of MRAP revealed an α-helical transmembrane domain, and an α-helical N-terminal LD(Y/I)L-motif. Further efforts concentrated on establishing the homodimeric conformation of MRAP in vitro. RAMP1 facilitates receptor trafficking and alters the ligand specificity of the GPCR Class B receptors calcitonin receptors and calcitonin receptor-like receptors. Moreover, RAMP1 is required to act as a Calcitonin-gene-related peptide (CGRP) receptor (RAMP1). RAMP1 has been shown to form stable parallel homodimers in the absence of its cognate receptor. Its dimerisation and the possible dimerisation motif PxxxxP-motif were studied extensively. With the goal of understanding the mechanism of dimerisation and the role of GPCR accessory proteins I have used solution-state NMR in detergent micelles as my main technique. NMR provides unique possibilities for understanding the structure and dynamics of such small membrane proteins.

The Fe(II) and 2-oxoglutarate dependent oxygenases (2OG oxygenases) catalyse a broad range of oxidative reactions in various organisms. 2OG oxygenases use 2OG and molecular oxygen to catalyse the oxidation of a variety of substrates including small molecules, fatty acids, nucleic acids and proteins. Several human 2OG oxygenases are implicated in diseases. The fat mass and obesity associated protein (FTO) is linked to obesity, whilst collagen prolyl-4-hydroxylases (CPHs), the procollagen lysyl hydroxylases (PLODs), and the hypoxia inducible factor hydroxylases (PHDs) are linked to cancer. Therefore, structure-based inhibition studies on FTO, CPH and related 2OG oxygenases are of significant therapeutic interest. The obesity-associated FTO is an mRNA N⁶-methyladenine (m⁶A) demethylase and a homologue of E. coli alkylated DNA repair protein (AlkB), a DNA repair enzyme that demethylates N¹-methyladenine (m¹A) and N³-methylcytosine (m³C) bases. Human AlkB homologue 5 (ALKBH5) has a similar substrate profile as FTO, i.e. ALKBH5 is another mRNA m⁶A demethylase, making it a target for structural and inhibition studies to improve inhibitor selectivity for FTO. The CPH and PLODs catalyse hydroxylation of collagen prolyl- and lysyl-residues; the resultant 4-hydroxyprolyl- and 4-hydroxylysyl-residues are important for the formation of the collagen triple helix and intermolecular crosslinks, respectively. Another 2OG oxygenase, the 2-oxoglutarate and iron-dependent oxygenase domain-containing protein 2 (OGFOD2), is related by sequence similarity to the PLODs but its biological role is unknown. This thesis describes crystallographic, biochemical and inhibition studies on five 2OG oxygenases: AlkB, FTO, ALKBH5, OGFOD2 and CPH. Three AlkB-inhibitor complexes were determined; taken together, these structures serve as proof-of-principle that the AlkB subfamily 2OG oxygenases are amenable to structure-based inhibition studies. Several classes of inhibitors of FTO were then identified and their binding modes were investigated through the determination of 7 crystal structures of FTO-inhibitor complexes. Subsequently, a crystal structure of ALKBH5 was determined, which provided insights into its substrate recognition mechanisms. The ALKBH5 structure also serves as a template for inhibitor design. Preliminary structural and biochemical data were also obtained for OGFOD2 and CPH. Overall, these results contribute to the development of a biophysical understanding of human 2OG oxygenases, and will help to enable the development of selective inhibitors of 2OG oxygenases involved in nucleic acid and collagen modifications.

Organic chemistry is a valuable tool for studying enzyme mechanisms. Upon incubation with a specific enzyme, synthetic substrate analogues labeled with heavy atoms or carrying extra functional groups can provide mechanistic insights. In the present work, new compounds were synthesised in order to study the mechanism and substrate selectivity of two enzymes: human γ-butyrobetaine hydroxylase and bacterial carboxymethylproline synthase. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyses the stereospecific hydroxylation of γ-butyrobetaine, the final step of L-carnitine (L-Car) biosynthesis in mammals. Substrate analogues were synthesised to probe BBOX specificity in vitro. Some of those unnatural substrates were oxidised by BBOX and the products identified using a range of analytical techniques. 3-(2,2,2-Trimethylhydrazinium)propionate (THP) is a clinically used BBOX inhibitor. Under standard assay conditions, THP was oxidised by BBOX. NMR studies have identified the products of this reaction to be malonic acid semialdehyde, formaldehyde, dimethylamine and 3-amino-4-(methylamino)butanoic acid. The formation of 3-amino-4-(methylamino)butanoic acid suggests that BBOX can catalyse a Stevens type rearrangement involving N-N bond cleavage and C-C bond formation. The proposed structures and mechanisms were confirmed by mass spectrometric and NMR analyses using [¹³C]-labeled THP as well as synthetic standards of both enantiomers of 3-amino-4-(methylamino)butanoic acid. Although the structure of the rearrangement product was confirmed, the stereochemistry remains unknown. Altogether, these studies revealed the unprecedented nature of a BBOX-catalysed C–C bond formation reaction upon THP oxidation and may inspire the design of improved inhibitors for BBOX and other 2OG oxygenases. Pectobacterium carotovorum CarB and Streptomyces cattleya ThnE are two carboxymethylproline synthases (CMPS) that catalyse an early step in carbapenem antibiotics biosynthesis. CMPS produces (2S,5S)-carboxymethylproline (t-CMP) from malonyl-CoA and L-glutamate semi-aldehyde. L-Glutamate semi-aldehyde exists in equilibrium with L-5-hydroxyproline and L-pyrroline-5-carboxylate in solution (collectively abbreviated L-GHP). Because of the high stereoselectivity of t-CMP formation and the growing interest in novel carbapenem antibiotics, CMPS is potentially an interesting biocatalyst. A series of L-GHP analogues were synthesised and tested as CMPS substrates in an attempt to produce unnatural t-CMP derivatives enzymatically. Methyl-substituted L-GHP analogues were accepted by CMPS and the t-CMP products could be further carried through to the corresponding bicyclic carbapenams using CarA, a β-lactam synthetase. These results demonstrate the versatility of the early carbapenem biosynthetic pathway and the possibility of introducing structural diversity using synthetic substrate analogues. A crystal structure of S. cattleya ThnE was obtained in complex with L-proline and coenzyme A, giving the first insight into substrate binding. This structural information will potentially allow further rational mutagenesis studies aiming to broaden the range of unnatural L-GHP analogues accepted by CMPS.