Although cytoglobin is widely considered a tumour suppressor, re-expression plays a role in disease progression in a subset of oral squamous cell carcinomas (OSC), but the mechanism of action is not understood. In this thesis, we developed a new OSC cell model to study the effects of cytoglobin over­ expression on cellular phenotype and resistance to cisplatin. Microarray analysis of cytoglobin­ expressing cells showed significantly altered transcripts related to stress response, adhesion and locomotion, and metabolism. Treatment of cytoglobin-expressing cells with cisplatin revealed a greater response in p53-regulated target expression (MAP3K5, NQOl, CDKN2A and GADD45A) compared to non-expressing cells. Further investigation showed this was associated with higher CHKl, p53 and p21 protein levels, suggesting enhanced activation of p53 signalling pathways. Furthermore, cytoglobin-expressing cells were more resistant to cisplatin-induced apoptosis and altered their cell cycle distribution. These changes were linked to reduced total cellular and mitochondrial superoxide. Collectively, these findings demonstrate for the first time that cytoglobin over- expression is associated with resistance to cisplatin-induced cytotoxicity and the mechanism involves p53 signalling. In conclusion, we propose expression of cytoglobin may afford tumours cells a survival advantage in the harsh environmental conditions of the developing tumour as well as resistance to drugs like cisplatin.

Membrane proteins are the gateways into cells and therefore a critical part of pharmaceutical research. Membrane proteins, in fact, have been shown to be the target of over 50% of all medicinal drugs. The development of drugs that acts on membrane proteins, however, has been limited by the lack of high resolution structural data. This lack of information results from a lack of a generic method to extract active membrane proteins from the cell membrane. We have developed a novel styrene maleic acid (SMA) / lipid particle system termed SMALP that preserves the structural and functional integrity of membrane proteins. We have optimised this system and used it to solubilise two membrane proteins with different architectures and functions. The first, human adenosine 2a receptor (A2aR), is a G-protein coupled receptor, a 7 transmembrane helix protein that is involved in cell signalling, whereas the second, FtsZ-interacting protein A (ZipA), is a protein involved in bacterial cell division that contains a single transmembrane domain. In this project, standardised purification protocols were developed that produced pure proteins without the need to add any detergents. The subsequent biophysical characterisation that included circular dichroism, mass spectrometry and sedimentation velocity analytical ultracentrifugation indicated that these purified SMALP solubilised proteins were natively folded once encapsulated. The activities of both proteins were then tested and shown to be close to that expected for the intact proteins in the cell membrane. Taken together these data suggest that the SMALP system offers the solution to membrane protein purification into the future.

Previous transcriptomic experiments have revealed that various Escherichia coli K-12 genes encoding proteins of unknown function are highly expressed during anaerobic growth in the presence of nitrate, or especially nitrite. Products of some of these genes, especially YeaR-YoaG, YgbA, YibIH and the hybrid cluster protein, Hcp, have been implicated in the response to nitrosative stress. The aims of this study were to investigate sources of nitrosative stress, and the possible roles of some of these proteins in protection against nitric oxide. The YtfE protein has been implicated in the repair of iron centres, especially in iron-sulphur proteins. The previously unexplained anaerobic growth defect of the ytfE strain LMS 4209 was shown to be due to a secondary 126-gene deletion rather than to the deletion of ytfE. At the start of the project, the transcription factor, NsrR, was known to respond to low concentrations of intracellular NO, and to repress expression of ytfE, hcp-hcr encoding the hybrid cluster protein and its reductase, and hmp that encodes the flavohaemoglobin, Hmp. In this work, a biochemical assay of hcp promoter activity was developed as a reporter of intracellular NO generation. This assay was used in combination with a range of mutants to show that the major source of intracellular NO is the reduction of nitrite by the cytoplasmic nitrate reductase, NarG. Although the periplasmic cytochrome c nitrite reductase, NrfAB, and the cytoplasmic nitrite reductase, NirBD, decrease nitrosative stress by reducing nitrite to ammonia, at least one additional source of NO production from nitrite remains unidentified. An assay for NO reduction using a Clark-Type electrode was validated. Rates of NO reduction were induced 2-fold in the presence of nitrate. Lowest rates of NO reduction were found in a mutant defective in nsrR. A quadruple mutant defective in Hmp, the flavorubredoxin, NorV, NrfAB and NirBD still reduced NO at more than half the rate of the parent. This residual activity was not due to YibIH, YeaR-YoaG or YgbA. Various hcp-hcr derivatives of strains defective in NO reductases revealed a severe growth defect under conditions of nitrosative stress, but Hcp was eliminated as a possible additional NO reductase. This growth defect was substantially suppressed by a further mutation in ytfE. Growth experiments with isogenic sets of mutant defective in all combinations of hcp and ytfE in addition to deletions in hmp, norVW and nrfAB implicated both YtfE and Hcp in repair of nitrosative damage. However, weaker phenotypes of these strains in absence of nitrate or nitrite are consistent with more general roles for these proteins in the repair of damage to protein iron centres.

A model system of Myzus persicae, Brevicoryne brassicae and Arabidopsis thaliana was used to investigate the effect of loss of function mutations in the plant amino acid transporter genes, AtAAP1 (Accession number: At1g58360) and AtAAP2 (Accession number: At5g09220). Homozygous mutant lines for each transporter were screened for phenotypic changes. Silique numbers and total silique seed mass were reduced for both aap1 and aap2 plants in comparison to wildtype plants (p < 0.05). Individual seed weight was also significantly reduced in aap2 plants (p < 0.05). Aphid probing behaviour, measured using EPG, indicated both aphid species took significantly longer attempting to locate a sieve element and reach sustained E2 feeding when on aap1 and aap2 plants (p < 0.05). The rate of aphid feeding was also significantly slower for both aphid species feeding on aap1 and aap2 (p < 0.05). M. persicae and B. brassicae feeding on aap1 and aap2 exhibited no change in aphid performance when compared to aphids on control plants (p > 0.05). Following antibiotic elimination of aphid symbionts in both species, aposymbiotic aphids were found to grow significantly slower on aap1 and aap2 plants in comparison with aposymbiotic aphids feeding on control plants (p < 0.005).

In recent years, it has become clear that pyrimidine metabolism is an excellent target for anti-protozoan drug development, with multiple enzymes genetically validated as essential. Pyrimidine nucleobase and nucleoside analogues with promising activity against Leishmania and Trypanosoma species can be readily identified. Well-known drugs like 5-fluorouracil and 5-fluoro-2’deoxyuridine are rapidly metabolised by the parasites and incorporated into RNA and metabolic intermediates such as 5F-2’dUMP and UDP-glucose. We thus see that pyrimidine salvage is a major vulnerability in protozoa, with non-redundant pathways sensitive to inhibition by small pyrimidine analogues. Currently, the main factor slowing down progress towards actual therapeutic exploitation of this weakness is the lack of information on the transport proteins for pyrimidines that need to efficiently internalise the pyrimidine analogues into the parasites. The De Koning laboratory cloned the first protozoan purine nucleobase transporter in 2003 and here we report strategies to similarly identify the pyrimidine transporter family. We characterized thymidine transport in L. major promastigotes in order to establish which transporter is responsible for its uptake. We found two separate thymidine transport activities represent LmajNT1.1 and LmajNT1.2, with Km values of 4.2 mM and 26.9 mM, respectively. In addition, to verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the L. major and L. mexicana ENT-family genes that are syntenic with the L. donovani nucleoside transporters, expressing each in T. brucei for individual characterisation. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated the uptake of [3H]-inosine but not [3H]-uridine. These results showed that the organisation of nucleoside transport is strictly conserved among Leishmania species - a conclusion that has important implications for the development of a nucleoside-based chemotherapy. Comparative sequencing of wild-type strains and 5-fluorouracil-resistant lines of T. b. brucei and L. mexicana using RNA-seq was performed in order to identify the candidate pyrimidine transporter genes downregulated in the resistant lines. We found 17 candidate pyrimidine transporter genes encoding for at least 3-TM domains that were down-regulated in both 5-fluorouracil-resistant lines. We overexpressed the most promising candidate genes in Tbb-5FURes and Lmex-5FURes cell lines, aiming to determine their contribution to the 5-FU uptake and resistance. Moreover, RNAi library screening for T. brucei for 5-FU and 6-AU resistance, with and without prior disruption of pyrimidine biosynthesis, was performed. Data generated by RIT-seq was compared to the RNA-seq results. Several candidate pyrimidine transporter genes were highlighted by the returned data. In conclusion, our results make several noteworthy contributions toward the eventual identification of the pyrimidine transporter gene and, by increasing our understanding of 5-FU toxicity in kinetoplastids, we gain insight into the complexities of pyrimidine metabolism in these parasites.

Pseudomonas aeruginosa is a Gram-negative species of bacteria that is of high clinical importance and is on the ESKAPE list of pathogens, causing the most serious nosocomial infections World-wide. Current antibiotics in clinical use against P. aeruginosa include carbenicillin, cefepime, ceftazidime, and the fluoroquinoline ciprofloxacin. The multi-drug resistant (MDR) phenotype exhibits widespread resistance to β-lactams and antibiotics from multiple classes often need to be administered in conjunction with β-lactamase inhibitors. Penicillin binding proteins (PBPs) are essential for cell viability and are important antibiotic targets, with the Class A bifunctional PBPs synthesising the principle component of the bacterial cell wall, peptidoglycan. The focus of this project was to investigate the kinetics of transglycosylation and transpeptidation mechanisms with a view to improving our understanding of bacterial cell wall synthesis, maintenance and regeneration. In this project, the function of Class A PBPs 1A and 1B from Escherichia coli and P. aeruginosa, with particular focus on PBP1A has been investigated. Transglycosylation and transpeptidation have been probed from multiple angles using orthogonal assays and fluorescently-labelled and native lipid substrates, as well as Lipid II structural variants. P. aeruginosa PBP1A transglycosylase and transpeptidase activities are demonstrated continuously, and compared to the model organism equivalent, E. coli PBP1A. The continuous monitoring of activity gave insights into the possibility of two catalytic sites for transglycosylation: a donor and an acceptor site. Data was fitted to different kinetic models to elucidate the best fit for extracting meaningful kinetic parameters. The data acquired over the course of this project suggests that P. aeruginosa PBP1A and E. coli PBP1A exhibit similar functional behaviour to each other, with E. coli PBP1B showing distinct activity from PBP1A. PaPBP1B activity was not detected at a level required to kinetically characterise the enzyme and it is possible that this PBP has an as yet undetermined stimulatory cofactor. The assays developed and optimised in this thesis will pave the way for further in-depth studies of P. aeruginosa PBP1A, which could be used to screen for putative inhibitor compounds and potentially identify certain characteristics required for antimicrobial efficacy. Over the past 4 years, major leaps in our knowledge of assay development, the transglycosylase and transpeptidase activities of Class A PBPs and substrate features and requirements for transglycosylase and transpeptidase donors and acceptors have thus far and will continue to unravel the unique and fascinating mechanistic intricacies of the bifunctionality of PBPs.

Nm23-H1 is elevated in acute myeloid leukaemia (AML) patient serum, where it is thought to enhance AML cell survival. However, the Nm23-H1 pro-survival mechanism remains poorly understood. Here it is demonstrated that AML samples are heterogeneous in their ability to bind Nm23-H1 and respond to the resultant survival signal. Although rNm23-H1 promoted the survival of the most primitive blasts within responding AMLs, it was not these cells that bound the protein. Instead, Nm23-H1 bound to more mature CD34\(^l\)\(^o\)/CD11b\(^+\)\(^v\)\(^e\) cells indicating that the survival effect on the blasts was indirect. In support of this, the survival of purified blast cells was enhanced by medium conditioned by more mature cells from the clone that had been stimulated by rNm23-H1. It is hypothesised that the AML clone subverts a signaling process between immature and more mature haemopoietic cells; a mechanism involved in controlling haemopoietic maturation.

This study examined the regulation of two genes, hcp and ogt, that are reported to be involved in protection from the mutagenic effects of reactive nitrogen species in Escherichia coli. Biochemical techniques were used to investigate promoter activity and the effects of the transcription factors that are reported to regulate expression of the hcp and ogt genes, namely NarL / NarP, FNR and NsrR. Transcription activation by NarL was then studied using semi-synthetic promoters. The hcp gene was found to be positively regulated by FNR and negatively regulated by NsrR. Contrary to previous reports, NarL and NarP were found to have little direct effect on expression of hcp, however, an indirect effect of NarL was detected. This study demonstrates that the indirect effect on hcp regulation of NarL is related to repression by NsrR and suggests that NarL is involved in the generation of reactive nitrogen species. The ogt gene was confirmed to be activated by NarL independently of FNR. Studies focused on characterising the different classes by which NarL / NarP can activate promoter activity independently of FNR. NarL was found to activate by class I, II and III mechanisms, whilst NarP was capable of class II activation only. Activation by NarL was studied and a library of alanine substitutions in the carboxyl terminal domain (CTD) of the α subunit of RNA polymerase was used to demonstrate direct interaction between NarL and RNA polymerase. NarL, the CTD of NarL, NarXL, NarP and the CTD of NarP were expressed from plasmids and transcription activation studied in cells lacking chromosomal narL and narP. Full-length NarL activated by Class I, II and III mechanisms, whilst the CTD of NarL only activated by class II mechanisms. Both the full-length NarP and the CTD of NarP only activated by class II mechanisms.

The human vasopressin receptors (V1aR, V1bR and V2R) are G-protein-coupled receptors (GPCR) and mediate the effects of [Arg8]vasopressin (AVP). The influence of G-protein selectivity on ligand binding was probed by exchanging G-protein selectivity of the V1aR and V2R. This was achieved by generating a V1aR chimera that was capable of coupling to Gs and a V2R chimera that signalled via Gq/11. A conserved Ar1-X-Ar2 motif in ECL 1 of Family A GPCRs was identified. The vasopressin receptors lack the second aromatic residue, however tryptophan (Trp2·64) is conserved at the beginning of ECL 1. Removal of the Trp2·64 sidechain in the V1aR resulted in loss of high affinity AVP binding. Transfer of tryptophan, to recreate the Ar1-X-Ar2 motif, did not recover high affinity AVP binding. Pharmacological chaperones are non-peptide ligands that can selectively rescue intracellularly retained receptors. As demonstrated herein, pharmacological chaperones are also useful laboratory tools for investigating low-expressing mutant receptors. Additionally, mutagenesis was utilised to investigate the chaperone capability of sub-type selective ligands by increasing the affinity of the ligand for the 'wrong' receptor subtype. A potential three-way interaction between the V1bR, CRFR1 and RAMP2 was probed. The study also investigated the effects of RAMP2 on two CRFR1 isoforms.

The production of biotherapeutics including antibodies and antibody fragments is a rapidly expanding market with an increasing number of products being approved for use. One of the major platforms used for production of such therapeutics is Escherichia coli, which offers a rapid production at low production costs. The favoured location for targeting these biotherapeutic is the periplasm of E. coli as this environment supports the formation of disulphide bonds and simplifies the purification process. There are a number of periplasmic release procedures currently practised in industry including osmotic shock. However their limitations call for the development of an improved generic periplasmic release method. This project demonstrates how the polymer poly(styrene-co-maleic acid) (SMA) can be applied as a novel and alternative periplasmic release agent. The amphipathic polymer self-assembles into discs encapsulating membrane proteins and thereby destabilises the outer membrane consequently releasing the periplasm. Data presented here show that SMA releases the model target proteins with a higher yield at equal or higher target purity than the conventional methods. Furthermore the developed methods was analysed and refined to be compatible with existing downstream and first steps for its adaptation on industrial scale were made.