The human-specific enteropathogenic Escherichia coli and the mouse-restricted Citrobacter rodentium colonise the host intestinal mucosa via attaching and effacing lesions, inducing severe diarrhoeal disease and transmissible murine colonic hyperplasia, respectively. C. rodentium is widely used as an in vivo model for EPEC infection, sharing a highly homologous arsenal of virulence factors and a similar infection strategy with EPEC. EPEC and C. rodentium pathogenesis relies on the locus of enterocyte effacement-encoded type III secretion system and delivery of effectors into the host cytosol to subvert host cell signalling including: actin dynamics, cell trafficking and immune signalling. The role of many effectors during infection remains unclear. We sought to identify the function of the non-LEE-encoded effector protein F (NleF). We discovered that EPEC infection of polarised epithelial cells activated a caspase-4-dependent non-canonical inflammasome response leading to the processing and secretion of IL-18, which was counteracted in a T3SS- and NleF-dependent manner. EPEC NleF interacts with the p20 and p10 subunits of caspase-4, associating with the substrate-binding domain, to inhibit its proteolytic activity and downstream inflammasome activation. Infection of mice with a C. rodentium nleF mutant enhanced IL-18 secretion and revealed that NleF is essential for the inhibition of the caspase-1/11-dependent inflammasome and recruitment of neutrophils in vivo. We further report that ectopically expressed NleF activated NF-κB nuclear translocation and the up-regulation of the pro-inflammatory chemokine IL-8. Infection of HeLa with an EPEC nleF deletion mutant abolished the T3SS-dependent activation of NF-κB and the expression of IL-8. NleF was identified to act upstream to IκBα activation and could be inhibited by the TAB2/3 inhibitor NleE1. However the mechanism of the pro-inflammatory role of NleF remains unclear. These findings identify novel roles for the T3SS effector NleF, furthering our understanding of the infection strategy of EPEC and the host epithelial cell inflammasome response.

Tuberculosis (TB) is a leading cause of death from a single infectious agent and infects one third of the world's population in a latent form. Latent TB is characterised by presence of TB antigens but a lack of symptoms of TB. Latent TB is associated with the persistent form of Mycobacterium tuberculosis, and is a reservoir from which symptomatic infection arises. Non-replicating persistence (NRP) is postulated to be a reversible state characterised by lack of replication, decreased metabolic activity and increased antimicrobial resistance. To achieve viable persistence, NRP cells have been postulated to require stabilisation of cellular structures needed for stress tolerance and for the transition from NRP to active replication. This study investigates the hypothesis that ribosome stabilisation assists in mycobacterial stress tolerance and persistence. RafS is a novel mycobacterial ribosome associated factor and putative ribosome stabilisation factor. The physiological roles and functional characteristics of RafS are investigated in this study. The role of RafS in M. smegmatis (Msm) and M. tuberculosis (Mtb) physiology were investigated. Competitive survival assays between wild type and ΔrafSMtb illustrated that RafSMtb confers a competitive advantage during survival under nutrient limitation. RafSMsm and RafHMtb were found to significantly inhibit in vitro translation. Furthermore, RafSMsm and RafHMtb inhibited in vitro translation of mRNA with and without Shine Dalgarno sequences. It was determined that RafSMsm is dispensable for growth and survival in several conditions and also for mature biofilm and pellicle formation. Also, RafSMsm is dispensable for tolerance of heat, acid and antibiotic stress. Ribosomal profiling indicated no significant effect of rafSMsm deletion on ribosomal subunit association in log phase and stationary phase rich media cultures. These findings are discussed in the context of mycobacterial growth, survival, stress tolerance and persistence mechanisms.

The amino acid sequence of the trans-membrane protein, PM19, unique to the plant kingdom has been highly conserved over 450 million years of evolution in all plant genera including ferns and mosses, but is not found in aqueous plants such as algae. Thus, the function of this protein is possibly linked to the plant ability to grow on land. We have investigated the PM19 protein using a number of molecular biological tools in Arabidopsis thaliana. Gene expression studies using bioinformatics, northern blotting and promoter-GUS fusions show that the AtPM19 gene is highly expressed in seeds and seedlings, in addition to expression observed in leaves under drought stress. A translational fusion with GFP reveals that the protein is located in the plasma membrane. T-DNA insertion mutants have a germination phenotype; the mutant is more sensitive to high levels of salts in the medium, and in addition, the mutant has a lower stomatal conductance indicative of reduced guard cell turgor. The predicted secondary structure of the protein and the mutant phenotype suggest that PM19 may be a cation transporter and this is being tested by functional complementation of yeast mutants.

To meet the challenges of protein price increases from animal sources, the development of new, sustainable and inexpensive proteins sources (nonanimal sources) is of great importance. Date palm (Phoenix dactylifera L.) seeds could be one of these sources. These seeds are considered a waste and a major problem to the food industry. In this thesis we report a physicochemical characterisation of date palm seed protein. Date palm seed was found to be composed of a number of components including protein and amino acids, fat, ash and fibre. The first objective of the project was to extract protein from date palm seed to produce a powder of sufficient protein content to test functional properties. This was achieved using several laboratory scale methods. Protein powders of varying protein content were produced depending on the method used. Most methods were based on solubilisation of the proteins in 0.1M NaOH. Using this method combined with enzymatic hydrolysis of seed polysaccharides (particularly mannans) it was possible to achieve a protein powder of about 40% protein (w/w) compared to a seed protein content of about 6% (w/w). Phenol/TCA extraction gave the protein powder with the highest protein percentage of 68.24% (w/w) and this powder was used for subsequent functional testing. Several factors were found to influence seed protein extraction such as pH, temperature, the extraction time, the solvent to sample ratio and the solvent concentration. Optimum conditions for extraction were found to be pH 10, 45˚C and extraction time of 60 min. The results showed that use of enzymes to hydrolyse and remove seed polysaccharides improved the extraction of date seed protein. Optimal improvement was obtained using Mannaway, which hydrolyses mannans and galactomannans, which gave a powder with 34.82% (w/w) protein compared to the control of 11.15% (w/w) protein. The proteins in the extracted date seed protein were profiled using LC/MSMS. Three-hundred and seventeen proteins were identified. The proteins belonged to all major functional categories. The most abundant proteins were glycinin and β-conglycinin, the two major seed storage proteins of plants. The functional properties of extracted date seed protein were investigated using a range of tests. The thermal properties of date seed proteins were consistent with a powder containing high levels of conglycinin and β-glycinin. The solubility had a similar pH profile to soy protein, but differed in absolute solubility due to differences in non-protein composition. Similarly, water holding and oil holding capacity of date seed protein was lower than for soy protein, probably because of compositional differences. Date seed proteins were able to emulsify oils and had a comparable emulsifying ability and emulsion stability to soy protein isolate. The date seed protein was not a good foaming agent compared to soy protein or whey protein concentrate.

Adipose tissue homeostasis is regulated by a combination of extracellular and intracellular signalling pathways. Activation of surface G protein-coupled receptors (GPCR) and insulin receptors, influence the rate of lipolysis within adipocytes, the pathway responsible for triacylglycerol (TAG) breakdown into non-esterified fatty acids (NEFA) and glycerol. A GPCR for short chain fatty acids (SCFA) has been identified on the surface of mature adipocytes and activation of this receptor by SCFA including acetate, butyrate and propionate has resulted in a decrease in lipolysis, measured as a reduction in NEFA and glycerol concentration in the media. However, evidence for a mechanism of action of SCFA within adipocytes has remained unclear. Therefore, this thesis has aimed to better understand the mechanism(s) by which the SCFA, acetate, regulates adipose tissue metabolism and function, and in particular the pathway of lipolysis. Through the development of experimental methods in vitro, it was determined that the dose of isoproterenol, a β-adrenergic receptor activator, required to stimulate lipolysis by 50 % in murine 3T3-L1 adipocytes, was 5 μM, and that no interference with metabolic assays was observed in the presence of any potential treatment condition, either in the basal or stimulated state. In 3T3-L1 cells, in the basal state, treatment of cells with the short chain fatty acid acetate (4 mM), significantly reduced lipolysis, as a measure of NEFA and glycerol (P = 0.004 and P = 0.020, respectively) after a 180 min incubation. Similarly, in the stimulated state, acetate also reduced NEFA significantly (P = 0.020), however, glycerol was not reduced (P = 0.529) compared with controls. To evaluate whether the metabolic changes in NEFA and glycerol concentration reflected an intracellular change to the pathway of lipolysis, phosphorylation of the key enzyme, hormone sensitive lipase (HSL), was also analysed. It was identified that in the stimulated state, phosphorylation of HSL, at serine residue 563, was reduced by 15 % in the presence of 4 mM acetate, compared with control, complimentary to metabolic data. However, treatment of isolated primary mouse mature adipocytes with acetate did not produce results comparable to those found in the literature. Instead, accumulation of NEFA and glycerol in the media were found to be negligible. Cell viability may have been a limiting factor regarding the outcome of these studies, and therefore development of a protocol to improve cell viability without compromising cell yield would prove useful. Furthermore, future work should consider the lack of reduction in glycerol in the media in the stimulated state from 3T3-L1 mature adipocytes. This may be accounted for by fatty acid re-esterification, as the NEFA:glycerol ratios were 1.5:1, compared with the expected 3:1 ratio. Similarly, to re-enforce the effects of acetate on stimulated lipolysis, other phosphorylation sites within HSL may be considered, including SER565. Overall, the results obtained in this thesis demonstrate that in the mature adipocyte cell line 3T3-L1, an increase in the availability of the SCFA, acetate, resulted in a change in the pathway of lipolysis. This was evidenced by a reduction in the phosphorylation of HSL(SER563) under sub-maximal stimulation with isoproterenol, similar to levels observed in the presence of insulin. Complimentary to these data, under the same treatment conditions, incubation with acetate also resulted in a small but significant reduction in NEFA concentration in the media.

Revival of the ABE fermentation will be enhanced by the ability of bacterial strains to utilise cheap, renewable substrates containing a range of fermentable carbohydrates. Development of an effective process will, however, depend on a detailed understanding of the mechanisms of uptake and metabolism of the available sugars. The predominant mechanism for uptake of sugars and sugar derivatives in the clostridia is the phosphoenolpyruvate (PEP) - dependent phosphotransferase system (PTS), which not only catalyses the concurrent uptake and phosphorylation of its substrate but also plays a central role in regulation of carbohydrate metabolism. Complete characterization of the PTS in the solventogenic clostridia will therefore be instrumental in developing strategies for constructing effective fermentation strains. The Clostridium beijerinckii 8052 genome encodes 43 complete phosphotransferase systems, including sixteen belonging to the glucose-glucoside family. Three of the PTSs are members of the glucose subgroup in a phylogenetic branch, and might therefore transport glucose. Since glucose has been shown to repress utilization of other sugars by Clostridium beijerinckii, these systems could also potentially be involved in glucose sensing and carbon catabolite repression (CCR). The cbei 0751 gene encoding a IICBA PTS permease was amplified by PCR, and cloned into Escherichia coli ZSC113, a mutant which cannot take up and phosphorylate glucose and mannose. Transformants showed a positive fermentation phenotype for glucose and mannose. Extracts showed glucose PTS activity, and cbei 0751 was therefore shown to be a functional glucose PTS. The activity was inhibited by mannose confirming that the system also recognises mannose as a substrate. The expression of this gene appeared to be constitutive although quantitative expression was not performed. Similar experiments were used to investigate the function of a second system encoded by cbei 4983 (IICB) and cbei 4982 (IIA). Although these genes were successfully cloned, their function could not be identified. Since the cbei 4984 gene encodes a putative glycoside hydrolase, this suggests that the primary function of this PTS may be to transport and phosphorylate a disaccharide, but further experimental analysis is required to identify the substrate of this system. Attempts to inactivate the two phosphotransferases to examine the effect on the cells were not successful.

Campylobacteriosis is common in both developing and developed countries. Although there had been numerous studies performed to gain a better understanding of the disease, much still remains to be unravel. In this project, the interactions between the bacteria and host cells or organisms were interrogated. Raman based imaging was initially investigated using an established Salmonella typhimurium and cell infection model. The results obtained showed the potential for real-time imaging. However, due to the instability of the laser system of the microscope, reproducible results could not be obtained. Therefore, this technique was not applied to Campylobacter jejuni. Zebrafish embryo was established as a new infection model suitable for C. jejuni studies. C. jejuni strains and mutants were screened using this model to determine their virulence. This model was used to screen two type six secretion system mutants constructed in this study. The results obtained showed that one of the mutants, Cj1DtssM(syn)::kanR_cas, was attenuated in the ZFE model. Subsequent competitive index challenges performed in piglets with C. jejuni wild-type and the two T6SS mutants, also showed that Cj1DtssM(syn)::kanR_cas was attenuated. Both T6SS mutants had shown increased adherence to macrophages and Cj1DtssM(syn)::kanR_cas also showed increased cell invasion. Together, these findings suggested that the T6SS is involved in establishment of infection in ZFE and piglet and as well as in adhesion and invasion of macrophages.

Saccharomyces cerevisiae provides an ideal model to study the regulation of cell cycle commitment due to the high conservation of signalling pathways and regulatory modules through to higher eukaryotes. My work investigates the interplay of cell cycle progression and arrest via the action of transcription factor regulation. Cell cycle commitment is controlled by the cyclin-dependent activation of transcription factor complexes, MBF and SBF. Here I describe the dynamics of SBF and MBF using new polyclonal anti-sera against the three key components Mbp1, Swi4 and Swi6, and their interaction with the inhibitor of SBF, Whi5, and the MBF co-repressor Nrm1. I identify epigenetic modifications that occur on histone proteins at promoters of SBF and MBF genes during the cell cycle. The histone deacetylase Rpd3 has also been investigated as to the role it plays in regulating G1/S transcription. Finally, I have identified a new class of G1/S genes, named switch genes, which are regulated independently by G1/S transcription factors during different phases of the cell cycle. Switch genes are regulated by SBF during G1 and MBF upon entry into S phase, and are enriched for dosage sensitive and replication induced G1/S genes. Switching from SBF-to-MBF allows genes to be activated in response to replication stress, via inactivation of Nrm1. In addition, through switching a potential defect in one of the transcriptional factor complexes will not result in overexpression of these genes. Detailed analysis of the prototypical switch gene TOS4 shows that it is regulated by SBF and MBF, accumulates in response to hydroxyurea, and delays cell cycle progression when over-expressed. The role Tos4 plays in the cell cycle and in response to checkpoint activation remains unclear, however, data suggests a role in modulating HDAC activity. The roles other switch genes play in response to checkpoint activation are yet to be investigated.

For all living organisms, macromolecular interactions facilitate most of their natural functions. Alterations to macromolecular structures through mutations, can affect the stability of their interactions, which may lead to unfavourable phenotypes and disease. Presented here, are a number of computational methods aimed at uncovering the principles behind complex stability - as described by binding affinity and dissociation rate constants. Several factors are known to govern the stability of protein-protein interactions, however, no one factor dominates, and it is the synergistic effect of a number of contributions, which amount to the affinity, and stability of a complex. The characterization of complex stability can thus be presented as a two-fold problem; modelling the individual factors and modelling the synergistic effect of the combination of such individual factors. Using machine learning as a central framework, empirical functions are designed for estimating affinity, dissociation rates and the effects of mutations on these properties. The performance of all models is in turn benchmarked on experimental data available from the literature and carefully curated datasets. Firstly, a wild-type binding free energy prediction model is designed, composed of a diverse set of stability descriptors, which account for flexibility and conformational changes undergone by the complex in question. Similarly, models for estimating the effects of mutations on binding affinity are also designed and benchmarked in a community-wide blind trial. Emphasis here is on the detection of a small subset of mutations that are able to enhance the stability of two de novo protein drugs targeting the flu virus hemagglutinin. Probing further the determinants of stability, a set of descriptors that link hotspot residues with the off-rate of a complex are designed, and applied to models predicting changes in off-rate upon mutation. Finally, the relationship between the distribution of hotspots at protein interfaces, and the rate of dissociation of such interfaces, is investigated.

Widespread antibiotic resistance among pathogenic bacteria and the low specificity of these drugs cause a pressing need for the development of novel antibiotics. Endolysins are proteins that are produced by bacteriophage infected cells and digest the bacterial cell wall for phage progeny release. These efficient enzymes are highly specific for the target bacteria without affecting other species. Development of resistance against endolysins is rare, because they evolved to target molecules that are essential for bacterial viability. Taken together, this makes them promising novel antibiotics. The development of recombinant endolysins as antibacterial agents requires an inexpensive and safe production platform. Microalgae emerged as an alternative expression platform in the last years. This study investigated therefore the production of endolysins in two distinct microalgal systems, the eukaryotic green microalga Chlamydomonas reinhardtii and the cyanobacterium Synechocystis sp. PCC 6803. C. reinhardtii is an attractive production platform for therapeutic proteins, due to the lack of endotoxins and infectious agents and its GRAS status (Generally Recognised as Safe). Synechocystis is a prokaryotic system and is natural transformable. Both offer established techniques for the expression of foreign genes and can be cultivated in full containment in simple photobioreactors. Transgenic lines of C. reinhardtii and Synechocystis expressing endolysins specific to the human pathogens Streptococcus pneumoniae and Staphylococcus aureus were created. The S. pneumoniae-specific endolysin Pal has been purified in an active form and its specificity and efficiency in killing the target bacterium demonstrated in vitro. Another endolysin specific to Propionibacterium acnes, a bacterium involved in the skin condition acne vulgaris, was synthesised in Synechocystis. The synthesis of the endolysins in the microalgae was analysed under the influence of different expression elements and at different growth stages, and the yields of recombinant protein were quantified to evaluate microalgae as a production platform for antibacterial and other protein therapeutics.