Mosquitoes are responsible for millions of deaths a year through the viruses and parasites they vector. Many of these vector species have successfully expanded their range into temperate climates due to a combination of climate change and the easy movement of goods and people around the world. The temperate climate of the U.K. is home to 34 native species, several of which bite humans and are capable of vectoring pathogens more commonly associated with warmer climates, therefore the threat of mosquito-borne illness in the U.K. is a very real possibility. Many vector mosquito species have evolved resistance to traditional chemical insecticides and the search for novel control strategies in endemic areas is a priority in vector control. Entomopathogenic nematodes (EPNs) are microscopic roundworms, which are obligate parasites of insects from the family Rhabditae. In particular, soil-dwelling nematodes from the genera Heterorhabditis and Steinernema. Presently EPNs are used in a range of plant-based industries as a chemical-pest control. However, previous laboratory research has shown that EPNs are capable of killing more than 250 species of insect including a selection of vector species and nuisance arthropods. This thesis is concerned with discovering whether commercially available and naturally occurring strains of EPNs from the U.K. could be used as an effective biocontrol agent for mosquito and chironomid species. This study includes a snapshot of the current EPN diversity in the U.K. which found four different Steinernema species, including the first molecular confirmation of Steinernema carpocapsae. EPNs from both field-collected and commercial sources were capable of killing and parasitizing two native and tropical mosquito species and Chironomus plumosus. Commercial strains were more effective at killing both, however, the native field-collected, mosquito species Ochlerotatus detritus was susceptible to field-caught EPNs, unlike the tropical, lab-reared Aedes aegypti. EPNs were found to be capable of tolerating the extremes of habitat that mosquito species can inhabit in laboratory tests. These studies have shown that with further research including viable field trials that EPNs could be very useful to add to a range of vector and nuisance control measures when used appropriately.

Healthcare associated infections (HCAIs) are a large and growing problem. Bacterial infections of patients and on the medical devices used to treat them represent a significant source of morbidity and mortality. There is also a significant economical impact to the healthcare system attributed to HCAIs. While bacterial infections per se are not a novel problem, the discovery of an adherent polymicrobial phenotype called a biofilm is. A biofilm is defined by its structure and the community of bacteria therein. This study investigated bacteria biofilms in a number of pertinent clinical scenarios. To achieve this, samples were taken from five different but related clinical areas where biofilms are known to infect or are suspected to, namely endotracheal tubes, tracheostomy tubes, burn wounds, chronic wounds and chronic wound dressings. Samples were analysed using microbiological and molecular analysis techniques, the latter included polymerase chain reactions, species-specific PCR and denaturing gradient gel electrophoresis to assess microbial diversity. Fluorescent in-situ hybridization was used subsequently to analyse species orientation and biofilm structure within the biofilm. This study showed a diverse bacterial population in all the samples, with the presence of oral biota in the ETT specimens, changing to commensal bacteria over time. Large threedimensional biofilm structures were present in the specimens confirming the presence of biofilms, and within one of the chronic wound dressings where a complex biofilm was visible within the matrix of the dressing itself. These findings have considerable significance clinically, not only in demonstrating the need for biofilm targeted diagnostic techniques, but also in highlighting the need for specific biofilm treatment modalities in critical care, burn services and chronic wound management.

The work detailed in this thesis aimed to address antibiotic resistance - a pressing health issue on a global scale - through three different approaches: identifying the mechanisms of bacterial resistance evolution; modifying antibiotic treatment strategies to limit, or redirect, the course of resistance evolution, and; developing an effective method to find potential compounds for new therapeutic agents. Initially, a systematic investigation was conducted to monitor the emergence of bacterial antibiotic resistance when bacteria were exposed to a single antibiotic. In order to study the dynamics of bacterial resistance emergence, we constructed a morbidostat device to continuously monitor bacterial growth, and regulate drug concentrations to maintain an antibiotic-induced selection pressure. The resistance evolution was then examined with eight antibiotics from different classes. During 12 days of antibiotic exposure, resistance levels increased dramatically, exhibiting various evolutionary patterns depending on which antibiotics were administered. The mechanisms underlying these various evolutionary changes were further investigated by genotypic and phenotypic characterisation experiments. Secondly, the effect of an alternating antibiotic treatment, in which antibiotics were administered sequentially with periodic switching, was examined. The intention was to identify a dosing regimen that could suppress or control the resistance evolution, using only existing antibiotics. Antibiotic-sensitive bacteria had their resistance evolution tested in response to a range of alternating treatments that employed seven antibiotics and three different cycling interval times. We found that the development of antibiotic resistance could be reduced, or even reversed, by using certain antibiotic pairs. Additionally, we also identified the optimal interval time to further increase the degree of resistance evolution suppression. Further genotypic and phenotypic assays were then conducted to gain insight into the population dynamics occurring during antibiotic cycling and the underlying mechanisms that enabled the manipulation of antibiotic resistance. Finally, the optimisation of antimicrobial peptide sequences (AMPs) was achieved, demonstrating the potential to rapidly discover new antibiotic candidates. The conventional approaches to drug discovery are extremely time and labour-intensive, and hence struggle to cope with the accelerated development of bacterial drug resistance. AMPs demonstrate great promise as a potential new class of antibiotics; they display a range of modes-of-action against bacterial pathogens, and there are an astronomical number of possible sequences available to explore (e.g. 20^13 ≈ 8×10^16 combinations for a 13-mer peptide). An evolution-based algorithm was developed to effectively find the most potent antimicrobial peptides whilst requiring only a small number of experimental evaluations. The algorithm was subsequently validated by conducting an optimisation experiment, whereby, a naturally-occurring 13-mer AMP was taken as a starting point, and optimised over three generations. Finally, the physicochemical properties of the identified potent peptides were examined to gain insights into the underlying mechanisms responsible for the improvement in antimicrobial activity.

Cardiovascular diseases are the leading cause of mortality worldwide, with the majority of these deaths being a result of the inflammatory pathology, atherosclerosis. A critical need for multi-parameter molecular imaging has been identified to facilitate improved atherosclerosis diagnosis and the understanding of local inflammatory pathways in humans. Established imaging modalities such as ultrasound and magnetic resonance imaging are being investigated as potential solutions to this clinical problem, however, inherent limitations with these technologies have resulted in the exploration of alternate imaging approaches. This thesis focuses on the development and testing of surface enhanced Raman spectroscopy (SERS), a promising and novel molecular imaging modality, for the molecular imaging of vascular inflammatory biomarkers in vitro, ex vivo and in vivo. SERS detects molecule specific vibrational signals which are enhanced when an analyte is excited with light in close proximity to a noble metal surface. To achieve molecular specificity and surface enhancement, we developed antibody functionalised gold nanoparticles (nanotags) designed to bind to our molecular targets of interest, the adhesion molecules, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and P-selectin, and produce a SERS signal detectable by spectroscopy and/or microscopy based approaches. In vitro, we demonstrate the simultaneous and quantifiable SERS detection of ICAM-1, VCAM-1 and P-selectin on TNFa stimulated human endothelial cells. We subsequently demonstrated the simultaneous SERS detection of ICAM-1, VCAM-1 and P-selectin in freshly isolated atherosclerotic human coronary artery ex vivo. Finally, we explored SERS imaging in a humanised mouse model, demonstrating non-invasive multiplex imaging of adhesion molecules in vivo. In summary, this proof of concept study demonstrates the suitability of SERS and nanotags for the non-invasive molecular imaging of vascular inflammation. We have tested this approach with increasing biological complexity and highlighted SERS as a potential molecular imaging tool for future clinical translation in the context of vascular inflammation, atherosclerosis and cardiovascular disease.

Short chain fatty acids (SCFA), including propionate, are produced by the bacterial fermentation of carbohydrates in the colon. Propionate has many potential roles in health, including inhibiting cholesterol synthesis, de novo lipogenesis and increasing satiety. The profile of SCFA produced is determined by both the substrate available and the bacteria present and may be influenced by environmental conditions within the lumen of the colon. Whilst it may be beneficial to increase colonic propionate production, dietary strategies to achieve this are unproven. Adding propionate to food leads to poorer organoleptic properties, and oral propionate is absorbed in the small intestine. The optimum way to selectively increase colonic propionate would be to select fermentable carbohydrates that selectively promote propionate production. To date, few studies have undertaken a systematic assessment of the factors leading to increased colonic propionate production making the selection of propiogenic carbohydrates challenging. The aim of this thesis was to identify the best carbohydrates for selectively increasing propionate production, and to explore the factors which control propionate production. This work started with a systematic review of the literature for evidence of candidate carbohydrates, which led to a screen of ‘propiogenic’ substrates using in vitro batch fermentations and mechanistic analysis of the impact of pH, bond linkage and orientation using a range of sugars, polysaccharides and fibre sources. A new unit for SCFA production was developed to allow comparison of results from in vitro studies encompassing a range different methodologies found in the literature. The systematic review found that rhamnose yielded the highest rate and proportion of propionate production whereas, for polysaccharides, β-glucan ranked highest for rate and guar gum ranked highest for molar production, but this was not replicated across all studies. Thus, no single NDC was established as highly propiogenic. Some substrates appeared more propiogenic than others and when these were screened in vitro. Laminarin, and other β-glucans ranked highest for propionate production. Legume fibre and mycoprotein fibre were also propiogenic. A full complement of glucose disaccharides were tested to examine the role glycosidic bond orientation and position on propionate production. Of the glucose disaccharides tested, β(1-4) bonding was associated with increased proportion of propionate and α(1-1) and β(1-4) increased the rate and proportion of butyrate production. In conclusion, it appears that for fibre to affect satiety, high intakes of fibre are needed, and which a major mechanism is thought to occur via propionate. Within this thesis it was identified that rather than selecting specific fibres, increasing overall intakes of highly fermentable carbohydrates is as effective at increasing propionate production. Selecting carbohydrates with beta-bonding, particularly laminarin and other β(1-4) fermentable carbohydrates leads to marginal increases in propionate production. Compared with targeted delivery of propionate to the colon, fermentable carbohydrates examined in this thesis have lesser and variable effects on propionate production. A more complete understanding of the impact of bond configurations in polysaccharides, rather than disaccharides, may help selection or design of dietary carbohydrates which selectively promote colonic propionate production substrates for inclusion in functional foods. Overall this study has concluded that few substrates are selectively propiogenic and the evidence suggests that similar changes in propionate production may be achieved by modest changes in dietary fibre intake.

Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are the main multi-drug resistant pathogens associated with deep wound infections which then may cause septicaemia. Treatment is problematic and re-infection is quite common. Free Living Amoebae (FLA), such as Acanthamoeba, are widely distributed in the environment and may also contaminate wounds. It is well known that Acanthamoeba feed on and protect bacteria. The role of Acanthamoeba in wound infections is not very well understood. It is possible that the presence of Acanthamoeba in wounds is one of the key factors for such re-infections. In this study, 140 wound swabs were collected to check for the presence of Acanthamoeba spp. Only one sample was positive for Acanthamoeba spp. Sequencing of the highly variable DF3 region of 18S rRNA gene for the sample showed that this isolate belongs to genotype T4. In addition, clinical isolates of MRSA and Pseudomonas from wound infections were used in this study. The results showed that MRSA and Pseudomonas were able to bind with, invade, survive and multiply inside Acanthamoeba species. One of the essential compounds for microorganisms to grow is iron. The role of iron chelators, including deferiprone and selected novel compounds based on hydroxyl pyridine moiety, was studied. Findings revealed that all novel iron chelators have an antimicrobial activity against both bacteria. In addition, all novel iron chelators were able to kill Acanthamoeba. Cytotoxic effects of MRSA, P. aeruginosa and Acanthamoeba were investigated using the KB epithelial cell line and mesenchymal stem cells (MSC) using a general caspase inhibitor. The results revealed that the ability of live bacteria to induce cell death was higher compared with heat-killed bacteria, bacteria conditioned medium (BCM) and Acanthamoeba conditioned media (CM). The exact trigger for the cell death in this study was not investigated but the relative contributions of apoptosis and necrosis were investigated using fluorescent technique, caspase inhibition and LDH assay. In conclusion, presence of Acanthamoeba in wounds could be the reason of prolong treatment and reinfection in wounds.

Haematopoietic stem cells (HSCs) can migrate to the injured kidney and aid in tissue repair, however clinical success remains poor and is partially attributed to limited HSC recruitment. This study determined the molecular mechanisms governing HSC recruitment to the ischaemia-reperfusion (IR) injured kidney, whether this recruitment could be enhanced and also any immuno-modulatory effects HSCs may be having on surrounding injured microvasculature. HSC adhesion was significantly enhanced to the IR injured kidney compared to sham; this recruitment was governed by CD49d/VCAM-1 and CD44/HA. KC or SDF-1α pre-treatment enhanced HSC adhesion to the IR kidney. KC and SDF-1α also increased CD44 and CD49d surface clusters on HSCs respectively, and therefore increased HSC adhesion to HA and VCAM-1. Following injection into IR injured mice, HSCs improved blood flow and kidney function in the injured kidney compared to sham. This may be related to inflammatory modulation, as neutrophil recruitment and number of platelet microthrombi were reduced following HSC administration. This is the first study to show that pre-treatment of HSCs increases their recruitment to the site of injury, and that recruitment of HSCs can reduce inflammatory cell infiltration following injury.

Urinary tract infections (UTIs) are one of the most common healthcare associated infections (HCAIs) accounting for 17.2% of the total HCAI’s in England. Some of the underlying issues associated with UTIs include recurrent infections, catheter associated UTIs and antibiotic resistance. These issues are responsible for prolonged hospital admissions, increased costs and significant morbidity. Another possible issue relates to the ubiquitous protozoa, Acanthamoeba. Although it is known to cause infections in humans, the amoeba has been isolated from apparently healthy people. Furthermore, Acanthamoeba is known to have an endosymbiotic relationship with bacteria. Therefore, it is reasonable to hypothesise that Acanthamoeba may possibly play an important role in UTIs. Clinical isolates of E. coli, K. pneumoniae and P. mirabilis were used in the current study. All uropathogens exhibited the ability to form biofilms in a nutrient dependent manner and complete the biofilm cycle within 24h. They also displayed the ability to form intracellular bacterial communities in urothelial cells and induce significant cytotoxicity. Moreover, they were able to associate, invade and survive within Acanthamoeba castellanii (T4). Furthermore, 200 urine samples from patients suspected of UTIs were collected from Colchester University Hospital NHS Trust and analysed for the presence of Acanthamoeba. Nineteen samples were positive for Acanthamoeba spp. (unclassified) and two samples for A. castellanii supporting our hypothesis that the amoeba possibly plays a role in UTIs. This is the first study in the UK to have confirmed the presence of Acanthamoeba in urine. This study also investigated the antimicrobial efficacy of cetylpyridinium chloride (CPC). CPC coated latex catheters were able to prevent biofilm formation at very low concentrations. This finding provides promising evidence for the potential application of CPC impregnated catheters in preventing CAUTIs. In conclusion, the findings from this study can be used to develop targeted interventions aimed at the underlying issues associated with UTIs.