Introduction: Interestingly, polyamidoamines (PAAs) are a group of biodegradable cationic polymers that exhibit a transfection efficiency comparable to that obtained with PEI but with a low cytotoxicity compared to other polycationic vectors. However, their cellular adverse effects have only been probed in assays indirectly measuring the cell metabolic activity based on the reduction of tetrazolium salts into coloured formazan derivatives by mitochondrial enzymes such as the MTT, MTS or XTT assays. There is therefore a need for a deeper insight into their cytotoxic profile before their potential as delivery agents in gene therapy can be fully appreciated. Furthermore, the intracellular fate of PAA polyplexes remains unclear to date, prompting questions around the mechanisms underlying their ability to mediate gene transfer. The present work explored cellular events induced by DNA polyplexes prepared using a linear PAA with a methylenebisacrylamide/dimethylethylenediamine (MBA-DMEDA) backbone of a molecular weight over 10kDa with the aim to confirm and better understand the absence of relationship between cytotoxicity and transfection efficiency reported with those vectors. Methods: Complexes were formed between pDNA and branched polyethyleneimine (BPEI, 25 kDa) or various PAAs including a homopolymer of a molecular weight >10 kDa (HP->10), a PEGylated Triblock Copolymer (CP) and a blend of CP and HP->10 to form a bioreducible cross-linked delivery system. Physicochemical properties of pDNA polyplexes were analysed by using simple techniques including gel electrophoresis, dynamic light scattering (DLS) and zeta potential measurement. The transfection efficiency of pDNA complexes was compared in A549 cells using a luciferase reporter gene assay. The impact of endo/lysosomal escape on transfectionefficiency of pDNA polyplexes was investigated by transfecting cells in presence of bafilomycin A1 or chloroquine, whereas the role of reactive oxygen species in transgene expression was evaluated by using hydroquinone as antioxidant. Cytotoxicity caused by the vectors was evaluated by measuring cell metabolic activity, lactate dehydrogenase release, formation of reactive oxygen species and changes in mitochondrial membrane potential. Results: Gel electrophoresis showed complete incorporation of pDNA by individual and cross linked PAA polyplexes at a similar polymer to DNA (RU:Nt) ratio of (2:1). In comparison, pDNA incorporation was achieved by BPEI at a 3:1 RU:Nt ratio. All polymers were able to condense pDNA into nanoparticles of ≤200 nm in diameter with a positive surface charge. Non-PEGylated PAA polyplexes showed higher luciferase activity than the PEGylated counterparts, but lower than that of BPEI. However, in contrast to BPEI vectors, PAA polyplexes caused negligible cytotoxic effects. The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine either enhanced or decreased transgene expression depending on the RU:Nt. No changes were found in the transfection of PAA polyplexes in the presence of hydroquinone compared to BPEI. Conclusions: PAA polyplexes displayed a pH-dependent endo/lysosomal escape which was not associated with cytotoxic events, unlike observed with BPEI polyplexes. This is likely due to their greater interactions with biological membranes at acidic than neutral pH. The presence of excess polymer is considered necessary for improving transfection efficiency of PAA polyplexes. PAA was identified as promising and safe candidate for in vitro gene delivery and has potential for future in vivo gene therapy.

In nature, several known proteins are glycosylated, and carbohydrate-cell receptor interactions mediate a plethora of key biological events, e.g. parasitisation and immune responses. Cell membranes display carbohydrate-binding proteins (lectins), which are able to selectively recognise specific sugar-ligands, decipher sugar-encoded instructions, and convert them into downstream biological processes. To overcome low binding affinities, which are typically observed for simple monosaccharides, protein-binding sugars are typically displayed within large multivalent ligands, where biological systems rely on multivalent interactions as a way to enhance selectivity and binding avidity. Glycoliposomes, phospholipid vesicles coated with sugars, can be designed to exploit this phenomenon through the presentation of multiple glycosylated ligands for actively targeting specific receptors, enhancing uptake of nanomedicines into target cells, or both. Among all the nanocarriers, liposomes are an advantageous strategy for drug delivery for their biocompatibility and high drug encapsulation efficiency. Functionalised liposomes have been developed over the last 30 years to optimise these nanocarriers by improving stability, circulation time and targeting. This thesis work focussed on the use of glycosylated liposomal systems for the treatment of intracellular bacterial infection in cells presenting lectin endocytic receptors. Antimicrobial resistance is one of the current main global healthcare challenges. In particular, antimicrobial resistance in Salmonella Enterica serotypes is responsible for 16 million cases of typhoid fever, 94 million cases of gastroenteritis and 600.000 deaths worldwide. Salmonella is able to survive in intracellular compartments of macrophages. Mannose Receptor (MR, CD206) is a potential access gate to Salmonella infected macrophages which could be selectively targeted with mannosylate-decorated liposomes. In this work, we present the design, the development and investigation of liposomal systems to deliver antimicrobials into Salmonella-infected macrophages. In order to investigate and identify the key structural parameters for efficient delivery of glycotargeted liposomes to selected cell targets (MR+ macrophages), two sets of monovalent glycoligands and two sets of multivalent polyglycosides - synthetic lipid-terminated glycopolymers - bearing a range of membrane-inserting anchors were synthesised. These synthesised membrane-inserting glycoligands have been used to formulate glycosylated liposomes with different glycosylation patterns and lipid composition through the Bangham method. Concanavalin A – a carbohydrate-binding protein – has been initially utilised as model protein target to study the surface presentation of the carbohydrate ligands. Firstly, the effect of lipid composition on the rate of liposomal clustering mediated by Concanavalin A (Con A) model lectin has been established. Our results showed that the binding properties of glycoliposomes are affected by the nature of both lipid constituents and carbohydrate ligands. Next, the uptake of glycosylated liposomes was investigated in salmonella infected macrophage-like cells. This in vitro infection model was used to evaluate the effect of different glycosylation patterns on the liposomal surface on mannose receptor (MR, CD206) targeting efficacy. Liposomes coated with mannose-containing glycopolymer significantly enhanced uptake compared to uncoated liposome control, and showed higher gentamicin delivery, resulting in reduction in internal infection.

Vitamins are micronutrients essential for life in an organism which must be obtained from diet or environment. Standard media often contains ten B-vitamins, which act as cofactors or precursors for co-enzymes. Clostridium autoethanogenum and Clostridium ljungdahlii are two obligately anaerobic acetogens which can utilize syngas (carbon dioxide, hydrogen and carbon monoxide) as a carbon source, generated from industrial waste gases or gasification of hydrocarbons. Clostridium autoethanogenum and Clostridium ljungdahlii use the Wood-Ljungdahl pathway to generate Acetyl-CoA from syngas. Acetyl-coA can be used by the bacterium for growth, production of energy and acetate, ethanol and 2,3-butanediol production. To increase the industrial attractiveness of producing chemicals via this route, the process must be as economical as possible and one way to increase the economic viability is to only add essential media components. This study attempted to define the exact B-Vitamin requirements of the two species, to show that the two species require only three “vitamins” – biotin, pantothenate and thiamine. Strains were created which have missing biotin and pantothenate pathway genes added in order to confer prototrophy for the vitamins and to determine the effects the addition of these genes had on viability, growth profile and product profile. A Continuous Stirred Tank reactor experiment was conducted in order to determine the effects of pantothenate, and therefore, Acetyl-CoA limitation on a continuously growing culture which mimicked an industrial reactor. Increased efficiency of the media could lead to a more economically attractive process for the sustainable production of the platform chemical than from fossil fuels leading us one step closer to decoupling our civilisation from oil.

Approximately 400 tonnes of antibiotics (including synthetic antibiotics) are used every year in treating infections in farm animals, and as prophylactics against infection. Antimicrobial resistance is a crucial problem that is now of great concern in public health, with food and food producing animals as a potential route for spread of these resistances, especially resistance to cephalosporins, which is increasing. The main aim of this study was to determine the prevalence and range of multidrug resistance (MDR) and extended spectrum β-lactamase (ESBL) or ampicillin C (AmpC) β-lactamase producing Escherichia coli within a commercial dairy farm, to understand the diversity of resistance to β-lactam antibiotics, and to determine if co-carriage of other antimicrobial resistance (AMR) was associated with ESBL/AmpC producers. This would allow a better understanding of the contributions that farms and farm slurry may make to the presence of AMR in the environment, and the reservoir of resistance in agriculture. In this study, E. coli strains were isolated from a single dairy farm (East Midlands, England, United Kingdom) on two visits, a preliminary isolation using TBX agar in 2012 and more targeted isolation using antibiotic supplemented TBX media in 2014. Confirmed E. coli (126 out of 155 selected strains) were genotyped using ERIC-PCR and analysis of the ERIC profiles showed that, in comparison to the 2014 isolates, the 2012 isolates were a quite distinct genetic population. Antimicrobial sensitivity tests were performed using a disk diffusion test for all the strains against 17 antimicrobials representing seven different antimicrobial groups: β-lactams, aminoglycosides, tetracyclines, sulphonamides, chloramphenicols, nitrofuran derivatives and quinolones. Antimicrobial resistance profiling showed 92% of isolates showed resistance to at least 1 antimicrobial, of which 27.8% of the isolates were isolated without antibiotic selection, and 57.9% of the isolates were multidrug resistant to between 3 and 15 antimicrobials, of which 43.6% of the isolates were isolated using antibiotic supplemented media. Two strains showed resistance to imipenem which appeared to be an unstable phenotype and was subsequently lost. The finding was unexpected and of concern as imipenem is not used in veterinary medicine. blaCTX-M, blaTEM and blaOXA genes were detected by PCR among the cephalosporin resistant strains. No plasmid ampC genes were detected. Four strains were fully sequenced and the genetic/genomic environment surrounding β-lactamase genes and analysis of some other AMR genes showed these genes are associated with transposable elements, such as ISEcp1, ISCR2, IS26-IS26, Tn2, Tn10 or within a class I integron carried by a Tn-21 like transposon. The association of AMR genes with these transposable elements might make the dissemination rate of these genes greater. Some of the insertion sequence-AMR gene combinations are thought to be novel, such as the unique insertion of ISEcp1- blaCTX_M14 unit into the fdeC chromosomal gene. This is the first study of this type performed on this dairy farm; the data showed a diverse range of resistance genes present in the E. coli population in the farm, including resistance to historically used antimicrobials as well as cephalosporins in contemporary use, and a high level of multidrug resistance. The spread of such highly resistant strains to the environment and possibly to humans could present a real threat to human health especially if they are pathogenic.

The standard preparation of valuable chemical compounds often exploits reactions that are plagued by suboptimal yields and a considerable amount of toxic waste, especially when it comes to asymmetric synthesis and metal catalysts. In the quest to achieve cleaner processes, enzymes are compelling alternatives; these natural devices offer "green" and efficient reactions. Furthermore, they are generally very enantioselective yielding optically pure products. Enzymes however have also many drawbacks: they especially suffer when forced to work under non-physiological conditions. A solution could be to exploit extremophile organisms, which are naturally adapted to work in “extreme” environments, as sources of new biocatalysts. Among the various classes of enzymes, aminotransferases (EC 2.6.1) are particularly interesting, since these biocatalysts are able to catalyze the amino transfer between two molecules, a reaction particularly difficult to perform with traditional synthetic approaches. An aminotransferase from Halomonas elongata (HEWT), a halotolerant organism, was isolated and characterized to verify its ability to work in the presence of organic solvents. Despite a broad substrate scope, HEWT was poorly active against ketones under standard condition. The investigation of the stereoelectronic effects of the reactions through a series of rationally engineered variants, led to a better understanding of the residues in the active pocket but produced only minimal improvement in activity. HEWT was therfore evolved towards the desired substrates applying iterative cycles of mutagenesis and screening to select the ameliorated protein. The development of an efficient high-throughput screening allowed isolation novel mutants with enhanced activity towards substituted acetophenones and small aliphatic ketones, with up to 60-fold improvement in the enzyme reactivity against para-CN-acetophenone. Furthermore, HEWT stability and tolerance were improved through protein immobilization, which showed excellent results especially in the presence of solvents. The methodology adopted was further developed by installing a protein spacer to preserve the integrity of the enzyme and prevent the significant loss of activity upon non-specific covalent linking. Immobilised HEWT was then applied in continuous flow biotransformations for the efficient production and isolation of amines. This cutting-edge approach showed about one order of magnitude higher synthetic efficiency with respect to batch processes and opened new avenues for the efficient application of enzymes in biotechnology.

Enzymes are promising stimuli for the development of responsive biomaterials for biomedical applications. Enzymes are inherently present in the biological environment thus cleverly designed materials for biomedical applications may require no external stimuli to ellicit the required material response. They have been targeted as stimuli in self assembly of bulk materials owing to the material changes in chemical composition afforded by the enzyme interaction. The first examples of autonomous self-regulated drug delivery systems have been reported via the development of reversible enzyme responsive materials that undergo a material change regulated by the enzymes in their environment. Although enzyme responsive surfaces have been reported there are no examples of reversible enzyme response surfaces. The surface is the first point of contact between the biological environment and a biomedical device/implant. Improving this interaction will improve the integration of these biomaterials in biological systems and it has been proposed that biomimetic surfaces are a promising method for full biomaterial integration in the biological environment. The body strives towards homeostasis and this is frequently achieved by enzymatic activating and deactivation of proteins in the body. This process is repeatable and reversible. Herein we address the absence of reversible and repeatable synthetic enzyme responsive surfaces towards the improvement of biomaterial integration. We aim to develop a truly autonomous system wherein enzymes present in the environment can interact with the modified surface to mediate a reversible material response. This goal was achieved by modifying surfaces with copolymers that contain the recognition sequence for Casein kinase II and Alkaline phosphatase to undergo enzymatic phosphorylation and dephosphorylation. Co and homo polymers of serine and glutamic acid were synthesised in solution and conformation/composition relationship was determined by analysis with NMR, GPC and FTIR. Polymerisation from the surface with NCA-Glu and NCA-Ser was achieved as characterised by FTIR, ToF SIMS, XPS and WCA. Enzymatic mediated phosphorylation (CKII) and dephosphorylation (AP) was monitored by surface analysis (ToF SIMS), by monitoring ATP to ADP conversion and phosphate cleavage from the surface using luminescence and colorimetric assays. Conformational changes mediated by enzymatic interactions with the surface was monitored indirectly using a FRET system incorporated in the surface modification. The modified surfaces were able to support cell culture and osteogenesis. This project has made advances in several fields, 1) The use of NCA-ROP as a method to modify surfaces with copolymers, in particular for a random/ alternating amino acid sequence. 2) The use of NCA-ROP as a method to develop stimuli responsive surfaces, specifically, this is the first report of an enzyme responsive surface prepared from NCA-amino acid derivatives. 3) The use of enzymes as stimuli, specifically, this is the first report of a reversible enzymatic responsive surface. In this system reversible phosphorlyation and dephosphorylation was monitored via changes in fluorescence output indicative of induced conformational changes.

The archaeon Haloferax volcanii is used to study DNA replication and repair, and it is unique amongst cellular organisms as it is able to grow in the absence of DNA replication origins. There are four DNA replication origins on the main circular chromosome (including the integrated mega-plasmid pHV4) and one on each of the other mega-plasmids pHV1 and pHV3. Replication origins are normally required for the initiation of DNA replication, however H. volcanii is able to grow faster when all chromosomal origins have been deleted. Therefore, H. volcanii must utilise other methods of DNA replication such as recombination-dependent replication. The origin found on pHV3 cannot be deleted from the episomal mega-plasmid, whereas the origin can be deleted from episomal pHV4. The pHV3 mega- plasmid can be integrated onto the main chromosome, which allows the pHV3 origin to be deleted from the chromosome. The pHV1 mega-plasmid origin can be deleted from the episomal mega-plasmid, and the entire mega-plasmid can be lost from the H. volcanii cell. This generates a viable, healthy strain, which shows that the pHV1 mega-plasmid is non- essential. It was also found that the pHV1 mega-plasmid exists in H. volcanii as a 6x concatemer which is ~510 kb in size, which may explain the reason for being able to delete the origin. To further investigate the mechanisms that recombination-dependent replication may use, replication machinery (MCM and GINS) were tagged and expressed. Due to time constraints, interactions were not seen. The mcm gene was put under the control of a tryptophan inducible promoter. A strain lacking chromosomal origins and therefore primarily using recombination-dependent replication was shown to require more MCM than a wild-type strain.

Phosphotransferase systems (PTS) represent an important method of sugar uptake in bacteria and have been well described in the past. However, research into PTS within the genus Clostridium has been mainly restricted to the non-pathogens. Analysis of the genome of Clostridium difficile 630 revealed over 40 intact PTS; this is over three times as many as in other pathogenic Clostridia, such as Clostridium perfringens and Clostridium botulinum. Previously, carbon catabolite repression has been shown to affect toxin production in C. difficile. Being capable of utilising different carbohydrates efficiently could be important for C. difficile to adapt to, grow, and survive in the human gut. So far, little work has been done to corroborate the role of individual PTS in carbohydrate uptake, sensing of environmental stimuli and regulation of virulence, i.e. toxin expression. A deeper understanding of the PTS in C. difficile, and their importance in virulence, could lead to the development of new drug targets. The aim of this study is to characterise the main PTS of C. difficile, determine their role in carbohydrate uptake, and their effect on regulation of virulence. To date, we have chosen the main candidates thought to be involved in mannitol and sorbitol uptake, and have inactivated these PTS using the ClosTron and in-frame deletion methods. Phenotypic characterisation of these mutants was undertaken to prove their role in uptake of the relevant sugar and to determine their role in virulence regulation. This study has demonstrated, by growth assays and HPLC, that the operons at CD630_0762-8 and CD630_2331-4 respectively encode PTS specific for sorbitol and mannitol uptake. In the case of the mannitol operon, it has been proved (through the use of cytotoxicity assays, which showed reduced bacterial toxicity in the presence of the sugar) that the suppression of toxin synthesis in the presence of mannitol is dependent upon uptake of the substrate via this operon. With sorbitol, toxin levels are, seemingly, not directly reliant upon uptake of the sugar, resulting in, mainly, an increase of toxin in sorbitol. Presently, it is not possible to say whether these systems have a distinct role or not in the motility of the organism.

Ubiquitination is a post-translational modification of protein by ubiquitin (Ub) and plays a vital role in the regulation of a number of cellular functions, including protein degradation via the ubiquitin proteasome system (UPS). These functions require recognition of specific ubiquitinated substrates by ubiquitin binding proteins (also known as ubiquitin receptors), which possess ubiquitin binding domains (UBDs) that interact directly with monoubiquitin and/or polyubiquitin chains. There are at least 16 different UBDs to date of which the ubiquitin associated domain (UBA) is an example. Unanchored polyubiquitin chains are a relatively new phenomenon. Studies suggest that these may be involved in the regulation of innate immunity, stress response and aggresome formation and disassembly. The level of unanchored polyubiquitin chains may be controlled by the action of specific ubiquitin ligases that synthesise them, e.g. E2-25K, or deubiquitinating enzymes that release unanchored polyubiquitin chains from polyubiquitinated substrate proteins, e.g. Isopeptidase T (IsoT). Major neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases are characterized by selective neurodegeneration and the formation of protein inclusions containing misfolded and aberrant proteins, and ubiquitin. UPS impairment has been implicated in the cause or progression of neurodegenerative disease. This thesis investigates the interaction of five different UBDs to synthetic ubiquitin; UBA of p62 and ubiqulin1 (UQ1), coupling of ubiquitin conjugation to endoplasmic reticulum degradation (CUE) of Vsp9, ubiquitin binding to ABIN and NEMO (UBAN) of NEMO and zinc finger domain (ZnFUBP) of IsoT. These studies are followed by investigating a mouse model of neurodegeneration caused by conditional genetic 26S proteasomal deletion in mouse forebrain neurons that shows accumulation of ubiquitin. I show in this thesis that the UBDs have different ubiquitin-binding properties. The p62 UBA and Vps9 CUE domains have similar binding affinity to ubiquitin; binding Lys48- and Lys63-linked, and linear polyubiquitin chains, but not to monoubiquitin. The UBAN domain of NEMO binds only to linear polyubiquitin chains. The IsoT ZnFUBP domain, which interacts with the C-terminus of proximal ubiquitin, only binds to unanchored/free ubiquitin. UQ1 did not show differential binding and bound to all species of ubiquitin investigated. Given the limited studies investigating linear and free chains in vivo, I used the UBAN domain of NEMO and the ZnFUBP domain of IsoT to investigate the abundance of linear and unanchored polyubiquitin chains respectively in the mouse brain cortex of control and 26S proteasome-deleted mice. Although I did not detect the presence of linear polyubiquitin in my studies, I demonstrate accumulation of unanchored polyubiquitin chains in the cortex and cortical mitochondria of 26S proteasome-depleted mice. I suggest that the accumulation of unanchored polyubiquitin chains in this mouse model may be due to increased de novo synthesis, disassembly of polyubiquitin chains from polyubiquitinated proteins by the action of deubiquitinating enzymes or inhibition of their degradation by the 26S proteasome. Further investigations of IsoT/USP5 and E2-25 levels did not show any significant differences that may explain the accumulation of unanchored polyubiquitin chains following 26S proteasome impairment. However, I show significantly decreased levels of p-TAK1 in the 26S proteasome-depleted mice compared to controls that will be further investigated in the future.

Age-Related Macular Degeneration (ARMD) is the most common cause of legal blindness in the elderly in the western world. One of the earliest signs of aging is the accumulation of lipid rich debris within and underneath the Retinal Pigmented Epithelium (RPE) cells, known as “drusen”. The disease is poorly understood - mainly because it occurs late in life as well as the lack of appropriate cell and animal models. RPE lipo-toxicity (the increased content of lipids within the RPE cell) is suggested to be a major factor affecting both the molecular mechanisms and the metabolic responses of the RPE cells leading to changes associated with drusen formation and ARMD pathology. To investigate this phenomenon, aged ARPE-19 cultures were induced to long term lipid loading with a free fatty acids (FFAs) mixture to ensures the increase of the intra-cellular lipid level within the cells. The accumulation of lipids was found to correlate with a destruction of the ARPE-19 monolayer integrity, an increase in VEGF-A secretion in media and most importantly the production of sub-RPE deposits positive for apolipoprotein E, vitronectin and Amyloid beta 1-42, all of which are prominent constituents of drusen, supporting the hypothesis of lipo-toxicity. To further investigate the effect of inflammation in ARMD, we introduced the aged ARPE-19 cell cultures to long term complement activation in the presence and absence of lipid loading. Complement activation showed protective response suggesting that the complement system plays a secondary modulating response role to a primary destructive initiator “lipid loading”. These findings suggest the use of aged ARPE-19 cell culture as a promising model for ocular aged related diseases study including drusen deposition mechanisms, while the use of ARPE-19 lipo-toxicity model will facilitate the analysis of molecular and cellular characteristics of ARMD pathogenesis, augmenting the therapeutic strategies for dry ARMD.