Limited fossil fuel resources and the environmental impacts of climate change are motivating the development of sustainable processes for the production of fuels and chemicals from renewable resources. The development of alternative energy sources, such as biofuels, will strengthen energy security and reduce dependence on fossil fuels. n-butanol is a biofuel and platform chemical. n-butanol is an advanced fuel with high energy content, compatible with existing infrastructure. Here the ability of microorganisms to use renewable resources for biofuel synthesis is exploited. In this work use of the thermophilic bacterium Geobacillus thermoglucosidasius is explored for the production of n-butanol. Geobacillus is considered a promising industrial process organism due to its high optimum growth temperature and ability to assimilate various substrates including both hexose and pentose sugars. As a relatively novel process organism, first the development of molecular tools was required to enable subsequent engineering of the host metabolism. Here four reporter assays were developed, three of which can be used simultaneously, providing for extensive analysis of qualitative and quantitative gene expression within the cell. A range of promoters and RBS’ were screened. Extension of the Geobacillus vector series, pMTL60000, with new component parts for each module enabled co-transformation of two plasmids into G. thermoglucosidasius. The molecular tools developed were then applied in Geobacillus metabolic engineering work, with the aim of producing the target molecule n-butanol. Initially a CoA dependent n-butanol pathway, based on naturally occurring production by ABE fermentation, was considered. Following introduction of the pathway further metabolic engineering was employed to improve pathway flux, creating a driving force through the pathway and increasing the substrate pool. Production of 0.137 mM (10.166 mg/l) n-butanol demonstrated proof of concept. Next, the use of genes native to Geobacillus were investigated for improved enzyme compatibility. This approach did not generate n-butanol here. Finally a CoA independent pathway utilising the host’s native fatty acid biosynthesis pathway was considered. Using this approach, butyric acid was produced. Butyric acid can subsequently be further converted to n-butanol however this was not demonstrated here. In addition to metabolite pathway introduction, host strain engineering was carried out with the aim of adaptation towards industrially desired properties. Directed evolution resulted in selection of a strain with an increased n-butanol tolerance of 2.5% (v/v). Such modifications resulted in an improved process organism for biotechnological application. This work provides the first reported production of n-butanol in thermophilic and aerobic conditions. Multiple approaches to n-butanol production are evaluated here. Use of heterologous and native genes are considered. Both CoA and ACP dependent pathways were introduced. Each approach presented advantages and drawbacks. A system compatible for use in Geobacillus has demonstrated proof of concept n-butanol production. Further development is required to increase production to industrially feasible quantities.

Clostridium difficile is an anaerobic, Gram-positive, endospore-forming, pathogenic bacterium which is the leading cause of antibiotic-associated diarrhoea, and causes a significant burden to healthcare facilities and communities, worldwide. Bacterial endospores are one of the most resilient forms of life, able to withstand exposures to wet-heat, desiccation, UV radiation, oxygen, and some disinfectants, which would otherwise kill the vegetative cell form. Thus, endospores of C. difficile are able to persist in the environment and contaminate surfaces within healthcare settings. Once ingested, these spores pass into the anaerobic lower intestines and in susceptible individuals find favourable conditions in which to germinate, generating the toxin-producing vegetative cells responsible for C. difficile associated disease. Consequently, spores are the infectious agent of this disease and both sporulation and germination processes are essential for disease. Whilst these processes have been well studied in Bacillus subtilis, it is only recently, with the development of appropriate reverse genetics tools for clostridia, that the mechanisms of sporulation and germination have begun to be described for C. difficile. This study uses the currently available mutagenesis tools of ClosTron and allelic exchange to generate mutant spores lacking spore-specific proteins, and through a range of assays characterises the sporulation, germination and resistance properties of these mutants, to understand the roles of these proteins in C. difficile endospores. Furthermore, these genetics tools are established in a novel C. difficile strain with beneficial properties for studying the processes of sporulation and germination in vitro. Finally, this study establishes CRISPR/Cas9 genome editing in C. difficile for the first time to overcome the major pitfalls associated with the previously available reverse genetics tools. This mutagenesis method was found to offer fast, highly efficient genome editing of two different C. difficile strains and will be the method of choice for future studies in C. difficile.

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.

Cloud Computing (CC), as defned by national Institute of Standards and Technology (NIST), is a new technology model for enabling convenient, on-demand network access to a shared pool of configurable computing resources such as networks, servers, storage, applications, and services that can be rapidly provisioned and released with minimal management effort or service-provider interaction. CC is a fast growing field; yet, there are major concerns regarding the detection of security threats, which in turn have urged experts to explore solutions to improve its security performance through conventional approaches, such as, Intrusion Detection System (IDS). In the literature, there are two most successful current IDS tools that are used worldwide: Snort and Suricata; however, these tools are not flexible to the uncertainty of intrusions. The aim of this study is to explore novel approaches to uplift the CC security performance using Type-1 fuzzy logic (T1FL) technique with IDS when compared to IDS alone. All experiments in this thesis were performed within a virtual cloud that was built within an experimental environment. By combining fuzzy logic technique (FL System) with IDSs, namely SnortIDS and SuricataIDS, SnortIDS and SuricataIDS for detection systems were used twice (with and without FL) to create four detection systems (FL-SnortIDS, FL-SuricataIDS, SnortIDS, and SuricataIDS) using Intrusion Detection Evaluation Dataset (namely ISCX). ISCX comprised two types of traffic (normal and threats); the latter was classified into four classes including Denial of Service, User-to-Root, Root-to-Local, and Probing. Sensitivity, specificity, accuracy, false alarms and detection rate were compared among the four detection systems. Then, Fuzzy Intrusion Detection System model was designed (namely FIDSCC) in CC based on the results of the aforementioned four detection systems. The FIDSCC model comprised of two individual systems pre-and-post threat detecting systems (pre-TDS and post-TDS). The pre-TDS was designed based on the number of threats in the aforementioned classes to assess the detection rate (DR). Based on the output of this DR and false positives of the four detection systems, the post-TDS was designed in order to assess CC security performance. To assure the validity of the results, classifier algorithms (CAs) were introduced to each of the four detection systems and four threat classes for further comparison. The classifier algorithms were OneR, Naive Bayes, Decision Tree (DT), and K-nearest neighbour. The comparison was made based on specific measures including accuracy, incorrect classified instances, mean absolute error, false positive rate, precision, recall, and ROC area. The empirical results showed that FL-SnortIDS was superior to FL-SuricataIDS, SnortIDS, and SuricataIDS in terms of sensitivity. However, insignificant difference was found in specificity, false alarms and accuracy among the four detection systems. Furthermore, among the four CAs, the combination of FL-SnortIDS and DT was shown to be the best detection method. The results of these studies showed that FIDSCC model can provide a better alternative to detecting threats and reducing the false positive rates more than the other conventional approaches.

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.

We encounter uncountable ephemeral phenomena in everyday life. Some of them are particularly appreciated for their ungraspable beauty and limited availability. From the outset, one strand of computing technology has evolved to encapsulate and preserve this transient experience. A myriad of digital devices has been developed to capture the fleeting moments and to store as digital files for later use, edit, share, and distribute. On the other hand, a portion of Human-Computer Interaction (HCI) research has engaged in adopting the transience of temporal phenomena in the design of interactive computing systems. Some computer and mobile applications metaphorically adopt the ephemerality in graphical elements or functions that resemble our real world experiences such as, forgetting and real-time conversation that naturally fades away immediately. Interactive artefacts or installations often incorporate ephemeral materials for abstract and artistic expression. Therefore, ephemeral artefacts or phenomena are often employed as a passive design element in ambient and peripheral interactions rather than in applications for practical purpose. However, ephemeral materials also engender experiences of a non-ambient nature. Some materials are physically fragile, only lasting for a brief moment, and therefore require constant care to retain their status, which might lead to highly focused attention, delicate interaction, and even a tense experience. This thesis aims to investigate how to harness the fleeting and irreversible feature of ephemeral artefacts in the design of practical products and services. This PhD builds on the methods of design-oriented HCI research. Thus, this thesis will present a research process that involves a series of challenges to initially frame a design problem in a fertile area for exploration; speculate a preferred situation; develop proof-of-concept prototypes to demonstrate the potential solution; and evaluate the prototypes through a user study. Contributions of this PhD have visualised by the outputs from multiple design studies. First, this thesis illustrates how the concept of ephemerality is currently understood in HCI. Then proposes a different approach to the use of ephemeral materials by shifting the focus to delicacy. The first design study introduces FugaciousFilm, a soap film based interactive touch display that shifted ephemerality from a user’s periphery to the focal point of interaction. The prototype is a platform for manifesting ephemeral interactions by inducing subtly delicate experiences. By demonstrating that ephemeral interactions reinforce user’s attention, delicacy was noticed as an attribute of user experience. By understanding of the use of delicacy, the research focus has moved from exploring how an individual ephemeral material can be utilised in interaction design, to harnessing delicacy of such materials in experience design that benefits Human-Computer Interaction. Thus, this thesis recaptures digital gift wrapping as a context by reviewing the current state of affairs in digital gifting in the field of HCI and design. A 5-stage gifting framework has been synthesised from the literature review and guided this PhD throughout the studies. The framework ought to be seen as a significant contribution in its own right. Based on this framework, a series of interviews was conducted to identify any weaknesses that reside in current media platforms, digital devices, and different modes of interaction. Hence, ‘unwrapping a digital gift’ has captured as a gap in the design space that could be reinforced by a delicate, ephemeral interaction. Therefore, this PhD proposes Hybrid Gift, a series of proof-of-concept prototypes that demonstrates digital gift wrappings. Hybrid Gift has been probed in a semi-structured design workshop to examine the use of delicacy and ephemerality in the design of digital gifting practices. The prototypes were designed to retrieve not only the unwrapping experience but also rituals around gift exchange. Therefore, this thesis discusses design implications of the findings that emerged throughout the study. Digital gifting is still an under-explored research area that is worthwhile to investigate through field works. Thus, the design implications and the framework are proposed to researchers and designers who wish to engage in the arena of digital gifting, also broadly in social user experience, and communication service and system design. From a macroscopic perspective, we are experiencing fleeting moments every second, minute, and day. However, they are rarely noticed unless we recognise that time passes irreversibly. This thesis extracted delicacy as a feature of ephemeral interactions and argued that it holds the potential to augment and enhance mundane experiences mediated by digital technology. In so doing, the series of design studies has conceptually influenced the design perspective to be shifted from material-oriented design to experience-focused design research. The design space of digital gifting would not have been recognised without the hands-on design practices in the process of this PhD. Finally, the proof-of-concept prototypes, framework, and design implications are thought to be of significance and value to the design students, researchers, and designers who want to employ similar methods and approaches in design research.

Our face is a key modality to convey emotions and infer intention. This makes face analysis an important factor in understanding the underlying mechanisms of interaction. Automatic solutions for facial expression recognition promise to deliver a significant fraction of the currently missing component of non-verbal communication to the human-machine interaction enabling more fulfilling experience closely modelling interpersonal communication. This thesis presents three major contributions aimed to overcome a number of issues currently preventing modern face analysis solutions from being applied in practice. The problem of reliable automatic discovery of facial actions is first considered from the point of view of manual feature craft, exploring ways to highlight features related to interpersonal commonalities in facial expression appearance, disregarding those corresponding to environmental conditions and subjective differences. It is then approached from the Multi-Task and Transfer learning perspective, presenting solutions for cost and performance efficient training of facial expression detection algorithms. Finally, a novel solution is proposed for multi-database heterogeneous data representation aimed to provide an environment for better generalisable face analysis solutions training and evaluation.

Wheat (Triticum spp.) is a particularly important crop for food security, providing 20% of worldwide calorie intake. Food production is not meeting the projected global demand of an increase of 2.4% p.a. Improvement of resource capture in wheat could help meet this demand. Nitrogen (N) is an essential macronutrient for plant growth and development; however, nitrogen use efficiency (NUE) for cereal production is only 33%. Domestication of modern varieties of wheat may have lost potentially beneficial agronomic traits, particularly in the root system. Optimisation of root system architecture could profoundly improve nitrogen uptake efficiency (NUpE) and in turn increase the yield potential of the crop. Using ancestral wheat germplasm and mapping populations, desirable traits may be identified and bred back into commercial wheat varieties to increase yield potential. Using a high-throughput hydroponic root phenotyping system, N-dependent root traits have been identified in wheat mapping populations. Using transcriptomic analyses, the gene expression profile of a candidate N-dependent root QTL has been identified. Using a new root phenotyping system, X-ray micro-computed tomography (μCT), a three-dimensional representation of wheat roots can now be imaged in soil. A selection of the same mapping lines have been used for 3D μCT analysis based on field NUpE parameters to identify promising root traits in both seedlings and mature plants.

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.

Johne’s disease (JD) is a severe wasting disease of ruminants, characterised by chronic enteritis, reduction in milk yield, and severe weight loss despite a maintained appetite. The causative agent is Mycobacterium avium subspecies paratuberculosis (MAP), a slow growing pathogen that can take up to 18 weeks for detection on solid culture. Control programmes rely on sensitive diagnostics to identify infected animals quickly so they can be either removed from the herd or managed differently to control the spread of disease. Unfortunately, the Gold Standard of detection is culture, which due to decontamination procedures, has low sensitivity. Enzyme-linked immunosorbent assays (ELISA) are used more often than faecal culture within control programmes as they are cheaper and quicker than culture methods. However, they only detect the animal’s immune response, rather than the causative agent. This can cause some issues with diagnosis as the immune response can be affected by other variables. Therefore, to effectively control disease, a new detection method needs to be developed. In this series of studies, phage-PCR was used within large scale on-farm sampling to establish its performance against the Gold Standard (liquid culture with ESP-trek) and MAP specific antibody milk ELISA (ab-ELISA). Phage-PCR is thought to be more sensitive than other methods due to its low limit of detection. It is also rapid and relatively inexpensive. Results suggest that phage-PCR can detect more animals shedding MAP into their milk than other methods, or in the least a different group of animals than the other methods. There was some evidence that animals who have had an ab-ELISA positive result in the last year are shedding less MAP into their milk, suggesting that the immune response is helping to control the disease in the short-term. However, this was not observed beyond one year. Phage-PCR had a better agreement with faecal culture than milk culture or ab-ELISA, but this was limited. There was also evidence that early detection could be achieved, as some animals were identified as faecal shedding with phage-PCR before they had seroconversion and detected with a-ELISA. However, it must be noted that these animals may not be infected and just passaging MAP through the GI tract from the contaminated environment. An investigation into the prevalence of MAP in pasteurised milk using phage-PCR was also carried out. There is thought to be an association between MAP and Crohn’s Disease, with milk highlighted by some as a key transmission vector. There was an increase in the proportion of samples containing viable MAP when compared to other surveys within the literature. However, this was thought to be due to the lower limit of detection that phage-PCR provides, rather than an increase in prevalence. Phage-PCR can be used effectively for large-scale on-farm sampling to identify animals shedding MAP into the milk. However, some changes to the assay and sample processing will have to be undertaken before this can be used within industry as its current format is laborious and not suited to automation. Until then, it could be used as a tool to further research and understanding into JD in dairy cattle.