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This thesis is located between. It is not quite about outdoor environmental education. It is not quite about research methodology. And it is not quite about the author's learning. These delineated categories exist on a different plane to the thesis, at least, to what the thesis becomes - is becoming. The thesis is thus a haecceity, a certain thisness that is no other. This is not meant to be a grand claim to the contribution or originality of the thesis. It is a statement about the nature of being that 'I' have been moved towards in exploring what practice might be in a world that is 'post' environment and 'post' methodology, and where the separation of theory and practice dissolves. The thesis is constituted of eight haecceities. The starting place is my concern, as (becoming) an outdoor education academic, for finding a pedagogy that might help mitigate environmental degradation. The search for this pedagogy takes up new materialist inclinations and particularly the concept of immanence as described by Deleuze and Guattari (2004). This in turn changes the thesis, and leads it to an exploration of various post-qualitative informed 'fuzzinesses' of research methodology; where methodology becomes a pedagogy and the notion of an educational practice that is separate from my life, a life, is troubled. From here, the thesis takes up an increasingly 'post' autoethnographic lilt to explore becoming a post environmental education academic. This exploration is carried by writing, and collaborative writing, as forms of inquiry by which various stories are told; stories in which the boundaries between environments, theory, practice, learning, and research become unclear. Throughout the thesis various concepts are created to help explore these tensions. These include the concept of the haecceitical self as the occurring process of, rather than a self being connected or in relation to something; the concept of becoming alien, as an attempt at unhumaning ourselves. i.e. to raise awareness of our belonging to something else, rather than the stable and quiddital (whatness) of the concept of the human; and lastly the concept of environing education. Environing education is difficult to define, but it is at the least the process of learning to live more ethically in response to the contradiction of caring about environmental degradation, whilst at the same time questioning the category of the environmental. The concepts are not monolithic and not necessarily transposable to other situations. Rather they live in their use within the thesis. Whilst the thesis originates from a place of trying to advocate for the environment, through the process of writing the 'environment' becomes troubled as a conceptual category, thus troubling the environmental as a category of moral value. Instead, towards the close of the thesis, I explore the competing lines of desire that function to produce the thesis, in the search for an immanent ethics. This ethics of affect is co-constitutive in the writing of the haecceities, to aesthetically explore post human/environment tensions of becoming an academic, and acknowledging the personal struggles that this entails. The contribution of the thesis lies in its exploration of writing an immanent ethics, given the destabilising effects of an immanent ontology on prevailing ethical orientation towards transcendent notions of the 'environment'. In this way, the writing is a form of post-ecobiography, whereby the contribution of the thesis is also its affective process, for me and (possibly) for the reader.

Hybrid materials have been found to be possessed with unique and novel properties, by superimposing the advantages of each component to provide material properties far superior to the individual constituents alone. These types of material are used throughout the field of bioengineering, and can facilitate biological interactions on both a molecular and cellular level, responding to biological and mechanical queues. With these inherent unique properties these materials provide novel platforms to help repair, replace and regenerate body tissues and function. This thesis explores the synthesis of new 'smart' hybrid hydrogels and their properties. Through the conjugation of self-assembling β-sheet peptide sequences to a naturally occurring poly (γ-glutamic acid) polymer backbone, peptide-polymer hybrid hydrogels were formed. These hybrid hydrogels were attributed with robust and tunable mechanical properties. Through the reassembly of the physical β-sheet crosslinks the hydrogels can respond to their mechanical environment, exhibiting 'self-healing' capabilities and a resistance to cyclic strain. Also, being composed entirely of natural peptide bonds they have excellent biocompatibility and are a promising platform for future tissue engineering scaffolds and biomedical applications. These hybrid hydrogels were further functionalised to detect for the activity of enzymatic biomarkers. A simple assay based on Förster resonance energy transfer was incorporated in to the hydrogel platform through the immobilisation of quantum dots modified with peptide substrates. The activity of matrix metalloproteinase-7 was targeted specifically, a marker for inflammation and immunity. This hydrogel sensing platform provides a basis for the in vivo sensing of enzymes and also, the potential to be used as a powerful tool to investigate biological processes in vitro.

This thesis examines the experience of gay male student nurses during their university course, which leads to registration as a nurse with the Nursing and Midwifery Council. Using in-depth qualitative interviews I focus on the student’s choice of nursing as a career and their performance of sexuality within the differing spaces of their clinical placements and the university. This thesis explores how these gay student nurses negotiate their gender, masculinity and gay sexuality within the professional boundaries of nursing. Furthermore, it identifies how these students negotiate issues of caring and the formation of therapeutic relationships with their patients, as men and gay men. The theoretical framing of the thesis draws upon Goffman's theories of presentation and performance of the self and Rubin’s 'charmed circle'. Alongside analysis of interview material, I explore the space of the hospital from a personal perspective and interrogate its gendered and desexualized organization through the lens of human geography. Moving between these two analytical frameworks, I examine and draw together the experiences of these students and examine their negotiation of the nursing role as gay men. I argue that the experience of these students and the negotiation of their sexuality as student nurses is fraught and precarious due to the complexities and boundaries of professional nursing roles in contemporary healthcare. Within the conclusion I address the implications of my research for gay nurses, patients, educators and for those who recruit nursing students.

Pescadero Marsh is a back-barrier wetland environment located on the central coast of California. In an attempt to reconstruct sub-annual trends in back-barrier environmental change over the late Holocene, driven by the presence and integrity of a coastal barrier system, carefully targeted sections of the sediment record from this site were analysed at high resolution (2mm) for their grain size and geochemical characteristics. Where possible, diatom analysis supplemented these data. The sediment record, and the methodology employed, proved to be sufficiently sensitive for this purpose by distinguishing between a series of barrier estuarine and lagoonal sedimentary environments. Grain size data were the primary basis for environmental interpretation. At this resolution, the grain size distribution curves of individual samples revealed sensitive environmental information regarding deposition through different transport processes, i.e. saltation vs suspension loads. Distinctions between samples were driven by the interaction of the lagoonal pool, tidal ingress, river flow and the integrity of the barrier system. Geochemical data indicated sediment provenance, distinguishing between samples primarily sourced by marine and terrestrial waters, and grain size related controls on many geochemical characteristics. Diatom assemblages reflected high energy inputs from the ocean and the feeding creeks whilst recording limited change in the salinity of the back-barrier environment. A confident chronology was constructed for Euro-American era sediments, with a sedimentation rate of 144.2 days per 2mm sample established above a 1963 'weapons testing' peak in 137Cs. A similar rate was demonstrated to continue to at least 1900 through the presence of geochemical markers of anthropogenic activity (pollution, catchment destabilisation and development of infrastructure). This represented a marked increase above the prehistoric rate (~0.8mm per year). During the late-Holocene period of relative sea-level stability, the environmental trends established are characteristic of a morphologically resilient coastal system. A barrier estuarine environment persisted, despite changing boundary conditions and a perturbation of very high magnitude/Iow frequency, and was observed to shift though a series of 'static equilibrium' operational states. From wavelet analysis, the ENSO phenomenon emerged as the likely principal driver of variability in the most recent of these static equilibra. The research presented greatly improves understanding of the sites prehistoric functioning and suggests significant anthropogenic impacts on the system during the Euro-American era. Such information can inform management of this and other analogous sites.

The development of collaborative interdisciplinary working is a key element of contemporary health policy. Future healthcare workers will need to work. individually, collaboratively and in teams if they are to meet the complex and changing needs of the patients they serve. The literature related to health professional team working identifies many barriers and sources of potential conflict, but there is also evidence that effective interdisciplinary teamwork can be achieved and is associated with improved health outcomes. The specialised and co-ordinated multidisciplinary team care provided in stroke units was considered to contribute directly to the improved patient outcomes seen in these units. However, the ways in which stroke unit team members co-ordinate their work was not clearly understood. This study utilised a grounded theory approach to develop an explanation of the ways in which health professionals in two stroke rehabilitation units in the North of England achieved teamwork. Data were generated through over 200 hours of participant observation and thirty four semistructured interviews with a range of team members. The findings of the study identified a basic social process which was common to team working in both stroke units; this process was termed 'opportunistic dialogue'. This represented an interactional process through which the division of labour in respect of specific rehabilitation activities was worked out and agreed by team members on a day-to-day basis. Co-location of most team members in both units led to repeated engagement in sharing and validating patient information and in exploring different perspectives. Opportunistic dialoguing contributed to mutual learning in the stroke unit teams and explained the shift in thinking and team culture which occurred as team members moved from concern with discrete disciplinary actions to dialogue and negotiations focused on collaboration to meet the needs of stroke patients. Negotiations played a major role in opportunistic dialogue and coming to agreement on the teams' rehabilitation work. The study findings emphasised the interrelatedness and interdependence of these concepts as core interactional processes contributing to the achievement of teamwork in stroke units. The study confirmed the utility of the negotiated order perspective in understanding and explaining workplace interactions, but identified that whilst negotiations were a key feature of opportunistic dialogue, other processes also contributed to achieving and maintaining teamwork. Focussing on dialogue demonstrated that patterned talk-in-interaction processes maximised the contribution of opportunistic dialogue to coordinating the skills and knowledge of the different disciplines participating in stroke rehabilitation. The achievement of teamwork in these units occurred through access to and participation in opportunistic dialogue.

Alcohol education programmes, based on mainstream psychological research, have been developed in an attempt to reduce the level of harm but have met with limited success. An alternative theoretical and methodological approach to the phenomenon of underage drinking was therefore proposed. Foucauldian discourse analysis, a qualitative methodology grounded in critical theory, was therefore used to analyse four different sets of data. These datasets were i) young people's talk around drinking (obtained through the use of focus groups), ii) educational leaflets targeted at young people, iii) a selection of educational sessions held in the classroom and iv) young people's own talk around alcohol education (again, obtained through the use of focus groups). Young people's talk around drinking was found to comprise dominant discourse around pleasure and the enjoyment of risk, all underpinned by a fundamental need to be able to lose control. Both sets of educational data, on the other hand, comprised discourses around the essential "vulnerability" of young people, and the need for young people to maintain control through the adoption of a self-monitoring , self-regulating identity (the Foucauldian self). Young people's talk around education appeared to show they had assimilated and accepted these latter discourses. However, the relative failure of educational programmes, coupled with the strength of the 'pleasure' discourses expressed by many young people in their talk around drinking, suggests this apparent success may be illusory. Educational programmes, rather than engaging in a convert programme of control, therefore need to acknowledge to a far greater degree the fundamental role alcohol use, particularly 'binge' drinking, plays in young people's lives. They need to find a way to connect with young people's experiences if they are to have any success in preventing some of the genuine harm that may arise as a result of such drinking.

We have characterised two biologically important protein complexes. Firstly, we have captured and analysed a complex of the biotin protein ligase (BPL) and the biotinylation domain (BCCPΔ67) of acetyl-CoA carboxylase (ACC) from the hyperthermophile Aquifex aeolicus. The genes encoding both BPL and BCCPΔ67 were overexpressed in E. coli and purified to homogeneity. Isolation of milligram amounts of both recombinant proteins allowed us to perform kinetic analysis of the BPL enzyme using steady-state techniques. Furthermore, a chemically crosslinked complex of BPL and BCCPΔ67 was isolated and a comprehensive mutational study has identified a salt bridge between the two proteins which is important for heterodimerisation. The role of a conserved ‘thumb-like’ motif in BCCPΔ67 was also investigated by mutagenesis. Our results suggest an interaction between the biotin moiety and the ‘thumb’ reduces the propensity of BPL and holo-BCCPΔ67 to heterodimerise. In a separate project we have investigate the relationship between the tertiary structure and biological activity of a novel β-defensin related peptide (Defrl). Defensins are cationic antimicrobial peptides which have a characteristic six cysteine motif and are important components of the innate immune system Defr1 is a polymorphism of mouse β-defensin 8 and contains only 5 cysteines. Against a panel of pathogens, we found that oxidized synthetic Defr1 had significantly higher activity than its reduced form, and the oxidized and reduced forms of its six-cysteine containing analogue (Defrl Y5C). Using non-denaturing gel electrophoresis and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) we observed Defrl and Defrl Y5C dimers.

Evidence relating to the use and veneration of the human head in a religious context is found repeatedly in the archaeological record and folklore of the British Isles. This has been documented from the earliest period, and manifests itself throughout prehistory and recent history in a variety of forms, from human skulls used as talismans to carved stone heads produced as part of a folk tradition of long standing. Until recently, much of the literature relating to the head as a sacred symbol has been produced from the perspective of Celtic studies, the material being interpreted as evidence for the existence of a "Celtic cult of the head" with roots in the pagan Iron Age. It has been claimed that this cult left an indelible mark both upon the archaeological record and the popular consciousness of later peoples, which has survived in superstition and folklore. This study aims to examine the evidence from archaeology, documentary sources and the folk tradition from outside the confines of the Celtic viewpoint, and to discuss the relationships between the different forms of material through a series of case studies. A cross-disciplinary approach is adopted as a method of interpreting this material using approaches from the viewpoints of both folklore and archaeology. Existing sources are complemented by original fieldwork, incorporating material collected from a wide range of continuing traditions surrounding the use of carved human heads and skulls. These were used for a variety of protective and luck-bringing purposes within living memory, many of them having been overlooked by previous studies. This study demonstrates the importance of integrating evidence from both archaeological and folkloric contexts as a method of understanding and interpreting ritual and religion from the past.

The experimental work described in this thesis was conducted in the surgical research laboratories of Dr Francis D. Moore in the Peter Bent Brigham Hospital (now the Brigham & Women's Hospital) Boston, Massachusetts, USA, between 1978-1979. It formed part of an ongoing programme of research into protein metabolism in man; specifically to measure total body nitrogen turnover and hence protein synthesis and breakdown, initially in normal volunteers receiving various intravenous feeding regimens, and subsequently in patients. The previous year, 1977, had seen the publication of 'Substrate Interaction in Intravenous Feeding' by Bruce Wolfe et al., from the same laboratories. This was an extensive piece of work incorporating many studies and compared nitrogen balance data together with biochemical, hormonal and plasma amino acid data in normal men fed intravenously with a variety of regimens. Shortly afterwards a series of protein turnover studies was embarked upon, using the uN glycine method, and in collaboration with Dr Vernon Young of the Massachusetts Institute of Technology. The first experiments were essentially a repeat of the studies described by Wolfe et al. (vide supra) but in addition nitrogen turnover, protein synthesis and breakdown were estimated using a continuous 60 hour infusion of uN glycine and measuring enrichment of urinary urea with uN when a plateau was reached. Normal volunteers were studied firstly on normal oral diet and then on a iv succession of intravenous regimens:- amino acids alone (AA), amino acids plus 'high dose' glucose (AA + HOG), amino acids plus fat emulsion (AA + FE), amino acids plus 'low dose' glucose (AA + LOG), amino acids, fat emulsion and low dose glucose (AA + LDG+ FE), and finally 'low dose' glucose alone (LOG). The studies on normal diet, AA and AA+HOG were conducted by Andrew Sim (a Glasgow/Harvard exchange fellow) and Bruce Wolfe. The author took no practical role in these experiments, but was responsible for analysis of the data and the protein metabolism calculations, and was a co-author when the work was published in 1979 (Sim et al., Glucose Promotes Whole-Body Protein Synthesis from Infused Aminoacids in Fasting Man, Lancet i, 68-71). Subsequently, the author did the experiments using AA + LOG + FE, AA + FE, and AA + LOG and LOG. The results on these four regimens were incorporated in a paper presented in 1979 at the Tripartite Meeting of the Surgical Research Society at Oxford under the title 'Isotope Studies of substrate interaction in parenteral nutrition', and also at the 2nd European Congress on Parenteral and Enteral Nutrition at Newcastle upon Tyne in 1980, and later published as 'The Effect of Fat Infusion on Protein Metabolism' (Acta. Chir. Scand., Suppl. 507, 475-484, 1981). When the studies on the various intravenous feeding regimens were completed, attention was turned to the possible distorting effects of variables such as exercise and diet v on the behaviour of the isotope equilibrium curve and plateau. Such effects, if present, might have significance when studies were carried out on patients at a later stage in the research programme. Because each study lasted 48-60 hours, changes might occur either unintentionally or as a result of the needs of clinical management, and if they affected the plateau would alter the resultant calculations of turnover, synthesis and breakdown. Such a potential source of error clearly needed investigation. A pilot study was done in two subjects, later repeated on each, to observe any effects on the curve and plateau of both doubling protein intake and bicycle exercise. Subsequently, more extensive studies were done varying the protein and energy intakes, both orally and intravenously, allowing a detailed analysis of curve perturbation, and introducing the concept of basal catabolic rate. Finally, protein turnover, synthesis and breakdown were estimated seven times in four seriously ill patients. All of the studies mentioned above form the basis of the thesis. Unfortunately, the gestation period of this thesis has been long. There are two main reasons for this. Firstly, the work done was part of a five-year programme of research, with the intention of publishing a paper in a scientific journal at the completion of each stage. This was done vi with the first three regimens (normal diet, AA and AA + HDG) but not with the last four (AA + FE, AA + LOO+ FE, AA + LOG, LDG), although the results were presented at two scientific meetings. Shortly after returning to the United Kingdom the author was appointed a consultant surgeon and this career move assumed priority. Secondly, although it was intended to publish the perturbation studies, it proved impossible to reduce the size of the text to a manageable level suitable for publication in the form of a scientific paper. However, despite the long interval since the experiments were done, no similar work has been published. In particular, virtually no attention has been paid to intentional perturbation. Also, whereas there was a spate of interest in protein turnover studies in the late 1970s and early 1980s, virtually no publications have appeared since 1985. It seems that the potential applications of the method are considered exhausted, and interest has been lost, rather as it was in the 1950s following a short flurry of activity exploring the first cumbersome technique. Hence, it seemed all the more pertinent, even at this late stage, to publish the work in the form of a thesis which could describe in chronological order the continuum of studies as briefly mentioned above. In order to preserve such a progression, the following Introduction contains, with few exceptions, only references up to the time that the experimental studies were commenced, 1978, but the subsequent Discussion(s) in the various sections will attempt to include the relevant literature up to the present time.

For most archaeologists today, barrow digging represents the archetypal antiquarian activity of the nineteenth century in particular; there has been less appreciation of its place in eighteenth-century work. Yet for all this acceptance of its importance there have been few attempts to understand the work of the barrow diggers in terms of their own aims and society. Fundamentally, then, this work has been undertaken in the hope that it will take us some of the way towards redressing the balance and, to this end, the writings of the barrow diggers have been allowed wherever possible to speak for themselves. Although the prime motivation in most barrow digging was the collection of the objects accompanying the burial it should not be supposed that other, often more overtly academic, aims were thereby excluded. In these other aspirations, we can more clearly determine the relationship of the barrow diggers to the broader intellectual aspirations of their day. A clear watershed is observable in the third, fourth and fifth decades of the nineteenth century when the previously secure links with the topographical tradition several centuries old were severed in favour of a more tenuous association with the newly emerging social sciences. This is particularly reflected in barrow digging by a weakening dependence upon classical sources and consequent increase in the use of ethnographic examples to explain the phenomena observed during barrow digging. Further there was an emphasis on the possibilities of meaningful racial determinations from the human skeletal remains, itself a result of the increasing racial concerns in a society seeking to generate new approaches to alien cultures and peoples with the collapse of the attitudes rooted in the acceptance of slavery. The appeal of racial analyses diminished in the face of the growth of social evolutionary theory which led in the latter years of the nineteenth century to the emergence of typology in archaeological analysis. In general, the approaches to excavation and analysis, though varied, show little innovative intention on the part of the barrow diggers whose aspirations were derived from outside views rather than generated by the demands of the material discovered. An important element in providing these views was the county societies and museums founded in the period after 1840 when antiquarian activities had become respectable in a way not known in the preceding century. The journals and other activities of these institutions both provided a wider diffusion of general aims and aspirations and enable us to determine the increasing tempo of antiquarian studies, including barrow digging, which was altogether less individualistic than it had been in the eighteenth century.

Chapter 1 presents a review on the reactions of electrophilic ketones with palladium and platinum compounds. Further reactions of various palladium(0) platinum(0) and platinum(II) complexes with the electrophilic ketones hexafluoropropan-2-one, chloropentafluoropropan- 2-one 1,3-dichlorotetrafluoropropan-2-one, 1,1,3-trichlorotrifluoropropan- 2-one, acetonedicarboxylic acid dimethyl ester and diethyl ketomalonate are described. Chapter 2 contains the results of some investigations into the reactivity of some iridium(I) olefin complexes towards hexafluorobut- 2-yne and dimethylacetylenedicarboxylate; hexafluorobut-2-yne reacts with complexes of the type [IrCl(cod)]2 and [Ir(dithio) (cod)] to produce previously unknown olefin-acetylene complexes. Chapter 3 discusses the preparation of the fluoro-olefin complexes [IrCl(cod) (CF2=CFX)] and [Ir(dithio) (cod) (CF2=CFX)] where X=F, Cl,Br,H,CF3. Studies of their 19F n.m.r. spectra indicates the presence of two isomers in many of these fluoro-olefin complexes.

Before the coming of the Methodists to the upper Eden valley in 1758, Celts, Romans, Anglians, Vikings, Normans and Scots had all left their mark on the area. Geographically, however, this part of Cumbria is somewhat mountainous and remote and their influences were not as significant as in other parts of England. A measure of consequent isolation was thus to affect the intensity of local religion, particularly that of later Methodism. Apart from a small number of Catholics and the Church of England, the immediate forerunners of the Methodists were the Quakers (from the seventeenth century) and the Inghamites (between 1755 and 1760). Methodism took root slowly but, by the middle of the nineteenth century, it was established in many of the hamlets and villages. By the time of a union of its various branches in 1932, preaching, fellowship, teaching, singing, missionary interest and abstinence from alcohol were already its distinctive emphases. Its impact on local society and the wider Church was most marked in its strong opposition to the use of alcohol and in continuing missionary interest. Such Methodism (which we have occasionally compared with the isolated Waldensian and Jewish religions) has declined in numbers but still seeks to retain its emphases. In an attempt to interpret the history and understand the nature and meaning of upper Eden Methodism, we apply twelve categories of the sacred. Thus sacred story, symbols of the sacred, sacred action, sacred time, sacred space, initiation, sacred persons, the deity, the sacred community, sacred word and writing, sacred silence and sacred world-view are all related to this particular community which experiences the predicament and potential of isolated religion. Such communities stagnate and decay unless they are activated by that love for the whole world which is supremely expressed in the life and death of the incarnate Deity.

Chapter 8 contains a brief discussion of two remaining issues arising from the linguistic analogy: the nature of the generative process in vivo by which the actor arrives at his performance, and the possible practical applications of a predictive model of social behaviour sequences in determining the outcome of a crucial course of events. A possible application of generative rules to the description of person and situation interactions in the determination of behaviour is also discussed.

Lipid metabolism and the actions of valproic acid, a human teratogen, have been studied in the organogenesis-stage rat conceptus, using whole embryo culture techniques. All major lipid classes, particularly phospholipids, cholesterol and triacylglycerols, were synthesized de novo from acetate by the conceptus. Oleic acid and glucose were also incorporated into complex lipids. The pattern of lipid biosynthesis was dependent on the developmental stage of the conceptus. Despite this lipid biosynthetic activity, cholesterol uptake experiments suggested that the rat conceptus derives most of its required lipid from exogenous sources. Valproic acid stimulated de novo synthesis of cholesterol, and concomitantly reduced cholesterol esterification, in the yolk sac during an eight hour exposure. These effects were also induced by butyric acid, which produces valproic acid-like dysmorphologies in vitro, but not by the non-teratogen valpromide. Some observations suggested that the initial action was inhibition of cholesterol uptake at the yolk sac. Subsequent studies disproved this hypothesis and also suggested that the increased levels of newly synthesized cholesterol were not responsible, per se, for the valproic acid teratogenicity. The specific actions of valproic acid on yolk sac cholesterol synthesis and esterification were, nevertheless, dose-dependent in culture, and were also induced in conceptuses exposed to valproic acid in utero. This contrasts with several other major biochemical pathways, previously studied in the conceptus, which are not affected by valproic acid. It is likely, therefore, that these changes in lipid metabolism are related to the initial teratogenic insult of valproic acid.

A study has been made of the dependence of diffusion coefficients on (a) sorbate concentration, (b) temperature and (c) chain length for the sorption of n-butane, n-hexane and n-nonane in a series of partially calcium-exchanged forms of Linde Sieve 4A, (NaA). The sorbents were regulated to give controlled uptake of n-paraffins by partially exchanging calcium ions for sodium ions in NaA; this removed sodium ions from the octagonal windows of the aluminosilicate framework and allowed sorbetes to diffuse into the zeolite.

The existing information concerning substituent effects in the mass spectra of aromatic compounds is reviewed. The syntheses of representative compounds of the series 4-Y C6H4 (C6H5)CHCH2R are described and their mass spectra discussed. Particular reference is made to the correlation between activation energies and Hammett substituent constants for the process 14-Y C6H4(06H5)CHCH2Ph ⟶ 4-Y C6H4(C6H5)CH+ + PhCH2. The preparation and mass spectra of several benzyloxycarbonyl derivatives of L-ornithine are discussed and a corresponding account of some derived piperid-2-ones is presented with a view to making a structural assignment to a. new type of lipid known to contain L-ornithine. The final section deals with the fragmentation of several acetals and fatty acid methyl esters, induced by photon- and electron-impact at room temperature and at 150°C. The significant observation was confirmed that the photon-impact spectra show enhancement of the relative abundance of the molecular ion.

Mixtures of non-electrolytes and water are frequently used as solvent media for chemical investigations. Many of these mixtures have sound absorption characteristics which indicate the presence of complex equilibria, which are often reflected in derived thermodynamic quantities. The present thesis deals with mixtures of butanol and water in particular. The relevant theory of ultrasonic relaxation is developed, and is frequently used in the ensuing discussion. A review is made of the properties of alcohol-water mixtures together with previous sound absorption investigations of these systems. Theories developed to explain the latter are reviewed and discussed. The equipment and technique involved in measuring sound absorption using the pulse technique is described. Investigations of the sound absorption properties of butanol-water mixtures at a fixed frequency as a function of solvent composition has revealed the existence of an initial "plateau" region. This is shown to indicate that in this region the solution exists in the form of a liquid clathrate. In the region of the P.S.A.C. the experimental data has been satisfactorily analysed in terms of three relaxation processes. The processes at higher frequencies are concentration dependent and have been interpreted in terms of the formation and dissociation of hydrophobic dimers and tetramers. The effect of urea on the concentration independent low relaxation frequency indicates that this is due to co-operative disruption of local water structure when hydrophobic interactions occur. The effect of temperature on the relaxation spectrum of a butanol-water mixture at X2=0"05, indicates the existence of a thermal anomaly over the region 40 ~ 60 °C, which is in agreement with spectroscopic investigations. For the diethylamine-water system the "plateau" region is absent. Instead, in dilute solutions of diethylamine a single concentration dependent relaxation frequency is observed, which has been associated with the existence of an acid-base equilibrium, Finally ultrasonic relaxation studies of alcoholic solutions of lithium chloride are reported. Here two relaxation processes are observed at concentrations 0.1 molar but only one relaxation process at lower concentrations. It is suggested that the results indicate the existence in solution of two types of ion-pair differing only in the configurations of their solvation sheaths.

The pathological hallmark of Parkinson's disease (PD) are Lewy bodies (LBs), insoluble inclusions observed in dopaminergic neurons in the brains of PD patients. The main protein component of LBs is alpha-synuclein (αSyn), a 140-residue intrinsically disordered protein. Around 10% of PD cases are associated with genetic mutations, including single-point variants of αSyn; and under physiological conditions, the protein carries a constitutive N-terminal acetylation modification. Thus, the structural and functional properties associated with αSyn are vitally important to investigate in order to further understanding of how this protein contributes to disease. Here, we report the first full biophysical characterisation and cross-comparison of wild-type (WT) αSyn and a panel of PD-associated variants, using circular dichroism spectroscopy, fluorescence aggregation assays, native mass spectrometry, and ion mobility-mass spectrometry (IM-MS). We uncover that the different variants occupy different conformational spaces in the gas phase, and that the monomeric proteins do not exhibit a completely unfolded structure, as expected for a disordered protein. The N-terminal acetylated variants of αSyn are a more physiologically relevant model, with the constitutive modification being important for the formation of a transient N-terminal α-helix which mediates the binding of αSyn to various cellular lipid membranes. Here, we studied the effect of this modification on the structure and function of the panel of αSyn variants. The native state of αSyn is highly disputed, with several reports proposing the existence of naturally occurring multimers of the protein that may be involved in the physiological role of αSyn. However, these species have not been studied extensively and their role is not fully understood. Here, IM-MS and native top-down fragmentation using electron capture dissociation were used to elucidate the structural properties associated with a stable dimeric species of αSyn. In addition, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal to noise ratio and/or reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. Using this new workflow, we were able to infer structural information about this previously unreported αSyn dimer interface. Overall, this body of work aims to highlight native mass spectrometry as an important tool for investigating challenging structural biology problems, such as intrinsically disordered and aggregating proteins. This work also represents a comprehensive structural study of physiologically relevant WT αSyn and PDassociated variants in the gas phase. We showed that the N-terminal acetylation of αSyn and various PD-associated variants alters all aspects of structure and function of the protein, highlighting the need for physiologically relevant modifications to be used in in vitro studies. We also provide conclusive evidence for a C-C terminal interaction between the monomer units forming the stable dimeric species of αSyn, presenting important structural data on endogenous αSyn multimers.

It is commonly recognised that mislocalisation and aggregation of Transactive response DNA- binding protein-43 (TDP-43) occurs in over 97% of amyotrophic lateral sclerosis (ALS) patients and around 50% of fronto-temporal dementia (FTD) patients. However, despite the commonality of this molecular phenotype, its precise role in disease onset and progression remains elusive. Although TDP-43 is an attractive target for therapeutic intervention and diagnostic testing, it has thus far not been possible to exploit either of these approaches. A core problem remains the lack of efficient detection and targeting probes for TDP- 43. A large variety of micrometre-scale TDP-43 inclusions have been described, however, smaller protein assemblies have not been studied in detail. Although traditional ensembleaveraging biochemical techniques fail to deliver information on single nanometre-scale aggregates, single-molecule and super-resolution (SR) methods can be used to circumvent these issues, as has been demonstrated in other neurodegenerative diseases involving protein aggregation. To date, the number of studies employing these techniques to examine TDP-43 is extremely limited. Highly specific and sensitive imaging probes are required for single-molecule measurements and would allow counting and characterisation of individual aggregates. Precise localisation of aggregates in tissue and the cellular milieu could enhance the understanding of TDP-43 relevance in a disease context. Meanwhile, separation of disease- relevant aggregates from physiological TDP-43 in biofluids could assist in the establishment of a reliable biomarker. Aptamers are artificial oligonucleotides capable of binding to specific molecular targets and are attractive alternatives to antibodies with regards to biomolecule labelling. In this study, aptamers targeting TDP-43's RNA recognition motifs (RRM1-2) were designed via the in silico algorithm, catRAPID, and validated in vitro. The highest affinity candidate aptamer, Apt-1, was labelled for fluorescent imaging and used to track the aggregation of a TDP-43 construct. Apt- 1 was capable of generating diffraction limited fluorescence images, as well as SR images at a resolution of 20 nm via Aptamer DNA-points accumulation in nanoscale topography (ADPAINT) and a newly developed technique, dubbed Aptamer-PAINT. To establish if Apt-1 could be used for imaging in biological samples of a complex composition, a comparative study of TDP-43 detection using gold-standard immunohistochemistry (IHC) and Apt-1 fluorescence imaging, was carried out. Despite low-agreement between both techniques, Apt-1 was capable of separating ALS cases from healthy controls based on the number of detected TDP-43 aggregates. Furthermore, Aptamer-PAINT SR images of aggregates could be acquired in tissue with a similar resolution to that achieved in vitro. TDP-43 can be detected in human biofluids, such as serum and cerebrospinal fluid (CSF). SR imaging of CSF samples using Apt-1 was carried out to establish if the number of detected species in samples from ALS patients with TDP-43 proteinopathy was higher than in ALS disease controls. Additionally, size characterisation of the detected species was used to determine differences in the relative distribution of aggregate populations. Overall, the work presented in this thesis demonstrates the development of a new TDP-43 imaging probe capable of characterising aggregate species individually in variety of in vitro and patient-relevant samples. Although these studies are exploratory, they form the basis of establishing a new class of detection probes in the field of ALS and FTD research, and show promise in the development of diagnostic tools. In this manner it is hoped that the developed aptamers will aid researchers and clinicians in understanding the mechanisms of TDP-43 pathology and assist in diagnosing patients.

In Nature, thousands of diverse, bioactive polyketides are assembled by a family of multifunctional "assembly line" enzyme complexes called polyketide synthases (PKS). Since the discovery of 6-deoxyerythronolide B synthase (DEBS) in the late 20th century, many attempts have been made to decode, re-arrange and "re-programme" the PKS assembly line to generate value chemicals such as biofuels and platform chemicals. Here, the first module from the MtPKS12 (hereby termed [M₁]) was repurposed towards the production of 2-methyltetradecanol (2-MTDOL), a valuable lubricant with wide applications in industry. Following a bottom-up, deconstructive study of the 440 kDa fully-reducing megasynthase, MtPKS12 [M1] was shown to condense 2(S)-methylmalonyl-CoA with C₁₂-CoA in a single-turnover assay to produce a 2-methyltetradecyl (2-MTD) scaffold, an important intermediate towards 2- MTDOL. The synthetic utility of MtPKS12 was further demonstrated with the successful production of C₁₃-C₂₁ methyl-branched scaffolds using C₁₀-C₁₈-CoA substrates. Following this, three trans-acting auxiliary enzymes (MtPapA5, UcFatB and the Mycobacterial protein Rv2047c) were investigated for the enzymatic release of polyketide products from MtPKS12. All three candidates presented significant barriers ranging from a lack of crosstalk to insolubility. The investigation concluded with the successful utilisation of ketosynthase (KS) - sourced from MtPKS12 module 2 ([M₂]) - to untether intermediates from MtPKS12 [M₁]. By exploiting the mutual recognition between the two MtPKS12 modules, the 2-MTD scaffold was hydrolysed to produce 2-methyltetradecanoic acid (2-MTDA). The challenge remains to improve substrate and intermediate flux through the PKS to benefit product titres, which presently remains very low. The study herein demonstrates the feasibility of repurposing a fully-reducing PKS module towards the manufacture of useful 2,2-dialkyl chemicals.

This thesis combines the disciplines of folklore studies and screen studies to examine the on-screen representation of folklore and contemporary legend in 1970s British television texts. Using original interviews with creators of film and television (older participants having worked in the 1970s and younger participants working in present-day media) alongside archive material, the thesis draws out influential examples from the study period that affected those making film and television today. This thesis makes an original contribution to knowledge by employing the notion of mass-mediated ostension, as an approach unique to folklore studies, to examine the cultural context of 1970s British television drama and account for the impact on audiences of unsettling, supernatural and extra-terrestrial stories. The importance of children's television in this era is also emphasised. This study considers the emergence of folk horror and its resurgence in the post-2000 period, particularly in the work of Generation X creatives, and sets out defining characteristics of 'hauntological' and 'wyrd' texts. Programme-makers' reproduction and reinterpretation of folklore and contemporary legend are situated as central to the way such texts are made to seem plausible for an audience. The work of Nigel Kneale is highlighted as having been especially influential in this regard. Screen studies analysis is applied to paradigmatic examples of television programmes (notably Quatermass, 1979, and Children of the Stones, 1977) to assess formal methods and techniques used to represent folkloric and contemporary-legend motifs. In conclusion, this thesis reflects upon the continuing influence of 1970s folkloric television texts: how this remains manifest in British media and how it shapes the communication of folklore and the future of the folk-horror genre itself.

The analysis of Scotch Whisky and other spirits is of great importance to the quality control, product protection and product development. The spirit industry uses various UKAS accredited analytical methods, such as those for chromatographic instru mentation that can be time consuming and require the use of multiple instruments. In this thesis we explore Nuclear Magnetic Resonance (NMR) spectroscopy as a possible method for providing multiple characteristics of spirits in few experiments and explore their accuracy. We also analyse composition of ethanol wood extracts and compare them with compounds found in a concentrated whisky sample. Quantitative NMR (qNMR) is a powerful analytic technique that is currently underutilised by the spirit industry. We have developed a method for quantification of 13 congeners applicable to high ethanol samples. The method uses a custom build library of individual compounds within the proprietary software program Chenomx. We were able to be confidently quantified these compounds using a single 1D ¹H NMR spectrum in 86 whisky sample (500 µl, 15 minutes per samples) within 6.4 % (standard deviation ± 5.0 % ) of their nominal concentration. We have conducted a chemometric analysis using NMR determined concentration of congeners (targeted analysis) achieving separation between single malt Scotch Whisky and Blended Scotch. Similar separation was realised through non-targeted analysis of 1D ¹H NMR spectra of the studied samples. Models were built using un-supervised (Principal Component Analysis) and supervised (Partial Least Squares - Discriminant Analysis) methods, which can be used as a method for classification of whisky samples and the identification of counterfeits. NMR spectroscopy was also used to study ethanol/water mixtures aiming to develop a methodology for the determination of the ethanol strength. Several NMR parameters, such as ¹³C and ¹H chemical shift differences between the CH₂ and CH₃ resonances of ethanol (∆ δ¹³C and ∆ δ¹H), one-bond and long-range proton-carbon coupling constants of ethanol were examined as a function of ethanol concentration. Regions of ethanol concentrations that produced a step change in these parameters agreed approximately with the literature data obtained by other methods. Several parameters were taken forward and tested on real samples of spirit. It was found that samples containing high levels of sugar directly influence these NMR parameters and further investigation will be needed to develop this method for use in spirits with (low) carbohydrate content. Due to the magnitude of the changes, the ∆ δ¹³C parameter (2.1 Hz/ 1 % ABV) is most promising for further exploration. Using an external standard, a quantitative NMR method utilising the absolute integral of the CH₂ and CH₃ peaks obtained in a 1 scan ¹H decoupled ¹³C spectrum of neat spirits was developed yielding accuracy of 1 % over the full range of analysed samples. The use of ¹H NMR spectroscopy using benchtop instruments was also explored and the ethanol strength was determined with an accuracy of ±X% using integrals of ¹H resonances of ethanol. Using an external standard, qNMR was explored for the determination of the site specific isotopic composition of ethanol. Both δ ¹³C and D/H ratios were investigated as these parameters are known to depend on the biosynthetic pathways of plant carbohydrates that are fermented to produce ethanol. A qNMR method using neat samples was developed that can determine site-specific D/H ratios to about 1 % accuracy, which is sufficient to link the origin of ethanol to C₃ or C₄/CAM biosynthetic pathways of plants. Chemometric analysis of the data was performed on range of samples containing different botanical origins, yielding a clear separation between C₃ and C₄/ CAM derived spirits. This method failed for δ ¹³C due to low accuracy of the integral intensities obtained from the ¹³C spectra. Wood extracts of American oak wood showings were partially separated by Solid Phase Extraction and concentrated fractions were studied using high resolution 2D NMR methods on 800 MHz NMR. Eighteen compounds were identified, including eleven carbohydrates, three cyclitols, 2-phenolethanol and a larger compound (8-(4- Hydroxy-3,5-dimethoxyphenyl)-6,7-bis(hydroxymethyl)-1,3-dimethoxy-5,6,7, 8-tetrahydro-2-naphthalenol) believed to be a degradation of lignin. In addition, two ethyl glycosides were identified. These however could be an artefact of samples preparption as their existence in whisky samples was not reported before.

Mass spectrometry (MS) has been extensively used to analyse biological samples and has evolved into an essential tool for proteomics research. Recent tremendous technological advancements, including improving instrument accuracy, resolution and sensitivity, and more freely accessible protein databases, have led to the growing importance of this technique. This study contributes to this growing area of research by developing novel orthogonal workflows that push the boundary of mass spectrometry capabilities, and allowed greater insight into the protein environment. Specifically, this research describes new methodology for de novo characterisation of disulfide bonding connectivity and de novo protein sequencing. Investigating protein 3D structure can provide essential information when addressing the issues in protein folding and function. One of the important structural features in proteins is the disulfide links between cysteine residue pairs, which play crucial roles in sustaining protein 3D structure. Although some conventional experimental techniques can provide information of the disulfide patterns within a specific proteoform, there has been little quantitative analysis due to cost and time limitations. Thus, there is a necessity for new approaches that increase confidence in disulfide mapping and maximise the protein sequence coverage obtained. Here, this study draws attention to the ability of a combination of pepsin and trypsin proteolysis and the usage of dual fragmentation of electron capture dissociation (ECD) and collision induced dissociation (CID) to assigning disulfide connectivity of proteins and maximise sequence coverage. This protease approach is based on the accurate mass measurement of proteins and high-resolution top-down fragmentation MS studies. Here we present our findings, which confirm that the developed method has significant advantages. Another main challenge in mass spectrometry-based proteomics is de novo protein sequencing, especially for novel proteins such as monoclonal antibodies for which genome information is often limited or not available. However, due to limitations in peptide fragmentation coupled with coverage and ambiguities in spectra interpretation, complete de novo assembly of unknown protein sequences remains challenging. Thus, there is a drive for new strategies that increase fragment ion assignment efficiency in top-down mass spectra and maximise the protein sequence coverage obtained. Here, we use a strategy for selective chemical labelling of the protein N-terminus using reductive alkylation and utilise this chemistry to introduce a halogen-based mass defect tag to the N-terminus of Insulin B chain, Ubiquitin, Myoglobin and Rnase A. We outline the potential advantages of using this simple chemical derivatisation in top-down de novo protein sequencing.

Lignin is one of the most abundant and diverse biopolymers on the planet, comprising approximately one third of all woody plant matter. This complex, non-repeating, heteroaromatic polymeric structure provides endless potential as an alternative, renewable feedstock to petrochemicals to produce fine chemicals. Lignin has been previously utilised to produce high value chemicals such as vanillin, however it is primarily considered a waste product and burned to fuel paper mill processes. As the world moves towards a circular economy, this abundant resource can no longer be neglected as a waste product and should be utilised as a replacement for fossil fuels in the synthesis of fine chemicals. In this regard, the use of ligninolytic enzymes has long been touted as the primary mechanism by which to achieve lignin valorisation because they act as more selective green catalysts that can perform an array of functions that are not always replicable by conventional chemistry. In Chapter 2, a laccase from Panus rudis was successfully expressed and purified using Pichia pastoris to obtain a highly active laccase for further downstream lignin degradation assays; however, only a low yield of protein was obtained. Owing to the inherent complexity of lignin, fractionation methodologies were utilised in Chapter 3 to simplify a frequently used, commercially available lignin into more uniform, lower molecular weight fractions for use in streamlined downstream depolymerisation. Uniformity was determined using nuclear magnetic resonance spectroscopy (NMR), which quantified β-O-4 linkage content, whilst Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) allowed the characterisation of the chemical diversity, identifying the presence of valuable monolignols. Chapter 4 is concerned with the characterisation of industrially relevant lignin fractions supplied by MetGen Oy, including the use of FT-ICR MS to characterise and compare the chemical diversity of an enzymatically fractioned lignin. This provided a basis for further enzymatic depolymerisation experiments. Subsequently, Chapter 5 focused on the treatment of a lignin fraction with an alkaliphilic laccase provided by MetGen Oy. FT-ICR MS highlighted the enzymatic targeting of compounds such as syringaresinol, which was almost wholly depleted after 24 hours, with NMR spectroscopy also indicating a >50% loss of β-β linkages. Ultimately this work aims to aid future developments in lignin research, by outlining strategies to improve, analyse and track the enzymatic depolymerisation of lignin.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a versatile biophysical technique used in a variety of applications within structural proteomics. The key advantages of HDX-MS are that it can provide insight into conformational changes in protein structure as well as allowing investigation of the details of protein-protein interactions, protein-small molecule interactions and protein folding. For these reasons it is an often-used, complementary technique when paired with other biomolecular techniques such as X-ray crystallography, cryogenic electron microscopy (Cryo-EM) and native mass spectrometry. Encapsulin nano compartments (Enc) or encapsulins are a family of bacterial proteins which form large icosahedral protein cages. These protein cages are of a large enough size (25-30 nm) to allow for the encapsulation of smaller cargo proteins. It has been found that compartmentalization plays an important physiological role within bacteria. One recently discovered ENC, encapsulated ferritin (EncFtn), is known to act as an iron storage system. Although details of Enc-EncFtn structure are beginning to emerge, several questions remain regarding dynamics and interactions of this complex. This thesis focuses on the application of HDX-MS for the study of these important bacterial nano-compartments. This body of work initially explores the assembly pathway of EncFtn derived from Haliangium ochraceum using a bottom-up HDX-MS workflow. It was previously shown that EncFtns characteristically exist as a decameric 'pentamer-of-dimers' arrangement. HDX was used to determine the details of the assembly pathway and the formation of the decameric assembly via recruitment of specific dimer subunits. This study's second aim was to analyse the structures of both a loaded encapsulin (Enc-Loaded) and empty encapsulin (Enc-Empty). Differential HDX-MS was used to reveal significant differences in deuteration exchange 6 between both the empty and loaded forms. The regions of higher deuterium uptake in Empty encapsulin were localized within the interior of the encapsulin and surrounded a hypothesised cargo binding sequence that been previously determined via Cryo-EM. This HDX analysis also highlighted the dynamic and flexible nature the putative entry pore within the ENC. Finally, a novel automated HDX method was developed for top-down HDX analysis of proteins. This strategy allows for single residue resolution of a deuterium uptake event with the potential of proteoform isolation and selection. Fourier Transform Ion Cyclotron Resonance (FT-ICR MS) was coupled with an automated HDX platform and Electron Capture Dissociation (ECD). This workflow was applied to study EncFtns, and direct comparison with traditional bottom-up data demonstrated the feasibility and potential advantages of this new workflow.

The human skin barrier is a biological imperative. Chronic inflammatory skin diseases, such as Atopic Dermatitis (AD), are characterised by a reduction in skin barrier function and an increased number of secondary infections. Staphyloccocus aureus (S. aureus) has an increased presence on AD lesional skin and contributes significantly to AD pathology. It was previously demonstrated that the damage induced by a virulence factor of S. aureus, V8 protease, which causes further breakdown in skin barrier function, can be reduced by induction of human β- defensin (HBD)2 (by IL-1β) or exogenous HBD2 application. Induction of this defensin is impaired in AD skin. This thesis examines the mechanism of HBD2-mediated barrier protection in vitro; demonstrating that in this system, HBD2 was not providing protection through direct protease inhibition, nor was it altering keratinocyte proliferation or migration, or exhibiting specific localisation within the monolayer. Proteomics data demonstrated that HBD2 did not induce expression of known antiproteases but suggested that HBD2 stimulation may function by modulating expression of extracellular matrix proteins, specifically collagen- IVα2 and Laminin-β-1. Alternative pathways of protection initiated by IL-1β and TNFα stimulation were also investigated, as well as their influence over generalised wound healing. Finally, novel 3D human skin epidermal models were used to better recapitulate the structure of human epidermis and examine alterations to skin barrier function in a more physiological system. These data validate the barrier-protective properties of HBD2 and extended our knowledge of the consequences of exposure to this peptide in this context.

Despite the impact of David Harvey's Marxist thought, his concept of historical-geographical materialism has attracted limited attention. Standing with Harvey, this dissertation argues that Marxism and geography need to maintain a symbiotic relationship in the shape of historical- geographical materialism, which gives processes that internalize time and space ontological priority, eschewing both historical determinism and spatial fetishism. Departing from Harvey, the thesis constructs historical-geographical materialism by means of Marx's materialist conception of history, through a reinterpreted historical geography of modes of production. With the discovery of Marx's anthropological notebooks on Asiatic society, and the re-emergence of Asian studies after the Asian economic "miracles" of the 1970s, a more systematic elaboration of the Asiatic mode of production (AMP) has become possible. Starting by scrutinizing the patterns of ownership and mode of surplus extraction peculiar to the Asiatic mode of production, the present work demonstrates the geographical dimension of historical materialism by illustrating and defending the particularity and reasonability of the AMP in the panorama of Marx's historical geography of modes of production. By comparing the AMP in late imperial China and feudal Europe, and incorporating the concept of relation of intercourse into the traditional two-aspect formulation of modes of production with the aid of Marx's underutilized Chronological Extracts, the thesis depicts the transformative dynamics of pre-capitalism as it transitions into capitalism. This leads to an analysis of theories of crisis and revolution in relation to Covid-19 and social movements such as Black Lives Matter. The thesis points out the necessity of crisis under the capitalist mode of production and its relation to revolution, which cannot succeed unless the heterogeneous multitude unites to organize mass action under the identity of the labourer. Moreover, it demonstrates that historical-geographical materialism concerns not only the past but the present and future prospects of global society.

A promising alternative to small molecular drugs is based on the RNA-targeting approaches which offer the potential for absolute selectivity, improved potency and reduced toxicity. Artificial ribonucleases (ARs) have been identified as novel agents capable of recognising and destroying pathogenic RNAs, reverting cellular pathways to their 'normal' state. However, metal-dependent ARs suffer from time-dependent leakage which can potentially lead to the destruction of non-target biopolymers and toxicity. Peptidyl-oligonucleotide conjugates (POCs) are metal-independent ARs consisting of an amphiphilic peptide and an antisense oligonucleotide recognition motif. Recent studies have indicated that, in principle, POCs are capable of multiple catalytic turnover. Nonetheless, all designed POCs to this date have exerted cleaving properties at two extremes: (i) base-specific cleavage with high catalytic turnover or (ii) sequence-specific cleavage in the absence of catalytic turnover. Henceforth, to make POCs suitable for therapeutic application, vital changes need to be implemented in their structural design to combine an absolute sequence-specificity with multiple catalytic turnover and produce highly-specific supramolecular catalysts. In this research, "smart" POCs have been designed to achieve precise and potent knockdown of RNAs. Firstly, a series of structurally diverse "single" bulge-loop inducing conjugates was designed. Comprehensive analysis of hybridisation and ribonuclease activity accompanied by dynamic molecular modelling simulations provided structural insights on how duplex-constrained cleavage occurs. Antisense recognition motifs of the conjugates were designed in such a way that a short region of the target was non-complementary upon hybridisation, forcing the target to adapt a bulge loop structure, a phenomenon which was substantiated by enzymatic probing. All type 2 conjugates were catalytically active, with the best structural candidate exhibiting up to 90% cleavage of the target tRNAPhe. Subsequently, the next generation bulge-loop inducing "bis" and "triple" conjugates demonstrated high catalytic efficacy (up to 100% cleavage in 24 hr). The addition of multiple peptide entities augmented ribonuclease activity without diminishing hybridisation capabilities. Furthermore, catalytic turnover studies demonstrated a true supramolecular catalyst. Detailed kinetic studies indicated the presence of complex inter- and intramolecular assembles in the reaction mixture. Avoidance of such non- productive conformations would improve the turnover number of the RNA substrates and may open the translational pathway to successful therapeutics. Ribonuclease activity in single and multiple turnover mode was preserved when the design was directed towards a biology relevant target (miR-21 and miR-17), which was monitored here using an in-house developed self-assembling peptide hydrogel-based 3D analytical device. We also discovered the ability of conjugates to recruit non-sequence specific RNase H and enhance cleavage efficiency and RNA substrate turnover number. Lastly, chemical modifications (2'OMe) were introduced in the conjugates. Complete modification of antisense recognition motif had a detrimental effect on ribonuclease activity, whereas partial modification improved hybridisation properties and catalytic activity. No statistically significant difference in serum stability was witnessed between all tested conjugates, indicating the inherent stability of the naked conjugate stemmed from the hairpin structure. The encouraging results presented here will provide a solid ground for the development of potent, highly selective nucleic acid therapeutics.

Background: An estimated eighty percent of stroke patients have difficulties with eating and drinking, with over fifty percent requiring some form of assistance. Evidence suggests those receiving nutrition and hydration via enteral or parenteral routes receive adequate nutrition. In contrast, stroke patients who require assistance to eat and drink via the oral route do not always meet their nutritional requirements, affecting their well-being and recovery from stroke. This study explores this phenomenon in contemporary stroke unit settings. Methods: A qualitative collective case study using non-participant observation, documentary analysis and semi-structured interviews was undertaken. The study gathered data from two sites incorporating acute and rehabilitation stroke services (cases). Forty-six multidisciplinary team (MDT) members, 31 patients and 13 informal carers (ICs) were recruited to the study. Data was thematically analysed using a six components approach developed by Braun and Clarke (2006). Findings: All MDT members considered adequate nutrition paramount to stroke patients' recovery with nursing staff responsible for supporting eating and drinking. However, patients did not receive consistently adequate support to eat and drink. This was due to multiple, interwoven factors including lack of formalised assessment of eating and drinking abilities unless under SLT management; poor communication and monitoring of patients' support requirements, nutritional intake and nutritional status; other work/ tasks given priority over supervision at mealtimes; inconsistent provision of accessible, nutritious, appealing food. Knowledge to support this activity varied hugely across the MDT, particularly with HCAs who reported variable levels of training but predominantly carried out this work unsupervised. Conclusion and implications for practice: Stroke patients do not receive consistent support with eating and drinking. Further research and the inclusion of specific recommendations for support with this activity in national guidance may promote this activity.

Sphingolipids (SLs) are a diverse class of lipid molecules derived from the amino acid L-serine and long chain fatty acids (e.g. carbon chain lengths C14-C26). When combined together these building blocks form a so-called "sphingoid", also known as a long-chain base (LCB). Decades of research into these enigmatic molecules has revealed that SLs are essential components of eukaryotic cell membranes and control many critical cellular functions. The SL biosynthetic pathway begins in all organisms with the Claisen-like, decarboxylative condensation of L-Ser and long-chain fatty acid acyl-CoA thioester (CoASH) substrates (most commonly C16/palmitoyl) to form the intermediate 3-ketodihydrosphingosine (3-KDS). This first key irreversible reaction is catalysed by the pyridoxal 5'-phosphate (PLP)-dependent serine palmitoyltransferase (SPT). The 3-KDS product is then reduced by a NAD(P)H-dependent enzyme (KDS reductase, KDSR) to generate dihydrosphingosine (DHS). Acylation of this molecule with a long-chain fatty acid by a ceramide synthase (CerS) leads to the formation of ceramide (Cer). Downstream, phosphorylation with a phosphate group can occur on the serine-derived head group and lead to the formation of sphingosine-1-phosphate (S-1-P). The balance between Cer and S-1-P concentrations has been proposed to control the cell survival rate in the host system. Alternatively, sugars such as glucose can be added to give the family of glycosphingolipids (GSLs). In contrast to higher order species, research into SL biosynthesis in bacteria has lagged much further behind. However recent studies have revealed a number of important microbes that produce a range of SLs and Cers. Interestingly, SLs from the human microbiota including those from Bacteroides fragilis, a Gram-negative commensal bacterium from the human gut, and from the oral pathogen Porphyromonas gingivalis have recently been shown to play an essential role in host/microbe communication. Of note is that these bacterial SLs and Cers display many similar structural features to their mammalian homologues. However, there appears to be "chemical signatures" distinct to those of bacterial origin. For example, B. fragilis SLs have iso-Me branched acyl chains which are also similar to those found in the round-worm Caenorhabditis elegans. This suggests a PLP-dependent branched-chain amino acid transferase (BCAT/IlvE) transfers iso-Me chains from amino acid precursors such as L-Leu to branched-chain keto acids. To fully understand the role of bacterial SLs and their metabolism detailed investigations of the enzymes, pathways and metabolism are required. In this thesis, studies of different types of the three key enzymes (SPT, IlvE/BCAT and KDSR) involved in the core microbial SL biosynthetic pathway have been presented. Firstly, both recombinant B. fragilis SPT (BfSPT, encoded by gene BF2461) and P. gingivalis (PgSPT, gene PG1780) were expressed in E. coli, purified and studied with protein UV-vis spectrometry, enzyme kinetics, inhibition assays, mass spectrometry and protein crystallization screening. 3-KDS products were detected derived from a range of straight-chain CoA substrates (C14-C18) and amino acids (Gly, L-Ala and L-Ser) produced by BfSPT and PgSPT. Mutagenesis of a conserved loop (PAVAP) in SPT homologues was found to be associated with the catalytic efficiency of PgSPT. Also the presence of a Val353 residue in BfSPT was shown to be essential to allow the enzyme to interact with the C16-CoA substrate. Moreover, data suggested that the position (N- or C- terminus) of the 6His-affinity tag used to purify SPT influenced substrate inhibition by C16-CoA. A hypothetical 3D structural model of the PgSPT PLP:L-Ser external aldimine complex was built in order to explore the active site and residues involved in substrate binding and catalysis. In collaboration with Prof. Mary-Ellen Davey (Florida), the role on SL biosynthesis in P. gingivalis was also explored and found that SLs impact on the way this pathogen interacts with human cells. Secondly, a branched acid transaminase P. gingivalis IlvE (PgIlvE, gene PG1290) was expressed, purified and studied using UV-vis spectrometry to investigate substrate binding and enzyme activity. A multi-enzyme coupled assay for PgIlvE was developed in both the 'forward' and 'reverse' direction, studied with inhibitors such as L- and D- cycloserine (LCS/DCS), as well as x-ray crystallography. In collaboration with Dr. Jon Marles-Wright (University of Newcastle) the crystal structures of four different forms of PgIlvE; the PLP-bound, internal aldimine form, LCS ring-opened ring-closed form and the PMP form, were obtained. Two residues (F56 and Y188) were identified which played a role in substrate binding and activity. In the final chapter, recombinant KDSR from the yeast Saccharomyces cerevisiae (ScKDSR) was isolated from E. coli. Kinetic parameters for a soluble, truncated form of the enzyme were determined using the substrates KDS and NADPH. The product C18 DHS derived from C18 KDS was detected with MALDI-ToF-MS. Recent studies of human KDSR revealed that patients with point mutations in this enzyme suffered from skin disorders (erythrokeratoderma). To investigate the impact of these mutations, a series of ScKDSR mutant mimics (G176S, Y180F and G263E) were prepared. To date there has been no crystal structure of a KDSR determined but it is a member of the short-chain dehydrogenases/reductases (SDR) superfamily. A homology model of the 3D structure of ScKDSR with three possible NADPH docking positions were constructed, and residues involved in substrate binding and catalysis were suggested. The results in this thesis shed light on the key enzymes involved in the core biosynthetic pathway of microbial SLs and lay down a blueprint for future studies.