Aspects of the biology of Lutraria lutraria (L.) (bivalvia : Mactracea)

The life history of a population of Lutraria lutraria in a depth of 7m at Hunterston, Ayrshire is discussed. Much of the present population Is thought to have settled in 1967. The functional morphology of Lutraria is described and related to its life as a large, deep-burrowing bivalve. Lutraria spawned in late spring and continued to do so through the summer in 1979 and 1980. Animals became spent in August and September. Unsuccessful attempts were made to induce spawning in the laboratory. Artificial fertilization was successful but development did not proceed beyond the ciliated gastrula stage. Larvae of Lutraria were not identified in plankton samples and young stages were not encountered in sieved sediment samples. The biochemical cycle of the total animal and five component parts (gonad and visceral mass, digestive gland, adductor muscle, siphon and 'other' tissue) is investigated. A marked increase in weight, reflected in an increase in weight of the component parts, was recorded in Autumn 1979. This is thought to be related to an exceptional increase in the phytoplankton at this time. Although a relationship between the biochemical cycle and reproductive cycle remains uncertain, definite seasonal changes were recorded in the respiration rate of Lutraria. At 10°C, the maximum rate of a standard 20g animal was 0.1283m1s 02/g. dry wt./hr. in May 1980 and the minimum rate was 0.O59mls 02/g. dry wt./hr. in October 1980. The effect of temperature on respiration rate was also investigated. Significant differences were recorded for five experimental temperatures (10°C, 15°C, 20°C, 25°C and 30 °C) in August and October but only between two temperatures (10 C and 30 C) in April. There was a decrease in respiration rate at 30 C in August and October, but an increase in April. Respiration rate is affected by a reduction in oxygen tension. A variety of responses were recorded with a small degree of regulation shown. Individuals of Lutraria were able to survive 48 hours under anaerobic conditions. In fully oxygenated conditions heart rate ranged from 4-15 beats per minute with an average of 8 beats per minute. Heart beat was markedly affected by changes in temperature and oxygen tension, increasing to a maximum 22 beats per minute at 25 C, and decreasing to a minimum 2 beats per minute in anaerobic conditions. Heart rate is reduced (12 beats per minute to 5 beats per minute) on exposure to air. Lutraria exhibits an intermittent pattern of pumping activity. Under normal conditions 35% of the time is spent pumping and this Increases as oxygen is reduced (3.00mls 02/litre) to 65% of the time spent pumping. 15. Under normal conditions the respiratory flow varies between 0.382 litres per hour and 1.023 litres per hxir. Adult Lutraria maintain their ability to burrow, albeit slowly.

Engineering the zinc finger recombinase for use in targeted genomic editing

No abstract available.

Cryptosporidiosis in farm livestock

Although diarrhoea caused by Cryptosporidium is prevalent in livestock species throughout the world relatively little is known about the species and subtypes of Cryptosporidium found in cattle on Scottish farms. In particular, little is known about the shedding profiles (age when calves become infected and duration of shedding) of the different species found in cattle and how calves become infected. There are several theories about how neonatal calves first become infected with the parasite but the role which adult cattle play in the transmission of the parasite has not been fully addressed. It was previously thought that adult cattle did not become infected with the same species of Cryptosporidium which causes disease in the young calves. Some studies have shown that this may not be true and with the advance of new techniques to discriminate species this is an area which should be revisited. In addition, it is known that it is possible for humans to become infected with Cryptosporidium and show clinical disease early in life and then again later in adulthood. In livestock however, diarrhoea caused by the parasite is generally only seen in neonatal livestock while older animals tend to be asymptomatic. It is not known if this resistance to clinical disease at an older age is due to changes in the host with an increase in age or if prior infection “immunises” the animal and provides protection against re-infection. It is also not known if infection with one isolate of C. parvum will provide protection against infection with another or if the protection formed is species/isolate specific. The main aims of this thesis were to: determine the species and subtypes of Cryptosporidium found in calves on a study farm over a one year period from birth; assess the role which adult cattle play in the transmission of the parasite to newborn calves; develop new typing tools to enable the rapid and easy differentiation of Cryptosporidium species found in cattle and to examine the host-pathogen interactions in animals given serial experimental challenges with distinct Cryptosporidium parvum isolates to determine if the resistance seen in older animals on farms is due to an increase in age or as a result of prior infection. iii A variety of different approaches were taken to achieve these aims. Longitudinal experiments carried out on a study farm revealed that in calves <9 weeks of age the most common species of Cryptosporidium is C. parvum and that all calves in the group became infected with Cryptosporidium within the first two weeks of life. Sample collection from the same animals later in life (at 6 months of age) showed that contrary to most previous studies the most common species detected at in this age group was also C. parvum although, interestingly, the subtype which the calves were shedding was not the same subtype that they were shedding previously. The longitudinal study which investigated the role of adult cattle in the transmission of Cryptosporidium also yielded some interesting results. It was found that most of the adult cattle on this farm were shedding Cryptosporidium albeit intermittently. Speciation of the positive samples revealed that, on this farm, the most predominant species of Cryptosporidium in adult cattle was also C. parvum. This is very unusual as most previous studies have not found this level of infection in older cattle and C. parvum is not usually found in this age group. A number of different subtypes were found in adult cattle and some animals shed more than one subtype over the course of the study. This contradicts prior findings which demonstrated that only one subtype is found on a single farm. The experimental infection trial involving infection of young (<1 week old) and older (6 week old) lambs with distinct C. parvum isolates demonstrated that an increase in age at primary infection reduces the effect of clinical disease. Animals which were infected at <1 week of age were re-challenged at 6 weeks of age with either a homologous or heterologous infection. Results revealed that previous exposure does not protect against re-infection with the same or a different isolate of C. parvum. This study also demonstrated that an increase in infective dose leads to a shorter pre-patent period and that there are variations in the clinical manifestations of different isolates of the same Cryptosporidium species.

Considering climate change in the development of marine protected areas

Marine ecosystems are facing a diverse range of threats, including climate change, prompting international efforts to safeguard marine biodiversity through the use of spatial management measures. Marine Protected Areas (MPAs) have been implemented as a conservation tool throughout the world, but their usefulness and effectiveness is strongly related to climate change. However, few MPA programmes have directly considered climate change in the design, management or monitoring of an MPA network. Under international obligations, EU, UK and national targets, Scotland has developed an MPA network that aims to protect marine biodiversity and contribute to the vision of a clean, healthy and productive marine environment. This is the first study to critically analyse the Scottish MPA process and highlight areas which may be improved upon in further iterations of the network in the context of climate change. Initially, a critical review of the Scottish MPA process considered how ecological principles for MPA network design were incorporated into the process, how stakeholder perceptions were considered and crucially what consideration was given to the influence of climate change on the eventual effectiveness of the network. The results indicated that to make a meaningful contribution to marine biodiversity protection for Europe the Scottish MPA network should: i) fully adopt best practice ecological principles ii) ensure effective protection and iii) explicitly consider climate change in the management, monitoring and future iterations of the network. However, this review also highlighted the difficulties of incorporating considerations of climate change into an already complex process. A series of international case studies from British Columbia, Canada; central California, USA; the Great Barrier Reef, Australia and the Hauraki Gulf, New Zealand, were then conducted to investigate perceptions of how climate change has been considered in the design, implementation, management and monitoring of MPAs. The key lessons from this study included: i) strictly protected marine reserves are considered essential for climate change resilience and will be necessary as scientific reference sites to understand climate change effects ii) adaptive management of MPA networks is important but hard to implement iii) strictly protected reserves managed as ecosystems are the best option for an uncertain future. This work provides new insights into the policy and practical challenges MPA managers face under climate change scenarios. Based on the Scottish and international studies, the need to facilitate clear communication between academics, policy makers and stakeholders was recognised in order to progress MPA policy delivery and to ensure decisions were jointly formed and acceptable. A Delphi technique was used to develop a series of recommendations for considering climate change in Scotland’s MPA process. The Delphi participant panel was selected for their knowledge of the Scottish MPA process and included stakeholders, policy makers and academics with expertise in MPA research. The results from the first round of the Delphi technique suggested that differing views of success would likely influence opinions regarding required management of MPAs, and in turn, the data requirements to support management action decisions. The second round of the Delphi technique explored this further and indicated that there was a fundamental dichotomy in panellists’ views of a successful MPA network depending upon whether they believed the MPAs should be strictly protected or allow for sustainable use. A third, focus group round of the Delphi Technique developed a feature-based management scenario matrix to aid in deciding upon management actions in light of changes occurring in the MPA network. This thesis highlights that if the Scottish MPA network is to fulfil objectives of conservation and restoration, the implications of climate change for the design, management and monitoring of the network must be considered. In particular, there needs to be a greater focus on: i) incorporating ecological principles that directly address climate change ii) effective protection that builds resilience of the marine and linked social environment iii) developing a focused, strong and adaptable monitoring framework iv) ensuring mechanisms for adaptive management.

Analysis and modelling of immune cell behaviour in lymph nodes based on multi-photon imaging

This thesis presents quantitative studies of T cell and dendritic cell (DC) behaviour in mouse lymph nodes (LNs) in the naive state and following immunisation. These processes are of importance and interest in basic immunology, and better understanding could improve both diagnostic capacity and therapeutic manipulations, potentially helping in producing more effective vaccines or developing treatments for autoimmune diseases. The problem is also interesting conceptually as it is relevant to other fields where 3D movement of objects is tracked with a discrete scanning interval. A general immunology introduction is presented in chapter 1. In chapter 2, I apply quantitative methods to multi-photon imaging data to measure how T cells and DCs are spatially arranged in LNs. This has been previously studied to describe differences between the naive and immunised state and as an indicator of the magnitude of the immune response in LNs, but previous analyses have been generally descriptive. The quantitative analysis shows that some of the previous conclusions may have been premature. In chapter 3, I use Bayesian state-space models to test some hypotheses about the mode of T cell search for DCs. A two-state mode of movement where T cells can be classified as either interacting to a DC or freely migrating is supported over a model where T cells would home in on DCs at distance through for example the action of chemokines. In chapter 4, I study whether T cell migration is linked to the geometric structure of the fibroblast reticular network (FRC). I find support for the hypothesis that the movement is constrained to the fibroblast reticular cell (FRC) network over an alternative 'random walk with persistence time' model where cells would move randomly, with a short-term persistence driven by a hypothetical T cell intrinsic 'clock'. I also present unexpected results on the FRC network geometry. Finally, a quantitative method is presented for addressing some measurement biases inherent to multi-photon imaging. In all three chapters, novel findings are made, and the methods developed have the potential for further use to address important problems in the field. In chapter 5, I present a summary and synthesis of results from chapters 3-4 and a more speculative discussion of these results and potential future directions.

Exploring complex chemical systems: insights from mass spectrometry, electrochemistry and continuous cell culture

The work presented herein covers a broad range of research topics and so, in the interest of clarity, has been presented in a portfolio format. Accordingly, each chapter consists of its own introductory material prior to presentation of the key results garnered, this is then proceeded by a short discussion on their significance. In the first chapter, a methodology to facilitate the resolution and qualitative assessment of very large inorganic polyoxometalates was designed and implemented employing ion-mobility mass spectrometry. Furthermore, the potential of this technique for ‘mapping’ the conformational space occupied by this class of materials was demonstrated. These claims are then substantiated by the development of a tuneable, polyoxometalate-based calibration protocol that provided the necessary platform for quantitative assessments of similarly large, but unknown, polyoxometalate species. In addition, whilst addressing a major limitation of travelling wave ion mobility, this result also highlighted the potential of this technique for solution-phase cluster discovery. The second chapter reports on the application of a biophotovoltaic electrochemical cell for characterising the electrogenic activity inherent to a number of mutant Synechocystis strains. The intention was to determine the key components in the photosynthetic electron transport chain responsible for extracellular electron transfer. This would help to address the significant lack of mechanistic understanding in this field. Finally, in the third chapter, the design and fabrication of a low-cost, highly modular, continuous cell culture system is presented. To demonstrate the advantages and suitability of this platform for experimental evolution investigations, an exploration into the photophysiological response to gradual iron limitation, in both the ancestral wild type and a randomly generated mutant library population, was undertaken. Furthermore, coupling random mutagenesis to continuous culture in this way is shown to constitute a novel source of genetic variation that is open to further investigation.

Unravelling metabolism of Leishmania by metabolomics

The leishmaniases are neglected tropical diseases with an urgent need for effective drugs. Better understanding of the metabolism of the causative parasites will hopefully lead to development of new compounds targeted at critical points of the parasite’s biochemical pathways. In my work I focused on the pentose phosphate pathway of Leishmania, specifically on transketolase, sugar utilisation, and comparison between insect and mammalian infective stages of the parasites. The pentose phosphate pathway (PPP) is the major cellular source of NADPH, an agent critical for oxidative stress defence. The PPP uses glucose, reduces the NADP+ cofactor and produces various sugar phosphates by mutual interconversions. One of the enzymes involved in this latter part is transketolase (TKT). A Leishmania mexicana cell line deleted in transketolase (Δtkt) was assessed regarding viability, sensitivity to a range of drugs, changes in metabolism, and infectivity. The Δtkt cell line had no obvious growth defect in the promastigote stage, but it was more sensitive to an oxidative stress inducing agent and most of the drugs tested. Most importantly, the Δtkt cells were not infective to mice, establishing TKT as a new potential drug target. Metabolomic analyses revealed multiple changes as a consequence of TKT deletion. Levels of the PPP intermediates upstream of TKT increased substantially, and were diverted into additional reactions. The perturbation triggered further changes in metabolism, resembling the ‘stringent metabolic response’ of amastigotes. The Δtkt cells consumed less glucose and glycolytic intermediates were decreased indicating a decrease in flux, and metabolic end products were diminished in production. The decrease in glycolysis was possibly caused by inhibition of fructose-1,6-bisphosphate aldolase by accumulation of the PPP intermediates 6-phosphogluconate and ribose 5-phosphate. The TCA cycle was fuelled by alternative carbon sources, most likely amino acids, instead of glucose. It remains unclear why deletion of TKT is lethal for amastigotes, increased sensitivity to oxidative stress or drop in mannogen levels may contribute, but no definite conclusions can be made. TKT localisation indicated interesting trends too. The WT enzyme is present in the cytosol and glycosomes, whereas a mutant version, truncated by ten amino acids, but retaining a C-terminal targeting sequence, localised solely to glycosomes. Surprisingly, cells expressing purely cytosolic or glycosomal TKT did not have different phenotypes regarding growth, oxidative stress sensitivity or any detected changes in metabolism. Hence, control of the subcellular localisation remains unclear as well as its function. However, these data are in agreement with the presumed semipermeable nature of the glycosome. Further, L. mexicana promastigote cultures were grown in media with different combinations of labelled glucose and ribose and their incorporation into metabolism was followed. Glucose was the preferred carbon source, but when not available, it could be fully replaced with ribose. I also compared metabolic profiles from splenic amastigotes, axenic amastigotes and promastigotes of L. donovani. Metabolomic analysis revealed a substantial drop in amino acids and other indications coherent with a stringent metabolic response in amastigotes. Despite some notable differences, axenic and splenic amastigotes demonstrated fairly similar results both regarding the total metabolic profile and specific metabolites of interest.

Development of radiotracers for nuclear imaging of poly(ADP-ribose) polymerase-1 and the translocator protein in glioblastoma.

Glioblastoma (GBM) is a highly aggressive and fatal brain cancer that is associated with a number of diagnostic, therapeutic, and treatment monitoring challenges. At the time of writing, inhibition of a protein called poly (ADP-ribose) polymerase-1 (PARP-1) in combination with chemotherapy was being investigated as a novel approach for the treatment of these tumours. However, human studies have encountered toxicity problems due to sub-optimal PARP-1 inhibitor and chemotherapeutic dosing regiments. Nuclear imaging of PARP-1 could help to address these issues and provide additional insight into potential PARP-1 inhibitor resistance mechanisms. Furthermore, nuclear imaging of the translocator protein (TSPO) could be used to improve GBM diagnosis, pre-surgical planning, and treatment monitoring as TSPO is overexpressed by GBM lesions in good contrast to surrounding brain tissue. To date, relatively few nuclear imaging radiotracers have been discovered for PARP-1. On the other hand, numerous tracers exist for TSPO many of which have been investigated in humans. However, these TSPO radiotracers suffer from either poor pharmacokinetic properties or high sensitivity to human TSPO polymorphism that can affect their binding to TSPO. Bearing in mind the above and the high attrition rates associated with advancement of radiotracers to the clinic, there is a need for novel radiotracers that can be used to image PARP-1 and TSPO. This thesis reports the pre-clinical discovery programme that led to the identification of two potent PARP-1 inhibitors, 4 and 17, that were successfully radiolabelled to generate the potential SPECT and PET imaging agents [123I]-4 and [18F]-17 respectively. Evaluation of these radiotracers in mice bearing subcutaneous human GBM xenografts using ex vivo biodistribution techniques revealed that the agents were retained in tumour tissue due to specific PARP-1 binding. This thesis also describes the pre-clinical in vivo evaluation of [18F]-AB5186, which is a novel radiotracer discovered previously within the research group with potential for PET imaging of TSPO. Using ex vivo autoradiography and PET imaging the agent was revealed to accumulate in intracranial human GBM tumour xenografts in good contrast to surrounding brain tissue, which was due to specific binding to TSPO. The in vivo data for all three radiolabelled compounds warrants further pre-clinical investigations with potential for clinical advancement in mind.

Natural and anthropogenic factors controlling algae growth in the Ythan Estuary, Aberdeenshire

The Ythan Estuary in Aberdeenshire is a Site of Special Scientific Interest due to the numerous protected species which incorporate it into their ecosystem. In recent years algae growth in the estuary has been seen to increase, causing concern over the integrity of the estuary as a habitat for wildlife. By examining sediment, river flow, nutrient levels and temperature it is possible to identify the driving factors behind this increased algal growth. Using data gathered in the field as well as historical climate data the contribution of each of these factors to algae growth can be determined.

Phylodynamic modelling of foot-and-mouth disease virus sequence data

The under-reporting of cases of infectious diseases is a substantial impediment to the control and management of infectious diseases in both epidemic and endemic contexts. Information about infectious disease dynamics can be recovered from sequence data using time-varying coalescent approaches, and phylodynamic models have been developed in order to reconstruct demographic changes of the numbers of infected hosts through time. In this study I have demonstrated the general concordance between empirically observed epidemiological incidence data and viral demography inferred through analysis of foot-and-mouth disease virus VP1 coding sequences belonging to the CATHAY topotype over large temporal and spatial scales. However a more precise and robust relationship between the effective population size (

Development of novel therapeutics and in vitro models for spinal cord injury

Spinal cord injury (SCI) is a devastating condition, which results from trauma to the cord, resulting in a primary injury response which leads to a secondary injury cascade, causing damage to both glial and neuronal cells. Following trauma, the central nervous system (CNS) fails to regenerate due to a plethora of both intrinsic and extrinsic factors. Unfortunately, these events lead to loss of both motor and sensory function and lifelong disability and care for sufferers of SCI. There have been tremendous advancements made in our understanding of the mechanisms behind axonal regeneration and remyelination of the damaged cord. These have provided many promising therapeutic targets. However, very few have made it to clinical application, which could potentially be due to inadequate understanding of compound mechanism of action and reliance on poor SCI models. This thesis describes the use of an established neural cell co-culture model of SCI as a medium throughput screen for compounds with potential therapeutic properties. A number of compounds were screened which resulted in a family of compounds, modified heparins, being taken forward for more intense investigation. Modified heparins (mHeps) are made up of the core heparin disaccharide unit with variable sulphation groups on the iduronic acid and glucosamine residues; 2-O-sulphate (C2), 6-O-sulphate (C6) and N-sulphate (N). 2-O-sulphated (mHep6) and N-sulphated (mHep7) heparin isomers were shown to promote both neurite outgrowth and myelination in the SCI model. It was found that both mHeps decreased oligodendrocyte precursor cell (OPC) proliferation and increased oligodendrocyte (OL) number adjacent to the lesion. However, there is a difference in the direct effects on the OL from each of the mHeps; mHep6 increased myelin internode length and mHep7 increased the overall cell size. It was further elucidated that these isoforms interact with and mediate both Wnt and FGF signalling. In OPC monoculture experiments FGF2 treated OPCs displayed increased proliferation but this effect was removed when co-treated with the mHeps. Therefore, suggesting that the mHeps interact with the ligand and inhibit FGF2 signalling. Additionally, it was shown that both mHeps could be partially mediating their effects through the Wnt pathway. mHep effects on both myelination and neurite outgrowth were removed when co-treated with a Wnt signalling inhibitor, suggesting cell signalling mediation by ligand immobilisation and signalling activation as a mechanistic action for the mHeps. However, the initial methods employed in this thesis were not sufficient to provide a more detailed study into the effects the mHeps have on neurite outgrowth. This led to the design and development of a novel microfluidic device (MFD), which provides a platform to study of axonal injury. This novel device is a three chamber device with two chambers converging onto a central open access chamber. This design allows axons from two points of origin to enter a chamber which can be subjected to injury, thus providing a platform in which targeted axonal injury and the regenerative capacity of a compound study can be performed. In conclusion, this thesis contributes to and advances the study of SCI in two ways; 1) identification and investigation of a novel set of compounds with potential therapeutic potential i.e. desulphated modified heparins. These compounds have multiple therapeutic properties and could revolutionise both the understanding of the basic pathological mechanisms underlying SCI but also be a powered therapeutic option. 2) Development of a novel microfluidic device to study in greater detail axonal biology, specifically, targeted axonal injury and treatment, providing a more representative model of SCI than standard in vitro models. Therefore, the MFD could lead to advancements and the identification of factors and compounds relating to axonal regeneration.

Filling in gaps of Drosophila melanogaster urate degradation metabolic pathway using metabolomics approaches: towards the core metabolome of the fruit fly

The primary goal of systems biology is to integrate complex omics data, and data obtained from traditional experimental studies in order to provide a holistic understanding of organismal function. One way of achieving this aim is to generate genome-scale metabolic models (GEMs), which contain information on all metabolites, enzyme-coding genes, and biochemical reactions in a biological system. Drosophila melanogaster GEM has not been reconstructed to date. Constraint-free genome-wide metabolic model of the fruit fly has been reconstructed in our lab, identifying gaps, where no enzyme was identified and metabolites were either only produced or consume. The main focus of the work presented in this thesis was to develop a pipeline for efficient gap filling using metabolomics approaches combined with standard reverse genetics methods, using 5-hydroxyisourate hydrolase (5-HIUH) as an example. 5-HIUH plays a role in urate degradation pathway. Inability to degrade urate can lead to inborn errors of metabolism (IEMs) in humans, including hyperuricemia. Based on sequence analysis Drosophila CG30016 gene was hypothesised to encode 5- HIUH. CG30016 knockout flies were examined to identify Malpighian tubules phenotype, and shortened lifespan might reflect kidney disorders in hyperuricemia in humans. Moreover, LC-MS analysis of mutant tubules revealed that CG30016 is involved in purine metabolism, and specifically urate degradation pathway. However, the exact role of the gene has not been identified, and the complete method for gap filling has not been developed. Nevertheless, thanks to the work presented here, we are a step closer towards the development of a gap-filling pipeline in Drosophila melanogaster GEM. Importantly, the areas that require further optimisation were identified and are the focus of future research. Moreover, LC-MS analysis confirmed that tubules rather than the whole fly were more suitable for metabolomics analysis of purine metabolism. Previously, Dow/Davies lab has generated the most complete tissue-specific transcriptomic atlas for Drosophila – FlyAtlas.org, which provides data on gene expression across multiple tissues of adult fly and larva. FlyAtlas revealed that transcripts of many genes are enriched in specific Drosophila tissues, and that it is possible to deduce the functions of individual tissues within the fly. Based on FlyAtlas data, it has become clear that the fly (like other metazoan species) must be considered as a set of tissues, each 2 with its own distinct transcriptional and functional profile. Moreover, it revealed that for about 30% of the genome, reverse genetic methods (i.e. mutation in an unknown gene followed by observation of phenotype) are only useful if specific tissues are investigated. Based on the FlyAtlas findings, we aimed to build a primary tissue-specific metabolome of the fruit fly, in order to establish whether different Drosophila tissues have different metabolomes and if they correspond to tissue-specific transcriptome of the fruit fly (FlyAtlas.org). Different fly tissues have been dissected and their metabolome elucidated using LC-MS. The results confirmed that tissue metabolomes differ significantly from each other and from the whole fly, and that some of these differences can be correlated to the tissue function. The results illustrate the need to study individual tissues as well as the whole organism. It is clear that some metabolites that play an important role in a given tissue might not be detected in the whole fly sample because their abundance is much lower in comparison to other metabolites present in all tissues, which prevent the detection of the tissue-specific compound.

Chiroptical spectroscopy of biomolecules using chiral plasmonic nanostructures

This thesis explores the potential of chiral plasmonic nanostructures for the ultrasensitive detection of protein structure. These nanostructures support the generation of fields with enhanced chirality relative to circularly polarised light and are an extremely incisive probe of protein structure. In chapter 4 we introduce a nanopatterned Au film (Templated Plasmonic Substrate, TPS) fabricated using a high through-put injection moulding technique which is a viable alternative to expensive lithographically fabricated nanostructures. The optical and chiroptical properties of TPS nanostructures are found to be highly dependent on the coupling between the electric and magnetic modes of the constituent solid and inverse structures. Significantly, refractive index based measurements of strongly coupled TPSs display a similar sensitivity to protein structure as previous lithographic nanostructures. We subsequently endeavour to improve the sensing properties of TPS nanostructures by developing a high through-put nanoscale chemical functionalisation technique. This process involves a chemical protection/deprotection strategy. The protection step generates a self-assembled monolayer (SAM) of a thermally responsive polymer on the TPS surface which inhibits protein binding. The deprotection step exploits the presence of nanolocalised thermal gradients in the water surrounding the TPS upon irradiation with an 8ns pulsed laser to modify the SAM conformation on surfaces with high net chirality. This allows binding of biomaterial in these regions and subsequently enhances the TPS sensitivity levels. In chapter 6 an alternative method for the detection of protein structure using TPS nanostructures is introduced. This technique relies on mediation of the electric/magnetic coupling in the TPS by the adsorbed protein. This phenomenon is probed through both linear reflectance and nonlinear second harmonic generation (SHG) measurements. Detection of protein structure using this method does not require the presence of fields of enhanced chirality whilst it is also sensitive to a larger array of secondary structure motifs than the measurements in chapters 4 and 5. Finally, a preliminary investigation into the detection of mesoscale biological structure is presented. Sensitivity to the mesoscale helical pitch of insulin amyloid fibrils is displayed through the asymmetry in the circular dichroism (CD) of lithographic gammadions of varying thickness upon adsorption of insulin amyloid fibril spherulites and fragmented fibrils. The proposed model for this sensitivity to the helical pitch relies on the vertical height of the nanostructures relative to this structural property as well as the binding orientation of the fibrils.

The influence of actions on auditory perception: cognitive and neural mechanisms

It is well known that self-generated stimuli are processed differently from externally generated stimuli. For example, many people have noticed since childhood that it is very difficult to make a self-tickling. In the auditory domain, self-generated sounds elicit smaller brain responses as compared to externally generated sounds, known as the sensory attenuation (SA) effect. SA is manifested in reduced amplitudes of evoked responses as measured through MEEG, decreased firing rates of neurons and a lower level of perceived loudness for self-generated sounds. The predominant explanation for SA is based on the idea that self-generated stimuli are predicted (e.g., the forward model account). It is the nature of their predictability that is crucial for SA. On the contrary, the sensory gating account emphasizes a general suppressive effect of actions on sensory processing, regardless of the predictability of the stimuli. Both accounts have received empirical support, which suggests that both mechanisms may exist. In chapter 2, three behavioural studies concerning the influence of motor activation on auditory perception were presented. Study 1 compared the effect of SA and attention in an auditory detection task and showed that SA was present even when substantial attention was paid to unpredictable stimuli. Study 2 compared the loudness perception of tones generated by others between Chinese and British participants. Compared to externally generated tones, a decrease in perceived loudness for others generated tones was found among Chinese but not among the British. In study 3, partial evidence was found that even when reading words that are related to action, auditory detection performance was impaired. In chapter 3, the classic SA effect of M100 suppression was replicated with MEG in study 4. With time-frequency analysis, a potential neural information processing sequence was found in auditory cortex. Prior to the onset of self-generated tones, there was an increase of oscillatory power in the alpha band. After the stimulus onset, reduced gamma power and alpha/beta phase locking were found. The three temporally segregated oscillatory events correlated with each other and with SA effect, which may be the underlying neural implementation of SA. In chapter 4, a TMS-MEG study was presented investigating the role of the cerebellum in adapting to delayed presentation of self-generated tones (study 5). It demonstrated that in sham stimulation condition, the brain can adapt to the delay (about 100 ms) within 300 trials of learning by showing a significant increase of SA effect in the suppression of M100, but not M200 component. Whereas after stimulating the cerebellum with a suppressive TMS protocol, the adaptation in M100 suppression disappeared and the pattern of M200 suppression reversed to M200 enhancement. These data support the idea that the suppressive effect of actions on auditory processing is a consequence of both motor driven sensory predictions and general sensory gating. The results also demonstrate the importance of neural oscillations in implementing SA effect and the critical role of the cerebellum in learning sensory predictions under sensory perturbation.

Biomimetic topography in orthopaedic ceramic

The primary objective of this research was to perform an in vitro assessment of the ability of microscale topography to alter cell behaviour, with specific regard to producing favourable topography in an orthopaedic ceramic material suitable for implantation in the treatment of arthritis. Topography at microscale and nanoscale alters the bioactivity of the material. This has been used in orthopaedics for some time as seen with optimal pore size in uncemented hip and knee implants. This level of topography involves scale in hundreds of micrometres and allows for the ingrowth of tissue. Topography at smaller scale is possible thanks to progressive miniaturisation of technology. A topographic feature was created in a readily available clinically licensed polymer, Polycaprolcatone (PCL). The effect of this topography was assessed in vitro. The same topography was transferred to the latest generation composite orthopaedic ceramic, zirconia toughened alumina (ZTA). The fidelity of reproduction of the topography was examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM). These investigations showed more accurate reproduction of the topography in PCL than ZTA with some material artefacts in the ZTA. Cell culture in vitro was performed on the patterned substrates. The response of osteoprogenitor cells was assessed using immunohistochemistry, real-time polymerase chain reaction and alizarin staining. These results showed a small effect on cell behaviour. Finally metabolic comparison was made of the effects created by the two different materials and the topography in each. The results have shown a reproducible topography in orthopaedic ceramics. This topography has demonstrated a positive osteogenic effect in both polycaprolactone and zirconia toughened alumina across multiple assessment modalities.