Bioengineering strategies to improve functional qualities of nisin

The abuse of antibiotics and the emergence of multi-drug resistant bacterial strains have created the need to explore alternative methods of controlling microbial pathogens. The bacteriocin family of antimicrobial peptides has been proposed as one such alternative to classic antibiotics. Nisin A belongs to the subgroup of bacteriocins called the lantibiotics, which contain several unusual amino acids as a consequence of enzyme-mediated post-translational modifications. As nisin is produced by generally regarded as safe (GRAS) microorganisms, it could potentially be applied in a clinical setting. However, as lantibiotics are naturally produced in such small quantities, this can hinder their industrial potential. In order to overcome this, several approaches can be utilised. For example, given the gene encoded nature of lantibiotics, genetic engineering approaches can be implemented in order to yield variants with enhanced properties. Here, the use of mutagenesis-based strategies was employed to obtain a derivative of nisin with enhanced bioactivity in vitro. Investigations with purified peptide highlighted the enhanced specific activity of this variant, nisin M21V, against food-borne Listeria monocytogenes strains. Furthermore, this specific enhanced bioactivity was evident in a mouse model of listeriosis. Reductions in bioluminescence and microbial counts in organs from infected mice were observed following treatment with nisin M21V compared to that of wild-type nisin A. Peptide bioengineering approaches were also implemented to obtain additional novel derivatives of nisin. The generation of “S5X” and “S33X” banks (representing a change of natural serines at positions 5 and 33 to all possible alternative residues) by a combination of site-saturation and site-directed mutagenesis led to the identification of several derivatives exhibiting improved stability. This allowed the rational design of variants with enhanced stability compared to that of wild type nisin. Another means of tackling issues associated with lantibiotic yield is to combine lantibiotics with other antimicrobials. This could circumvent the need for enhanced production while also reducing concentrations of the peptide antimicrobials. We observed that combinations of nisin variants and low levels of plant essential oils (thymol, carvacrol, trans-cinnamaldehyde) significantly controlled Gram negative foodborne pathogens in in vitro assays compared to nisin A-essential oil combinations. This enhanced control was also evident in model food systems. Nisin variants used in conjunction with carvacrol significantly reduced numbers of E. coli O157:H7 in apple juice while a commercial nisin preparation used in combination with citric acid significantly controlled C. sakazakii in infant milk formula. It is noteworthy that while nisin is generally associated with Gram positive targets, upon combination with plant essential oils the spectrum of inhibition was broadened to Gram negative targets.

The impacts of past land-use on the ecology of an ancient woodland in south-west Ireland

A multi-disciplinary study was conducted to compare stands of ancient and secondary origin within a single wood, the Gearagh woodland, County Cork. These sites were compared with adjacent areas of grassland, which provided a reference for the former land-use (pasture) of the secondary woodland. A historical study confirmed that while the core of the Gearagh has been subject to minimal human interference, other sections have been cleared in the past for agricultural purposes. Investigations into soil structure and composition showed that soil properties in these secondary woodland areas were significantly altered by this past woodland clearance and conversion to agriculture, while the soil of the ancient woodland showed little signs of disturbance. The vegetation community also differed between the two woodland areas, partly due to altered environmental conditions. Many of the ancient woodland plant species were unable to form a persistent seed bank, while there was increased representation of species associated with more open-habitat conditions in the seed bank of the secondary woodland. While germination of woodland species was low in all sites, overall, seeds tended to germinate more successfully in the ancient woodland. The ancient woodland also provided a suitable habitat for many soil and ground detritivores, most notably enchytraeids, although earthworms were not abundant. Past agricultural use, however, changed the decomposer community considerably, with increased representation of earthworm species and a decline in the abundance of enchytraeids in the secondary stands. In conclusion, the legacies of historical agricultural activities can continue to significantly affect the structure and composition of present-day woodlands so that they may differ considerably from undisturbed ancient woodland stands, even within the same woodland. A greater understanding of the origin, development and ecological functioning of ancient woodlands should aid in determining future conservation and management requirements.

Biodiversity change in the Irish uplands - the effects of grazing management

As a prominent form of land use across much of upland Europe, extensive livestock grazing may hold the key to the sustainable management of these landscapes. Recent agricultural policy reform, however, has resulted in a decline in upland sheep numbers, prompting concern for the biodiversity value of these areas. This study quantifies the effects of varying levels of grazing management on plant, ground beetle and breeding bird diversity and assemblage in the uplands and lowlands of hill sheep farms in County Kerry, Ireland. Farms represent a continuum of light to heavy grazing, measured using a series of field indicators across several habitats, such as the internationally important blanket bog, home to the ground beetle, Carabus clatratus. Linear mixed effects modelling and non-metric multidimensional scaling are employed to disentangle the most influential management and environmental factors. Grazing state may be determined by the presence of Molinia caerulea or Nardus stricta, and variables such as % traditional ewes, % vegetation litter and % scrub prove valuable indicators of diversity. Measures of ecosystem functioning, e.g. plant biomass (nutrient cycling) and % vegetation cover (erosion rates) are influenced by plant diversity, which is influenced by grazing management. Levels of the ecosystem service, soil organic carbon, vary with ground beetle abundance and diversity, potentially influencing carbon sequestration and thereby climate change. The majority of species from all three taxa are found in the lowlands, with the exception of birds such as meadow pipit and skylark. The scale of measurement should be determined by the size and mobility of the species in question. The challenge is to manage these high nature value landscapes using agri-environment schemes which enhance biodiversity by maintaining structural heterogeneity across a range of scales, altitudes and habitats whilst integrating the decisions of people living and working in these marginal areas.

Environmental and biological characteristics of lagoons on the southwest coast of Ireland

Coastal lagoons are defined as shallow coastal water bodies partially separated from the adjacent sea by a restrictive barrier. Coastal lagoons are protected under Annex I of the European Habitats Directive (92/43/EEC). Lagoons are also considered to be “transitional water bodies” and are therefore included in the “register of protected areas” under the Water Framework Directive (2000/60/EC). Consequently, EU member states are required to establish monitoring plans and to regularly report on lagoon condition and conservation status. Irish lagoons are considered relatively rare and unusual because of their North Atlantic, macrotidal location on high energy coastlines and have received little attention. This work aimed to assess the physicochemical and ecological status of three lagoons, Cuskinny, Farranamanagh and Toormore, on the southwest coast of Ireland. Baseline salinity, nutrient and biological conditions were determined in order to provide reference conditions to detect perturbations, and to inform future maintenance of ecosystem health. Accumulation of organic matter is an increasing pressure in coastal lagoon habitats worldwide, often compounding existing eutrophication problems. This research also aimed to investigate the in situ decomposition process in a lagoon habitat together with exploring the associated invertebrate assemblages. Re-classification of the lagoons, under the guidelines of the Venice system for the classifications of marine waters according to salinity, was completed by taking spatial and temporal changes in salinity regimes into consideration. Based on the results of this study, Cuskinny, Farranamanagh and Toormore lagoons are now classified as mesohaline (5 ppt – 18 ppt), oligohaline (0.5 ppt – 5 ppt) and polyhaline (18 ppt – 30 ppt), respectively. Varying vertical, longitudinal and transverse salinity patterns were observed in the three lagoons. Strong correlations between salinity and cumulative rainfall highlighted the important role of precipitation in controlling the lagoon environment. Maximum effect of precipitation on the salinity of the lagoon was observed between four and fourteen days later depending on catchment area geology, indicating the uniqueness of each lagoon system. Seasonal nutrient patterns were evident in the lagoons. Nutrient concentrations were found to be reflective of the catchment area and the magnitude of the freshwater inflow. Assessment based on the Redfield molar ratio indicated a trend towards phosphorus, rather than nitrogen, limitation in Irish lagoons. Investigation of the decomposition process in Cuskinny Lagoon revealed that greatest biomass loss occurred in the winter season. Lowest biomass loss occurred in spring, possibly due to the high density of invertebrates feeding on the thick microbial layer rather than the decomposing litter. It has been reported that the decomposition of plant biomass is highest in the preferential distribution area of the plant species; however, no similar trend was observed in this study with the most active zones of decomposition varying spatially throughout the seasons. Macroinvertebrate analysis revealed low species diversity but high abundance, indicating the dominance of a small number of species. Invertebrate assemblages within the lagoon varied significantly from communities in the adjacent freshwater or marine environments. Although carried out in coastal lagoons on the southwest coast of Ireland, it is envisaged that the overall findings of this study have relevance throughout the entire island of Ireland and possibly to many North Atlantic coastal lagoon ecosystems elsewhere.

Efficient delivery of scalable media streaming over lossy networks

Recent years have witnessed a rapid growth in the demand for streaming video over the Internet, exposing challenges in coping with heterogeneous device capabilities and varying network throughput. When we couple this rise in streaming with the growing number of portable devices (smart phones, tablets, laptops) we see an ever-increasing demand for high-definition videos online while on the move. Wireless networks are inherently characterised by restricted shared bandwidth and relatively high error loss rates, thus presenting a challenge for the efficient delivery of high quality video. Additionally, mobile devices can support/demand a range of video resolutions and qualities. This demand for mobile streaming highlights the need for adaptive video streaming schemes that can adjust to available bandwidth and heterogeneity, and can provide us with graceful changes in video quality, all while respecting our viewing satisfaction. In this context the use of well-known scalable media streaming techniques, commonly known as scalable coding, is an attractive solution and the focus of this thesis. In this thesis we investigate the transmission of existing scalable video models over a lossy network and determine how the variation in viewable quality is affected by packet loss. This work focuses on leveraging the benefits of scalable media, while reducing the effects of data loss on achievable video quality. The overall approach is focused on the strategic packetisation of the underlying scalable video and how to best utilise error resiliency to maximise viewable quality. In particular, we examine the manner in which scalable video is packetised for transmission over lossy networks and propose new techniques that reduce the impact of packet loss on scalable video by selectively choosing how to packetise the data and which data to transmit. We also exploit redundancy techniques, such as error resiliency, to enhance the stream quality by ensuring a smooth play-out with fewer changes in achievable video quality. The contributions of this thesis are in the creation of new segmentation and encapsulation techniques which increase the viewable quality of existing scalable models by fragmenting and re-allocating the video sub-streams based on user requirements, available bandwidth and variations in loss rates. We offer new packetisation techniques which reduce the effects of packet loss on viewable quality by leveraging the increase in the number of frames per group of pictures (GOP) and by providing equality of data in every packet transmitted per GOP. These provide novel mechanisms for packetizing and error resiliency, as well as providing new applications for existing techniques such as Interleaving and Priority Encoded Transmission. We also introduce three new scalable coding models, which offer a balance between transmission cost and the consistency of viewable quality.

Totality of experience: sound, music and mythology in Gus Van Sant’s “Death Quartet”

Reflecting on Gus Van Sant’s films Gerry (2003) Elephant (2004) and Last Days (2005), the director’s long-term sound-designer Leslie Shatz observed that “You have to get into the totality of the experience and not just the dialogue”. Shatz’s comment expresses something fundamental about the experimental approach to cinema and to soundscapes undertaken by Van Sant in these three films, unofficially known as the “Death Trilogy”. This thesis contends that Van Sant makes deliberate aesthetic choices which do indicate a distinctly “auteurist” leaning. However, I also argue that intertextual elements, prior knowledge, and audience participation in meaningmaking enhance the experience of, and reveal the nuances in, the soundtracks themselves. This thesis aims to contribute to a growing body of work within filmmusic scholarship concerned with resisting a traditional bias in the field: that film music should be understood as a means of characterisation and as emotional signifier. The films of the “Death Quartet”, which includes Paranoid Park (2007), I believe, offer fertile ground on which to explore these new approaches. It is my contention that these films deconstruct the traditional approach to soundtracking and the relationship between soundtrack and character, and that only an approach sensitive to the aesthetic and philosophical functions of music and sound can adequately acknowledge their unique cinematic qualities.

Monstrative acts and becoming-monster: on identity, bodies, and the feminine other

Using two examples of literary monsters, the Creature in Mary Shelley’s Frankenstein (1818), and Grendel’s Mother in Beowulf, this thesis demonstrates the bearing fictional identities have on “real” bodies, through an examination of two further literary texts, David Henry Hwang’s play, M. Butterfly (1986) and J. M. Coetzee’s novel, Disgrace (1999). Western definitions of Being have historically divided body and mind, favouring the mind as formative of subjective experience and denigrating the body as secondary and impure. This thesis demonstrates that this mind/body binary is symptomatic of the masculine ontological imperative to disown the body and its effects on Being, simultaneously ridding itself of the feminine it believes is its irrational opposite. Using recent feminist reviews of the canon, which emphasise the body’s importance to ontology and demonstrate the conceptual association between the feminine and the corporeal, this thesis links performative identity practices to theories of monstrosity, explaining how fictional qualities adhere to monstrous bodies by proposing a new theoretical category, the “monstrative.” The monstrative is a performative force that makes the Other into a living sign of Otherness; however, unlike earlier theories of Othering, the monstrative accounts for the Other’s being other to herself. This thesis also attempts to read the misrepresented body of the Other as a possible site for more empowered identity performances, where the monstrous “I” is interpreted as a potentially positive model for identity practice, through the conceptualisation of identity as a process of Becoming rather than Being. The transferal from a noun to a verb not only emphasises the performativity of identity, but also suggests fluidity and multiplicity in identity practice, which always already indicates a monstrosity at work. Thus, while monstrative acts constitute bodies as monstrous, Becoming-monster is an empathetic response to the Other’s monstrosity.

Artificial opal photonic crystals and inverse opal structures - fundamentals and applications from optics to energy storage

Photonic crystals (PhCs) influence the propagation of light by their periodic variation in dielectric contrast or refractive index. This review outlines the attractive optical qualities inherent to most PhCs namely the presence of full or partial photonic band gaps and the possibilities they present towards the inhibition of spontaneous emission and the localization of light. Colloidal self-assembly of polymer or silica spheres is one of the most favoured and low cost methods for the formation of PhCs as artificial opals. The state of the art in growth methods currently used for colloidal self-assembly are discussed and the use of these structures for the formation of inverse opal architectures is then presented. Inverse opal structures with their porous and interconnected architecture span several technological arenas - optics and optoelectronics, energy storage, communications, sensor and biological applications. This review presents several of these applications and an accessible overview of the physics of photonic crystal optics that may be useful for opal and inverse opal researchers in general, with a particular emphasis on the recent use of these three-dimensional porous structures in electrochemical energy storage technology. Progress towards all-optical integrated circuits may lie with the concepts of the photonic crystal, but the unique optical and structural properties of these materials and the convergence of PhC and energy storage disciplines may facilitate further developments and non-destructive optical analysis capabilities for (electro)chemical processes that occur within a wide variety of materials in energy storage research.