An analysis of selected secretion systems of Pseudomonas species

Direct secretion systems which deliver molecules from one cell to another have huge significance in shaping bacterial communities or in determining the outcome of bacterial associations with eukaryotic organisms. This work examines the roles of the Type III Secretion System (T3SS) and the Type VI Secretion System (T6SS) systems of Pseudomonas, a widespread genus including clinical pathogens and biocontrol strains. Bioinformatic analysis of T6SS phylogeny and associated gene content within Pseudomonas identified several T6SS phylogenetic groups, and linked T6SS components VgrG and Hcp encoded outside of T6SS gene loci with their cognate T6SS phylogenetic groups. Remarkably, such “orphan” vgrG and hcp genes were found to occur in diverse, horizontally transferred, operons often containing putative T6SS accessory components and effectors. The prevalence of a widespread superfamily of T6SS lipase effectors (Tle) was assessed in metagenomes from various environments. The abundance of the Tle superfamily and individual families varied between niches, suggesting there is niche specific selection and specialisation of Tle. Experimental work also discovered that P. fluorescens F113 uses the SPI-1 T3SS to avoid amoeboid grazing in mixed populations. This finding may represent a significant aspect of F113 rhizocompetence, and the rhizocompetence of other Rhizobacteria.

Childhood obesity treatment: integrating mobile health technology into a paediatric obesity service

Background: The management of childhood obesity is challenging. Aims: Thesis, i) reviews the evidence for lifestyle treatment of obesity, ii) explores cardiometabolic burden in childhood obesity, iii) explores whether changes in body composition predicts change in insulin sensitivity (IS), iv) develops and evaluates a lifestyle obesity intervention; v) develops a mobile health application for obesity treatment and vi) tests the application in a clinical trial. Methods: In Study 1, systematic reviews and meta-analyses of the 12‐month effects of lifestyle and mHealth interventions were conducted. In Study 2, the prevalence of cardiometabolic burden was estimated in a consecutive series of 267 children. In Study 3, body composition was estimated with bioelectrical impedance analysis (BIA) and dual x-ray absorptiometry (DXA) and linear regression analyses were used to estimate the extent to which each methods predicted change in IS. Study 4 describes the development of the Temple Street W82GO Healthy Lifestyle intervention for clinical obesity in children and a controlled study of treatment effect in 276 children is reported. Study 5 describes the development and testing of the Reactivate Mobile Obesity Application. Study 6 outlines the development and preliminary report from a clinical effectiveness trial of Reactivate. Results: In Study 1, meta--‐analyses BMI SDS changed by -0.16 (-0.24,‐0.07, p<0.01) and -0.03 (-0.13, 0.06, p=0.48). In study 2, cardiometabolic comorbidities were common (e.g. hypertension in 49%) and prevalence increased as obesity level increased. In Study 3, BC changes significantly predicted changes in IS. In Study 4, BMI SDS was significantly reduced in W82GO compared to controls (p<0.001). In Study 5, the Reactivate application had good usability indices and preliminary 6‐month process report data from Study 6, revealed a promising effect for Reactivate. Conclusions: W82GO and Reactivate are promising forms of treatment.

Education - research - practice

Through this paper we will look at links between architecture education, research and practice, using a current project as a vehicle to cover aspects of building, pilot and live project. The first aspect, the building project consists of the refurbishment and extension of a Parnell Cottage for a private client and is located near Cloyne, in East Cork, Ireland. The pilot project falls within the NEES Project, investigating the use of materials and services based on natural or recycled materials to improve the energy performance of new and existing buildings. The live project aims to hold a series of on site workshops and seminars for students of Architecture, Architects and interested parties, demonstrating the integration of the NEES best practice materials and techniques within the built project. The workshops, seminars and key project documents will be digitally recorded for dissemination through a web based publication. The small scale of the building project allowed for flexibility in the early conceptual design stages and the integration of the research and educational aspects.

A facilities maintenance management process based on degradation prediction using sensed data

Energy efficiency and user comfort have recently become priorities in the Facility Management (FM) sector. This has resulted in the use of innovative building components, such as thermal solar panels, heat pumps, etc., as they have potential to provide better performance, energy savings and increased user comfort. However, as the complexity of components increases, the requirement for maintenance management also increases. The standard routine for building maintenance is inspection which results in repairs or replacement when a fault is found. This routine leads to unnecessary inspections which have a cost with respect to downtime of a component and work hours. This research proposes an alternative routine: performing building maintenance at the point in time when the component is degrading and requires maintenance, thus reducing the frequency of unnecessary inspections. This thesis demonstrates that statistical techniques can be used as part of a maintenance management methodology to invoke maintenance before failure occurs. The proposed FM process is presented through a scenario utilising current Building Information Modelling (BIM) technology and innovative contractual and organisational models. This FM scenario supports a Degradation based Maintenance (DbM) scheduling methodology, implemented using two statistical techniques, Particle Filters (PFs) and Gaussian Processes (GPs). DbM consists of extracting and tracking a degradation metric for a component. Limits for the degradation metric are identified based on one of a number of proposed processes. These processes determine the limits based on the maturity of the historical information available. DbM is implemented for three case study components: a heat exchanger; a heat pump; and a set of bearings. The identified degradation points for each case study, from a PF, a GP and a hybrid (PF and GP combined) DbM implementation are assessed against known degradation points. The GP implementations are successful for all components. For the PF implementations, the results presented in this thesis find that the extracted metrics and limits identify degradation occurrences accurately for components which are in continuous operation. For components which have seasonal operational periods, the PF may wrongly identify degradation. The GP performs more robustly than the PF, but the PF, on average, results in fewer false positives. The hybrid implementations, which are a combination of GP and PF results, are successful for 2 of 3 case studies and are not affected by seasonal data. Overall, DbM is effectively applied for the three case study components. The accuracy of the implementations is dependant on the relationships modelled by the PF and GP, and on the type and quantity of data available. This novel maintenance process can improve equipment performance and reduce energy wastage from BSCs operation.

Sex offender risk assessment and management in Ireland

This research examined sex offender risk assessment and management in Ireland. It focused on the statutory agencies with primary responsibility (Garda Síochána and the Probation Service). The goal was to document the historical, contextual and current systems, in addition to identifying areas of concern/improvements. The research was a mixed-methods approach. Eight studies were conducted. This incorporated documentary reviews of four Commission to Inquire Reports, qualitative interviews/focus groups with Garda staff, Probation Service staff, statutory agencies, community stakeholders, various Non-Governmental Organisations (NGOs) and sex offenders. Quantitative questionnaires were also administered to Garda staff. In all over 70 interviews were conducted and questionnaires were forwarded to 270 Garda members. The overall findings are: •Sex offender management in Ireland has become formal only since 2001. Knowledge, skills and expertise is in its infancy and is still evolving. •Mixed reviews and questions regarding fitness for purpose of currently used risk assessments tools were noted. •The Sex Offender Act 2001 requires additional elements to ensure safe sex offender monitoring and public protection. A judicial review of the Sex Offender Act 2001 was recommended by many respondents. •Interagency working under SORAM was hugely welcomed. The sharing of information has been welcomed by managing agencies as the key benefit to improving sex offender management. •Respondents reported that in practice, sex offender management in Ireland is fragmented and unevenly implemented. The research concluded that an independent National Sex Offender Authority should be established as an oversight and regulatory body for policy, strategy and direction in sex offender management. Further areas of research were also highlighted: ongoing evaluation and audits of the joint agency process and systems in place; recidivism studies tracking the risk assessment ratings and subsequent offending; and an evaluation of the current status of sex offender housing in Ireland.

The impact of maternal inflammation and maternal stress in the regulation of neurodevelopment and physiological function

The mechanisms governing fetal development follow a tightly regulated pattern of progression such that interference at any one particular stage is likely to have consequences for all other stages of development in the physiological system that has been affected thereafter. These disturbances can take the form of many different events but two of the most common and widely implicated in causing detrimental effects to the developing fetus are maternal immune activation (MIA) and maternal stress. MIA has been shown to cause an increase in circulating proinflammatory cytokines in both the maternal and fetal circulation. This increase in proinflammatory mediators in the fetus is thought to occur by fetal production rather than through exchange between the maternal-fetal interface. In the case of maternal stress it is increased levels of stress related hormones such as cortisol/corticosterone which is thought to elicit the detrimental effects on fetal development. In the case of both maternal infection and stress the timing and nature of the insult generally dictates the severity and type of effects seen in affected offspring. We investigated the effect of a proinflammatory environment on neural precursor cells of which exposure resulted in a significant decrease in the normal rate of proliferation of NPCs in culture but did not have any effect on cell survival. These effects were seen to be age dependent. Using a restraint stress model we investigated the effects of prenatal stress on the development of a number of different physiological systems in the same cohort of animals. PNS animals exhibited a number of aberrant changes in cardiovascular function with altered responses to stress and hypertension, modifications in respiratory responses to hypercapnic and hypoxic challenges and discrepancies in gastrointestinal innervation. Taken together these findings suggest that both maternal infection and maternal stress are detrimental to the normal development of the fetus.

Intersections: proceedings of the youth work and music education symposium

The ‘Intersections: Youth Work and Music Education’ Symposium took place on Friday, 27th June 2014 in University College Cork. This event, held in association with the Institute of Social Sciences in the 21st Century (ISS21), was made possible thanks to funding from UCC’s Strategic Research Fund. The principle aim of this seed funding is to generate new research interests and this presented the ideal opportunity for developing collaborative relationships between youth work and music education lecturers, researchers and practitioners across the island of Ireland. This led to the formation of a new ‘Intersections’ research cluster, comprising representatives from four third-level institutions, each of which offers both youth work and music education undergraduate and/or postgraduate programmes, namely: University College Cork; National University of Ireland, Maynooth; Dundalk Institute of Technology; and, University of Ulster. This document presents some preliminary findings from primary research conducted through each of the participating institutions in their local areas. Data was also collected during the symposium, through engaging in small group discussions populated by the event’s participants. The publication and dissemination of this document was included in the original Strategic Research Fund proposal. Its intended audience includes youth workers, music educators, community-based practitioners, policy-makers and academics who are motivated and enthused by the possibilities of music-oriented youth work in Ireland and beyond.

Bioengineering of nisin to enhance functionality against dairy pathogens

The bacteriocin class of antimicrobial peptides have emerged as a viable alternative to at least partially fill the void created by the end of the golden age of antibiotic discovery. Along with this potential use in a clinical setting, bacteriocins also play an important role as bio-preservatives in the food industry. This thesis focuses on a specific bacteriocin group, the lantibiotics (Lanthionine-containing antibiotics). Their numerous methods of appliance in a food setting and how their gene-encoded nature can be modified to improve on overall bioactivity and functionality are explored here. The use of a lantibiotic (lacticin 3147) producing starter culture to control the Crohn’s disease-linked pathogen Mycobacterium paratuberculosis was assessed in a raw milk cheese. Although lacticin 3147 production did not effectively control the pathogen, the study provided an impetus to employ a variety of PCR-based mutagenesis techniques with a view to the creation of enhanced lantibiotic derivatives. Through the use of these techniques, a number of enhanced derivatives were generated from the ‘hinge’ region of the nisin peptide. Furthermore, a derivative in which the three hinge amino acids were replaced with three alanines represents the first enhanced derivative of nisin to have been designed through a rational process. This derivative also formed the backbone for the creation of an active, trypsin resistant, variant. Through the employment of further mutagenesis methods a derivative was created with potential use as an oral anti-bacterial in the future. Finally a number of lead nisin derivatives were investigated to assess their anti- Streptococcus agalactiae ability, a mastitis associated pathogen. Also a system was developed to facilitate the large scale production of these candidates, or other nisin derivatives, from dairy substrates.

Catalyst and substrate effects in enantioselective C–H insertion reactions of α-diazosulfones

This thesis describes a systematic investigation of the mechanistic and synthetic aspects of intramolecular reactions of a series of α-diazo-β-oxo sulfone derivatives using copper and, to a lesser extent, rhodium catalysts. The key reaction pathways explored were C–H insertion and cyclopropanation, with hydride transfer competing in certain instances. Significantly, up to 98% ee has been achieved in the C–H insertion processes using copper-NaBARF-bisoxazoline catalysts, with the presence of the additive NaBARF critical to the efficiency of the transformations. This novel synthetic methodology provides access to a diverse range of enantioenriched heterocyclic compounds including thiopyrans, sulfolanes, β- and γ-lactams, in addition to carbocycles such as fused cyclopropanes. The synthesis of the α-diazosulfones required for subsequent investigations is initially described. Of the twenty seven diazo sulfones described, nineteen are novel and are fully characterised in this work. The discussion is subsequently focused on a study of the copper and rhodium catalysed reactions of the α-diazosulfones with Chapter Four concentrated on highly enantioselective C–H insertion to form thiopyrans and sufolanes, Chapter Five focused on C–H insertion to form fused sulfolanes, Chapter Six focused on C–H insertion in sulfonyl α-diazoamides where both lactam formation and / or thiopyran / sulfolane formation can result from competing C–H insertion pathways, while Chapter Seven focuses on cyclopropanation to yield fused cyclopropane derviatives. One of the key outcomes of this work is an insight into the steric and / or electronic factors on both the substrate and the catalyst which control regio-, diastereo- and enantioselectivity patterns in these synthetically powerful transformations. Full experimental details for the synthesis and spectral characterisation of the compounds are included at the end of each Chapter, with details of chiral stationary phase HPLC analysis and assignment of absolute stereochemistry included in the appendix.

Experimental analysis of novel telecom source materials and devices

Incumbent telecommunication lasers emitting at 1.5 µm are fabricated on InP substrates and consist of multiple strained quantum well layers of the ternary alloy InGaAs, with barriers of InGaAsP or InGaAlAs. These lasers have been seen to exhibit very strong temperature dependence of the threshold current. This strong temperature dependence leads to a situation where external cooling equipment is required to stabilise the optical output power of these lasers. This results in a significant increase in the energy bill associated with telecommunications, as well as a large increase in equipment budgets. If the exponential growth trend of end user bandwidth demand associated with the internet continues, these inefficient lasers could see the telecommunications industry become the dominant consumer of world energy. For this reason there is strong interest in developing new, much more efficient telecommunication lasers. One avenue being investigated is the development of quantum dot lasers on InP. The confinement experienced in these low dimensional structures leads to a strong perturbation of the density of states at the band edge, and has been predicted to result in reduced temperature dependence of the threshold current in these devices. The growth of these structures is difficult due to the large lattice mismatch between InP and InAs; however, recently quantum dots elongated in one dimension, known as quantum dashes, have been demonstrated. Chapter 4 of this thesis provides an experimental analysis of one of these quantum dash lasers emitting at 1.5 µm along with a numerical investigation of threshold dynamics present in this device. Another avenue being explored to increase the efficiency of telecommunications lasers is bandstructure engineering of GaAs-based materials to emit at 1.5 µm. The cause of the strong temperature sensitivity in InP-based quantum well structures has been shown to be CHSH Auger recombination. Calculations have shown and experiments have verified that the addition of bismuth to GaAs strongly reduces the bandgap and increases the spin orbit splitting energy of the alloy GaAs1−xBix. This leads to a bandstructure condition at x = 10 % where not only is 1.5 µm emission achieved on GaAs-based material, but also the bandstructure of the material can naturally suppress the costly CHSH Auger recombination which plagues InP-based quantum-well-based material. It has been predicted that telecommunications lasers based on this material system should operate in the absence of external cooling equipment and offer electrical and optical benefits over the incumbent lasers. Chapters 5, 6, and 7 provide a first analysis of several aspects of this material system relevant to the development of high bismuth content telecommunication lasers.

Statistical methods for polyhedral shape classification with incomplete data - application to cryo-electron tomographic images

A certain type of bacterial inclusion, known as a bacterial microcompartment, was recently identified and imaged through cryo-electron tomography. A reconstructed 3D object from single-axis limited angle tilt-series cryo-electron tomography contains missing regions and this problem is known as the missing wedge problem. Due to missing regions on the reconstructed images, analyzing their 3D structures is a challenging problem. The existing methods overcome this problem by aligning and averaging several similar shaped objects. These schemes work well if the objects are symmetric and several objects with almost similar shapes and sizes are available. Since the bacterial inclusions studied here are not symmetric, are deformed, and show a wide range of shapes and sizes, the existing approaches are not appropriate. This research develops new statistical methods for analyzing geometric properties, such as volume, symmetry, aspect ratio, polyhedral structures etc., of these bacterial inclusions in presence of missing data. These methods work with deformed and non-symmetric varied shaped objects and do not necessitate multiple objects for handling the missing wedge problem. The developed methods and contributions include: (a) an improved method for manual image segmentation, (b) a new approach to 'complete' the segmented and reconstructed incomplete 3D images, (c) a polyhedral structural distance model to predict the polyhedral shapes of these microstructures, (d) a new shape descriptor for polyhedral shapes, named as polyhedron profile statistic, and (e) the Bayes classifier, linear discriminant analysis and support vector machine based classifiers for supervised incomplete polyhedral shape classification. Finally, the predicted 3D shapes for these bacterial microstructures belong to the Johnson solids family, and these shapes along with their other geometric properties are important for better understanding of their chemical and biological characteristics.

Nanofabrication towards biophotonics

This thesis explores methods for fabrication of nanohole arrays, and their integration into a benchtop system for use as sensors or anti-counterfeit labels. Chapter 1 gives an introduction to plasmonics and more specifically nanohole arrays and how they have potential as label free sensors compared to the current biosensors on the market. Various fabrication methods are explored, including Focused Ion Beam, Electron Beam Lithography, Nanoimprint lithography, Template stripping and Phase Shift Lithography. Focused Ion Beam was chosen to fabricate the nanohole arrays due to its suitability for rapid prototyping and it’s relatively low cost. In chapter 2 the fabrication of nanohole arrays using FIB is described, and the samples characterised. The fabricated nanohole arrays are tested as bulk refractive index sensors, before a bioassay using whole molecule human IgG antibodies and antigen is developed and performed on the senor. In chapter 3 the fabricated sensors are integrated into a custom built system, capable of real time, multiplexed detection of biomolecules. Here, scFv antibodies of two biomolecules relevant to the detection of pancreatic cancer (C1q and C3) are attached to the nanohole arrays, and detection of their complementary proteins is demonstrated both in buffer (10 nM detection of C1q Ag) and human serum. Chapter 4 explores arrays of anisotropic (elliptical) nanoholes and shows how the shape anisotropy induces polarisation sensitive transmission spectra, in both simulations and fabricated arrays. The potential use of such samples as visible and NIR tag for anti-counterfeiting applications is demonstrated. Finally, chapter 5 gives a summary of the work completed and discusses potential future work in this area.

Stabilisation of dehydrated nanoemulsions using sugar - protein matrices

There are numerous review papers discussing liquid nanoemulsions and how they compare to other emulsion systems. Little research is available on dried nanoemulsions. The objectives of this research were to (i) study the effect of varying the continuous phase of nanoemulsions with different carbohydrate/protein ratios on subsequent emulsion stability, and (ii) compare the physicochemical properties, lactose crystallisation properties, microstructure, and lipid oxidation of spray dried nanoemulsions compared to spray dried conventional emulsions having different water and sugar contents. Nanoemulsions containing sunflower oil (10% w/w), β-casein (2.5–10% w/w) and lactose or trehalose (10–17.5%) were produced following optimisation of the continuous phase by maximising and minimising viscosity and glass transition temperature (Tg’) using mixture design software. Increasing levels of β-casein from caused a significant increase in viscosity, particle size, and nanoemulsion stability, while resulting in a decrease in Tg’. Powders were made from spray drying emulsions/nanoemulsions consisting of lactose or a 70:30 mixture of lactose:sucrose (23.9%), sodium caseinate (5.1%) and sunflower oil (11.5%) in water. Nanoemulsions, produced by microfluidisation (100 MPa), had higher stability and lower viscosity than control emulsions (homogenization at 17 MPa) with lower solvent extractable free fat in the resulting powder. Partial replacement of lactose with sucrose decreased Tg and delayed Tcr. DVS and PLM showed that in powdered nanoemulsions, lactose crystallises faster than in powdered conventional emulsions. Microstructure of both powders (CLSM and cryo-SEM) showed different FGS in powders and different structure post lactose crystallisation. Powdered nanoemulsions had lower pentanal and hexanal (indicators of lipid oxidation) after 24 months storage due to their lower free fat and porosity, measured using a validated GC HS-SPME method, This research has shown the effect of altering the continuous phase of nanoemulsions on microstructure of spray dried nanoemulsions, which affects physical properties, sugar crystallisation, and lipid oxidation.

Genomic diversity of Lactobacillus salivarius

Lactobacillus salivarius is unusual among the lactobacilli due to its multireplicon genome architecture. The circular megaplasmids harboured by L. salivarius strains encode strain-specific traits for intestinal survival and probiotic activity. L. salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. In terms of probiotic strain selection, it is important to have an understanding of the level of genomic diversity present in this species. Comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) were employed to assess the level of genomic diversity in L. salivarius. The wellcharacterised probiotic strains L. salivarius UCC118 was employed as a genetic reference strain. The group of test strains were chosen to reflect the range of habitats from which L. salivarius strains are frequently recovered, including human, animal, and environmental sources. Strains of L. salivarius were found to be genetically diverse when compared to the UCC118 genome. The most conserved strains were human GIT isolates, while the greatest level of divergence were identified in animal associated isolates. MLST produced a better separation of the test strains according to their isolation origins, than that produced by CGHbased strain clustering. The exopolysaccharide (EPS) associated genes of L. salivarius strains were found to be highly divergent. The EPS-producing phenotype was found to be carbonsource dependent and inversely related to a strain's ability to produce a biofilm. The genome of the porcine isolate L. salivarius JCM1046 was shown by sequencing to harbour four extrachromosomal replicons, a circular megaplasmid (pMP1046A), a putative chromid (pMP1046B), a linear megaplasmid (pLMP1046) and a smaller circular plasmid (pCTN1046) which contains an integrated Tn916-like element (Tn6224), which carries the tetracycline resistance gene tetM. pLMP1046 represents the first sequence of a linear plasmid in a Lactobacillus species. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable, and the identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications. In summary, this thesis used a comparative genomics approach to examine the level of genotypic diversity in L. salivarius, a species which contains probiotic strains. The genome sequence of strain JCM1046 provides additional insight into the spectrum of extrachromosomal replicons present in this species.