Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

High-efficiency photovoltaics through mechanically stacked integration of solar cells based on the InP lattice constant

Solar Energy is a clean and abundant energy source that can help reduce reliance on fossil fuels around which questions still persist about their contribution to climate and long-term availability. Monolithic triple-junction solar cells are currently the state of the art photovoltaic devices with champion cell efficiencies exceeding 40%, but their ultimate efficiency is restricted by the current-matching constraint of series-connected cells. The objective of this thesis was to investigate the use of solar cells with lattice constants equal to InP in order to reduce the constraint of current matching in multi-junction solar cells. This was addressed by two approaches: Firstly, the formation of mechanically stacked solar cells (MSSC) was investigated through the addition of separate connections to individual cells that make up a multi-junction device. An electrical and optical modelling approach identified separately connected InGaAs bottom cells stacked under dual-junction GaAs based top cells as a route to high efficiency. An InGaAs solar cell was fabricated on an InP substrate with a measured 1-Sun conversion efficiency of 9.3%. A comparative study of adhesives found benzocyclobutene to be the most suitable for bonding component cells in a mechanically stacked configuration owing to its higher thermal conductivity and refractive index when compared to other candidate adhesives. A flip-chip process was developed to bond single-junction GaAs and InGaAs cells with a measured 4-terminal MSSC efficiency of 25.2% under 1-Sun conditions. Additionally, a novel InAlAs solar cell was identified, which can be used to provide an alternative to the well established GaAs solar cell. As wide bandgap InAlAs solar cells have not been extensively investigated for use in photovoltaics, single-junction cells were fabricated and their properties relevant to PV operation analysed. Minority carrier diffusion lengths in the micrometre range were extracted, confirming InAlAs as a suitable material for use in III-V solar cells, and a 1-Sun conversion efficiency of 6.6% measured for cells with 800 nm thick absorber layers. Given the cost and small diameter of commercially available InP wafers, InGaAs and InAlAs solar cells were fabricated on alternative substrates, namely GaAs. As a first demonstration the lattice constant of a GaAs substrate was graded to InP using an InxGa1-xAs metamorphic buffer layer onto which cells were grown. This was the first demonstration of an InAlAs solar cell on an alternative substrate and an initial step towards fabricating these cells on Si. The results presented offer a route to developing multi-junction solar cell devices based on the InP lattice parameter, thus extending the range of available bandgaps for high efficiency cells.

Impurity profiling and synthesis of precursors to the hallucinogenic amphetamine DOB

DOB (4‐bromo‐2,5‐dimethoxyamphetamine) is a newly emerging hallucinogenic amphetamine that sparked serious health warnings in Ireland, following its first seizure back in 2003. Known more commonly as “snowball”, this drug is highly potent and may be used as a substitute to ecstasy (MDMA) and lysergic acid diethylamide (LSD). To date, the work carried out on the impurity profiling of DOB is limited in comparison to amphetamine, methamphetamine and MDMA. In this work, the impurity profile of 4‐bromo‐2,5‐dimethoxyphenyl‐2‐propanone (4‐Br‐2,5‐P2P) is explored. This ketone is a direct precursor to DOB. Its more versatile non‐bromo analogue, 2,5‐ dimethoxyphenyl‐2‐propanone (2,5‐P2P) is also examined, as in addition to DOB, it may be used in the synthesis of a range of several other hallucinogenic amphetamines. A number of different routes to both 2,5‐P2P and 4‐Br‐2,5‐P2P were investigated. For each of these routes, the impurities produced were carefully isolated. Following isolation, the impurities were fully characterised (by 1H‐NMR/13C‐NMR spectroscopy, IR, MS), in order to aid structure elucidation. Compounds not easily resolved by flash column chromatography were analysed by LC‐MS and/or independently synthesised for the purpose of attaining reference standards. Adaptation of the well‐known ‘phenylacetic acid route’ to synthesis of both 2,5‐P2P and 4‐Br‐2,5‐P2P, was found to provide low yields of the expected ketone products. Four impurities were isolated during the preparation of both ketones. The yield of one of these impurities (possessing a dibenzylketone core), was greatly influenced by the amount of acetic anhydride reagent used during the reaction. Having carried out the reaction with several different equivalents of acetic anhydride, it was found that formation of the ‘dibenzylketone’ could not be eliminated. This may increase its likelihood of being detected in the final drug product. The ‘Darzens route’, having very recently emerged as a synthetic route to amphetamine and MDMA precursors, was discovered to be a viable route for manufacture of 2,5‐P2P and 4‐Br‐2,5‐P2P. Despite execution of the reaction being more tedious, the route provides superior yields (≈50–60%) to those achieved using the ‘phenylacetic acid route’ (≈35–38%). Incorporation of a bromine atom (at the aromatic 4‐position) is required at some stage during synthesis of DOB. The bromination of many intermediates/starting materials was therefore also examined in detail. Bromination of the acid starting material 2,5‐dimethoxyphenylacetic acid (2,5‐PAA) was found to be clean and high yielding. This was in stark contrast to the bromination of the benzaldehyde starting material, the ketone precursor 2,5‐P2P and the dibenzylketone‐based impurity. Numerous brominated products were isolated from each of these reactions, many of which were novel compounds, and previously unreported as impurities in the literature. The unpredictable/nondescript nature of these brominations is likely to have a significant impact on the impurity profile of illicitly produced DOB.

A recessionary debate: who suffered more? A study of sectoral well-being and economic insecurity in Ireland

The aim of this thesis is to identify the relationship between subjective well-being and economic insecurity for public and private sector workers in Ireland using the European Social Survey 2010-2012. Life satisfaction and job satisfaction are the indicators used to measure subjective well-being. Economic insecurity is approximated by regional unemployment rates and self-perceived job insecurity. Potential sample selection bias and endogeneity bias are accounted for. It is traditionally believed that public sector workers are relatively more protected against insecurity due to very institution of public sector employment. The institution of public sector employment is made up of stricter dismissal practices (Luechinger et al., 2010a) and less volatile employment (Freeman, 1987) where workers are subsequently less likely to be affected by business cycle downturns (Clark and Postal-Vinay, 2009). It is found in the literature that economic insecurity depresses the well-being of public sector workers to a lesser degree than private sector workers (Luechinger et al., 2010a; Artz and Kaya, 2014). These studies provide the rationale for this thesis in testing for similar relationships in an Irish context. Sample selection bias arises when a selection into a particular category is not random (Heckman, 1979). An example of this is non-random selection into public sector employment based on personal characteristics (Heckman, 1979; Luechinger et al., 2010b). If selection into public sector employment is not corrected for this can lead to biased and inconsistent estimators (Gujarati, 2009). Selection bias of public sector employment is corrected for by using a standard Two-Step Heckman Probit OLS estimation method. Following Luechinger et al. (2010b), the propensity for individuals to select into public sector employment is estimated by a binomial probit model with the inclusion of the additional regressor Irish citizenship. Job satisfaction is then estimated by Ordinary Least Squares (OLS) with the inclusion of a sample correction term similar as is done in Clark (1997). Endogeneity is where an independent variable included in the model is determined within in the context of the model (Chenhall and Moers, 2007). The econometric definition states that an endogenous independent variable is one that is correlated with the error term (Wooldridge, 2010). Endogeneity is expected to be present due to a simultaneous relationship between job insecurity and job satisfaction whereby both variables are jointly determined (Theodossiou and Vasileiou, 2007). Simultaneity, as an instigator of endogeneity, is corrected for using Instrumental Variables (IV) techniques. Limited Information Methods and Full Information Methods of estimation of simultaneous equations models are assed and compared. The general results show that job insecurity depresses the subjective well-being of all workers in both the public and private sectors in Ireland. The magnitude of this effect differs among sectoral workers. The subjective well-being of private sector workers is more adversely affected by job insecurity than the subjective well-being of public sector workers. This is observed in basic ordered probit estimations of both a life satisfaction equation and a job satisfaction equation. The marginal effects from the ordered probit estimation of a basic job satisfaction equation show that as job insecurity increases the probability of reporting a 9 on a 10-point job satisfaction scale significantly decreases by 3.4% for the whole sample of workers, 2.8% for public sector workers and 4.0% for private sector workers. Artz and Kaya (2014) explain that as a result of many austerity policies implemented to reduce government expenditure during the economic recession, workers in the public sector may for the first time face worsening perceptions of job security which can have significant implications for their well-being (Artz and Kaya, 2014). This can be observed in the marginal effects where job insecurity negatively impacts the well-being of public sector workers in Ireland. However, in accordance with Luechinger et al. (2010a) the results show that private sector workers are more adversely impacted by economic insecurity than public sector workers. This suggests that in a time of high economic volatility, the institution of public sector employment held and was able to protect workers against some of the well-being consequences of rising insecurity. In estimating the relationship between subjective well-being and economic insecurity advanced econometric issues arise. The results show that when selection bias is corrected for, any statistically significant relationship between job insecurity and job satisfaction disappears for public sector workers. Additionally, in order to correct for endogeneity bias the simultaneous equations model for job satisfaction and job insecurity is estimated by Limited Information and Full Information Methods. The results from two different estimators classified as Limited Information Methods support the general findings of this research. Moreover, the magnitude of the endogeneity-corrected estimates are twice as large as those not corrected for endogeneity bias which is similarly found in Geishecker (2010, 2012). As part of the analysis into the effect of economic insecurity on subjective well-being, the effects of other socioeconomic variables and work-related variables are examined for public and private sector workers in Ireland.