Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions.

Inclusive education policy, the general allocation model and dilemmas of practice in primary schools

Background: Inclusive education is central to contemporary discourse internationally reflecting societies’ wider commitment to social inclusion. Education has witnessed transforming approaches that have created differing distributions of power, resource allocation and accountability. Multiple actors are being forced to consider changes to how key services and supports are organised. This research constitutes a case study situated within this broader social service dilemma of how to distribute finite resources equitably to meet individual need, while advancing inclusion. It focuses on the national directive with regard to inclusive educational practice for primary schools, Department of Education and Science Special Education Circular 02/05, which introduced the General Allocation Model (GAM) within the legislative context of the Education of Persons with Special Educational Needs (EPSEN) Act (Government of Ireland, 2004). This research could help to inform policy with ‘facts about what is happening on the ground’ (Quinn, 2013). Research Aims: The research set out to unearth the assumptions and definitions embedded within the policy document, to analyse how those who are at the coalface of policy, and who interface with multiple interests in primary schools, understand the GAM and respond to it, and to investigate its effects on students and their education. It examines student outcomes in the primary schools where the GAM was investigated. Methods and Sample The post-structural study acknowledges the importance of policy analysis which explicitly links the ‘bigger worlds’ of global and national policy contexts to the ‘smaller worlds’ of policies and practices within schools and classrooms. This study insists upon taking the detail seriously (Ozga, 1990). A mixed methods approach to data collection and analysis is applied. In order to secure the perspectives of key stakeholders, semi-structured interviews were conducted with primary school principals, class teachers and learning support/resource teachers (n=14) in three distinct mainstream, non-DEIS schools. Data from the schools and their environs provided a profile of students. The researcher then used the Pobal Maps Facility (available at www.pobal.ie) to identify the Small Area (SA) in which each student resides, and to assign values to each address based on the Pobal HP Deprivation Index (Haase and Pratschke, 2012). Analysis of the datasets, guided by the conceptual framework of the policy cycle (Ball, 1994), revealed a number of significant themes. Results: Data illustrate that the main model to support student need is withdrawal from the classroom under policy that espouses inclusion. Quantitative data, in particular, highlighted an association between segregated practice and lower socioeconomic status (LSES) backgrounds of students. Up to 83% of the students in special education programmes are from lower socio-economic status (LSES) backgrounds. In some schools 94% of students from LSES backgrounds are withdrawn from classrooms daily for special education. While the internal processes of schooling are not solely to blame for class inequalities, this study reveals the power of professionals to order children in school, which has implications for segregated special education practice. Such agency on the part of key actors in the context of practice relates to ‘local constructions of dis/ability’, which is influenced by teacher habitus (Bourdieu, 1984). The researcher contends that inclusive education has not resulted in positive outcomes for students from LSES backgrounds because it is built on faulty assumptions that focus on a psycho-medical perspective of dis/ability, that is, placement decisions do not consider the intersectionality of dis/ability with class or culture. This study argues that the student need for support is better understood as ‘home/school discontinuity’ not ‘disability’. Moreover, the study unearths the power of some parents to use social and cultural capital to ensure eligibility to enhanced resources. Therefore, a hierarchical system has developed in mainstream schools as a result of funding models to support need in inclusive settings. Furthermore, all schools in the study are ‘ordinary’ schools yet participants acknowledged that some schools are more ‘advantaged’, which may suggest that ‘ordinary’ schools serve to ‘bury class’ (Reay, 2010) as a key marker in allocating resources. The research suggests that general allocation models of funding to meet the needs of students demands a systematic approach grounded in reallocating funds from where they have less benefit to where they have more. The calculation of the composite Haase Value in respect of the student cohort in receipt of special education support adopted for this study could be usefully applied at a national level to ensure that the greatest level of support is targeted at greatest need. Conclusion: In summary, the study reveals that existing structures constrain and enable agents, whose interactions produce intended and unintended consequences. The study suggests that policy should be viewed as a continuous and evolving cycle (Ball, 1994) where actors in each of the social contexts have a shared responsibility in the evolution of education that is equitable, excellent and inclusive.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms

Immunostimulatory effects of different aspects of aquaculture on the host response in the edible sea urchin, paracentrotus lividus

Aquaculture is a fast-growing industry contributing to global food security and sustainable aquaculture, which may reduce pressures on capture fisheries. The overall objective of this thesis was to look at the immunostimulatory effects of different aspects of aquaculture on the host response of the edible sea urchin, Paracentrotus lividus, which are a prized delicacy (roe) in many Asian and Mediterranean countries. In Chapter 1, the importance of understanding the biology, ecology, and physiology of P. lividus, as well as the current status in the culture of this organism for mass production and introducing the thesis objectives for following chapters is discussed. As the research commenced, the difficulties of identifying individuals for repeat sampling became clear; therefore, Chapter 2 was a tagging experiment that indicated PIT tagging was a successful way of identifying individual sea urchins over time with a high tag retention rate. However, it was also found that repeat sampling via syringe to measure host response of an individual caused stress which masked results and thus animals would be sampled and sacrificed going forward. Additionally, from personal observations and discussion with peers, it was suggested to look at the effect that diet has on sea urchin immune function and the parameters I measured which led to Chapter 3. In this chapter, both Laminaria digitata and Mytilus edulis were shown to influence measured immune parameters of differential cell counts, nitric oxide production, and lysozyme activity. Therefore, trials commencing after Trial 5 in Chapter 4, were modified to include starvation in order to remove any effect of diet. Another important aspect of culturing any organism is the study of their immune function and its response to several immunostimulatory agents (Chapter 4). Zymosan A was shown to be an effective immunostimulatory agent in P. lividus. Further work on handled/stored animals (Chapter 5) showed Zymosan A reduced the measured levels of some immune parameters measured relative to the control, which may reduce the amount of stress in the animals. In Chapter 6, animals were infected with Vibrio anguillarum and, although V. anguillarum, impacted immune parameters of P. lividus, it did not cause mortality as predicted. Lastly, throughout this thesis work, it was noted that the immune parameters measured produced different values at different times of the year (Chapter 7); therefore, using collated baseline (control) data, results were compiled to observe seasonal effects. It was determined that both seasonality and sourcing sites influenced immune parameter measurements taken at different times throughout the year. In conclusion, this thesis work fits into the framework of development of aquaculture practices that affect immune function of the host and future research focusing on the edible sea urchin, P. lividus.

Global surface net-radiation at 5 km from MODIS Terra

Reliable and fine resolution estimates of surface net-radiation are required for estimating latent and sensible heat fluxes between the land surface and the atmosphere. However, currently, fine resolution estimates of net-radiation are not available and consequently it is challenging to develop multi-year estimates of evapotranspiration at scales that can capture land surface heterogeneity and are relevant for policy and decision-making. We developed and evaluated a global net-radiation product at 5 km and 8-day resolution by combining mutually consistent atmosphere and land data from the Moderate Resolution Imaging Spectroradiometer (MODIS) on board Terra. Comparison with net-radiation measurements from 154 globally distributed sites (414 site-years) from the FLUXNET and Surface Radiation budget network (SURFRAD) showed that the net-radiation product agreed well with measurements across seasons and climate types in the extratropics (Wilmott’s index ranged from 0.74 for boreal to 0.63 for Mediterranean sites). Mean absolute deviation between the MODIS and measured net-radiation ranged from 38.0 ± 1.8 W∙m−2 in boreal to 72.0 ± 4.1 W∙m−2 in the tropical climates. The mean bias was small and constituted only 11%, 0.7%, 8.4%, 4.2%, 13.3%, and 5.4% of the mean absolute error in daytime net-radiation in boreal, Mediterranean, temperate-continental, temperate, semi-arid, and tropical climate, respectively. To assess the accuracy of the broader spatiotemporal patterns, we upscaled error-quantified MODIS net-radiation and compared it with the net-radiation estimates from the coarse spatial (1° × 1°) but high temporal resolution gridded net-radiation product from the Clouds and Earth’s Radiant Energy System (CERES). Our estimates agreed closely with the net-radiation estimates from the CERES. Difference between the two was less than 10 W•m−2 in 94% of the total land area. MODIS net-radiation product will be a valuable resource for the science community studying turbulent fluxes and energy budget at the Earth’s surface.