Mammalian cell cultures as a model system for the determination of cytotoxicity induced by cholesterol oxidation products

Oxysterols are products of cholesterol oxidation, which may be produced endogenously or may be absorbed from the diet where they are commonly found in foods of animal origin. Oxysterols are known to be cyctotoxic to cells in culture and mode of toxicity has been identified as apoptosis in certain cell lines. The cytotoxicity of the oxysterols 25-hydroxycholesterol (25-OH) and 7β-hydroxycholesterol (7β-OH) was examined in two human cell lines, HepG2, a hepatoma cell line, and U937, a monocytic cell line. Both 25-OH and 7β-OH were cytotoxic to the HepG2 cell line but apoptotic cells were not detected and it was concluded that cells underwent necrosis. 25-OH was not cytotoxic to the U937 cell line but it was found to have a cytostatic effect. 7β-OH was shown to induce apoptosis in the U937 line. The mechanism of oxysterol-induced apoptosis has not yet been fully elucidated, however the generation of an oxidative stress and the depletion of glutathione have been associated with the initial stages of the apoptotic process. The concentration of cellular antioxidant enzyme, superoxide dismutase (SOD) was increased in association with 7β-OH induced apoptosis in the U937 cell line. There was no change in the glutathione concentration or the SOD activity of HepG2 cells, which underwent necrosis in the presence of 7β-OH. Many apoptotic pathways center on the activation of caspase-3, which is the key executioner protease of apoptosis. Caspase-3 activity was also shown to increase in association with 7β-OH-induced apoptosis in U937 cells but there was no significant increase in caspase-3 activity in HepG2 cells. DNA fragmentation is regarded as the biochemical hallmark of apoptosis, therefore the comet assay as a measure of DNA fragmentation was assessed as a measure of apoptosis. The level of DNA fragmentation induced by 7β-OH, as measured using the comet assay, was similar for both cell lines. Therefore, it was concluded that the comet assay could not be used to distinguish between 7β-OH-induced apoptosis in U937 cells and 7β-OH-induced necrosis in HepG2 cells. The cytotoxicity and apoptotic potency of oxysterols 25-OH, 7β-OH, cholesterol- 5a,6a-epoxide (a-epoxide), cholesterol-5β,6β-epoxide (β-epoxide), 19-hydroxy-cholesterol (19-OH), and 7-ketocholesterol (7-keto) was compared in the U937 cell line. 7 β-OH, β-epoxide and 7-keto were found to induce apoptosis in U937 cells. 7β-OH-induced apoptosis was associated with a decrease in the cellular glutathione concentration and an increase in SOD activity, 7-keto and β-epoxide did not affect the glutathione concentration or the SOD activity of the cells.a-Epoxide, 19-OH and 25-OH were not cytotoxic to the U937 cell line.

Uniformly convergent finite element methods for singularly perturbed parabolic partial differential equations

This thesis is concerned with uniformly convergent finite element methods for numerically solving singularly perturbed parabolic partial differential equations in one space variable. First, we use Petrov-Galerkin finite element methods to generate three schemes for such problems, each of these schemes uses exponentially fitted elements in space. Two of them are lumped and the other is non-lumped. On meshes which are either arbitrary or slightly restricted, we derive global energy norm and L2 norm error bounds, uniformly in the diffusion parameter. Under some reasonable global assumptions together with realistic local assumptions on the solution and its derivatives, we prove that these exponentially fitted schemes are locally uniformly convergent, with order one, in a discrete L∞norm both outside and inside the boundary layer. We next analyse a streamline diffusion scheme on a Shishkin mesh for a model singularly perturbed parabolic partial differential equation. The method with piecewise linear space-time elements is shown, under reasonable assumptions on the solution, to be convergent, independently of the diffusion parameter, with a pointwise accuracy of almost order 5/4 outside layers and almost order 3/4 inside the boundary layer. Numerical results for the above schemes are presented. Finally, we examine a cell vertex finite volume method which is applied to a model time-dependent convection-diffusion problem. Local errors away from all layers are obtained in the l2 seminorm by using techniques from finite element analysis.

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.

Generation and characterisation of biologically active milk-derived protein and peptide fractions

In recent years, extensive research has been carried out on the health benefits of milk proteins and peptides. Biologically active peptides are defined as specific protein fragments which have a positive impact on the physiological functions of the body; such peptides are produced naturally in vivo, but can also be generated by physical and/or chemical processes, enzymatic hydrolysis and/or microbial fermentation. The aims of this thesis were to investigate not only the traditional methods used for the generation of bioactive peptides, but also novel processes such as heat treatment, and the role of indigenous milk proteases, e.g., in mastitic milk, in the production of such peptides. In addition, colostrum was characterised as a source of bioactive proteins and peptides. Firstly, a comprehensive study was carried out on the composition and physical properties of colostrum throughout the early-lactation period. Marked differences in the physico-chemical properties of colostrum compared with milk were observed. Various fractions of colostrum were also tested for their effect on the secretion of pro- and anti-inflammatory cytokines from a macrophage cell line and bone marrow dendritic cells, as well as insulin secretion from a pancreatic beta cell line. A significant reduction in the secretion of the pro-inflammatory cytokines, TNF-α, IL-6, IL-1β and IL-12, a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, as well as a significant increase in insulin secretion were observed for various colostrum fractions. Another study examined the early proteomic changes in the milk of 8 cows in response to infusion with the endotoxin lipopolysaccharide (LPS) at quarter level in a model mastitic system; marked differences in the protein and peptide profile of milk from LPS challenged cows were observed, and a pH 4.6-soluble fraction of this milk was found to cause a substantial induction in the secretion of IL-10 from a murine macrophage cell line. Heat-induced hydrolysis of sodium caseinate was investigated from the dual viewpoints of protein breakdown and peptide formation, and, a peptide fraction produced in this manner was found to cause a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, from a murine macrophage cell line. The effects of sodium caseinate hydrolysed by chymosin on the gut-derived satiety hormone glucagon-like peptide-1 (GLP-1) were investigated; the resulting casein-derived peptides displayed good in vitro and in vivo secretion of GLP-1. Overall, the studies described in this thesis expand on current knowledge and provide good evidence for the use of novel methods for the isolation, generation and characterisation of bioactive proteins and/or peptides.