Cereal products for specific dietary requirements. Evaluation and improvement of technological and nutritional properties of gluten free raw materials and end products

Coeliac disease is one of the most common food intolerances worldwide and at present the gluten free diet remains the only suitable treatment. A market overview conducted as part of this thesis on nutritional and sensory quality of commercially available gluten free breads and pasta showed that improvements are necessary. Many products show strong off-flavors, poor mouthfeel and reduced shelf-life. Since the life-long avoidance of the cereal protein gluten means a major change to the diet, it is important to also consider the nutritional value of products intending to replace staple foods such as bread or pasta. This thesis addresses this issue by characterising available gluten free cereal and pseudocereal flours to facilitate a better raw material choice. It was observed that especially quinoa, buckwheat and teff are high in essential nutrients, such as protein, minerals and folate. In addition the potential of functional ingredients such as inulin, β-glucan, HPMC and xanthan to improve loaf quality were evaluated. Results show that these ingredients can increase loaf volume and reduce crumb hardness as well as rate of staling but that the effect diverges strongly depending on the bread formulation used. Furthermore, fresh egg pasta formulations based on teff and oat flour were developed. The resulting products were characterised regarding sensory and textural properties as well as in vitro digestibility. Scanning electron and confocal laser scanning microscopy was used throughout the thesis to visualise structural changes occurring during baking and pasta making

Development of a toolbox for the reduction of hordeins in barley malt beers

Gluten sensitive consumers and people suffering from coeliac disease account for up to 6% of the general population (Catassi et al., 2013). These consumers must avoid foods which contain gluten and related proteins found in wheat, rye or barley. Beer is produced from barley malt and therefore contains hordeins, (gluten like proteins). Beers labelled as gluten-free must contain below 10 mg/kg hordeins (10 mg/kg hordeins = 20 mg/kg gluten under current regulations) to be considered safe for gluten sensitive consumers. Currently there are a limited number of methods available for reducing beer hordeins, the studies outlined in this thesis provide a range of tools for the beverage industry to reduce the hordein content of beer It is well known, that during malting and brewing hordeins are reduced, but they still remain in beer at levels above 10 mg/kg. During malting, hordeins are broken down to form new proteins in the growing plant. Model malting and brewing systems were developed and used to test, how the modification of the malting process could be used to reduce beer hordeins. It was shown, that by using a controlled malting and brewing regime, a range of barley cultivars produced beer with significant differences in levels of hordeins. Beer hordeins ranged from 10 mg/kg to 60 mg/kg. Another study revealed that when malting was prolonged, to maximise breakdown of proteins, beer hordeins can be reduced by up to 44%. The natural breakdown of hordein during malting enhanced in a further study, when a protease was added to support the hordein degradation during steeping and germination. The enzyme addition resulted in a 46% reduction in beer hordeins 2 when compared to the control. All of the malt treatments had little or no impact on malt quality. The hordein levels can also be reduced during the beer stabilisation process. Levels of beer hordein were tested after stabilisation using two different concentrations of silica gel and tannic acid. Silica gel was very effective in reducing beer hordeins, 90% of beer hordeins were removed compared to the control beer. Beer hordeins could be reduced to below 10 mg/kg and the beer qualities such as foam, colour and flavour were not affected. Tannic acid also reduced beer hordein by up to 90%, but it reduced foam stability and affected beer flavours. A further study described treatment of beer with microbial transglutaminase (mTG), to create bonds between hordein proteins, which increased particle size and allowed removal during filtration. The addition of the mTG led to a reduction of the beer hordein by up to 96% in beer, and the impact on the resulting beer quality was minimal. These studies provide the industry with a toolbox of methods leading to the reduction of hordein in the final beer without negatively affecting beer quality.

Established cardiovascular disease and CVD risk factors in a primary care population of middle-aged Irish men and women

Contemporary Irish data on the prevalence of major cardiovascular disease (CVD) risk factors are sparse. The primary aims of this study were (1) to estimate the prevalence of major cardiovascular disease risk factors, including Type 2 Diabetes Mellitus, in the general population of men and women between the ages of 50 and 69 years; and (2) to estimate the proportion of individuals in this age group at high absolute risk of cardiovascular disease events on the basis of pre-existing cardiovascular disease or as defined by the Framingham equation. Participants were drawn from the practice lists of 17 general practices in Cork and Kerry using stratified random sampling. A total of 1018 people attended for screening (490 men, 48%) from 1473 who were invited, a response rate of 69.1%. Cardiovascular disease risk factors and glucose intolerance are common in the population of men and women aged between 50 and 69 years. Almost half the participants were overweight and a further quarter met current international criteria for obesity, one of the highest recorded prevalence rates for obesity in a European population sample. Forty per cent of the population reported minimal levels of physical activity and 19% were current cigarette smokers. Approximately half the sample had blood pressure readings consistent with international criteria for the diagnosis of hypertension, but only 38% of these individuals were known to be hypertensive. Eighty per cent of the population sample had a cholesterol concentration in excess of 5 mmol/l. Almost 4% of the population had Type 2 Diabetes Mellitus, of whom 30% were previously undiagnosed. A total of 137 participants (13.5%) had a history or ECG findings consistent with established cardiovascular disease. Of the remaining 881 individuals in the primary prevention population, a total of 20 high-risk individuals (19 male) had a risk of a coronary heart disease event 30% over ten years according to the Framingham risk equation, giving an overall population prevalence of 2.0% (95% CI 1.3 - 3.0). At a risk level 20% over ten years, an additional 91 individuals (8.9%) were identified. Thus a total of 24.4% of the population were at risk either through pre-existing CVD (13.5%) or an estimated 10-year risk exceeding 20% according to the Framingham risk equation (10.9%). Thus a substantial proportion of middle-aged men are at high risk of CVD. The findings emphasise the scale of the CVD epidemic in Ireland and the need for ongoing monitoring of risk factors at the population level and the need to develop preventive strategies at both the clinical and societal level.

Synecology in soft sediment bivalves: the influence of parasites, physiological processes, and environmental stressors on health and disease

Ecosystem goods and services provided by estuarine and near coastal regions are being increasingly recognised for their immense value, as is the biodiversity in these areas and these near coastal communities have been identified as sentinels of climate change also. Population structure and reproductive biology of two bivalve molluscs, Cerastoderma edule and, Mytilus edulis were assessed at two study sites over a 16-month study period. Following an anomalously harsh winter, advancement of spawning time was observed in both species. Throughout Ireland and Europe the cockle has experienced mass surfacings in geographically distinct regions, and a concurrent study of cockles was undertaken to explore this phenomenon. Surfaced and buried cockles were collected on a monthly basis and their health compared. Age was highlighted as a source of variation between dying and healthy animals with a parasite threshold being reached possibly around age three. Local factors dominated when looking at the cause of surfacing at each site. The health of mussels was explored too on a temporal and seasonal basis in an attempt to assess what constitutes a healthy organism. In essence external drivers can tip the balance between “acceptable” levels of infection where the mussel can still function physiologically and “unacceptable” where prevalence and intensity of infection can result in physiological impairment at the individual and population level. Synecological studies of intertidal ecosystems are lacking, so all bivalves encountered during the sampling were assessed in terms of population structure, reproduction, and health. It became clear, that some parasites might specialize on one host species while others are not so specific in host choice. Furthermore the population genetics of the cockle, its parasite Meiogymnophallus minutus, and its hyperparasite Unikaryon legeri were examined too. A small nucleotide polymorphism was detected upon comparison of Ireland and Morocco.

Expression and function of the neurotrophic factors GDF5 and GDNF in the nigrostriatal system during development and in rat models of Parkinson's disease

Growth/differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) are neurotrophic factors that promote the survival of midbrain dopaminergic neurons in vitro and in vivo. Both factors have potent neurotrophic and neuroprotective effects in rat models of Parkinson's disease (PD), and may represent promising new therapies for PD. The aim of the present study was to investigate the endogenous expression and function of GDF5 and GDNF in the nigrostriatal dopaminergic system during development and in rat models of PD. Examination of the temporal expression patterns of endogenous GDF5, GDNF, and their respective receptors, in the developing and adult nigrostriatal dopaminergic system suggest that these factors play important roles in promoting the survival and maturation of midbrain dopaminergic neurons during the period of postnatal programmed cell death. The relative levels of GDF5 and GDNF mRNAs in the midbrain and striatum, and their individual temporal expression patterns during development, suggest that their modes of actions are quite distinct in vivo. Furthermore, the sustained expression of GDF5, GDNF, and their receptors into adulthood suggest roles for these factors in the continued support and maintenance of mature nigrostriatal dopaminergic neurons. The present study found that endogenous GDF5, GDNF, and their receptors are differentially expressed in two 6-hydroxydopamine-induced lesion adult rat models of PD. In both terminal and axonal lesion models of PD, GDF5 mRNA levels in the striatum increased at 10 days post-lesion, while GDNF mRNA levels in the nigrostriatal system decreased at 10 and 28 days post-lesion. Thus, despite the fact that exogenous GDF5 and GDNF have similar effects on midbrain dopaminergic neurons in vitro and in vivo, their endogenous responses to a neurotoxic injury are quite distinct. These results highlight the importance of studying the temporal dynamic changes in neurotrophic factor expression during development and in animal models of PD.

Vascular calcification and mineral bone disorder in chronic kidney disease

Chronic Kidney Disease (CKD), osteoporosis and mild hyponatremia are all prevalent chronic conditions that may coexist and are often under-recognized. Mineral-Bone Disorder begins early in the natural history of CKD and results in complex abnormalities of bone which ultimately confers a well-established increased risk of fragility fractures in End Stage Kidney Disease. Hyponatremia is a novel, usually renal mediated metabolic perturbation, that most commonly occurs independently of the stage of renal dysfunction but which may also predispose to increased fracture risk. The extent -if any- to which either early stages of renal dysfunction or the presence of hyponatremia contribute to fracture occurrence in the general population, independently of osteoporosis, is unclear. Renal transplantation is the treatment of choice for ESKD and although it restores endogenous renal function it typically fails to normalize either the long term cardiovascular or fracture risk. One potential mechanism contributing to these elevated long-term risks and to diminished Health Related Quality of Life is persistent, post-transplant hyperparathyroidism. In this study we retrospectively examine the association of renal function and serum sodium with Bone Mineral Density and fracture occurrence in a retrospective cohort of 1930 female members of the general population who underwent routine DXA scan. We then prospectively recruited a cohort of 90 renal transplant recipients in order to examine the association of post transplant parathyroid hormone (PTH) level with measures of CKD Mineral Bone Disorder, including, DXA Bone Mineral Density, Vascular Calcification (assessed using both abdominal radiography and CT techniques, as well as indirectly by carotid-femoral Pulse Wave Velocity) and Quality of Life (using the Short Form-12 and a PTH specific symptom score). In the retrospective DXA cohort, moderate CKD (eGFR 30-59ml/min/1.73m2) and hyponatremia (<135mmol/L) were associated with fracture occurrence, independently of BMD, with an adjusted Odds Ratio (95% Confidence Interval), of 1.37 (1.0, 1.89) and 2.25 (1.24, 4.09) respectively. In the renal transplant study, PTH was independently associated with the presence of osteoporosis, adjusted Odds Ratio (95% Confidence Interval), 1.15 (per 10ng/ml increment), (1.04, 1.26). The presence of osteoporosis but not PTH was independently associated with measures of vascular calcification, adjusted ß (95% Confidence Interval), 12.45, (1.16, 23.75). Of the eight quality-of-life domains examined, post-transplant PTH (per 10ng/ml increment), was only significantly and independently associated with reduced Physical Functioning, (95% Confidence Interval), 1.12 (1.01, 1.23). CKD and hyponatremia are both common health problems that may contribute to fracture occurrence in the general population, a major on-going public health concern. PTH and decreased Bone Mineral Density may signal sub-optimal long-term outcomes post renal transplantation, influencing bone and vascular health and to a limited extent long term Health Related Quality of Life

Symptom burden in individuals with inflammatory bowel disease: a mixed methods study

To investigate the symptom burden experiences of individuals with inflammatory bowel disease (IBD). An explanatory sequential mixed methods study was conducted. A cross-sectional, correlational survey was first undertaken. Symptom burden was measured using a modified disease specific version of the Memorial Symptom Assessment Scale, which was administered to a consecutive sample of individuals with IBD (n = 247) at an IBD Outpatients department in one urban teaching hospital in Ireland. Disease activity was determined using clinical disease activity indices, which were completed by the consulting physician. A sequential qualitative, descriptive study was then conducted aimed at explaining noteworthy quantitative findings. A criterion-related purposeful sample of seven participants from the quantitative study was recruited. Semi-structured face to face interviews were conducted using an interview guide and data were analysed using content analysis. Findings revealed that participants experienced a median of 10 symptoms during the last week, however as many as 16 symptoms were experienced during active disease. The most burdensome symptoms were lack of energy, bowel urgency, diarrhoea, feeling bloated, flatulence and worry. Total symptom burden was found to be low with a mean score of 0.56 identified out of a possible range from 0 to 4. Participants with active disease (M = 0.81, SD = 0.48; n = 68) had almost double mean total symptom burden scores than participants with inactive disease (M = 0.46, SD = 0.43; n = 166) (p < 0.001). Mean total psychological symptom burden was found to be significantly greater than mean total physical symptom burden (rho = 0.73, n = 247, p < 0.001). Self-reported disease control, gender, number of flare ups in the last two years, and smoking status was found to be significant predictors of total symptom burden, with self-reported disease control identified as the strongest predictor. Qualitative data revealed tiredness, pain, bowel symptoms, worry and fear as being burdensome. Furthermore, symptom burden experiences were described in terms of its impact on restricting aspects of daily activities, which accumulated into restrictions on general life events. Psychological symptom burden was revealed as more problematic than physical symptom burden due to its constant nature, with physical and psychological symptoms described to occur in a cyclical manner. Participants revealed that disease control was evaluated not only in terms of symptoms, but also in terms of their abilities to control the impact of symptoms on their lives. This study highlights the considerable number of symptoms and the most burdensome symptoms experienced by individuals with IBD, both during active and inactive disease. This study has important implications on symptom assessment in terms of the need to encompass both physical and psychological symptoms. In addition, greater attention needs to be placed on psychological aspects of IBD care.

The role of mitogen activated protein kinase phosphatase-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons: relevance to Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterised by the loss of midbrain dopaminergic neurons from the substantia nigra pars compacta(SNpc), which results in motor, cognitive and psychiatric symptoms. Evidence supports a role for the mitogen-activated protein kinase p38 in the demise of dopaminergic neurons, while mitogen-activated protein kinase phosphatase-1 (MKP-1), which negatively regulates p38 activity, has not yet been investigated in this context. Inflammation may also be associated with the neuropathology of PD due to evidence of increased levels of proinflammatory cytokines such as interleukin-1β (IL-1β) within the SNpc. Because of the specific loss of dopaminergic neurons in a discreet region of the brain, PD is considered a suitable candidate for cell replacement therapy but challenges remain to optimise dopaminergic cell survival and morphological development. The present thesis examined the role of MKP-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons. We show that MKP-1 is expressed in dopaminergic neurons cultured from embryonic day (E) 14 rat ventral mesencephalon (VM). Inhibition of dopaminergic neurite growth induced by treatment of rat VM neurons with the dopaminergic neurotoxin 6- hydroxydopamine (6-OHDA) is mediated by p38, and is concomitant with a significant and selective decrease in MKP-1 expression in these neurons. Dopaminergic neurons transfected to overexpress MKP-1 displayed a more complex morphology and contributed to neuroprotection against the effects of 6-OHDA. Therefore, MKP-1 expression can promote the growth and elaboration of dopaminergic neuronal processes and can help protect them from the neurotoxic effects of 6-OHDA. Neural precursor cells (NPCs) have emerged as promising alternative candidates to fetal VM for cell replacement strategies in PD. Here we show that phosphorylated (and thus activated) p38 and MKP-1 are expressed at basal levels in untreated E14 rat VM NPCs (nestin, DCX, GFAP and DAT-positive cells) following proliferation as well as in their differentiated progeny (DCX, DAT, GFAP and βIII-tubulin) in vitro. Challenge with 6-OHDA or IL-1β changed the expression of endogenous phospho-p38 and MKP-1 in these cells in a time-dependent manner, and so the dynamic balance in expression may mediate the detrimental effects of neurotoxicity and inflammation in proliferating and differentiating NPCs. We demonstrate that there was an up-regulation in MKP-1 mRNA expression in adult rat midbrain tissue 4 days post lesion in two rat models of PD; the 6-OHDA medial forebrain bundle (MFB) model and the four-site 6-OHDA striatal lesion model. This was concomitant with a decrease in tyrosine hydroxylase (TH) mRNA expression at 4 and 10 days post-lesion in the MFB model and 10 and 28 days post-lesion in the striatal lesion model. There was no change in mRNA expression of the pro-apoptotic gene, bax and the anti-apoptotic gene, bcl-2 in the midbrain and striatum. These data suggest that the early and transient upregulation of MKP-1 mRNA in the midbrain at 4 days post-6-OHDA administration may be indicative of an attempt by dopaminergic neurons in the midbrain to protect against the neurotoxic effects of 6-OHDA at later time points. Collectively, these findings show that MKP-1 is expressed by developing and adult dopaminergic neurons in the midbrain, and can promote their morphological development. MKP-1 also exerts neuroprotective effects against dopaminergic neurotoxins in vitro, and its expression in dopaminergic neurons can be modulated by inflammatory and neurotoxic insults both in vitro and in vivo. Thus, these data contribute to the information needed to develop therapeutic strategies for protecting midbrain dopaminergic neurons in the context of PD.

Novel insights into the inflammatory basis of gastrointestinal disease

It has become clear that inflammation is beneficial to man, there are situations though that the inflammatory response causes damage to the host that is harmful to health. When the inflammatory response fails or is too strong, the health of the host is damaged and disease can occur. The implication of intestinal disease caused by an ineffective immune response is of great social and economic burden to society. The overarching purpose of this thesis is to assess inflammatory signalling targets associated with immune mediated disorders such as IBD, IBS and inflammatory liver disease. By assessing these targets and modifying their function I hope to contribute and expand further the pre-existing information on these disorders and improve the therapeutic interventions available in these debilitating conditions. I will assess the role of inflammation in disorders of the GI tract and liver IBD, IBS, hepatic inflammatory injury and furthermore, I will use pharmaceutical agents to activate and suppress components of the immune system. I will examine the inflammatory response in experimental models of disease for IBD and liver injury, I will attempt to alter these pathways using pharmaceutical intervention to delineate the disease causing mechanism that may lead to clinically relevant therapeutic interventions. In regards to IBS, I will attempt to improve the existing knowledge that exists in relation to the pathogenesis of this functional bowel disorder. I will attempt to define a mechanism by which the low grade mucosal inflammation that has been demonstrated by others arises and what this inflammation is induced by. The overall aim of this thesis is to attempt to further understand the mechanisms behind GI and liver disease. Looking at the inflammatory response in these specific conditions and how they can be altered may lead to exciting new therapies for inflammatory conditions in the gastrointestinal tract.

Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

An assessment of health in post-medieval Ireland: ‘One vast Lazar house filled with famine, disease and death’

Three indicators of health and diet were selected to examine the health status in three socioeconomic groups in post-medieval Ireland. The aim was to examine the reliability of traditional skeletal markers of health in highly contextualised populations. The link between socio-economic status and health was examined to determine if traditional linking of poor health with poverty was evident in skeletal samples. The analysis indicated that this was indeed the case and that health was significantly compromised in populations of low socio-economic status. Thus it indicated that status intimately influences the physical body form. Sex was also found to be a major defining factor in the response of an individual to physiological stress. It was also evident that contemporary populations may suffer from different physiological stresses, and their responses to those stresses may differ. Adaptation was a key factor here. This has implications for studies of earlier populations that may lack detailed contextual data in terms of blanket applications of interpretations. The results also show a decline in health from the medieval through to the post-medieval period, which is intimately linked with the immense social changes and all the related effects of these. The socio-economic structure of post-medieval Ireland was a direct result of the British policies in Ireland. The physical form of the Irish may be seen to have occurred as a result of those policies, with the Irish poor in particular suffering substantial health problems, even in contrast to the poor of Britain. This study has enriched the recorded historical narrative of this period of the recent past, and highlights more nuanced narratives may emerge from the osteoarchaeological analysis when sound contextual information is available. It also examines a period in Irish history that, until very recently, had been virtually untouched in terms of archaeological study.

Akt signal transduction dysfunction in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder, accounting for over 60% of all cases of dementia. The primary risk factor for AD is age, however several genetic and environmental factors are also involved. The pathological characteristics of AD include extracellular deposition of the beta-amyloid peptide (Aβ) and intraneuronal accumulation of neurofibrillary tangles (NFTs) made of aggregated paired helical filaments (PHFs) of the hyperphosphorylated tau protein, along with synaptic loss and neuronal death. There are numerous biochemical mechanisms involved in AD pathogenesis, however the reigning hypothesis points to toxic oligomeric Aβ species as the primary causative factor in a cascade of events leading to neuronal stress and dyshomeostasis that initiate abnormal regulation of tau. The insulin and IGF-1 receptors (IR, IGF-1R) are the primary activators of PI3- K/Akt through which they regulate cell growth, development, glucose metabolism, and learning and memory. Work in our lab and others shows increased Akt activity and phosphorylation of its downstream targets in AD brain, along with insulin and insulin-like growth factor-1 signalling (IIS) dysfunction. This is supported by studies of AD models in vivo and in vitro. Our group and others hypothesise that Aβ activates Akt through IIS to initiate a negative feedback mechanism that desensitises neurons to insulin/IGF-1, and sustains activation of Akt. In this study the functions of endogenous Akt, IR, and the insulin receptor substrate (IRS-1) were examined in relationship to Aβ and tau pathology in the 3xTg-AD mouse model, which contains three mutant human transgenes associated with familial AD or dementia. The 3xTg-AD mouse develops Aβ and tau pathology in a spatiotemporal manner that best recapitulates the progression of AD in human brain. Western blotting and immunofluorescent microscopy techniques were utilised in vivo and in vitro, to examine the relationship between IIS, Akt, and AD pathology. I first characterised in detail AD pathology in 3xTg-AD mice, where an age-related accumulation of intraneuronal Aβ and tau was observed in the hippocampal formation, amygdala, and entorhinal cortex, and at late stages (18 months), extracellular amyloid plaques and NFTs, primarily in the subiculum and the CA1 layer of the hippocampal formation. Increased activity of Akt, detected with antibody to phosphoSer473-Akt, was increased in 3xTg-AD mice compared to age-matched non-transgenic mice (non-Tg), and in direct correlation to the accumulation of Aβ and tau in neuronal somatodendritic compartments. Akt phosphorylates tau at residue Ser214 within a highly specific consensus sequence for Akt phosphorylation, and phosphoSer214-tau strongly decreases microtubule (MT) stabilisation by preventing tau-MT binding. PhosphoSer214-tau increased concomitantly with this in the same age-related and region-specific fashion. Polarisation of tau phosphorylation was observed, where PHF-1 (tauSer396/404) and phosphoSer214-tau both appeared early in 3xTg-AD mice in distinct neuronal compartments: PHF-1 in axons, and phosphoSer214-tau in neuronal soma and dendrites. At 18 months, phosphoSer214-tau strongly colocalised with NFTs positive for the PHF- 1 and AT8 (tauSer202/Thr205) phosphoepitopes. IR was decreased with age in 3xTg-AD brain and in comparison to age-matched non-Tg, and this was specific for brain regions containing Aβ, tau, and hyperactive Akt. IRS-1 was similarly decreased, and both proteins showed altered subcellular distribution. Phosphorylation of IRS-1Ser312 is a strong indicator of IIS dysfunction and insulin resistance, and was increased in 3xTg-AD mice with age and in relation to pathology. Of particular note was our observation that abberant IIS and Akt signalling in 3xTg-AD brain related to Aβ and tau pathology on a gross anatomical level, and specifically localised to the brain regions and circuitry of the perforant path. Finally, I conducted a preliminary study of the effects of synthetic Aβ oligomers on embryonic rat hippocampus neuronal cultures to support these results and those in the literature. Taken together, these novel findings provide evidence for IIS and Akt signal transduction dysfunction as the missing link between Aβ and tau pathogenesis, and contribute to the overall understanding of the biochemical mechanisms of AD.

Towards the enantioselective synthesis of Plakortide P and its application to the development of drugs for the treatment of Chagas disease

The primary focus of this thesis was the asymmetric peroxidation of α,β-unsaturated aldehydes and the development of this methodology to include the synthesis of bioactive chiral 1,2-dioxane and 1,2-dioxalane rings. In Chapter 1 a review detailing the new and improved methods for the acyclic introduction of peroxide functionality to substrates over the last decade was discussed. These include a detailed examination of metal-mediated transformations, chiral peroxidation using organocatalytic means and the improvements in methodology of well-established peroxidation pathways. The second chapter discusses the method by which peroxidation of our various substrates was attempted and the optimisation studies associated with these reactions. The method by which the enantioselectivity of our β-peroxyaldehydes was determined is also reviewed. Chapters 3 and 4 focus on improving the enantioselectivity associated with our asymmetric peroxidation reaction. A comprehensive analysis exploring the effect of solvent, concentration and temperature on enantioselectivity was examined. The effect that different catalytic systems have on enantioselectivity and reactivity was also investigated in depth. Chapter 5 details the various transformations that β-peroxyaldehydes can undergo and the manipulation of these transformations towards the establishment of several routes for the formation of chiral 1,2-dioxane and 1,2-dioxalane rings. Chapter 6 details the full experimental procedures, including spectroscopic and analytical data for the compounds prepared during this research.

Identification and analysis of mechanisms involved in a broad-spectrum disease resistant mutant of the winter wheat cv. Guardian

M66 an X-ray induced mutant of winter wheat (Triticum aestivum) cv. Guardian exhibits broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. tritici), yellow rust (Puccinia striiformis f. sp. tritici), and leaf rust (Puccinia recondita f. sp. tritici), along with partial resistance to stagnonospora nodorum blotch (caused by the necrotroph Stagonosporum nodorum) and septoria tritici blotch (caused by the hemibiotroph Mycosphaerella graminicola) compared to the parent plant ‘Guardian’. Analysis revealed that M66 exhibited no symptoms of infection following artificial inoculation with Bgt in the glasshouse after adult growth stage (GS 45). Resistance in M66 was associated with widespread leaf flecking which developed during tillering. Flecking also occurred in M66 leaves without Bgt challenge; as a result grain yields were reduced by approximately 17% compared to ‘Guardian’ in the absence of disease. At the seedling stage, M66 exhibited partial resistance. M66, along with Tht mutants (Tht 12, Tht13), also exhibit increased tolerance to environmental stresses (abiotic), such as drought and heat stress at seedling and adult growth stages, However, adult M66 exhibited increased susceptibility to the aphid Schizaphis graminum compared to ‘Guardian’. Resistance to Bgt in M66 was characterized with increased and earlier H2O2 accumulation at the site of infection which resulted in increased papilla formation in epidermal cells, compared to ‘Guardian’. Papilla formation was associated with reduced pathogen ingress and haustorium formation, indicating that the primary cause of resistance in M66 was prevention of pathogen penetration. Heat treatment at 46º C prior to challenge with Bgt also induced partial disease resistance to Blumeria graminis f. sp. tritici in ‘Guardian’ and M66 seedlings. This was characterized by a delay in primary infection, due to increased production of ROS species, such as hydrogen peroxide, ROS-scavenging enzymes and Hsp70, resulting in cross-linking of cell wall components prior to inoculation. This actively prevented the fungus from penetrating the epidermal cell wall. Proteomics analysis using 2-D gel electrophoresis identified primary and secondary disease resistance effects in M66 including detection of ROS scavenging enzymes (4, 24 hai), such as ascorbate peroxidase and a superoxidase dismutase isoform (CuZnSOD) in M66 which were absent from ‘Guardian’. Chitinase (PR protein) was also upregulated (24 hai) in M66 compared to ‘Guardian’.Monosomic and ditelosomic analysis of M66 revealed that the mutation in M66 is located on the long arm of chromosome 2B (2BL). Chromosome 2BL is known to have key genes involved in resistance to pathogens such as those causing stripe rust and powdery mildew. The TaMloB1 gene, an orthologue of the barley Mlo gene, is also located on chromosome 2BL. Sanger sequencing of part of the coding sequence revealed no deletions in the TaMloB1 gene between ‘Guardian’ and M66.

Survival analysis of adult tuberculosis disease

Background: We conducted a survival analysis of all the confirmed cases of Adult Tuberculosis (TB) patients treated in Cork-City, Ireland. The aim of this study was to estimate Survival time (ST), including median time of survival and to assess the association and impact of covariates (TB risk factors) to event status and ST. The outcome of the survival analysis is reported in this paper. Methods: We used a retrospective cohort study research design to review data of 647 bacteriologically confirmed TB patients from the medical record of two teaching hospitals. Mean age 49 years (Range 18–112). We collected information on potential risk factors of all confirmed cases of TB treated between 2008–2012. For the survival analysis, the outcome of interest was ‘treatment failure’ or ‘death’ (whichever came first). A univariate descriptive statistics analysis was conducted using a non- parametric procedure, Kaplan -Meier (KM) method to estimate overall survival (OS), while the Cox proportional hazard model was used for the multivariate analysis to determine possible association of predictor variables and to obtain adjusted hazard ratio. P value was set at <0.05, log likelihood ratio test at >0.10. Data were analysed using SPSS version 15.0. Results: There was no significant difference in the survival curves of male and female patients. (Log rank statistic = 0.194, df = 1, p = 0.66) and among different age group (Log rank statistic = 1.337, df = 3, p = 0.72). The mean overall survival (OS) was 209 days (95%CI: 92–346) while the median was 51 days (95% CI: 35.7–66). The mean ST for women was 385 days (95%CI: 76.6–694) and for men was 69 days (95%CI: 48.8–88.5). Multivariate Cox regression showed that patient who had history of drug misuse had 2.2 times hazard than those who do not have drug misuse. Smokers and alcohol drinkers had hazard of 1.8 while patients born in country of high endemicity (BICHE) had hazard of 6.3 and HIV co-infection hazard was 1.2. Conclusion: There was no significant difference in survival curves of male and female and among age group. Women had a higher ST compared to men. But men had a higher hazard rate compared to women. Anti-TNF, immunosuppressive medication and diabetes were found to be associated with longer ST, while alcohol, smoking, RICHE, BICHE was associated with shorter ST.