An exploration of the positive and negative relationships associated with the development of asthma and atopic disorders in primary school children in Cork

Childhood asthma, allergic rhinitis and eczema are complex heterogenic chronic inflammatory allergic disorders which constitute a major burden to children, their families. The prevalence of childhood allergic disorders is increasing worldwide and merely rudimentary understanding exists regarding causality, or the influence of the environment on disease expression. Phase Three of the International Study of Asthma and Allergy in Childhood (ISAAC) reported that Irish adolescents had the 4th highest eczema and rhinoconjunctivitis prevalence and 3rd highest asthma prevalence in the world. There are no ISAAC data pertaining to young Irish children. In 2002, Sturley reported a high prevalence of current asthma in Cork primary school children aged 6-9 years. This thesis comprises of three cross-sectional studies which examined the prevalence of and associations with childhood allergy and a quasi-retrospective cohort study which observed the natural history of allergy from 6-9 until 11-13 years. Although not part of ISAAC, data was attained by parentally completed ISAAC-based questionnaires, using the ISAAC protocol. The prevalence, natural history and risk factors of childhood allergy in Ireland, as described in this thesis, echo those in worldwide allergy research. The variations of prevalence in different populations worldwide and the recurring themes of associations between childhood allergy and microbial exposures, from farming environments and/or gastrointestinal infections, as shown in this thesis, strengthen the mounting evidence that microbial exposure on GALT may hold the key to the mechanisms of allergy development. In this regard, probiotics may be an area of particular interest in allergy modification. Although their effects in relation to allergy, have been investigated now for several years, our knowledge of their diversity, complex functions and interactions with gut microflora, remain rudimentary. Birth cohort studies which include genomic and microbiomic research are recommended in order to examine the underlying mechanisms and the natural course of allergic diseases.

Mesenchymal stem cell therapy for the treatment of myocardial infarction

Mesenchymal stem cells (MSCs) are currently under investigation as repair agents in the preservation of cardiac function following myocardial infarction (MI). However concerns have emerged regarding the safety of acute intracoronary (IC) MSC delivery specifically related to mortality, micro-infarction and microvascular flow restriction post cell therapy in animal models. This thesis aimed to firstly identify an optimal dose of MSC that could be tolerated when delivered via the coronary artery in a porcine model of acute MI (AMI). Initial dosing studies identified 25x106 MSC to be a safe MSC cell dose, however, angiographic observations from these studies recognised that on delivery of MSC there was a significant adverse decrease in distal blood flow within the artery. This observation along with additional supportive data in the literature (published during the course of this thesis) suggested MSC may be contributing to such adverse events through the propagation of thrombosis. Therefore further studies aimed to investigate the innate prothrombotic activity of MSC. Expression of the initiator of the coagulation cascade initiator tissue factor (TF) on MSC was detected in high levels on the surface of these cells. MSC-derived TF antigen was catalytically active, capable of supporting thrombin generation in vitro and enhancing platelet-driven thrombus deposition on collagen under flow. Infusion of MSC via IC route was associated with a decreased coronary flow reserve when delivered but not when coadministered with an antithrombin agent heparin. Heparin also reduced MSC-associated in situ thrombosis incorporating platelets and VWF in the microvasculature. Heparin-assisted MSC delivery reduced acute apoptosis and significantly improved infarct size, left ventricular ejection fraction, LV volumes, wall motion and scar formation at 6 weeks post AMI. In addition, this thesis investigated the paracrine factors secreted by MSC, in particular focusing on the effect on cardiac repair of a novel MSC-paracrine factor SPARCL1. In summary this work provides new insight into the mechanism by which MSC may be deleterious when delivered by an IC route and a means of abrogating this effect. Moreover we present new data on the MSC secretome with elucidation of the challenges encountered using a single paracrine factor cardiac repair strategy.

Studies on quinoa (Chenopodium quinoa) for novel food and beverage applications

Quinoa (Chenopodium quinoa) is a seed crop native to the Andes, that can be used in a variety of food product in a similar manner to cereals. Unlike most plants, quinoa contains protein with a balanced amino acid profile. This makes it an interesting raw material for e.g. dairy product substitutes, a growing market in Europe and U.S. Quinoa can however have unpleasant off-flavours when processed into formulated products. One means of improving the palatability is seed germination. Also, the increased activities of hydrolytic enzymes can have a beneficial influence in food processing. In this thesis, the germination pattern of quinoa was studied, and the influence of quinoa malt was evaluated in a model product. Additionally, to explore its potential for dairy-type products, quinoa protein was isolated from an embryo-enriched milling fraction of non-germinated quinoa and tested for functional and gelation properties. Quinoa seeds imbibed water very rapidly, and most seeds showed radicle protrusion after 8-9 h. The α-amylase activity was very low, and started to increase only after 24 hours of germination in the starchy perisperm. Proteolytic activity was very high in dry ungerminated seeds, and increased slightly over 24 h. A significant fraction of this activity was located in the micropylar endosperm. The incorporation of germinated quinoa in gluten-free bread had no significant effect on the baking properties due to low α-amylase activity. Upon acidification with glucono-δ-lactone, quinoa milk formed a structured gel. The gelation behaviour was further studied using a quinoa protein isolate (QPI) extracted from an embryoenriched milling fraction. QPI required a heat-denaturation step to form gel structures. The heating pH influenced the properties drastically: heating at pH 10.5 led to a dramatic increase in solubility, emulsifying properties, and a formation of a fine-structured gel with a high storage modulus (G') when acidified. Heating at pH 8.5 varied very little from the unheated protein in terms of functional properties, and only formed a randomly aggregated coagulum with a low G'. Further study of changes over the course of heating showed that the mechanism of heat-denaturation and aggregation indeed varied largely depending on pH. The large difference in gelation behaviour may be related to the nature of aggregates formed during heating. To conclude, germination for increased enzyme activities may not be feasible, but the structure-forming properties of quinoa protein could possibly be exploited in dairy-type products.

Optimised crystal morphologies for active pharmaceutical ingredients and related studies

The majority of active pharmaceutical ingredients (APIs) are crystalline solids in their pure forms. Crystalline solids have definable morphologies, i.e. shape and size. Crystal morphology is determined by both the internal structure of the crystals and external factors during growth from solution. The morphology of a crystal batch can affect key processes during manufacturing. Companies generally accept whatever morphology the manufacturing process provides and deal with any subsequent problems by costly trouble‒shooting. Rational design of optimised morphologies for crystalline pharmaceutical solids would be a very significant technical and commercial advance. Chapter one introduces the concept of crystal nucleation and growth. The phenomenon of polymorphism alongside the causes and impact is discussed. A summary of the scope of instrumentation used in the investigation of crystal polymorphism and morphology, including crystal size distribution (CSD), is also included. Chapter two examines the research carried out during an exploration of the optimum crystallisation parameters of phenacetin. Following a morphological study, the impact this induces on particle density and flow properties is examined. The impact of impurities on the crystallisation properties of phenacetin is investigated. Significantly, the location of impurities within individual crystals is also studied. The third chapter describes an industrial collaboration looking at the resolution and polymorphic study of trometamol and lysine salts of ketoprofen and 2‒phenylpropionic acid (2‒PPA). Chapter four incorporates a solid state study on three separate compounds: 2‒chloro‒4‒nitroaniline, 4‒hydroxy‒N‒phenylbenzenesulfonamide and N‒acetyl‒D‒glucosamine‒6‒O‒sulfate. 2‒Chloro‒4‒nitroaniline and 4‒hydroxy‒N‒phenylbenzenesulfonamide both produced interesting, extreme morphologies which warranted further investigation as part of a collaborative study. Following a summarisation of results in chapter five, chapter six contains the full experimental details, incorporating spectral and other analytical data for all compounds synthesised during the course of the research.