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

The expression and regulation of pregnancy-specific glycoproteins in the mouse

Pregnancy-Specific Glycoproteins (PSG) are the most abundant fetally expressed proteins in the maternal bloodstream at term. This multigene family are immunoglobulin superfamily members and are predominantly expressed in the syncytiotrophoblast of human placenta and in giant cells and spongiotrophoblast of rodent placenta. PSGs are encoded by seventeen genes in the mouse and ten genes in the human. Little is known about the function of this gene family, although they have been implicated in immune modulation and angiogenesis through the induction of cytokines such as IL-10 and TGFβ1 in monocytes, and more recently, have been shown to inhibit the platelet-fibrinogen interaction. I provide new information concerning the evolution of the murine Psg genomic locus structure and organisation, through the discovery of a recent gene inversion event of Psg22 within the major murine Psg cluster. In addition to this, I have performed an examination of the expression patterns of individual Psg genes in placental and non-placental tissues. This study centres on Psg22, which is the most abundant murine Psg transcript detected in the first half of pregnancy. A novel alternative splice variant transcript of Psg22 lacking the protein N1-domain was discovered, and similar to the full length isoform induces TGFβ1 in macrophage and monocytic cell lines. The identification of a bidirectional antisense long non-coding RNA transcript directly adjacent to Psg22 and its associated active local chromatin conformation, suggests an interesting epigenetic gene-specific regulatory mechanism that may be responsible for the high level of Psg22 expression relative to the other Psg family members upon trophoblast giant cell differentiation

Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs.

Preclinical atherosclerosis in rheumatoid arthritis

Introduction: There is accumulating evidence of an increased risk of cardiovascular morbidity and mortality in rheumatoid arthritis patients. A combination of both traditional cardiovascular risks and rheumatoid specific factors appear to be responsible for driving this phenomenon. Rheumatoid arthritis has been an orphan of cardiologists in the past and rheumatologists themselves are not good at CVD screening. Identifying the extent of preclinical atherosclerosis in RA patients will help us to appreciate the magnitude of this serious problem in an Irish population. Methods: We undertook a cross-sectional study of 63 RA patients and 48 OA controls and compared the 2 groups with respect to 1) traditional CV risks factors, 2) serum biomarkers of inflammation, including CRP, TNFα, IL6 and PAI-1, 3) carotid intima-media thickness (cIMT), carotid plaque and ankle-brachial index (ABI) as markers of pre-clinical atherosclerosis, 4) biochemical and ultrasonic measures of endothelial dysfunction and 5) serum and echocardiographic measures of diastolic dysfunction. Within the RA group, we also investigated for associations between markers of inflammation, subclinical atherosclerosis and diastolic dysfunction. Results: Prevalence of traditional CV risks was similar in the RA and OA groups. A number of biomarkers of inflammation were significantly higher in the RA group: CRP, fibrinogen, IL- 2, -4, -6, TNFα. PAI-1, a marker of thrombosis, correlated with disease activity and subclinical atherosclerosis in RA patients. With regard to subclinical atherosclerosis measures, RA patients had a significantly lower ABI than OA patients. Carotid plaque and cIMT readings were similar in RA and OA patients. Assessment of endothelial function revealed that RA patients had significantly higher concentrations of adhesion molecules, in particular sero-positive RA patients and RA smokers. Adhesion molecule concentrations were associated with markers of diastolic dysfunction in RA. Urine PCR, another marker of endothelial dysfunction also correlated with diastolic dysfunction in RA. Assessment of endothelial function with flow mediated dilatation (FMD) found no difference between the RA and OA groups. Disease activity scores in RA patients were associated with endothelial dysfunction, as assessed by FMD. Conclusions: We did not find significant differences in measures of subclinical atherosclerosis, flow mediated dilatation or diastolic function between RA and OA patients. This is most likely in part due to the fact that there is increasing evidence that OA has an inflammatory component to its pathogenesis and is associated with metabolic syndrome and increased CV risk. We reported a significant association between urinary PCR and measures of diastolic dysfunction. Urinary PCR may be a useful screening tool for diastolic dysfunction in RA. The association between RA disease activity and measures of vascular function supports the theory that the excess cardiovascular burden in RA is linked to uncontrolled inflammation.

A cultural study on communication and technology with specific reference to media in China

This dissertation examines the role of communications technology in social change. It examines secondary data on contemporary China arguing that many interpretations of events in China are unsuitable at best and at worst conceptually damages our understanding of social change in China. This is especially the case in media studies under the ‘democratic framework’. It proposes that there is an alternative framework in studying the media and social change. This alternative conceptual framework is termed a zone of interpretative development offering a means by which to discuss events that take place in a mediated environment. Taking a theoretical foundation using the philosophy of Mikhail Bakhtin this dissertation develops a platform with which to understand communication technology from an anthropological perspective. Three media events from contemporary China are examined. The first examines the Democracy Wall event and the implications of using a public sphere framework. The second case examines the phenomenon of the Grass Mud Horse, a symbol that has gained popular purchase as a humorous expression of political dissatisfaction and develops the problems seen in the first case but with some solutions. Using a modification of Lev Vygotskiĭ’s zone of proximal development this symbol is understood as an expression of the collective recognition of a shared experience. In the second example from the popular TV talent show contests in China further expressions of collective experience are introduced. With the evidence from these media events in contemporary China this dissertation proposes that we can understand certain modes of communication as occurring in a zone of interpretative development. This proposed anthropological feature of social change via communication and technology can fruitfully describe meaning-formation in society via the expression and recognition of shared experiences.

Identification of ribosomal proteins specific to higher eukaryotic organisms

This report describes the identification of a novel protein named PS1D (Genbank accession number ), which is composed of an S1-like RNA-binding domain, a (cysteine)x3-(histidine) CCCH-zinc finger, and a very basic carboxyl domain. PS1D is expressed as two isoforms, probably resulting from the alternative splicing of mRNA. The long PS1D isoform differs from the short one by the presence of 48 additional amino acids at its amino-terminal extremity. Analysis of PS1D subcellular distribution by cell fractionation reveals that this protein belongs to the core of the eukaryotic 60S ribosomal subunit. Interestingly, PS1D protein is a highly conserved protein among mammalians as murine, human, and simian PS1D homologues share more than 95% identity. In contrast, no homologous protein is found in lower eukaryotes such as yeast and Caenorhabditis elegans. These observations indicate that PS1D is the first eukaryotic ribosomal protein that is specific to higher eukaryotes.

A variant of SCID with specific immune responses and predominance of gamma delta T cells.

We describe here a patient with a clinical and molecular diagnosis of recombinase activating gene 1-deficient (RAG1-deficient) SCID, who produced specific antibodies despite minimal B cell numbers. Memory B cells were detected and antibodies were produced not only against some vaccines and infections, but also against autoantigens. The patient had severely reduced levels of oligoclonal T cells expressing the alphabeta TCR but surprisingly normal numbers of T cells expressing the gammadelta TCR. Analysis at a clonal level and TCR complementarity-determining region-3 spectratyping for gammadelta T cells revealed a diversified oligoclonal repertoire with predominance of cells expressing a gamma4-delta3 TCR. Several gammadelta T cell clones displayed reactivity against CMV-infected cells. These observations are compatible with 2 non-mutually exclusive explanations for the gammadelta T cell predominance: a developmental advantage and infection-triggered, antigen-driven peripheral expansion. The patient carried the homozygous hypomorphic R561H RAG1 mutation leading to reduced V(D)J recombination but lacked all clinical features characteristic of Omenn syndrome. This report describes a new phenotype of RAG deficiency and shows that the ability to form specific antibodies does not exclude the diagnosis of SCID.

Evaluation of a WHO-Validated Serotype-Specific Serological Assay for the Diagnosis of Pneumococcal Etiology in Children with Community-Acquired Pneumonia.

BACKGROUND: The etiologic diagnosis of community-acquired pneumonia (CAP) remains challenging in children because blood cultures have low sensitivity. Novel approaches are needed to confirm the role of Streptococcus pneumoniae. METHODS: In this study, pneumococcal aetiology was determined by serology using a subset of blood samples collected during a prospective multicentre observational study of children <15 years of age hospitalised in Belgium with X-ray-confirmed CAP. Blood samples were collected at admission and 3-4 weeks later. Pneumococcal (P)-CAP was defined in the presence of a positive blood or pleural fluid culture. Serotyping of Streptococcus pneumoniae isolates was done with the Quellung reaction. Serological diagnosis was assessed for nine serotypes using World Health Organization validated IgG and IgA serotype-specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: Paired admission/convalescent sera from 163 children were evaluated by ELISA (35 with proven P-CAP and 128 with non proven P-CAP). ELISA detected pneumococci in 82.8% of patients with proven P-CAP. The serotypes identified were the same as with the Quellung reaction in 82% and 59% of cases by IgG ELISA and IgA ELISA, respectively. Overall, ELISA identified a pneumococcal aetiology in 55% of patients with non-proven P-CAP. Serotypes 1 (51.6%), 7F (19%), and 5 (15.7%) were the most frequent according to IgG ELISA. CONCLUSIONS: In conclusion, the serological assay allows recognition of pneumococcal origin in 55% of CAP patients with negative culture. This assay should improve the diagnosis of P-CAP in children and could be a useful tool for future epidemiological studies on childhood CAP etiology.