Exploring oxidative modifications of tyrosine:an update on mechanisms of formation, advances in analysis and biological consequences

Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

Neuropathology of the sporadic and variant subtypes of Creutzfeldt-Jakob disease:a comparative quantitative study

The objective of the present study was to compare quantitatively the neuropathology of two subtypes of Creutzfeldt-Jakob disease (CJD), viz., sporadic CJD (sCJD) and variant CJD (vCJD). The vacuolation (‘spongiform change’), surviving neurons, glial cell nuclei, and deposits of the disease form of prion protein (PrPsc) were quantified in histological sections of the cerebral cortex, hippocampus, and cerebellum in 11 cases of sCJD and 15 cases of vCJD. Three aspects of the quantitative pathology of each histological feature were studied: overall abundance (density or coverage), spatial distribution parallel to the tissue boundary, and laminar distribution across gyri of the cerebral cortex. Overall vacuole density was greater in sCJD than in vCJD in some regions while overall neuronal densities were greater in vCJD. In cerebral cortex, vacuoles and PrPsc deposits were distributed in clusters which exhibited a regular distribution parallel to the pia mater, this type of spatial pattern being more frequent in sCJD than in vCJD. In some cortical gyri there were differences in laminar distribution between subtypes, viz. the vacuolation was more generally distributed across cortical laminae in sCJD, neuronal loss was often greater in upper laminae in vCJD but in lower laminae in sCJD, and PrPsc deposits were more frequently distributed in upper laminae in vCJD but in lower laminae in sCJD. A significant gliosis affected lower cortical laminae in both sCJD and vCJD. Hence, there were differences in degeneration of cerebral cortex, hippocampus, and cerebellum in sCJD and vCJD, which may reflect variations in disease aetiology and propagation of PrPsc through the brain.

Comparative quantitative neuropathology of the sporadic and variant subtypes of Creutzfeldt-Jakob disease

The objective of the present study was to compare quantitatively the neuropathology of two subtypes of Creutzfeldt-Jakob disease (CJD), viz., sporadic CJD (sCJD) and variant CJD (vCJD). The vacuolation (‘spongiform change’), surviving neurons, glial cell nuclei, and deposits of the disease form of prion protein (PrPsc) were quantified in histological sections of the cerebral cortex, hippocampus, and cerebellum in 11 cases of sCJD and 15 cases of vCJD. Three aspects of the quantitative pathology of each histological feature were studied: overall abundance (density or coverage), spatial distribution parallel to the tissue boundary, and laminar distribution across gyri of the cerebral cortex. Overall vacuole density was greater in sCJD than in vCJD in some regions while overall neuronal densities were greater in vCJD. In cerebral cortex, vacuoles and PrPsc deposits were distributed in clusters which exhibited a regular distribution parallel to the pia mater, this type of spatial pattern being more frequent in sCJD than in vCJD. In some cortical gyri there were differences in laminar distribution between subtypes, viz. the vacuolation was more generally distributed across cortical laminae in sCJD, neuronal loss was often greater in upper laminae in vCJD but in lower laminae in sCJD, and PrPsc deposits were more frequently distributed in upper laminae in vCJD but in lower laminae in sCJD. A significant gliosis affected lower cortical laminae in both sCJD and vCJD. Hence, there were differences in degeneration of cerebral cortex, hippocampus, and cerebellum in sCJD and vCJD, which may reflect variations in disease aetiology and propagation of PrPsc through the brain.

Characterizations of the major coral diseases of the Philippines: Ulcerative white spot disease and novel growth anomalies of Porites

Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases. Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.

Characterizations of the Major Coral Diseases of the Philippines: Ulcerative White Spot Disease and Novel Growth Anomalies of Porites

Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases. Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.

A Central Role for Monocyte-Platelet Interactions in Heart Failure

Heart failure (HF) is an increasingly prevalent and costly multifactorial syndrome with high morbidity and mortality rates. The exact pathophysiological mechanisms leading to the development of HF are not completely understood. Several emerging paradigms implicate cardiometabolic risk factors, inflammation, endothelial dysfunction, myocardial fibrosis, and myocyte dysfunction as key factors in the gradual progression from a healthy state to HF. Inflammation is now a recognized factor in disease progression in HF and a therapeutic target. Furthermore, the monocyte-platelet interaction has been highlighted as an important pathophysiological link between inflammation, thrombosis, endothelial activation, and myocardial malfunction. The contribution of monocytes and platelets to acute cardiovascular injury and acute HF is well established. However, their role and interaction in the pathogenesis of chronic HF are not well understood. In particular, the cross talk between monocytes and platelets in the peripheral circulation and in the vicinity of the vascular wall in the form of monocyte-platelet complexes (MPCs) may be a crucial element, which influences the pathophysiology and progression of chronic heart disease and HF. In this review, we discuss the role of monocytes and platelets as key mediators of cardiovascular inflammation in HF, the mechanisms of cell activation, and the importance of monocyte-platelet interaction and complexes in HF pathogenesis. Finally, we summarize recent information on pharmacological inhibition of inflammation and studies of antithrombotic strategies in the setting of HF that can inform opportunities for future work. We discuss recent data on monocyte-platelet interactions and the potential benefits of therapy directed at MPCs, particularly in the setting of HF with preserved ejection fraction.

Genome-wide association study reveals greater polygenic loading for schizophrenia in cases with a family history of illness

Genome-wide association studies (GWAS) of schizophrenia have yielded more than 100 common susceptibility variants, and strongly support a substantial polygenic contribution of a large number of small allelic effects. It has been hypothesized that familial schizophrenia is largely a consequence of inherited rather than environmental factors. We investigated the extent to which familiality of schizophrenia is associated with enrichment for common risk variants detectable in a large GWAS. We analyzed single nucleotide polymorphism (SNP) data for cases reporting a family history of psychotic illness (N = 978), cases reporting no such family history (N = 4,503), and unscreened controls (N = 8,285) from the Psychiatric Genomics Consortium (PGC1) study of schizophrenia. We used a multinomial logistic regression approach with model-fitting to detect allelic effects specific to either family history subgroup. We also considered a polygenic model, in which we tested whether family history positive subjects carried more schizophrenia risk alleles than family history negative subjects, on average. Several individual SNPs attained suggestive but not genome-wide significant association with either family history subgroup. Comparison of genome-wide polygenic risk scores based on GWAS summary statistics indicated a significant enrichment for SNP effects among family history positive compared to family history negative cases (Nagelkerke's R(2 ) = 0.0021; P = 0.00331; P-value threshold <0.4). Estimates of variability in disease liability attributable to the aggregate effect of genome-wide SNPs were significantly greater for family history positive compared to family history negative cases (0.32 and 0.22, respectively; P = 0.031). We found suggestive evidence of allelic effects detectable in large GWAS of schizophrenia that might be specific to particular family history subgroups. However, consideration of a polygenic risk score indicated a significant enrichment among family history positive cases for common allelic effects. Familial illness might, therefore, represent a more heritable form of schizophrenia, as suggested by previous epidemiological studies.

Development of Imaging Mass Spectrometry Analysis of Lipids in Biological and Clinically Relevant Applications

La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.

Saúde oral e cuidados primários em Odontopediatria

Introdução: A cárie dentária é uma doença multifatorial, infeciosa, transmissível e dependente da dieta, que provoca a desmineralização das estruturas dentárias. Devido a esta natureza etiológica multifatorial é fundamental o aumento da sensibilização e educação das crianças e pais, quer por profissionais de saúde, quer pelos professores em ambiente escolar. A aquisição de hábitos de higiene oral e nutricionais adequados pela criança é um fator imperativo para a manutenção de uma boa saúde oral. Objetivos: Avaliação da Saúde Oral e cuidados primários num grupo de crianças e adolescentes pertencentes ao Agrupamento de Escolas de Alijó. Método: A amostra é constituída por 216 alunos do Agrupamento de Escolas de Alijó, agrupados em três escalões etários: 6-9 anos, 10-14 anos e 15-16 anos. Foi realizada uma observação oral com base no índice de higiene oral simplificado e no índice de cárie (CPOd/cpod- Dentes Cariados, Perdidos e Obturados). Após esta observação foi aplicado um questionário escrito sobre diversos temas relacionados com a saúde oral. Resultados: A percentagem de crianças livres de lesões cariosas aos 6, 12 e 15 anos foi de 33,3%, 28,4% e 16,2%, respetivamente. Os valores da média de número de escovagens diárias aumentam de acordo com a faixa etária. O número de escovagens médias aos 6-9anos, 10-14anos e 15-16anos foi 1,37; 1,48 e 1,55 no sexo masculino, no caso de sexo feminino estes valores passam para 1,58; 1,80 e 1,89 respetivamente. Discussão: Os números registados no presente estudo são semelhantes aos do I Estudo Nacional de Prevalência da Cárie Dentária, onde se verificou percentagens de 33%, 27% e 18,9% para os escalões referentes aos 6 anos, 9 anos e 15 anos. Existe uma melhoria significativa em cada um destes escalões nos dois estudos seguintes realizados pela DGS. Em 2008 estas percentagens foram de 51%, 44% e 28%, respetivamente para cada um dos escalões. Em 2015 existiu apenas um pequeno acréscimo, com percentagens registadas de 54,8%, 53% e 32,4%, respetivamente. A última percentagem, de 32,4%, refere-se neste caso ao escalão dos 18 anos, uma vez que a DGS substituiu o escalão de estudo dos 15 anos de idade. Quando nos referimos ao número de escovagens médio, os números registados neste estudo têm uma evolução semelhante aos encontrados no III Estudo Nacional de Prevalência da Cárie Dentária, publicado em 2015 pela DGS, que refere que aos 6 anos 53% dos inquiridos escova duas vezes ao dia e 25,7% apenas uma vez, enquanto que aos 12 anos nos deparamos com uma descida para 19,9% dos que escovam 1x por dia e um aumento para 69,9% dos que escovam duas vezes. O mesmo estudo afirma ainda que as jovens escovam mais vezes os dentes, tal como no presente estudo. Conclusão: Os últimos anos têm-se revelado determinantes na melhoria da Saúde Oral dos portugueses e a aposta na prevenção, nomeadamente nos Cuidados Primários de Saúde Oral tem apresentado uma mais-valia inquestionável. Como em todos os percursos que se iniciam, a monitorização dos resultados e a sua melhoria contínua podem conduzir à otimização das estratégias e no seu conjunto contribuir para fazer ainda mais e melhor. Este estudo permitiu aferir, que a realidade, em contextos de Saúde Oral, no Concelho de Alijó é preocupante sugerindo que existem assimetrias em Portugal e cabe a todos os intervenientes em Saúde Oral diminuí-las. O envolvimento de cada Médico Dentista neste processo é fundamental.

Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea. The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum. GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification.

Ecological drivers of a vector borne pathogen: distribution and abundance of Borrelia burgdorferi sensu lato and its vector Ixodes ricinus in Scotland

Vector-borne disease emergence in recent decades has been associated with different environmental drivers including changes in habitat, hosts and climate. Lyme borreliosis is among the most important vector-borne diseases in the Northern hemisphere and is an emerging disease in Scotland. Transmitted by Ixodid tick vectors between large numbers of wild vertebrate host species, Lyme borreliosis is caused by bacteria from the Borrelia burgdorferi sensu lato species group. Ecological studies can inform how environmental factors such as host abundance and community composition, habitat and landscape heterogeneity contribute to spatial and temporal variation in risk from B. burgdorferi s.l. In this thesis a range of approaches were used to investigate the effects of vertebrate host communities and individual host species as drivers of B. burgdorferi s.l. dynamics and its tick vector Ixodes ricinus. Host species differ in reservoir competence for B. burgdorferi s.l. and as hosts for ticks. Deer are incompetent transmission hosts for B. burgdorferi s.l. but are significant hosts of all life-stages of I. ricinus. Rodents and birds are important transmission hosts of B. burgdorferi s.l. and common hosts of immature life-stages of I. ricinus. In this thesis, surveys of woodland sites revealed variable effects of deer density on B. burgdorferi prevalence, from no effect (Chapter 2) to a possible ‘dilution’ effect resulting in lower prevalence at higher deer densities (Chapter 3). An invasive species in Scotland, the grey squirrel (Sciurus carolinensis), was found to host diverse genotypes of B. burgdorferi s.l. and may act as a spill-over host for strains maintained by native host species (Chapter 4). Habitat fragmentation may alter the dynamics of B. burgdorferi s.l. via effects on the host community and host movements. In this thesis, there was lack of persistence of the rodent associated genospecies of B. burgdorferi s.l. within a naturally fragmented landscape (Chapter 3). Rodent host biology, particularly population cycles and dispersal ability are likely to affect pathogen persistence and recolonization in fragmented habitats. Heterogeneity in disease dynamics can occur spatially and temporally due to differences in the host community, habitat and climatic factors. Higher numbers of I. ricinus nymphs, and a higher probability of detecting a nymph infected with B. burgdorferi s.l., were found in areas with warmer climates estimated by growing degree days (Chapter 2). The ground vegetation type associated with the highest number of I. ricinus nymphs varied between studies in this thesis (Chapter 2 & 3) and does not appear to be a reliable predictor across large areas. B. burgdorferi s.l. prevalence and genospecies composition was highly variable for the same sites sampled in subsequent years (Chapter 2). This suggests that dynamic variables such as reservoir host densities and deer should be measured as well as more static habitat and climatic factors to understand the drivers of B. burgdorferi s.l. infection in ticks. Heterogeneity in parasite loads amongst hosts is a common finding which has implications for disease ecology and management. Using a 17-year data set for tick infestations in a wild bird community in Scotland, different effects of age and sex on tick burdens were found among four species of passerine bird (Chapter 5). There were also different rates of decline in tick burdens among bird species in response to a long term decrease in questing tick pressure over the study. Species specific patterns may be driven by differences in behaviour and immunity and highlight the importance of comparative approaches. Combining whole genome sequencing (WGS) and population genetics approaches offers a novel approach to identify ecological drivers of pathogen populations. An initial analysis of WGS from B. burgdorferi s.s. isolates sampled 16 years apart suggests that there is a signal of measurable evolution (Chapter 6). This suggests demographic analyses may be applied to understand ecological and evolutionary processes of these bacteria. This work shows how host communities, habitat and climatic factors can affect the local transmission dynamics of B. burgdorferi s.l. and the potential risk of infection to humans. Spatial and temporal heterogeneity in pathogen dynamics poses challenges for the prediction of risk. New tools such as WGS of the pathogen (Chapter 6) and blood meal analysis techniques will add power to future studies on the ecology and evolution of B. burgdorferi s.l.

Engraulis encrasicolus (Linnaeus, 1758): technical report about extraction and recognition of the digestive tract contents in early stages of the life cycle

The study of ichthyio-plankton stages and its relations with the environment and other organisms is therefore crucial for a correct use of fishery resources. In this context, the extraction and the analysis of the content of the digestive tract, is a key method for the identification of the diet in early larval stages, the determination of the resources they rely on and possibly a comparison with the diet of other species. Additionally this approach could be useful in determination on occurrence of species competition. This technique is preceded by the analysis of morphometric data (Blackith & Reyment, 1971; Marcus, 1990), that is the acquisition of quantitative variables measured from the morphology of the object of study. They are linear distances, count, angles and ratios. The subsequent application of multivariate statistical methods, aims to quantify the changes in morphological measures between and within groups, relating them to the type and size of prey and evaluate if some changes appear in food choices along the larvae growth.

Lifestyle factors, adiposity and cardiometabolic risk factors in South Asian women living in Scotland

South Asians migrating to the Western world have a 3 to 5-fold higher risk of developing type 2 diabetes and double the risk of cardiovascular disease (CVD) than the background population of White European descent, without exhibiting a proportional higher prevalence of conventional cardiometabolic risk factors. Notably, women of South Asian descent are more likely to be diagnosed with type 2 diabetes as they grow older compared with South Asian men and, in addition, they have lost the cardio-protective effects of being females. Despite South Asian women in Western countries being a high risk group for developing future type 2 diabetes and CVD, they have been largely overlooked. The aims of this thesis were to compare lifestyle factors, body composition and cardiometabolic risk factors in healthy South Asian and European women who reside in Scotland, to examine whether ethnicity modifies the associations between modifiable environmental factors and cardiometabolic risks and to assess whether vascular reactivity is altered by ethnicity or other conventional and novel CVD risks. I conducted a cross-sectional study and recruited 92 women of South Asian and 87 women of White European descent without diagnosed diabetes or CVD. Women on hormone replacement therapy or hormonal contraceptives were excluded too. Age and body mass index (BMI) did not differ between the two ethnic groups. Physical activity was assessed and with self-reported questionnaires and objectively with the use of accelerometers. Cardiorespiratory fitness was quantified with the predicted maximal oxygen uptake (VO2 max) during a submaximal test (Chester step test). Body composition was assessed with skinfolds measured at seven body sites, five body circumferences, measurement of abdominal subcutaneous (SAT) and visceral adipose tissue (VAT) with the use of magnetic resonance imaging (MRI) and liver fat with the use MR spectroscopy. Dietary density was assessed with food frequency questionnaires. Vascular response was assessed by measuring the response to acetylcholine and sodium nitroprusside with the use of Laser Doppler Imaging with Iontophoresis (LDI-ION) and the response to shear stress with the use of Peripheral Arterial Tonometry (EndoPAT). The South Asian women exhibited a metabolic profile consistent with the insulin resistant phenotype, characterised by greater levels of fasting insulin, lower levels of high density lipoprotein (HDL) and higher levels of triglycerides (TG) compared with their European counterparts. In addition, the South Asians had greater levels of glycated haemoglobin (HbA1c) for any given level of fasting glucose. The South Asian women engaged less time weekly with moderate to vigorous physical activity (MVPA) and had lower levels of cardiorespiratory fitness for any given level of physical activity than the women of White descent. In addition, they accumulated more fat centrally for any given BMI. Notably, the South Asians had equivalent SAT with the European women but greater VAT and hepatic fat for any given BMI. Dietary density did not differ among the groups. Increasing central adiposity had the largest effect on insulin resistance in both ethic groups compared with physical inactivity or decreased cardiorespiratory fitness. Interestingly, ethnicity modified the association between central adiposity and insulin resistance index with a similar increase in central adiposity having a substantially larger effect on insulin resistance index in the South Asian women than in the Europeans. I subsequently examined whether ethnic specific thresholds are required for lifestyle modifications and demonstrated that South Asian women need to engage with MVPA for around 195 min.week-1 in order to equate their cardiometabolic risk with that of the Europeans exercising 150 min.week-1. In addition, lower thresholds of abdominal adiposity and BMI should apply for the South Asians compared with the conventional thresholds. Although the South Asians displayed an adverse metabolic profile, vascular reactivity measured with both methods did not differ among the two groups. An additional finding was that menopausal women with hot flushing of both ethnic groups showed a paradoxical vascular profile with enhanced skin perfusion (measured with LDI-ION) but decreased reactive hyperaemia index (measured with EndoPAT) compared with asymptomatic menopausal women. The latter association was independent of conventional CVD risk factors. To conclude, South Asian women without overt disease who live in Scotland display an adverse metabolic profile with steeper associations between lifestyle risk factors and adverse cardiometabolic outcomes compared with their White counterparts. Further work in exploring ethnic specific thresholds in lifestyle interventions or in disease diagnosis is warranted.

Effects of supplemental osteopontin on intestinal development and serum antibody responses to rotavirus vaccination in piglets

Milk contains numerous bioactive substances including immunoglobulins, cytokines, growth factors and components that exert antibiotic and prebiotic activity (Field, 2005). Little is known about the biological effects of individual milk bioactives, despite the fact that natural milk improves intestinal development and immune system functions in neonates (Donovan et al., 1994; Field, 2005) relative to milk formula. Characterization of the biological effects of such components is important for optimal production of infant milk formulas to be used when mother’s milk is not available. Milk components with preliminary evidence of positive effects on the intestinal growth and mucosal immunity include osteopontin (OPN). Osteopontin is a phosphorylated acidic glycoprotein expressed by a number of different immune and non-immune cells and tissues (Sodek et al., 2000). It is also present in body fluids including blood, bile and milk (Sodek et al., 2000). Osteopontin is a multifunctional protein that is implicated in a wide number of biological processes including cell survival, bone remodeling, and immune modulatory functions (Sodek et al., 2000). Furthermore, Schack and colleagues (2009) demonstrated that the concentration of OPN in human milk is considerably higher than in bovine milk and infant formulas. Taken together, it is likely that OPN plays a role in the early development of gastrointestinal tract and mucosal immune responses in infants. Since the neonatal pig shares anatomical, physiological, immunological, and metabolic similarities with the human infants (Moughan, et al., 1992), they were selected as the animal model in our studies. Our first aim was to investigate the effects of OPN on piglet intestinal development. Newborn, colostrum-deprived piglets (n=27) were randomized to receive three treatments: formula with bovine OPN (OPN; 140 mg/L); formula alone (FF); or sow reared (SR) for 21 days. Body weight, intestinal weight and length, mucosal protein and DNA content, disaccharidase activity, villus morphology, and crypt cell proliferation were measured. Statistical significance was assigned at P<0.05. No significant effects of OPN were observed for body weight, intestinal weight and length. Mucosal protein content of SR piglets was lower than FF and OPN piglets in the duodenum, but higher than FF and OPN piglets in the ileum. No significant effects of diet in mucosal DNA content were detected for the three regions of the small intestine. Lactase and sucrase activities of SR piglets were higher than the two formula-fed groups in the duodenum, lower in the ileum. No significant effects of diet on lactase and sucrase activities were noted between two formula-fed groups in the duodenum and ileum. Jejunal lactase activity of FF piglets was higher than SR piglets, whereas no significant effect of diet was observed in jejunal sucrase activity among the three groups. Duodenal and ileal villus height and villus area of SR piglets were lower than two formula-fed groups, while OPN piglets did not differ from FF piglets. There was a significant effect of diet (P<0.0001) on jejunal crypt cell proliferation, with proliferation in OPN piglets being intermediate between that of FF and SR. In summary, supplemental OPN increased jejunal crypt cell proliferation, independent of evident morphological growth, and had a minor impact on disaccharidase activity in the small intestine of neonatal piglets. Rotavirus (RV) is the most common viral cause of severe gastroenteritis in infants and young children worldwide (Parashar et al., 2006). Maeno et al. (2009) reported that OPN knockout (OPN-KO) suckling mice were more susceptible to RV infection compared to wild-type (WT) suckling mice. To detect the role of OPN in intestinal immune responses of neonates, the goal of the second study was to evaluate whether supplemental OPN influenced the serum antibody responses to RV vaccination in neonatal piglets. Newborn, colostrum-deprived piglets were randomized into two dietary groups: formula with bovine OPN (OPN; 140 mg/L) and formula alone (FF) for 35 days. On d7, piglets in each dietary group were further randomized to receive rotavirus (RV) vaccination (Rotarix®) (FF+RV and OPN+RV) or remained non-vaccinated (FF+NV and OPN+NV). Booster vaccination was provided on d14. Blood samples were collected on d7, 14, 21, 28 and 35. RV-specific serum immunoglobulin (Ig) G, IgA, IgM and total serum IgG, IgA, IgM were measured by ELISA. Statistical significance was assigned at P<0.05, with trends reported as P<0.10. Body weight gain was unaffected by diet and/or vaccination. No significant effect of oral OPN supplementation was observed for RV-specific antibody responses and total Igs levels. After the combination of dietary groups, RV piglets had significantly higher RV-specific IgM concentrations compared to NV piglets. Although there were higher means of RV-specific IgG and RV-specific IgA concentrations in RV group than their counterparts in NV group, the difference did not reach statistical significance. RV-specific IgM reached a peak at d7 post booster vaccination (PBV), whereas the RV-specific IgG and IgA peaked later at PBV 14 or 21. Total Igs were unaffected by RV vaccination but were significantly increased over time, following similar pattern as RV-specific Igs. In summary, neonatal piglets generated weak antibody responses to RV vaccination. Supplemental OPN did not enhance RV-specific serum antibody responses and total serum Igs levels in neonatal piglets with or without RV vaccination. In conclusion, we observed normal developmental changes in the small intestine and serum Igs levels in neonatal piglets over time. Oral OPN supplementation showed minimal impacts on intestinal development and no effect on serum Igs levels. The role of supplemental OPN on the growth and development of infants is still inconclusive. Future studies should measure other physiological and immunological parameters by using different models of vaccination or infection.

Test-Retest Reliability of Resting-State Magnetoencephalography Power in Sensor and Source Space

Several studies have reported changes in spontaneous brain rhythms that could be used asclinical biomarkers or in the evaluation of neuropsychological and drug treatments in longitudinal studies using magnetoencephalography (MEG). There is an increasing necessity to use these measures in early diagnosis and pathology progression; however, there is a lack of studies addressing how reliable they are. Here, we provide the first test-retest reliability estimate of MEG power in resting-state at sensor and source space. In this study, we recorded 3 sessions of resting-state MEG activity from 24 healthy subjects with an interval of a week between each session. Power values were estimated at sensor and source space with beamforming for classical frequency bands: delta (2–4 Hz), theta (4–8 Hz), alpha (8–13 Hz), low beta (13–20 Hz), high beta (20–30 Hz), and gamma (30–45 Hz). Then, test-retest reliability was evaluated using the intraclass correlation coefficient (ICC). We also evaluated the relation between source power and the within-subject variability. In general, ICC of theta, alpha, and low beta power was fairly high (ICC > 0.6) while in delta and gamma power was lower. In source space, fronto-posterior alpha, frontal beta, and medial temporal theta showed the most reliable profiles. Signal-to-noise ratio could be partially responsible for reliability as low signal intensity resulted inhigh within-subject variability, but also the inherent nature of some brain rhythms in resting-state might be driving these reliability patterns. In conclusion, our results described the reliability of MEG power estimates in each frequency band, which could be considered in disease characterization or clinical trials.