Neuroblastoma is a malignant childhood cancer, originating from sympathetic neuroblasts of the peripheral nervous system. Neuroblastoma is a heterogenous group of tumours, while some are highly malignant others can spontaneosly mature into a more benign form or regress. Less than half of the patients survive and this statistics has improved only modestly over the past 20 years. SH-SY5Y is a human neuroblastoma cell line established from a highly malignant tumour. The cells have retained a capacity to differentiate in vitro in response to low concentrations of the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. Differentiated cells are characterised by neurite formation and upregulation of neuronal marker genes. SH-SY5Y are unresponsive to nerve growth factor (NGF), but when transfected to express the NGF-receptor TrkA, they differentiate in response to NGF. Protein kinase C (PKC) is pivotal for the differentiation response to take place. We have investigated the role of signaling through the Ras-MAPK pathway in differentiating SH-SY5Y, with respect to neurite formation, expression of neuronal marker genes and growth control. Our results show that differentiation-promoting treatment induced a sustained activation and nuclear accumulation of the MAPK ERK in SH-SY5Y. The nuclear accumulation of ERK was PKC-dependent. However, nuclear accumulation of ERK was not sufficient for a differentiation response to take place in these cells, but ERK activity was needed for the characteristic upregulation of NPY and GAP-43 induced by TPA. ERK activity did not induce neurite formation, neither was it necessary for TPA-induced neurite formation. Instead, stimulation of a pathway distinct from MEK/ERK, but downstream of Ras, was needed for morphological differentiation. We could also show that differentiated cells still entered S-phase and that there was no correlation between expression of the CKI p21cip1 (an ERK target), BrdU-incorporation or neurite formation.

名古屋大学博士学位論文 学位の種類:博士（医療技術学） (課程) 学位授与年月日　平成22年3月25日

This thesis consists of two parts: a report on the use of a binocular Shack-Hartmann (SH) sensor to study the dynamic correlation of ocular aberrations; and the application of an adaptive optics (AO) system to investigate the effect of the manipulation of aberrations on the accommodation control. The binocular SH sensor consists of one laser source and one camera to reduce system cost and complexity. Six participants took part in this study. Coherence function analysis showed that coherence values were dependent on the subject, aberration and frequency component. Inter-ocular correlations of the aberration dynamics were fairly weak for all participants. Binocular and monocular viewing conditions produced similar wavefront error dynamics. The AO system has a dual wavefront sensing channel. The extra sensing channel permits direct measurement of the eye's aberrations independent of the deformable mirror. Dynamic correction of aberrations during steady-state fixation did not affect the accommodation microfluctuations, possibly due to the prior correction of the static aberration level and/or the limited correction bandwidth. The inversion of certain aberrations during dynamic accommodation affected the gain and latency of accommodation response (AR), suggesting that the eye used the aberrations to guide its initial path of accommodative step response. Corrections of aberrations at various temporal locations of AR cycle produced subject- and aberration-dependent results. The gain and phase lag of the AR to a sinusoidally moving target were unaffected by aberration correction. The predictable nature of the target had been suggested as the reason for its failure to produce any significant effect on the AR gain and phase lag.

Verticillium wilt is a serious disease in a wide range of economic crops worldwide. Verticillium wilt of potato is caused, primarily, by the fungus Verticillium dahliae. Disease management requires understanding of V. dahliae pathogenesis and interactions with potato, which was the main objective of this study. A differential potato-V. dahliae pathosystem was established where pathogenicity of four V. dahliae isolates with different levels of aggressiveness was evaluated on two potato cultivars, Kennebec (susceptible) and Ranger Russet (moderately resistant). External and internal symptoms and growth measurements revealed that isolates Vd1396-9 and Vs06-14 are highly and weakly aggressive, respectively. These two isolates were selected for transcriptomics and proteomics investigations to identify pathogenicity-related factors. Transciptomics analysis was conducted in both isolates after elicitation by root extracts from either Kennebec or Ranger Russet using a combinational approach involving subtractive hybridization and cDNA-AFLP. A total of 573 differentially expressed transcripts were detected in one or the other isolate. Among them, 185 transcripts of interest were recovered, re-amplified, sequenced and searched against NCBI and the Broad Institute V. dahliae genome databases for identification. The two contrasting-aggressiveness isolates were used for a comparative proteomics investigation. The first proteomic map of V. dahliae was established. The proteomics analysis was carried out using 2-Dimentional electrophoresis and mass spectrometry. Twenty five proteins were differentially expressed and identified in one or the other isolate. Many of the identified genes/proteins showed potential involvement in pathogenesis of V. dahliae or other fungi. Genes of stress response regulator A (oxidative stress tolerance factor), isochorismatase hydrolase (potential plant defense suppressor) and tetrahydroxynaphthalene reductase (involved in melanin and microsclerotia formation) were isolated from both isolates and cloned. Sequence analysis of these genes showed many differences that may explain their differential expression in the two isolates. Given that some of the identified genes/proteins are potentially involved in overcoming and suppressing plant defense, phenolics were profiled in Kennebec-inoculated with Vd1396-9 or Vs06-14 isolate. Chlorogenic, caffeic, ferulic acids, cis-jasmone and rutin accumulation showed variations after inoculation. The results obtained from this study will help understanding the V. dahliae-potato interactions and develop efficient strategies to control Verticillium wilt disease.

Over 7.8 km of seismic reflection data and 2 km of electrical resistivity data were acquired, processed, and interpreted during this multi-method geophysical study. Objectives included the definition of geologic conditions underlying a contaminant plume in McCracken County, western Kentucky, and the determination of the potential for structural control on the rate and direction of plume migration. Both geophysical methods indicate the presence of multiple high-angle normal faults outlining a series of asymmetric grabens ranging in width from 160 m to almost 300 m and striking between N40°E and N45°E. There was agreement between the two methods on fault location and degree of near-surface offset, with offsets of 1 to 2 m observed at 10 to 20 m below ground surface and 3 to 8 m observed at 20 to 30 m depth. Bedrock displacement was generally 2 to 3 times larger, with offsets of 10 to 26 m observed. The faults appear to have originated in the Paleozoic with predominantly normal reactivation occurring as recently as the Pleistocene. The fault strikes generally approximate the orientation of the northwestern contaminant plume. Observed offset of the Regional Gravel Aquifer may form a preferential flow path for contaminant migration.

Verticillium wilt is a serious disease in a wide range of economic crops worldwide. Verticillium wilt of potato is caused, primarily, by the fungus Verticillium dahliae. Disease management requires understanding of V. dahliae pathogenesis and interactions with potato, which was the main objective of this study. A differential potato-V. dahliae pathosystem was established where pathogenicity of four V. dahliae isolates with different levels of aggressiveness was evaluated on two potato cultivars, Kennebec (susceptible) and Ranger Russet (moderately resistant). External and internal symptoms and growth measurements revealed that isolates Vd1396-9 and Vs06-14 are highly and weakly aggressive, respectively. These two isolates were selected for transcriptomics and proteomics investigations to identify pathogenicity-related factors. Transciptomics analysis was conducted in both isolates after elicitation by root extracts from either Kennebec or Ranger Russet using a combinational approach involving subtractive hybridization and cDNA-AFLP. A total of 573 differentially expressed transcripts were detected in one or the other isolate. Among them, 185 transcripts of interest were recovered, re-amplified, sequenced and searched against NCBI and the Broad Institute V. dahliae genome databases for identification. The two contrasting-aggressiveness isolates were used for a comparative proteomics investigation. The first proteomic map of V. dahliae was established. The proteomics analysis was carried out using 2-Dimentional electrophoresis and mass spectrometry. Twenty five proteins were differentially expressed and identified in one or the other isolate. Many of the identified genes/proteins showed potential involvement in pathogenesis of V. dahliae or other fungi. Genes of stress response regulator A (oxidative stress tolerance factor), isochorismatase hydrolase (potential plant defense suppressor) and tetrahydroxynaphthalene reductase (involved in melanin and microsclerotia formation) were isolated from both isolates and cloned. Sequence analysis of these genes showed many differences that may explain their differential expression in the two isolates. Given that some of the identified genes/proteins are potentially involved in overcoming and suppressing plant defense, phenolics were profiled in Kennebec-inoculated with Vd1396-9 or Vs06-14 isolate. Chlorogenic, caffeic, ferulic acids, cis-jasmone and rutin accumulation showed variations after inoculation. The results obtained from this study will help understanding the V. dahliae-potato interactions and develop efficient strategies to control Verticillium wilt disease.

UK consumers bought just under 500 thousand tonnes of seafood in 2010, at a cost of £3.8bn. Despite rising prices, consumption is on a general upwards path, with the average UK adult now eating 2% more seafood than they would have eaten a generation ago, and demand predicted to grow by a further 17% by 2030. However, this demand is increasingly restricted to a narrow range of imported and wild-caught species (Cod, Haddock, Tuna) and farmed products (Salmon and Prawns) over locally-available species with the consequence that between 60% and 80% of UK domestic landings are currently exported and 80% of all the seafood eaten in the UK is one of either Cod, Haddock, Salmon, Tuna or Prawns – the so-called Big 5. The shortage of local markets for native fish species is arguably reducing the relative viability of small scale, over large-scale, fisheries in the UK. It also increases pressure upon wild stocks of commercially valuable species and is driving the rapid expansion of fish-farming operations which can have negative environmental and social implications. Considering the above, it is suggested that UK consumers could make a positive contribution to the UK economy and marine environment if they chose to buy native, locally-caught species, over farmed and exotic imports. In order to achieve this, however, significant behavioural change would need to take place; and for behavioural change initiatives to be successful, it is argued that it is first necessary to understand why these consumption patterns have developed, i.e. Why the Big 5? Accepting that no single theory has been found that can fully explain behaviours from intentions, this research used mixed methods to develop a consumer-centric view of the full range of factors that might be driving these unsustainable consumption patterns. Regular consumers of seafood from four contrasting localities in England were recruited to complete surveys, maintain shopping diaries and to take part in group and one-to-one interviews to understand their reasons for eating seafood in general and the Big 5 in particular. Drawing on the Literature on seafood consumption, demographic and geographic-induced differences in consumption were explored; and consumer understanding of, and concern for, the sustainability of their seafood was assessed. Retail behaviour was also examined by undertaking an on-line review of the seafood offered for sale by the UK’s five largest retailers. Consumers were generally ill informed and confused about the sustainability of their seafood and had little to no awareness of labelling. In their confusion/apathy, they tended to revert to habitual behaviours and safe choices tending towards the Big 5. They felt strongly that retailers should be making it easier for them to make sustainable choices. The evidence from this study is that Retail is failing in this respect. Interest in and demand for local seafood was very high, with consumers equating local with sustainable, even though the evidence to support this assertion is currently lacking. Consumer definitions of “local” and “sustainable” were found to vary from accepted policy and academic understanding, presenting the possibility of adding to consumer confusion when communicating about sustainable seafood; further, “sustainable” possessed negative connotations for these consumers who, in stark contrast to the average UK consumer, were found to score highly for Hedonism. In total, twenty eight distinct variables were identified as influencing unsustainable UK seafood consumer behaviour. Key amongst these were consumer ignorance/apathy regarding sustainability; retail behaviour; and habit – factors that are presented in the Trifold Model of Unsustainable Consumer Behaviour. This model brings much needed clarity to a complex and poorly understood area of consumer behaviour and marks a significant contribution to three areas of academic study: Sustainable Consumption; Consumer Behaviour; and Business Ethics. The Trifold Model is presented for further testing. Recommendations for policy and industry are highlighted as are areas for further research.

The research focusses on the river Lugg – a cross-border catchment and major tributary of the river Wye, the most important Atlantic salmon river in England and Wales. The problem of declining Atlantic salmon populations in the catchment is addressed through investigating recruitment from egg fertilization to the emergent life stage and beyond using multiple field-based and laboratory techniques. The approach adopted is multidisciplinary and addresses the need for holistic approaches to habitat degradation which is increasingly recognised as systemic in nature; often with multiple stressors acting interactively. The initial premise of deleterious fine sediment infiltration into spawning gravels was addressed by a sediment fingerprinting study to ascertain the provenance of infiltrated redd sediment from a range of land-use types. In addition, nine artificial redd sites were constructed and assessed for fine sediment infiltration, intragravel dissolved oxygen levels, intragravel flow velocity and other hyporheic pore water characteristics, in relation to survival to emergence over two field seasons. A study examining the quality of emergent fry was also carried out using fitness tests and individual stress levels. Additionally, a study on long-residence groundwater infiltration into the incubation environment was carried out. The main fine sediment contributor was derived from agricultural sources, particularly during wetter periods. The average contribution of fine sediment from agricultural sources was 60%. Survival ranged from 12% to 70% during the 2008 flood season and from 76% - 93% during the 2009 dry season. Fine sediment mass as a stand-alone index was only weakly correlated with survival but is thought to influence other factors; medium strength correlations of survival with dissolved oxygen, intragravel flow velocity and oxygen supply in particular were observed. Evidence of groundwater-surface water interactions were detected at two of three sites investigated and is proposed as an additional controlling mechanism for embryonic survival in the catchment. Sublethal fitness tests demonstrated variations between cohorts in the 2009 period despite a relatively small range of oxygen concentrations. The results highlight both temporal and spatial variations in spawning habitat quality, which influence not only survival to hatch but also posthatch fitness.

An improved object tracking algorithm based Kalman filtering is developed in this thesis. The algorithm uses a median filter and morphological operations during tracking. The problem created by object shadows is identified and the primary focus is to incorporate shadow detection and removal to improve tracking multiple objects in complex scenes. It is shown that the Kalman filter, without the improvements, fails to remove shadows that connect different objects. The application of the median filter helps the separation of different objects and thus enables the tracking of multiple objects individually. The performances of the Kalman filter and the improved tracking algorithm were tested on a highway video sequence of moving cars and it is shown that the proposed algorithm yields better performance in the presence of shadows.

O açaí (Euterpe oleracea) é um fruto proveniente do açaizeiro, uma palmeira nativa da Amazônia Brasileira, considerado uma “superfruta” devido ao seu alto valor nutricional, com destaque para as antocianinas, associadas a efeitos benéficos à saúde. Uma vez que genótipos de frutos desenvolvidos por programas de melhoramento têm sido avaliados a fim de identificar frutos com composição fitoquímica melhorada e potencial antioxidante, o objetivo deste trabalho foi investigar o efeito do melhoramento genético na composição química, perfil de compostos bioativos e atividade antioxidante de seis genótipos de açaí (E. oleracea). Análises de composição química, perfil de antocianinas e carotenoides e de atividade antioxidante em diversos sistemas in vitro (método ABTS, oxidação da glutationa, teste da desoxirribose e células SH-SY5Y) foram realizadas em 6 genótipos e 1 amostra comercial utilizada como padrão. Com relação a composição química, de maneira geral, os genótipos apresentaram elevados teores de lipídeos (36,30 – 47,44%), fibras totais (11,31 – 15,88%), proteínas (7,78-9,50%) e cinzas (3,49 – 4,44%). Adicionalmente, as antocianinas cianidina 3-glicosídeo (12-43%) e cianidina 3-rutinosídeo (57-88%) foram identificadas nos genótipos avaliados e o genótipo L22P13 apresentou um incremento de 85% no teor de antocianinas totais. Com relação ao perfil de carotenoides, luteína, zeaxantina, α-caroteno e β-caroteno foram os principais carotenoides identificados nos genótipos avaliados, sendo que os genótipos BRS-PAMISTA e L22P13 apresentaram os teores mais elevados de carotenoides totais (125,39μg/g e 118μg/g). Com relação a atividade antioxidante, todos os genótipos reduziram a geração do radical ABTS em maior ou menor grau, embora diferenças significativas entre os genótipos e a amostra comercial não tenham sido observadas (IC50 = 73,98 – 175,7 vs. 121,7 μg/g). Entretanto, houve associação moderada e inversa entre a geração do radical ABTS e níveis de antocianinas e carotenoides. Ausência de diferenças significativas entre os extratos hidroetanólicos dos genótipos e a amostra comercial também foi observada no ensaio da desoxirribose (IC50 = 361,5 – 497,7 vs. 520,1 μg/g). Em cultura de células SH-SY5Y, os extratos hidroetanólicos na concentração de 50 μg/mL apresentaram um efeito protetor contra as espécies reativas geradas pelo H2O2 (ensaio DCFH-DA) e esse resultado foi associado ao teor de antocianinas dos extratos. Os resultados apontam genótipos promissores, com elevados teores de antocianinas e carotenoides, com potencial atividade antioxidante in vitro e um possível efeito protetor de células frente as espécies reativas. / The açaí (Euterpe oleracea) is a fruit of the açaizeiro, a native palm of the Brazilian Amazon, considered a "superfruit" due to its high nutritional value, especially anthocyanins, associated with beneficial health effects. Since fruit genotypes developed by breeding programs have been evaluated to identify fruits with improved phytochemical composition and antioxidant potential, the aim of this study was to investigate the effect of genetic improvement in the chemical composition, bioactive compounds profile and antioxidant activity of six açaí (E. oleracea) genotypes. The analysis of chemical composition, anthocyanins and carotenoids profile and antioxidant activity in various in vitro systems (ABTS method, glutathione oxidation, deoxyribose test and SH-SY5Y cells) were performed in six genotypes of açaí and one commercial sample that was used as standard. Regarding the chemical composition, in general, the genotypes showed high levels of lipids (36.30 – 47.44%), total fiber (11.31 – 15.88%), protein (7.78-9.50%) and ashes (3.49 – 4.44%). In addition, the anthocyanins cyanidin 3-glucoside (12-43%) and cyanidin 3-rutinoside (57-88%) were identified in evaluated genotypes and L22P13 genotype showed an increase of 85% in total anthocyanin content. Regarding the carotenoid profile, lutein, zeaxanthin, α-carotene and β-carotene were the main carotenoid identified in genotypes, being that BRS-PAMISTA and L22P13 genotypes showed the highest levels of total carotenoids (125.39 μg/g and 118 μg/g). Regarding the antioxidant activity, all genotypes reduced ABTS radical generation in greater or lesser extension, although there were no significant differences between hydroethanolic extracts of different genotypes and the commercial sample (IC50 = 73.98 - 175.7 vs. 121.7 μg/g). However, an inverse and moderate association between radical ABTS generation and anthocyanins and carotenoids content was observed. Absence of significant differences between hydroethanolic extracts of evaluated genotypes and the commercial sample was observed also in the deoxyribose assay (IC50 = 361.5 to 497.7 vs. 520.1 μg/g). In culture of SH-SY5Y cells, hydroethanolic extracts at concentration of 50 μg/mL had a protective effect against the reactive species generated by H2O2 (DCFH-DA assay) and this result was associated with the anthocyanins content of the extracts. The results showed promising genotypes with high levels of anthocyanins and carotenoids with antioxidant potential in vitro and a possible protective effect of cells against reactive species.