The integration of proteomics and systems approaches to map regulatory mechanisms underpinning platelet function.

Platelets in the circulation are triggered by vascular damage to activate, aggregate and form a thrombus that prevents excessive blood loss. Platelet activation is stringently regulated by intracellular signalling cascades, which when activated inappropriately lead to myocardial infarction and stroke. Strategies to address platelet dysfunction have included proteomics approaches which have lead to the discovery of a number of novel regulatory proteins of potential therapeutic value. Global analysis of platelet proteomes may enhance the outcome of these studies by arranging this information in a contextual manner that recapitulates established signalling complexes and predicts novel regulatory processes. Platelet signalling networks have already begun to be exploited with interrogation of protein datasets using in silico methodologies that locate functionally feasible protein clusters for subsequent biochemical validation. Characterization of these biological systems through analysis of spatial and temporal organization of component proteins is developing alongside advances in the proteomics field. This focused review highlights advances in platelet proteomics data mining approaches that complement the emerging systems biology field. We have also highlighted nucleated cell types as key examples that can inform platelet research. Therapeutic translation of these modern approaches to understanding platelet regulatory mechanisms will enable the development of novel anti-thrombotic strategies.

New approaches to gene discovery with animal models of obesity and diabetes

DNA-based approaches to the discovery of genes contributing to the development of type 2 diabetes have not been very successful despite substantial investments of time and money. The multiple gene-gene and gene-environment interactions that influence the development of type 2 diabetes mean that DNA approaches are not the ideal tool for defining the etiology of this complex disease. Gene expression-based technologies may prove to be a more rewarding strategy to identify diabetes candidate genes. There are a number of RNA-based technologies available to identify genes that are differentially expressed in various tissues in type 2 diabetes. These include differential display polymerase chain reaction (ddPCR), suppression subtractive hybridization (SSH), and cDNA microarrays. The power of new technologies to detect differential gene expression is ideally suited to studies utilizing appropriate animal models of human disease. We have shown that the gene expression approach, in combination with an excellent animal model such as the Israeli sand rat (Psammomys obesus), can provide novel genes and pathways that may be important in the disease process and provide novel therapeutic approaches. This paper will describe a new gene discovery, beacon, a novel gene linked with energy intake. As the functional characterization of novel genes discovered in our laboratory using this approach continues, it is anticipated that we will soon be able to compile a definitive list of genes that are important in the development of obesity and type 2 diabetes.

Multicultural places and the idea of home

Place identification in urban sociology has traditionally be associated with a sense of ‘being at home’ and connected to the formation of stable and fixed identities. The rise in transnational migration and the increasing number of refugees around the world has made particular regions and communities, within many western nations, culturally diverse. This has led to a re-conceptualisation and re-examination of the relationship between place and home. In light of this new paradigm I explore the existence of multicultural places and investigate the ways, if any, we can speak of ‘being at home’ in these diverse urban places. If home has been traditionally associated with order, sameness and identity while multicultural places are conceptualised in terms of fluidity, contingency, heterogeneity and difference then there seems to be an inherent tension between these two ideas. Are the ideas of home and multiculturalism mutually exclusive? I maintain that they are dialectically interwoven, especially when we acknowledge that otherness and home should not be conceived in binary terms. In order to examine this complex relationship the paper provides a brief discussion of home within the discourses of modernity and postmodernity and then links these discourses to phenomenological and sociological approaches to home. The concluding section demonstrates how home and otherness are expressed in intercultural moments where sameness and diversity rub against each other causing occasional friction but also moments of intercultural dialogue.

Synthesis of Biologically Active Small Molecules: Different Approaches to Drug Design

In the past years, genome biology had disclosed an ever-growing kind of biological targets that emerged as ideal points for therapeutic intervention. Nevertheless, the number of new chemical entities (NCEs) translated into effective therapies employed in the clinic, still not observed. Innovative strategies in drug discovery combined with different approaches to drug design should be searched for bridge this gap. In this context organic synthetic chemistry had to provide for effective strategies to achieve biologically active small molecules to consider not only as potentially drug candidates, but also as chemical tools to dissect biological systems. In this scenario, during my PhD, inspired by the Biology-oriented Synthesis approach, a small library of hybrid molecules endowed with privileged scaffolds, able to block cell cycle and to induce apoptosis and cell differentiation, merged with natural-like cores were synthesized. A synthetic platform which joined a Domino Knoevenagel-Diels Alder reaction with a Suzuki coupling was performed in order to reach the hybrid compounds. These molecules can represent either antitumor lead candidates, or valuable chemical tools to study molecular pathways in cancer cells. The biological profile expressed by some of these derivatives showed a well defined antiproliferative activity on leukemia Bcr-Abl expressing K562 cell lines. A parallel project regarded the rational design and synthesis of minimally structurally hERG blockers with the purpose of enhancing the SAR studies of a previously synthesized collection. A Target-Oriented Synthesis approach was applied. Combining conventional and microwave heating, the desired final compounds were achieved in good yields and reaction rates. The preliminary biological results of the compounds, showed a potent blocking activity. The obtained small set of hERG blockers, was able to gain more insight the minimal structural requirements for hERG liability, which is mandatory to investigate in order to reduce the risk of potential side effects of new drug candidates.

Chronic hepatitis B: A critical appraisal of current approaches to therapy

Background & Aims: Treatment of chronic hepatitis B (CHB) involves a number of complex and controversial issues. Expert opinions may differ from those of practicing hepatologists and gastroenterologists. We aimed to explore this issue further after a critical review of the literature. Methods: A panel of 14 international experts graded the strength of evidence for 16 statements addressing 3 content areas: patient selection, therapeutic end points, and treatment options. Available data relating to the statements were reviewed critically in 3 small work groups. After discussion of each statement with the entire panel, the experts voted anonymously to accept or reject statements based on the strength of evidence and their experience. A total of 241 members of the American Association for the Study of Liver Diseases (AASLD) responded to the same statements and their responses were compared with those of the experts. A discordant response was defined as a difference of more than 20% in any of the 5 graded levels of response (accept or reject) between the 2 groups. Results: With the exception of 2 statements, the experts’ responses were relatively uniform. However, the responses of the AASLD members were discordant from the experts in 12 statements, spanning all 3 content areas. Conclusions: Several areas of disagreement on the management of CHB exist between experts and AASLD members. Our results indicate a potential knowledge gap among practicing hepatologists. Better educational efforts are needed to meet the challenge of managing this complex disorder in which even expert opinion occasionally may disagree.

Approaches to issues of job regulation. A study at branch level in a white collar, public sector trade union. Available in 2 volumes.

This study examines the internal dynamics of white collar trade union branches in the public sector. The effects of a number of internal and external factors on branch patterns of action are evaluated. For the purposes of the study branch action is taken to be the approach to issues of job regulation, as expressed along the five dimensions of dependence on the outside trade union, focus in issues adopted, initiation of issues, intensity of action in issue pursuit and representativeness. The setting chosen for the study is four branches drawn from the same geographical area of the National and Local Government Officers Association. Branches were selected to give a variety in industry settings while controlling for the potentially influential variables of branch size, density of trade union membership and possession of exclusive representational rights in the employing organisation. Identical methods of data collection were used for each branch. The principal findings of the study are that the framework of national agreements and industry collective bargaining structures are strongly related to the industrial relations climate in the employing organisation and the structures of representation within the branch. Where agreements and collective bargaining structures formally restrict branch job regulation roles, there is a degree of devolution of bargaining authority from branch level negotiators to autonomous shop stewards at workplace level. In these circumstances industrial relations climate is characterised by a degree of informality in relationships between management and trade union activists. In turn, industrial relations climate and representative structures together with actor attitudes, have strong effects on all dimensions of approach to issues of job regulation.

The redoxomics of PTEN: walking a fine line between damage and signaling:mass spectrometry-based approaches to study the effect of oxidation on PTEN function, structure, and protein-protein interactions

The research described in this PhD thesis focuses on proteomics approaches to study the effect of oxidation on the modification status and protein-protein interactions of PTEN, a redox-sensitive phosphatase involved in a number of cellular processes including metabolism, apoptosis, cell proliferation, and survival. While direct evidence of a redox regulation of PTEN and its downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on PTEN structure and interactome is still poorly defined. In a first study, GST-tagged PTEN was directly oxidized over a range of hypochlorous acid (HOCl) concentration, assayed for phosphatase activity, and oxidative post-translational modifications (oxPTMs) were quantified using LC-MS/MS-based label-free methods. In a second study, GSTtagged PTEN was prepared in a reduced and reversibly H2O2-oxidized form, immobilized on a resin support and incubated with HCT116 cell lysate to capture PTEN interacting proteins, which were analyzed by LC-MS/MS and comparatively quantified using label-free methods. In parallel experiments, HCT116 cells transfected with a GFP-tagged PTEN were treated with H2O2 and PTENinteracting proteins immunoprecipitated using standard methods. Several high abundance HOCl-induced oxPTMs were mapped, including those taking place at amino acids known to be important for PTEN phosphatase activity and protein-protein interactions, such as Met35, Tyr155, Tyr240 and Tyr315. A PTEN redox interactome was also characterized, which identified a number of PTEN-interacting proteins that vary with the reversible inactivation of PTEN caused by H2O2 oxidation. These included new PTEN interactors as well as the redox proteins peroxiredoxin-1 (Prdx1) and thioredoxin (Trx), which are known to be involved in the recycling of PTEN active site following H2O2-induced reversible inactivation. The results suggest that the oxidative modification of PTEN causes functional alterations in PTEN structure and interactome, with fundamental implications for the PTEN signaling role in many cellular processes, such as those involved in the pathophysiology of disease and ageing.

Development of effective approaches to the large-scale aerodynamic testing of low-rise buildings

Low-rise buildings are often subjected to high wind loads during hurricanes that lead to severe damage and cause water intrusion. It is therefore important to estimate accurate wind pressures for design purposes to reduce losses. Wind loads on low-rise buildings can differ significantly depending upon the laboratory in which they were measured. The differences are due in large part to inadequate simulations of the low-frequency content of atmospheric velocity fluctuations in the laboratory and to the small scale of the models used for the measurements. A new partial turbulence simulation methodology was developed for simulating the effect of low-frequency flow fluctuations on low-rise buildings more effectively from the point of view of testing accuracy and repeatability than is currently the case. The methodology was validated by comparing aerodynamic pressure data for building models obtained in the open-jet 12-Fan Wall of Wind (WOW) facility against their counterparts in a boundary-layer wind tunnel. Field measurements of pressures on Texas Tech University building and Silsoe building were also used for validation purposes. The tests in partial simulation are freed of integral length scale constraints, meaning that model length scales in such testing are only limited by blockage considerations. Thus the partial simulation methodology can be used to produce aerodynamic data for low-rise buildings by using large-scale models in wind tunnels and WOW-like facilities. This is a major advantage, because large-scale models allow for accurate modeling of architectural details, testing at higher Reynolds number, using greater spatial resolution of the pressure taps in high pressure zones, and assessing the performance of aerodynamic devices to reduce wind effects. The technique eliminates a major cause of discrepancies among measurements conducted in different laboratories and can help to standardize flow simulations for testing residential homes as well as significantly improving testing accuracy and repeatability. Partial turbulence simulation was used in the WOW to determine the performance of discontinuous perforated parapets in mitigating roof pressures. The comparisons of pressures with and without parapets showed significant reductions in pressure coefficients in the zones with high suctions. This demonstrated the potential of such aerodynamic add-on devices to reduce uplift forces.

A review of the game-centred approaches to teaching and coaching literature since 2006

Background: In 2006, Oslin and Mitchell published a review of the game-centred approaches (GCAs) to teaching and coaching literature highlighting a number of core concepts thought to provide justification for the use of GCAs including (a) its potential to enhance participant motivation, (b) potential for tactical transfer, and (c) development of decision-making skills and effective decision-makers. Oslin and Mitchell also suggested recommendations for future GCA research.Purpose: The purpose of this paper was threefold: (a) to present a review of Anglophone research into GCAs building on the previous review of Oslin and Mitchell published in 2006; (b) to identify new trends in research since 2006; and (c) to investigate the extent to which the initial suggestions and future research directions suggested by Oslin and Mitchell have been addressed.Data collection: GCA literature since 2006 was searched systematically using a three-phase approach. Phase 1 included initial searches of the EBSCO database using terms associated with GCAs and their acronyms (e.g. TGfU (teaching games for understanding), GS (Game Sense), etc.). Phase 2 expanded the search adopting more generic terms from keywords located in the recent literature (e.g. teaching games, tactical development, game performance, etc.). Multiple searches through the EBSCO database were conducted, whereby key terms were cross-referenced until a saturation point was reached. Phase 3 involved removing those publications that were not empirical, peer reviewed, intervention studies or published in English.Findings: Forty-four studies on GCA implementation were identified and the methodological and substantive nature of these studies was examined. The review noted two positive trends: (a) the expansion of research which included the growth of research on GCAs in Europe and Southeast Asia and (b) an increased amount of research in the affective domain. The review found, however, that a number of key challenges remain within GCA research, which include (a) the need for improved articulation of GCA verification procedures; (b) further assessment of tactical awareness development; (c) extended inquiry about GCAs in coaching contexts; (d) more research into ‘newer’ GCAs (i.e. PP (play practice), IGCM (invasion game competence model) and TDLM (tactical decision learning model)); (e) use of longitudinal research designs; (f) inadequate length of GCA induction and training for teachers and coaches, and (g) examination of GCAs in terms of fitness and special populations.Conclusions: GCA pedagogies are of significant importance as they have the potential to promote change within current adult-centric cultures of youth sport and encourage engagement in physical activity over the life course. To meet these needs, it is recommended that GCA research undergo continued expansion with the use of research designs and data collection techniques that aid the examination of different philosophical understandings of GCAs (e.g. ethnographic, phenomenological and psycho-phenomenological). These are paramount to the exploration of ‘who the individual is’ and ‘how the learner is motivated to continue to participate’ and further permit the in-depth, contextual and ecological analysis of GCA interventions that Oslin and Mitchell recommended in their previous review.

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

The influences of conceptions of learning and learner self-concept on high school students' approaches to learning

This study investigated the mediating effect of learner selfconcept between conceptions of learning and students' approaches to learning using structural equation modelling. Data were collected using a modified version of Biggs' Learning Process Questionnaire, together with the recently developed 'What is Learning Survey' and 'Learner Self-Concept Scale'. A sample of 355 high school students participated in the study. Results indicate that learner self-concept does mediate between conceptions of meaning and approaches to learning. Students who adopted a deep approach liked learning new things and indirectly viewed learning as experiential, involving social interaction and directly viewed learning as personal development. Implications for teachers are discussed, with consideration given to appropriate classroom practice.

Students’ perceptions of teaching and learning: the influence of students’ approaches to learning and teachers’ approaches to teaching

Nearly 500 secondary students in 24 classes were surveyed and four students in each class interviewed concerning their approaches to learning and perceptions of their classroom environment. While interviewed students with deep approaches to learning generally demonstrated a more sophisticated understanding of the learning opportunities offered to them than did students with surface approaches, teaching strategies also influenced students' perceptions. When teachers focused strongly on actively engaging students and creating a supportive environment, students with both deep and surface approaches focused on student-centred aspects of the class. In contrast, when traditional expository teaching methods were used exclusively, students with deep and surface approaches both focused on transmission and reproduction.