Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I.

The RER retains a specific subset of ER proteins, many of which have been shown to participate in the translocation of nascent secretory and membrane proteins. The mechanism of retention of RER specific membrane proteins is unknown. To study this phenomenon in yeast, where no RER-specific membrane proteins have yet been identified, we expressed the human RER-specific protein, ribophorin I. In all mammalian cell types examined, ribophorin I has been shown to be restricted to the membrane of the RER. Here we ascertain that yeast cells correctly target, assemble, and retain ribophorin I in their RER. Floatation experiments demonstrated that human ribophorin I, expressed in yeast, was membrane associated. Carbonate (pH = 11) washing and Triton X-114 cloud-point precipitations of yeast microsomes indicated that ribophorin I was integrated into the membrane bilayer. Both chromatography on Con A and digestion with endoglycosidase H were used to prove that ribophorin I was glycosylated once, consistent with its expression in mammalian cells. Proteolysis of microsomal membranes and subsequent immunoblotting showed ribophorin I to have assumed the correct transmembrane topology. Sucrose gradient centrifugation studies found ribophorin I to be included only in fractions containing rough membranes and excluded from smooth ones that, on the basis of the distribution of BiP, included smooth ER. Ribosome removal from rough membranes and subsequent isopycnic centrifugation resulted in a shift in the buoyant density of the ribophorin I-containing membranes. Furthermore, the rough and density-shifted fractions were the exclusive location of protein translocation activity. Based on these studies we conclude that sequestration of membrane proteins to rough domains of ER probably occurs in a like manner in yeast and mammalian cells

KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing

Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated

Key exploited species as surrogates for coastal conservation in an oceanic archipelago: insights from topshells and limpets from Madeira (NE Atlantic Ocean)

As lapas e os caramujos estão entre os herbívoros mais bem adaptados ao intertidal do Atlântico Nordeste. Estas espécies-chave fornecem serviços ecossistémicos valiosos, desempenhando um papel fundamental no equilíbrio ecológico do intertidal e têm um elevado valor económico, estando sujeitas a altos níveis de exploração e representando uma das atividades económicas mais rentáveis na pesca de pequena escala no arquipélago da Madeira. Esta dissertação visa preencher as lacunas existentes na história de vida e dinâmica populacional destas espécies, e aferir os efeitos da regulamentação da apanha nos mananciais explorados. A abordagem conservacionista implícita ao longo desta tese pretende promover: (i) a regulamentação adequada da apanha de caramujos (Phorcus sauciatus) e (ii) a avaliação dos efeitos da regulamentação da apanha de lapas nas populações exploradas (Patella aspera, Patella candei). Atualmente, os mananciais de lapas e caramujos são explorados perto do rendimento máximo sustentável, e a monitorização e fiscalização são fundamentais para evitar a futura sobre-exploração. A regulamentação da apanha de lapas produziu um efeito positivo nas espécies de lapas exploradas, com um aumento no tamanho, na proporção de indivíduos reprodutores, no tamanho de maturação e num maior equilíbrio na proporção de sexos. A apanha de caramujos não está regulamentada e com o atual nível de exploração ocorrem alterações na estrutura de tamanhos, abundância e potencial reprodutivo das populações exploradas, pelo que urge implementar a regulamentação da apanha desta espécie, por forma a mitigar os efeitos negativos desta atividade. O efeito da proximidade das populações humanas e acessibilidade costeira na estrutura de tamanhos e abundância de gastrópodes explorados mostrou que a proporção de reprodutores e a abundância eram geralmente menores em áreas mais próximas das populações humanas e em áreas mais acessíveis. Os efeitos das Áreas Marinhas Protegidas na proteção das populações de lapas resultaram num aumento diferencial do tamanho, da maturidade sexual e da captura por unidade de esforço de acordo com o grau de proteção. O esclarecimento e envolvimento das comunidades locais, reguladores, decisores políticos e partes interessadas, baseados em informação e educação, são cruciais para uma gestão eficaz e sustentável destes gastrópodes marinhos e ecossistemas a médio e longo prazo.Limpets and topshells are among the most successful intertidal grazers in the North-eastern Atlantic. These keystone species play a pivotal role in structuring rocky shores communities, and provoding valuable ecosystem services. Than have an important economic value, being subject to high levels of exploitation and representing one of the most profitable economic activities in small-scale fisheries in the archipelago of Madeira. This thesis aims to fill the gaps on the life-traits and population dynamics of these species, and assess the effects of harvesting regulations on the exploited stocks. A focus on conservation is implicit throughout this thesis since it addresses the promotion of: (i) proper regulation of the unregulated harvesting of topshells (Phorcus sauciatus) and (ii) provide additional information on the effects of harvesting regulations on limpets (Patella aspera, Patella candei). Currently, limpets and topshells stocks are being exploited near the maximum sustainable yield and monitoring and enforcement must be accomplished to avoid future overexploitation. Conservation measures prompted a positive effect on both exploited limpet species with an increase in length, reproductive individuals, size-at-first maturity and a more balanced sex-ratio after harvesting regulations. The harvesting of topshells is not regulated and with the current level of exploitation there are changes in the size structure, abundance and reproductive potential of the exploited populations, so it is imperative to implement the harvesting regulations for this species, in order to mitigate the negative effects of harvesting. The effect of proximity to human settlements and coastal accessibility on the size-structure and abundance of exploited gastropods showed that the mean-size, proportion of reproductive individuals and abundance were generally smaller in areas closer to human settlements and in more accessible areas. The effects of protection from the Marine Protected Areas on limpet populations resulted in a differential increase on size, size-at-first maturity and catch-per-unit-effort according to the degree of protection. The understanding and commitment of local communities, regulators, policymakers and stakeholders, based on information and education are crucial to the effective management and to ensure the sustainability of these marine gastropods and ecosystems at medium and long term

Glucocorticoids rapidly inhibit cell migration through a novel, non-transcriptional HDAC6 pathway

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture

Climate change adaptation in European river basins

This paper contains an assessment and standardized comparative analysis of the current water management regimes in four case-studies in three European river basins: the Hungarian part of the Upper Tisza, the Ukrainian part of the Upper Tisza (also called Zacarpathian Tisza), Alentejo Region (including the Alqueva Reservoir) in the Lower Guadiana in Portugal, and Rivierenland in the Netherlands. The analysis comprises several regime elements considered to be important in adaptive and integrated water management: agency, awareness raising and education, type of governance and cooperation structures, information management and—exchange, policy development and—implementation, risk management, and finances and cost recovery. This comparative analysis has an explorative character intended to identify general patterns in adaptive and integrated water management and to determine its role in coping with the impacts of climate change on floods and droughts. The results show that there is a strong interdependence of the elements within a water management regime, and as such this interdependence is a stabilizing factor in current management regimes. For example, this research provides evidence that a lack of joint/participative knowledge is an important obstacle for cooperation, or vice versa. We argue that there is a two-way relationship between information management and collaboration. Moreover, this research suggests that bottom-up governance is not a straightforward solution to water management problems in large-scale, complex, multiple-use systems, such as river basins. Instead, all the regimes being analyzed are in a process of finding a balance between bottom-up and top–down governance. Finally, this research shows that in a basin where one type of extreme is dominant—like droughts in the Alentejo (Portugal) and floods in Rivierenland (Netherlands)—the potential impacts of other extremes are somehow ignored or not perceived with the urgency they might deserv

The politicisation of evaluation: constructing and contesting EU policy performance

Although systematic policy evaluation has been conducted for decades and has been growing strongly within the European Union (EU) institutions and in the member states, it remains largely underexplored in political science literatures. Extant work in political science and public policy typically focuses on elements such as agenda setting, policy shaping, decision making, or implementation rather than evaluation. Although individual pieces of research on evaluation in the EU have started to emerge, most often regarding policy “effectiveness” (one criterion among many in evaluation), a more structured approach is currently missing. This special issue aims to address this gap in political science by focusing on four key focal points: evaluation institutions (including rules and cultures), evaluation actors and interests (including competencies, power, roles and tasks), evaluation design (including research methods and theories, and their impact on policy design and legislation), and finally, evaluation purpose and use (including the relationships between discourse and scientific evidence, political attitudes and strategic use). The special issue considers how each of these elements contributes to an evolving governance system in the EU, where evaluation is playing an increasingly important role in decision making

Microarray Method for the Rapid Detection of Glycosaminoglycan–Protein Interactions

Glycosaminoglycans (GAGs) perform numerous vital functions within the body. As major components of the extracellular matrix, these polysaccharides participate in a diverse array of cell-signaling events. We have developed a simple microarray assay for the evaluation of protein binding to various GAG subclasses. In a single experiment, the binding to all members of the GAG family can be rapidly determined, giving insight into the relative specificity of the interactions and the importance of specific sulfation motifs. The arrays are facile to prepare from commercially available materials

Stable isotope analysis provides new information on winter habitat use of declining avian migrants that is relevant to their conservation

Winter habitat use and the magnitude of migratory connectivity are important parameters when assessing drivers of the marked declines in avian migrants. Such information is unavailable for most species. We use a stable isotope approach to assess these factors for three declining African-Eurasian migrants whose winter ecology is poorly known: wood warbler Phylloscopus sibilatrix, house martin Delichon urbicum and common swift Apus apus. Spatially segregated breeding wood warbler populations (sampled across a 800 km transect), house martins and common swifts (sampled across a 3,500 km transect) exhibited statistically identical intra-specific carbon and nitrogen isotope ratios in winter grown feathers. Such patterns are compatible with a high degree of migratory connectivity, but could arise if species use isotopically similar resources at different locations. Wood warbler carbon isotope ratios are more depleted than typical for African-Eurasian migrants and are compatible with use of moist lowland forest. The very limited variance in these ratios indicates specialisation on isotopically restricted resources, which may drive the similarity in wood warbler populations' stable isotope ratios and increase susceptibility to environmental change within its wintering grounds. House martins were previously considered to primarily use moist montane forest during the winter, but this seems unlikely given the enriched nature of their carbon isotope ratios. House martins use a narrower isotopic range of resources than the common swift, indicative of increased specialisation or a relatively limited wintering range; both factors could increase house martins' vulnerability to environmental change. The marked variance in isotope ratios within each common swift population contributes to the lack of population specific signatures and indicates that the species is less vulnerable to environmental change in sub-Saharan Africa than our other focal species. Our findings demonstrate how stable isotope research can contribute to understanding avian migrants' winter ecology and conservation status