Nuclear organisation and epigenetic regulation of gene expression in Dictyostelium discoideum

A series of vectors for the over-expression of tagged proteins in Dictyostelium were designed, constructed and tested. These vectors allow the addition of an N- or C-terminal tag (GFP, RFP, 3xFLAG, 3xHA, 6xMYC and TAP) with an optimized polylinker sequence and no additional amino acid residues at the N or C terminus. Different selectable markers (Blasticidin and gentamicin) are available as well as an extra chromosomal version; these allow copy number and thus expression level to be controlled, as well as allowing for more options with regard to complementation, co- and super-transformation. Finally, the vectors share standardized cloning sites, allowing a gene of interest to be easily transfered between the different versions of the vectors as experimental requirements evolve. The organisation and dynamics of the Dictyostelium nucleus during the cell cycle was investigated. The centromeric histone H3 (CenH3) variant serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. A number of Dictyostelium histone H3-domain containing proteins as GFP-tagged fusions were expressed and it was found that one of them functions as CenH3 in this species. Like CenH3 from some other species, Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins. The targeting domain, comprising α-helix 2 and loop 1 of the histone fold is required for targeting CenH3 to centromeres. Compared to the targeting domain of other known and putative CenH3 species, Dictyostelium CenH3 has a shorter loop 1 region. The localisation of a variety of histone modifications and histone modifying enzymes was examined. Using fluorescence in situ hybridisation (FISH) and CenH3 chromatin-immunoprecipitation (ChIP) it was shown that the six telocentric centromeres contain all of the DIRS-1 and most of the DDT-A and skipper transposons. During interphase the centromeres remain attached to the centrosome resulting in a single CenH3 cluster which also contains the putative histone H3K9 methyltransferase SuvA, H3K9me3 and HP1 (heterochromatin protein 1). Except for the centromere cluster and a number of small foci at the nuclear periphery opposite the centromeres, the rest of the nucleus is largely devoid of transposons and heterochromatin associated histone modifications. At least some of the small foci correspond to the distal telomeres, suggesting that the chromosomes are organised in a Rabl-like manner. It was found that in contrast to metazoans, loading of CenH3 onto Dictyostelium centromeres occurs in late G2 phase. Transformation of Dictyostelium with vectors carrying the G418 resistance cassette typically results in the vector integrating into the genome in one or a few tandem arrays of approximately a hundred copies. In contrast, plasmids containing a Blasticidin resistance cassette integrate as single or a few copies. The behaviour of transgenes in the nucleus was examined by FISH, and it was found that low copy transgenes show apparently random distribution within the nucleus, while transgenes with more than approximately 10 copies cluster at or immediately adjacent to the centromeres in interphase cells regardless of the actual integration site along the chromosome. During mitosis the transgenes show centromere-like behaviour, and ChIP experiments show that transgenes contain the heterochromatin marker H3K9me2 and the centromeric histone variant H3v1. This clustering, and centromere-like behaviour was not observed on extrachromosomal transgenes, nor on a line where the transgene had integrated into the extrachromosomal rDNA palindrome. This suggests that it is the repetitive nature of the transgenes that causes the centromere-like behaviour. A Dictyostelium homolog of DET1, a protein largely restricted to multicellular eukaryotes where it has a role in developmental regulation was identified. As in other species Dictyostelium DET1 is nuclear localised. In ChIP experiments DET1 was found to bind the promoters of a number of developmentally regulated loci. In contrast to other species where it is an essential protein, loss of DET1 is not lethal in Dictyostelium, although viability is greatly reduced. Loss of DET1 results in delayed and abnormal development with enlarged aggregation territories. Mutant slugs displayed apparent cell type patterning with a bias towards pre-stalk cell types.

The role of ligneous vegetation for livestock nutrition in the sub-Sahelian and Sudanian zones of West Africa: Potential effects of climate change

Climate change and variability in sub-Saharan West Africa is expected to have negative consequences for crop and livestock farming due to the strong dependence of these sectors on rainfall and natural resources, and the low adaptive capacity of crops farmers, agro-pastoralist and pastoralists in the region. The objective of this PhD research was to investigate the anticipated impacts of expected future climate change and variability on nutrition and grazing management of livestock in the prevailing extensive agro-pastoral and pastoral systems of the Sahelian and Sudanian zones of Burkina Faso. To achieve this, three studies were undertaken in selected village territories (100 km² each) in the southern Sahelian (Taffogo), northern Sudanian (Nobere, Safane) and southern Sudanian (Sokouraba) zone of the country during 2009 and 2010. The choice of two villages in the northern Sudanian zone was guided by the dichotomy between intense agricultural land use and high population density near Safane, and lower agricultural land use in the tampon zone between the village of Nobere and the National Park Kaboré Tambi of Pô. Using global positioning and geographical information systems tools, the spatio-temporal variation in the use of grazing areas by cattle, sheep and goats, and in their foraging behaviour in the four villages was assessed by monitoring three herds each per species during a one-year cycle (Chapter 2). Maximum itinerary lengths (km/d) were observed in the hot dry season (March-May); they were longer for sheep (18.8) and cattle (17.4) than for goats (10.5, p<0.05). Daily total grazing time spent on pasture ranged from 6 - 11 h with cattle staying longer on pasture than small ruminants (p<0.05). Feeding time accounted for 52% - 72% of daily time on pasture, irrespective of species. Herds spent longer time on pasture and walked farther distances in the southern Sahelian than the two Sudanian zones (p<0.01), while daily feeding time was longer in the southern Sudanian than in the other two zones (p>0.05). Proportional time spent resting decreased from the rainy (June - October) to the cool (November - February) and hot dry season (p<0.05), while in parallel the proportion of walking time increased. Feeding time of all species was to a significantly high proportion spent on wooded land (tree crown cover 5-10%, or shrub cover >10%) in the southern Sahelian zone, and on forest land (tree crown cover >10%) in the two Sudanian zones, irrespective of season. It is concluded that with the expansion of cropland in the whole region, remaining islands of wooded land, including also fields fallowed for three or more years with their considerable shrub cover, are particularly valuable pasturing areas for ruminant stock. Measures must be taken that counteract the shrinking of wooded land and forests across the whole region, including also active protection and (re)establishment of drought-tolerant fodder trees. Observation of the selection behaviour of the above herds of cattle and small ruminant as far as browse species were concerned, and interviews with 75 of Fulani livestock keepers on use of browse as feed by their ruminant stock and as remedies for animal disease treatment was undertaken (Chapter 3) in order to evaluate the consequence of climate change for the contribution of browse to livestock nutrition and animal health in the extensive grazing-based livestock systems. The results indicated that grazing cattle and small ruminants do make considerable use of browse species on pasture across the studied agro-ecological zones. Goats spent more time (p<0.01) feeding on browse species than sheep and cattle, which spent a low to moderate proportion of their feeding time on browsing in any of the study sites. As far as the agro-ecological zones were concerned, the contribution of browse species to livestock nutrition was more important in the southern Sahelian and northern Sudanian zone than the southern Sudanian zone, and this contribution is higher during the cold and hot dry season than during the rainy season. A total of 75 browse species were selected on pasture year around, whereby cattle strongly preferred Afzelia africana, Pterocarpus erinaceus and Piliostigma sp., while sheep and goats primarily fed on Balanites aegyptiaca, Ziziphus mauritiana and Acacia sp. Crude protein concentration (in DM) of pods or fruits of the most important browse species selected by goats, sheep and cattle ranged from 7% to 13% for pods, and from 10% to 18% for foliage. The concentration of digestible organic matter of preferred browse species mostly ranged from 40% to 60%, and the concentrations of total phenols, condensed tannins and acid detergent lignin were low. Linear regression analyses showed that browse preference on pasture is strongly related to its contents (% of DM) of CP, ADF, NDF and OM digestibility. Interviewed livestock keepers reported that browse species are increasingly use by their grazing animals, while for animal health care use of tree- and shrub-based remedies decreased over the last two decades. It is concluded that due to climate change with expected negative impact on the productivity of the herbaceous layer of communal pastures browse fodder will gain in importance for animal nutrition. Therefore re-establishment and dissemination of locally adapted browse species preferred by ruminants is needed to increase the nutritional situation of ruminant stock in the region and contribute to species diversity and soil fertility restoration in degraded pasture areas. In Chapter 4 a combination of household surveys and participatory research approaches was used in the four villages, and additionally in the village of Zogoré (southern Sahelian zone) and of Karangasso Vigué (northern Sudanian zone) to investigate pastoralists’ (n= 76) and agro-pastoralists’ (n= 83) perception of climate change, and their adaptation strategies in crop and livestock production at farm level. Across the three agro-ecological zones, the majority of the interviewees perceived an increase in maximum day temperatures and decrease of total annual rainfall over the last two decades. Perceptions of change in climate patterns were in line with meteorological data for increased temperatures while for total rainfall farmers’ views contrasted the rainfall records which showed a slight increase of precipitation. According to all interviewees climate change and variability have negative impacts on their crop and animal husbandry, and most of them already adopted some coping and adaptation strategies at farm level to secure their livelihoods and reduce negative impacts on their farming system. Although these strategies are valuable and can help crop and livestock farmers to cope with the recurrent droughts and climate variability, they are not effective against expected extreme climate events. Governmental and non-governmental organisations should develop effective policies and strategies at local, regional and national level to support farmers in their endeavours to cope with climate change phenomena; measures should be site-specific and take into account farmers’ experiences and strategies already in place.

Lean Transition of Emerging Industrial Capability

This volume of the final report documents the technical work performed from December 1998 through December 2002 under Cooperative Agreement F33615-97-2-5153 executed between the U.S. Air Force, Air Force Research Laboratory, Materials and Manufacturing Directorate, Manufacturing Technology Division (AFRL/MLM) and the McDonnell Douglas Corporation, a wholly-owned subsidiary of The Boeing Company. The work was accomplished by The Boeing Company, Phantom Works, Huntington Beach, St. Louis, and Seattle; Ford Motor Company; Integral Inc.; Sloan School of Management in the Massachusetts Institute of Technology; Pratt & Whitney; and Central State University in Xenia, Ohio and in association with Raytheon Corporation. The LeanTEC program manager for AFRL is John Crabill of AFRL / MLMP and The Boeing Company program manager is Ed Shroyer of Boeing Phantom Works in Huntington Beach, CA. Financial performance under this contract is documented in the Financial Volume of the final report.

Effects of Air Pollution Control on Climate

Urban air pollution and climate are closely connected due to shared generating processes (e.g., combustion) for emissions of the driving gases and aerosols. They are also connected because the atmospheric lifecycles of common air pollutants such as CO, NOx and VOCs, and of the climatically important methane gas (CH4) and sulfate aerosols, both involve the fast photochemistry of the hydroxyl free radical (OH). Thus policies designed to address air pollution may impact climate and vice versa. We present calculations using a model coupling economics, atmospheric chemistry, climate and ecosystems to illustrate some effects of air pollution policy alone on global warming. We consider caps on emissions of NOx, CO, volatile organic carbon, and SOx both individually and combined in two ways. These caps can lower ozone causing less warming, lower sulfate aerosols yielding more warming, lower OH and thus increase CH4 giving more warming, and finally, allow more carbon uptake by ecosystems leading to less warming. Overall, these effects significantly offset each other suggesting that air pollution policy has a relatively small net effect on the global mean surface temperature and sea level rise. However, our study does not account for the effects of air pollution policies on overall demand for fossil fuels and on the choice of fuels (coal, oil, gas), nor have we considered the effects of caps on black carbon or organic carbon aerosols on climate. These effects, if included, could lead to more substantial impacts of capping pollutant emissions on global temperature and sea level than concluded here. Caps on aerosols in general could also yield impacts on other important aspects of climate beyond those addressed here, such as the regional patterns of cloudiness and precipitation.

Implementation of global make to inventory strategy at BASF SE

La optimización y armonización son factores clave para tener un buen desempeño en la industria química. BASF ha desarrollado un proyecto llamada acelerador. El objetivo de este proyecto ha sido la armonización y la integración de los procesos de la cadena de suministro a nivel mundial. El proceso básico de manejo de inventarios se quedó fuera del proyecto y debía ser analizado. El departamento de manejo de inventarios en BASF SE ha estado desarrollando su propia estrategia para la definición de procesos globales de manufactura. En este trabajo se presentará un informe de las fases de la formulación de la estrategia y establecer algunas pautas para la fase de implementación que está teniendo lugar en 2012 y 2013.

A comparative study of two Casas de la Cultura in Bogota: Tunjuelito and Usaquen

Es un estudio descriptivo/exploratorio de dos Casas de la Cultura en Bogotá (Tunjuelito/Usaquén), basado en la revisión de documentos y recolección de datos a través de herramientas cualitativas, con aspectos cuantitativos en la presentación de datos por medio de cuadros de comparación. Presenta resultados de las siguientes categorías: grupos de interés, aspectos administrativos y productos.

Incidencia de conflicto turcochipriota en las relaciones políticas de Turquía y la Unión Europea (2005-2010)

Las relaciones políticas entre Turquía y la Unión Europea tienen un historial de altos y bajos marcado por los intereses comerciales de cada uno. Sin embargo, en 2005 con la postulación de Turquía como candidato a país miembro de la Unión Europea, el conflicto en la isla de Chipre entre comunidades griegas y turcas se convierte en el principal protagonista de las relaciones bilaterales.

Self-organized sociopolitical interactions as the best way to achieve organized patterns in human social systems: going beyond the top-down control of classical political regimes

La monografía presenta la auto-organización sociopolítica como la mejor manera de lograr patrones organizados en los sistemas sociales humanos, dada su naturaleza compleja y la imposibilidad de las tareas computacionales de los regímenes políticos clásico, debido a que operan con control jerárquico, el cual ha demostrado no ser óptimo en la producción de orden en los sistemas sociales humanos. En la monografía se extrapola la teoría de la auto-organización en los sistemas biológicos a las dinámicas sociopolíticas humanas, buscando maneras óptimas de organizarlas, y se afirma que redes complejas anárquicas son la estructura emergente de la auto-organización sociopolítica.

Effect of the human papillomavirus (HPV) quadrivalent vaccine in a subgroup of women with cervical and vulvar disease: Retrospective pooled analysis of trial data

Objectives To determine the effect of human papillomavirus (HPV) quadrivalent vaccine on the risk of developing subsequent disease after an excisional procedure for cervical intraepithelial neoplasia or diagnosis of genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia. Design Retrospective analysis of data from two international, double blind, placebo controlled, randomised efficacy trials of quadrivalent HPV vaccine (protocol 013 (FUTURE I) and protocol 015 (FUTURE II)). Setting Primary care centres and university or hospital associated health centres in 24 countries and territories around the world. Participants Among 17 622 women aged 15–26 years who underwent 1:1 randomisation to vaccine or placebo, 2054 received cervical surgery or were diagnosed with genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia. Intervention Three doses of quadrivalent HPV vaccine or placebo at day 1, month 2, and month 6. Main outcome measures Incidence of HPV related disease from 60 days after treatment or diagnosis, expressed as the number of women with an end point per 100 person years at risk. Results A total of 587 vaccine and 763 placebo recipients underwent cervical surgery. The incidence of any subsequent HPV related disease was 6.6 and 12.2 in vaccine and placebo recipients respectively (46.2% reduction (95% confidence interval 22.5% to 63.2%) with vaccination). Vaccination was associated with a significant reduction in risk of any subsequent high grade disease of the cervix by 64.9% (20.1% to 86.3%). A total of 229 vaccine recipients and 475 placebo recipients were diagnosed with genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia, and the incidence of any subsequent HPV related disease was 20.1 and 31.0 in vaccine and placebo recipients respectively (35.2% reduction (13.8% to 51.8%)). Conclusions Previous vaccination with quadrivalent HPV vaccine among women who had surgical treatment for HPV related disease significantly reduced the incidence of subsequent HPV related disease, including high grade disease.

Four year efficacy of prophylactic human papillomavirus quadrivalent vaccine against low grade cervical, vulvar, and vaginal intraepithelial neoplasia and anogenital warts: randomised controlled trial

Objectives: To evaluate the prophylactic efficacy of the human papillomavirus (HPV) quadrivalent vaccine in preventing low grade cervical, vulvar, and vaginal intraepithelial neoplasias and anogenital warts (condyloma acuminata). Design: Data from two international, double blind, placebo controlled, randomised efficacy trials of quadrivalent HPV vaccine (protocol 013 (FUTURE I) and protocol 015 (FUTURE II)). The trials were to be 4 years in length, and the results reported are from final study data of 42 months' follow-up. Setting: Primary care centres and university or hospital associated health centres in 24 countries and territories around the world. Participants: 17 622 women aged 16-26 years enrolled between December 2001 and May 2003. Major exclusion criteria were lifetime number of sexual partners (>4), history of abnormal cervical smear test results, and pregnancy. Intervention: Three doses of quadrivalent HPV vaccine (for serotypes 6, 11, 16, and 18) or placebo at day 1, month 2, and month 6. Main outcome measures: Vaccine efficacy against cervical, vulvar, and vaginal intraepithelial neoplasia grade I and condyloma in a per protocol susceptible population that included subjects who received all three vaccine doses, tested negative for the relevant vaccine HPV types at day 1 and remained negative through month 7, and had no major protocol violations. Intention to treat, generally HPV naive, and unrestricted susceptible populations were also studied. Results: In the per protocol susceptible population, vaccine efficacy against lesions related to the HPV types in the vaccine was 96% for cervical intraepithelial neoplasia grade I (95% confidence interval 91% to 98%), 100% for both vulvar and vaginal intraepithelial neoplasia grade I (95% CIs 74% to 100%, 64% to 100% respectively), and 99% for condyloma (96% to 100%). Vaccine efficacy against any lesion (regardless of HPV type) in the generally naive population was 30% (17% to 41%), 75% (22% to 94%), and 48% (10% to 71%) for cervical, vulvar, and vaginal intraepithelial neoplasia grade I, respectively, and 83% (74% to 89%) for condyloma. Conclusions: Quadrivalent HPV vaccine provided sustained protection against low grade lesions attributable to vaccine HPV types (6, 11, 16, and 18) and a substantial reduction in the burden of these diseases through 42 months of follow-up. Trial registrations: NCT00092521 and NCT00092534.

Land Restitution and Prevention of Forced Displacement in Colombia

The armed conflict in Colombia, which has generated over three million internally displaced persons, has dramatic humanitarian consequences and raises serious issues regarding the protection of displaced peoples’ rights. The underlying reasons for the displacement often lie in the dynamics associated with territorial control and land seizures undertaken for strategic, military or purely economic purposes. Domestic and international legal provisions have established the victims’ right to the restitution of their homes and property as the “preferred remedy” in cases of displacement. However, policies dealing with displacement, both those of the Colombian government and of several international institutions, fail to take this sufficiently into account. A comprehensive reparation policy for victims must necessarily entail the reversion of lands, territories and goods seized in Colombia under the pretext of the internal armed conflict.

The French Constitutional Council as the Rottweiler of the Republican Ideal in the Language Field: Does Jurisprudence Really Reflect Reality? EDAP 03/2012, 2012

France is known for being a champion of individual rights as well as for its overt hostility to any form of group rights. Linguistic pluralism in the public sphere is rejected for fear of babelization and Balkanization of the country. Over recent decades the Conseil Constitutionnel (CC) has, together with the Conseil d’État, remained arguably the strongest defender of this Jacobin ideal in France. In this article, I will discuss the role of France’s restrictive language policy through the prism of the CC’s jurisprudence. Overall, I will argue that the CC made reference to the (Jacobin) state-nation concept, a concept that is discussed in the first part of the paper, in order to fight the revival of regional languages in France over recent decades. The clause making French the official language in 1992 was functional to this policy. The intriguing aspect is that in France the CC managed to standardise France’s policy vis-à-vis regional and minority languages through its jurisprudence; an issue discussed in the second part of the paper. But in those regions with a stronger tradition of identity, particularly in the French overseas territories, the third part of the paper argues, normative reality has increasingly become under pressure. Therefore, a discrepancy between the ‘law in courts’ and the compliance with these decisions (‘law in action’) has been emerging over recent years. Amid some signs of opening of France to minorities, this contradiction delineates a trend that might well continue in future.

Short-term Response of Breeding Barred Owls to Forestry in a Boreal Mixedwood Forest Landscape

Forestry and other activities are increasing in the boreal mixedwood of Alberta, with a concomitant decrease in older forest. The Barred Owl (Strix varia) is an old-growth indicator species in some jurisdictions in North America. Hence, we radio-tagged Barred Owls in boreal mixedwood in Alberta to determine whether harvesting influenced habitat selection. We used three spatial scales: nest sites, i.e., nest tree and adjacent area of 11.7 m radius around nests, nesting territory of 1000 m radius around nests, and home range locations within 2000 m radius of the home range center. Barred Owls nested primarily in balsam poplar (Populus balsamifera) snags > 34 cm dbh and nest trees were surrounded by large, > 34 cm dbh, balsam poplar trees and snags. Nesting territories contained a variety of habitats including young < 80-yr-old, deciduous-dominated stands, old deciduous and coniferous-dominated stands, treed bogs, and recent clear-cuts. However, when compared to available habitat in the study area, they were more likely to contain old conifer-dominated stands and recent cutblocks. We assumed this is because all of the recent harvest occurred in old stands, habitat preferred by the owls. When compared with random sites, locations used for foraging and roosting at the home range scale were more likely to be in young deciduous-dominated stands, old conifer-dominated stands and cutblocks > 30 yr old, and less likely to occur in old deciduous-dominated stands and recent cutblocks. Hence, although recent clearcuts occurred in territories, birds avoided these microhabitats during foraging. To meet the breeding requirements of Barred Owls in managed forests, 10–20 ha patches of old deciduous and mixedwood forest containing large Populus snags or trees should be maintained. In our study area, nest trees had a minimum dbh of 34 cm. Although cut areas were incorporated into home ranges, the amount logged was low, i.e., 7%, in our area. Hence more research is required to determine harvest levels tolerated by owls over the long term.

Understanding Demographic and Behavioral Mechanisms that Guide Responses of Neotropical Migratory Birds to Urbanization: a Simulation Approach

Although studies often report that densities of many forest birds are negatively related to urbanization, the mechanisms guiding this pattern are poorly understood. Our objective was to use a population simulation to examine the relative influence of six demographic and behavioral processes on patterns of avian abundance in urbanizing landscapes. We constructed an individual-based population simulation model representing the annual cycle of a Neotropical migratory songbird. Each simulation was performed under two landscape scenarios. The first scenario had similar proportions of high- and low-quality habitat across the urban to rural gradient. Under the first scenario, avian density was negatively related to urbanization only when rural habitats were perceived to be of higher quality than they actually were. The second landscape scenario had declining proportions of high-quality habitat as urbanization increased. Under the second scenario, each mechanism generated a negative relationship between density and urbanization. The strongest effect on density resulted when birds preferentially selected habitats in landscapes from which they fledged or were constrained from dispersing. The next strongest patterns occurred when birds directly evaluated habitat quality and accurately selected the highest-quality available territories. When birds selected habitats based on the presence of conspecifics, the density–urbanization relationship was only one-third the strength of other habitat selection mechanisms and only occurred under certain levels of population survival. Although differences in adult or nest survival in the face of random habitat selection still elicited reduced densities in urban landscapes, the relationships between urbanization and density were weaker than those produced by the conspecific attraction mechanism. Results from our study identify key predictions and areas for future research, including assessing habitat quality in urban and rural areas in order to determine if habitats in urban areas are underutilized.

Devolution and the political representation of business interests in the UK

The devolution of political power in Scotland, Wales and Northern Ireland and the developing regional agenda in England are widely read as a significant reconfiguration of the institutions and scales of economic governance. The process is furthest developed in Scotland while Wales and Northern Ireland, in their own distinct ways, provide intermediate cases. Devolution is least developed in England where regional political identities are generally weak and the historical legacy of regional institutions is limited. Within the overall context of devolution government policy has continued to emphasize partnership forms of. governance. Accordingly, the political representation of business interests has a particular salience in the new arrangements. This paper reports on findings from a study designed to examine the relationship between devolution and changes in the political representation of business interests in the territories and regions of the UK. It highlights a number of changes in the nature and extent of business representation. While some of these are significant the evidence suggests that they fail to mark a fundamental shift in the institutional foundation for sub-national business interest representation in the UK. Indeed the political geography of business representation remains dominated by an overarching centralism that is likely to provide a significant check on the further devolution of political power and democratic authority. (c) 2004 Elsevier Ltd. All rights reserved.