Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.

Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^

Anelastic strain recovery and elastic properties of ODP Leg 123 oceanic basalt samples

A knowledge of rock stress is fundamental for improving our understanding of oceanic crustal mechanisms and lithospheric dynamic processes. However, direct measurements of stress in the deep oceans, and in particular stress magnitudes, have proved to be technically difficult. Anelastic strain recovery measurements were conducted on 15 basalt core samples from Sites 765 and 766 during Leg 123. Three sets of experiments were performed: anelastic strain recovery monitoring, dynamic elastic property measurements, and thermal azimuthal anisotropy observations. In addition, a range of other tests and observations were recorded to characterize each of the samples. One common feature of the experimental results and observations is that apparently no consistent orientation trend exists, either between the different measurements on each core sample or between the same sets of measurements on the various core samples. However, some evidence of correspondence between velocity anisotropy and anelastic strain recovery exists, but this is not consistent for all the core samples investigated. Thermal azimuthal anisotropy observations, although showing no conclusive correlations with the other results, were of significant interest in that they clearly exhibited anisotropic behavior. The apparent reproducibility of this behavior may point toward the possibility of rocks that retain a "memory" of their stress history, which could be exploited to derive stress orientations from archived core. Anelastic strain recovery is a relatively new technique. Because use of the method has extended to a wider range of rock types, the literature has begun to include examples of rocks that contracted with time. Strong circumstantial evidence exists to suggest that core-sample contractions result from the slow diffusion of pore fluids from a preexisting microcrack structure that permits the rock to deflate at a greater rate than the expansion caused by anelastic strain recovery. Both expansions and contractions of the Leg 123 cores were observed. The basalt cores have clearly been intersected by an abundance of preexisting fractures, some of which pass right through the samples, but many are intercepted or terminate within the rock matrix. Thus, the behavior of the core samples will be influenced not only by the properties of the rock matrix between the fractures, but also by how these macro- and micro-scale fractures mutually interact. The strain-recovery curves recorded during Leg 123 for each of the 15 basalt core samples may reflect the result of two competing time dependent processes: anelastic strain recovery and pore pressure recovery. Were these the only two processes to influence the gauge responses, then one might expect that given the additional information required, established theoretical models might be used to determine consistent stress orientations and reliable stress magnitudes. However, superimposed upon these competing processes is their respective interaction with the preexisting fractures that intersect each core. Evidence from our experiments and observations suggests that these fractures have a dominating influence on the characteristics of the recovery curves and that their effects are complex.

Fission track analyses of ODP Hole 129-800A samples

Four models of fission track annealing in apatite are compared with measured fission track lengths in samples from Site 800 in the East Mariana Basin, Ocean Drilling Program Leg 129, given an independently determined temperature history. The temperature history of Site 800 was calculated using a one-dimensional, compactive, conductive heat flow model assuming two end-member thermal cases: one for cooling of Jurassic ocean crust that has experienced no subsequent heating, and one for cooling of Cretaceous ocean crust. Because the samples analyzed were only shallowly buried and because the tectonic history of the area since sample deposition is simple, resolution of the temperature history is high. The maximum temperature experienced by the sampled bed is between 16°-21°C and occurs at 96 Ma; temperatures since the Cretaceous have dropped in spite of continued pelagic sediment deposition because heat flow has continued to decay exponentially and bottom-water temperatures have dropped. Fission tracks observed within apatite grains from the sampled bed are 14.6 +/- 0.1 µm (1 sigma) long. Given the proposed temperature history of the samples, one unpublished and three published models of fission track annealing predict mean track lengths from 14.8 to 15.9 µm. These models require temperatures as much as 40°C higher than the calculated paleotemperature maximum of the sampled bed to produce the same degree of track annealing. Measured and predicted values are different because annealing models are based on extrapolation of high temperature laboratory data to geologic times. The model that makes the closest prediction is based on the greatest number of experiments performed at low temperature and on an apatite having composition closest to that of the core samples.

The development of professor's professional knowledge in Psychology

This work belongs to the Project "Teaching and learning Psychology: a study of the educational practice in professors and beginners". It is part of an investigation program developed in the context of the subject "Didactic and Practical Planning of Psychology teaching" in which we have been working since 1998. This subject is also considered as the final stage of the formative itinerary for Psychology s professors. The reached results show the incidence that the beliefs and professors' representations have in the teaching processes. By getting into the studies centered in the educational knowledge we guide the inquiry around the development of the professor's professional knowledge and particularly the development of their formation. Our proposal contemplates a formation device which incorporates the observation, reflection, evaluation and self-evaluation of the practice in order to facilitate the reconstruction, significance and re-significance of the teaching and learning processes by giving relevance to the development of the teacher professional knowledge. The analysis of teaching practices process reveals that the mediations that cross the knowledge in the being taught instances are constituted in reflections axes. This involves the future professor's relationship with the knowledge and their projection in the construction of the professional identity.

The development of professor's professional knowledge in Psychology

This work belongs to the Project "Teaching and learning Psychology: a study of the educational practice in professors and beginners". It is part of an investigation program developed in the context of the subject "Didactic and Practical Planning of Psychology teaching" in which we have been working since 1998. This subject is also considered as the final stage of the formative itinerary for Psychology s professors. The reached results show the incidence that the beliefs and professors' representations have in the teaching processes. By getting into the studies centered in the educational knowledge we guide the inquiry around the development of the professor's professional knowledge and particularly the development of their formation. Our proposal contemplates a formation device which incorporates the observation, reflection, evaluation and self-evaluation of the practice in order to facilitate the reconstruction, significance and re-significance of the teaching and learning processes by giving relevance to the development of the teacher professional knowledge. The analysis of teaching practices process reveals that the mediations that cross the knowledge in the being taught instances are constituted in reflections axes. This involves the future professor's relationship with the knowledge and their projection in the construction of the professional identity.

The development of professor's professional knowledge in Psychology

This work belongs to the Project "Teaching and learning Psychology: a study of the educational practice in professors and beginners". It is part of an investigation program developed in the context of the subject "Didactic and Practical Planning of Psychology teaching" in which we have been working since 1998. This subject is also considered as the final stage of the formative itinerary for Psychology s professors. The reached results show the incidence that the beliefs and professors' representations have in the teaching processes. By getting into the studies centered in the educational knowledge we guide the inquiry around the development of the professor's professional knowledge and particularly the development of their formation. Our proposal contemplates a formation device which incorporates the observation, reflection, evaluation and self-evaluation of the practice in order to facilitate the reconstruction, significance and re-significance of the teaching and learning processes by giving relevance to the development of the teacher professional knowledge. The analysis of teaching practices process reveals that the mediations that cross the knowledge in the being taught instances are constituted in reflections axes. This involves the future professor's relationship with the knowledge and their projection in the construction of the professional identity.

Modeling the sensitivity of precipitation oxygen isotopes to the Last Glacial Maximum boundary conditions: A study using Community Atmosphere Model (CAM3.0)

To understand the validity of d18O proxy records as indicators of past temperature change, a series of experiments was conducted using an atmospheric general circulation model fitted with water isotope tracers (Community Atmosphere Model version 3.0, IsoCAM). A pre-industrial simulation was performed as the control experiment, as well as a simulation with all the boundary conditions set to Last Glacial Maximum (LGM) values. Results from the pre-industrial and LGM simulations were compared to experiments in which the influence of individual boundary conditions (greenhouse gases, ice sheet albedo and topography, sea surface temperature (SST), and orbital parameters) were changed each at a time to assess their individual impact. The experiments were designed in order to analyze the spatial variations of the oxygen isotopic composition of precipitation (d18Oprecip) in response to individual climate factors. The change in topography (due to the change in land ice cover) played a significant role in reducing the surface temperature and d18Oprecip over North America. Exposed shelf areas and the ice sheet albedo reduced the Northern Hemisphere surface temperature and d18Oprecip further. A global mean cooling of 4.1 °C was simulated with combined LGM boundary conditions compared to the control simulation, which was in agreement with previous experiments using the fully coupled Community Climate System Model (CCSM3). Large reductions in d18Oprecip over the LGM ice sheets were strongly linked to the temperature decrease over them. The SST and ice sheet topography changes were responsible for most of the changes in the climate and hence the d18Oprecip distribution among the simulations.

Biological, physical and chemical characteristics of water and sediment samples from the Volga River delta, northern Caspian

Based on the data of synchronous observations of hydrophysical and biogeochemical parameters in the near-mouth and shallow-water areas of the northern Caspian in 2000-2001, the scale of spatiotemporal variability in the following characteristics of the water-bottom system was estimated (1) flow velocity and direction within vortex structures formed by the combined effect of wind, discharge current, and the presence of higher aquatic plants; (2) dependence of the spatial distribution of the content and composition of suspended particulate matter on the hydrodynamic regime of waters and development of phytoplankton; (3) variations in the grain-size, petrographic, mineralogical, and chemical compositions of the upper layer of bottom sediments at several sites in the northern Caspian related to the particular local combination of dominant natural processes; and (4) limits of variability in the group composition of humus compounds in bottom sediments. The acquired data are helpful in estimating the geochemical consequences of a sea level rise and during the planning of preventive environmental protection measures in view of future oil and gas recovery in this region.

Time-series of seagrass and land cover maps from 1972 to 2010, in South East Queensland, Australia, with links to GeoTIFFs

Long term global archives of high-moderate spatial resolution, multi-spectral satellite imagery are now readily accessible, but are not being fully utilised by management agencies due to the lack of appropriate methods to consistently produce accurate and timely management ready information. This work developed an object-based remote sensing approach to map land cover and seagrass distribution in an Australian coastal environment for a 38 year Landsat image time-series archive (1972-2010). Landsat Multi-Spectral Scanner (MSS), Thematic Mapper (TM) and Enhanced Thematic Mapper (ETM+) imagery were used without in situ field data input (but still using field knowledge) to produce land and seagrass cover maps every year data were available, resulting in over 60 map products over the 38 year archive. Land cover was mapped annually using vegetation, bare ground, urban and agricultural classes. Seagrass distribution was also mapped annually, and in some years monthly, via horizontal projected foliage cover classes, sand and deep water. Land cover products were validated using aerial photography and seagrass maps were validated with field survey data, producing several measures of accuracy. An average overall accuracy of 65% and 80% was reported for seagrass and land cover products respectively, which is consistent with other studies in the area. This study is the first to show moderate spatial resolution, long term annual changes in land cover and seagrass in an Australian environment, created without the use of in situ data; and only one of a few similar studies globally. The land cover products identify several long term trends; such as significant increases in South East Queensland's urban density and extent, vegetation clearing in rural and rural-residential areas, and inter-annual variation in dry vegetation types in western South East Queensland. The seagrass cover products show that there has been a minimal overall change in seagrass extent, but that seagrass cover level distribution is extremely dynamic; evidenced by large scale migrations of higher seagrass cover levels and several sudden and significant changes in cover level. These mapping products will allow management agencies to build a baseline assessment of their resources, understand past changes and help inform implementation and planning of management policy to address potential future changes.

Respiration and ingestion rates of calanoid copepod in the northern Benguela Current System measured ex situ during the RSS Discovery D356 cruise in 2010

Respiration rates of 16 calanoid copepod species from the northern Benguela upwelling system were measured on board RRS Discovery in September/October 2010 to determine their energy requirements and assess their significance in the carbon cycle. Copepod species were sampled by different net types. Immediately after the hauls, samples were sorted to species and stages (16 species; females, males and C5 copepodids) according to Bradford-Grieve et al. (1999). Specimens were kept in temperature-controlled refrigerators for at least 12 h before they were used in experiments. Respiration rates of different copepod species were measured onboard by optode respirometry (for details see Köster et al., 2008) with a 10-channel optode respirometer (PreSens Precision Sensing Oxy-10 Mini, Regensburg, Germany) under simulated in situ conditions in temperature-controlled refrigerators. Experiments were run in gas-tight glass bottles (12-13 ml). For each set of experiments, two controls without animals were measured under exactly the same conditions to compensate for potential bias. The number of animals per bottle depended on the copepods size, stage and metabolic activity. Animals were not fed during the experiments but they showed natural species-specific movements. Immediately after the experiments, all specimens were deep-frozen at - 80 °C for later dry mass determination (after lyophilisation for 48 h) in the home lab. The carbon content (% of dry mass) of each species was measured by mass-spectrometry in association with stable isotope analysis and body dry mass was converted to units of carbon. For species without available carbon data, the mean value of all copepod species (44% dry mass) was applied. For the estimation of carbon requirements of copepod species, individual oxygen consumption rates were converted to carbon units, assuming that the expiration of 1 ml oxygen mobilises 0.44 mg of organic carbon by using a respiratory quotient (RQ) of 0.82 for a mixed diet consisting of proteins (RQ = 0.8-1.0), lipids (RQ = 0.7) and carbohydrates (RQ = 1.0) (Auel and Werner, 2003). The carbon ingestion rates were calculated using the energy budget and the potential maximum ingestion rate approach. To allow for physiological comparisons of respiration rates of deep- and shallow-living copepod species without the effects of ambient temperature and different individual body mass, individual respiration rates were temperature- (15°C, Q10=2) and size-adjusted. The scaling coefficient of 0.76 (R2=0.556) is used for the standardisation of body dry mass to 0.3 mg (mean dry mass of all analysed copepods), applying the allometric equation R= (R15°C/M0.76)×0.30.76, where R is respiration and M is individual dry mass in mg.

Radiometric age determinations for basements of ODP Sites 123-765 and 123-766

The results of experiments in 40Ar/39Ar age dating using fresh basement material from Sites 765 and 766 of Leg 123 of the Ocean Drilling Program are inconsistent and cannot be used to constrain the basement age of the Argo Abyssal Plain in the Indian Ocean. However, a celadonite sample, which was precipitated during a low-temperature alteration event that affected the basement at Site 765, yielded a K-Ar age of 155.3 ±3.4 Ma. Celadonites, which have been dated using Rb-Sr methods for basement in the Atlantic Ocean (Staudigel et al., 1981, doi:10.1016/0012-821X(81)90186-2) and by K-Ar methods for the Troodos Ophiolite (Staudigel et al., 1986, doi:10.1130/0091-7613(1986)14<72:AASAOC>2.0.CO;2), and for sediments from the Pacific Ocean (Peterson et al., 1986, doi:10.2973/dsdp.proc.92.132.1986) yield ages that are up to 15 Ma younger than the age for the formation of basement. Thus, the celadonite age is retained as a reliable minimum age for basement at Site 765. This radiometric age is inconsistent with biostratigraphic ages, which indicate a maximum of late Berriasian (approximately 140 Ma) for Site 765, but is consistent with geophysical interpretations of marine magnetic anomalies and with the early north-south seafloor spreading history of the Argo Abyssal Plain region of the Indian Ocean.

A continent revealed - The European Geotraverse - Link to Atlas of compiled data

The continent of Europe has a complex geological history of successive tectonic events. Over several thousand million years these have formed the present day configuration of major tectonic provinces. A Continent Revealed unravels this history by presenting and interpreting the results of the European Geotraverse (EGT) a unique study of the continent of Europe and the first comprehensive cross section of continental lithosphere. This illustrated book has been put together by key workers in the EGT project. It uses the wealth of information yielded by the ten years of experiments, study centres and workshops to provide a concise and thought provoking account of the geological processes that created the European continent. It provides a summary of the European Geotraverse, and at the same time a starting point for further work.

Registry of samples and environmental context from the Ocean Sampling Day 2014

The Ocean Sampling Day (OSD) is a simultaneous sampling campaign of the world's oceans which took place (for the first time) on the summer solstice (June 21st) in the year 2014. These cumulative samples, related in time, space and environmental parameters, provide insights into fundamental rules describing microbial diversity and function and contribute to the blue economy through the identification of novel, ocean-derived biotechnologies. We see OSD data as a reference data set for generations of experiments to follow in the coming decade. The present data set includes a description of each sample collected during the Ocean Sampling Day 2014 and provides contextual environmental data measured concurrently with the collection of water samples for genomic analyses.

Ship manoeuvrability: full scale trials of colombian navy riverine support patrol vessel

Methodology and results of full scale maneuvering trials for Riverine Support Patrol Vessel “RSPV”, built by COTECMAR for the Colombian Navy are presented. !is ship is equipped with a “Pump – Jet” propulsion system and the hull corresponds to a wide-hull with a high Beam – Draft ratio (B/T=9.5). Tests were based on the results of simulation of turning diameters obtained from TRIBON M3© design software, applying techniques of Design of Experiments “DOE”, to rationalize the number of runs in di"erent conditions of water depth, ship speed, and rudder angle. Results validate the excellent performance of this class of ship and show that turning diameter and other maneuvering characteristics improve with decreasing water depth.