Stability and potential application of spliced nuclear pre -mRNA introns

A major portion of this thesis work was dedicated to study the nature and significance of spliced introns. The initial work was focused on studying the IVS1$\sb{\rm C\beta 1}$ intron from a T-cell receptor (TCR)-$\beta$ gene. Compared to an intron lariat control from adenovirus pre-mRNA that was spliced in vitro, IVS1$\sb{\rm C\beta 1}$ was debranched less efficiently by HeLa S100 extracts, although IVS1$\sb{\rm C\beta 1}$ also used the consensus branchpoint in vivo. Subcellular-fractionation analysis showed that most IVS1$\sb{\rm C\beta 1}$ lariats cofractionated with pre-mRNA in the nucleus, consistent with the possibility that intron degradation releases splicing factors which will be available for further rounds of splicing. The half-life of IVS1$\sb{\rm C\beta 1}$ from the endogenous TCR-$\beta$ gene was measured using the general transcription inhibitor actinomycin D to be about $\sim$15 min, which was similar to that of unstable mRNAs such as c-myc mRNA.^ The general transcription inhibitor DRB was also used for intron stability analysis. Unexpectedly, DRB decreased intron and pre-mRNA levels only initially, it later increased the levels of intron-containing RNAs. Inhibition of transcription initiation appeared to be the major early effect (the reduction phase); whereas enhanced premature transcription termination was dominant later (the induction phase).^ Having established the procedures for studying in vivo spliced introns, this approach was applied to study the mechanism of nonsense-mediated downregulation (NMD), a phenomena in which premature termination codons (PTCs) decrease the levels of mRNAs. In this study, the novel intron-oriented approach was applied to study the mechanism of NMD. The levels of spliced introns immediately upstream and downstream of a PTC-bearing exon in a TCR-$\beta$ gene were identified and analyzed along with their pre-mRNA. Although PTC reduced the mRNA levels by 4 to 9 fold, the steady-state levels of spliced introns and the pre-mRNA-to-intron ratios were not significantly altered, indicating that the PTC did not significantly inhibit TCR-$\beta$ RNA splicing. Consistent with this conclusion, the half-lives of the PTC$\sp+$ and PTC$\sp-$ pre-mRNA were similar. The protein synthesis inhibitor cyclohexmide (CHX) upregulated the levels of the PTC$\sp+$ mRNA over 10 fold without affecting the levels of the spliced introns, suggesting that the reversal effect of CHX was through stabilization, not production. These results indicated that inhibition of splicing could not be the major mechanism for the NMD pathway of the TCR-$\beta$ gene, instead, suggesting that mRNA destabilization may be more important. (Abstract shortened by UMI.) ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^

Molecular mechanisms involved in the regulation of cell polarity and genome stability by the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The essential p21-activated kinase (PAK), Shk1, is a critical component of a Ras/Cdc42/PAK complex required for cell viability, normal cell polarity, proper regulation of cytoskeletal dynamics, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. While cellular functions of PAKs have been described in eukaryotes from yeasts to mammals, the molecular mechanisms of PAK regulation and function are poorly understood. This study has characterized a novel Shk1 inhibitor, Skb15, and, in addition, identified the cell polarity regulator, Tea1, as a potential biological substrate of Shk1 in S. pombe. Skb15 is a highly conserved WD repeat protein that was discovered from a two-hybrid screen for proteins that interact with the catalytic domain of Shk1. Molecular data indicate that Skb15 negatively regulates Shk1 kinase activity in S. pombe cells. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution. ^ Our laboratory has recently demonstrated that Shk1, in addition to regulating actin cytoskeletal organization, is required for proper regulation of microtubule dynamics in S. pombe cells. The Shk1 protein localizes to interphase and mitotic microtubules, the septum-forming region, and cell ends. This pattern of localization overlaps with that of the cell polarity regulator, Tea1, in S. pombe cells. The tea1 gene was identified by Paul Nurse's laboratory from a screen for genes involved in the control of cell morphogenesis in S. pombe. In contrast to wild type S. pombe cells, which are rod shaped, tea1 null cells are often bent and/or branched in shape. The Tea1 protein localizes to the cell ends, like Shk1, and the growing tips of interphase microtubules. Thus, experiments were performed to investigate whether Tea1 interacts with Shk1. The tea1 null mutation strongly suppresses the loss of function of Skb15, an essential inhibitor of Shk1 function. All defects associated with the skb15 mutation, including defects in F-actin organization, septation, spindle elongation, and chromosome segregation, are suppressed by tea1Δ, suggesting that Tea1 may function in these diverse processes. Consistent with a role for Tea1 in cytokinesis, tea1Δ cells have a modest cell separation defect that is greatly exacerbated by a shk1 mutation and, like Shk1, Tea1 localizes to the septation site. Molecular analyses showed that Tea1 phosphorylation is significantly dependent on Shk1 function in vivo and that bacterially expressed Tea1 protein is directly phosphorylated by recombinant Shk1 kinase in vitro. Taken together, these results identify Tea1 as a potential biological substrate of Shk1 in S. pombe. ^ In summary, this study provides new insights into a conserved regulatory mechanism for PAKs, and also begins to uncover the molecular mechanisms by which the Ras/Cdc42/PAK complex regulates the microtubule and actin cytoskeletons and cell growth polarization in fission yeast. ^

A magneto-mineralogical study of basalt at DSDP Leg 73 Holes

The basalts in Holes 519A, 522B, and 524 were studied for intensity of natural remanent magnetization, magnetic hysteresis, magnetic susceptibility, stability of isothermal remanence, and thermomagnetic behavior. Some of these properties are sensitive to both the composition and the microstructure of the magnetic minerals, others to composition only. Thus it is possible to separate the two effects and to trace the variation of effective magnetic grain size and degree of alteration within a lithologic unit or over a yet larger distance or time interval. The flow in Hole 519A is highly maghemitized at the top, the degree of maghemitization decreasing with depth in the flow. Effective grain size increases with increasing depth. Electron microprobe analysis of the titanomaghemite grains in these samples provides no support for the leaching out of iron during alteration. The pillows and flows in Hole 522B are distributed among a number of cooling units, and no systematic downhole variations are apparent. The inferred magneto-petrology is consistent with the cooling and alteration history that might be expected within the units. The upper and lower sills in Hole 524 are more uniform and have a larger concentration of well-developed magnetic mineral grains than the pillows and flows in Holes 519A and 522B. Maghemitization appears to have developed from the boundaries of the sills that are in contact with the sediments between the sills.

Effects of seawater pCO2 and temperature on shell growth, shell stability, condition and cellular stress of Western Baltic Sea Mytilus edulis (L.) and Arctica islandica (L.)

Acidification of the World's oceans may directly impact reproduction, performance and shell formation of marine calcifying organisms. In addition, since shell production is costly and stress in general draws on an organism's energy budget, shell growth and stability of bivalves should indirectly be affected by environmental stress. The aim of this study was to investigate whether a combination of warming and acidification leads to increased physiological stress (lipofuscin accumulation and mortality) and affects the performance [shell growth, shell breaking force, condition index (Ci)] of young Mytilus edulis and Arctica islandica from the Baltic Sea. We cultured the bivalves in a fully-crossed 2-factorial experimental setup (seawater (sw) pCO2 levels "low", "medium" and "high" for both species, temperature levels 7.5, 10, 16, 20 and 25 °C for M. edulis and 7.5, 10 and 16 °C for A. islandica) for 13 weeks in summer. Mytilus edulis and A. islandica appeared to tolerate wide ranges of sw temperature and pCO2. Lipofuscin accumulation of M. edulis increased with temperature while the Ci decreased, but shell growth of the mussels only sharply decreased while its mortality increased between 20 and 25 °C. In A. islandica, lipofuscin accumulation increased with temperature, whereas the Ci, shell growth and shell breaking force decreased. The pCO2 treatment had only marginal effects on the measured parameters of both bivalve species. Shell growth of both bivalve species was not impaired by under-saturation of the sea water with respect to aragonite and calcite. Furthermore, independently of water temperatures shell breaking force of both species and shell growth of A. islandica remained unaffected by the applied elevated sw pCO2 for several months. Only at the highest temperature (25 °C), growth arrest of M. edulis was recorded at the high sw pCO2 treatment and the Ci of M. edulis was slightly higher at the medium sw pCO2 treatment than at the low and high sw pCO2 treatments. The only effect of elevated sw pCO2 on A. islandica was an increase in lipofuscin accumulation at the high sw pCO2 treatment compared to the medium sw pCO2 treatment. Our results show that, despite this robustness, growth of both M. edulis and A. islandica can be reduced if sw temperatures remain high for several weeks in summer. As large body size constitutes an escape from crab and sea star predation, this can make bivalves presumably more vulnerable to predation with possible negative consequences on population growth. In M. edulis, but not in A. islandica, this effect is amplified by elevated sw pCO2. We follow that combined effects of elevated sw pCO2 and ocean warming might cause shifts in future Western Baltic Sea community structures and ecosystem services; however, only if predators or other interacting species do not suffer as strong from these stressors.

The stakes of politics and electoral administration : a comparative study of Southeast Asian democracies

Elections play a crucial role in political stability in post-democratization, and electoral administrations are the key to the electoral process. However, not all newly democratized countries have established reliable electoral administration. New democracies in Southeast Asia, such as the Philippines, Indonesia and Thailand, have independent election commissions which have different characteristics, especially in terms of neutrality. Based on three cases, this paper claims that the stakes of politics are the major determinant of the variations in neutrality. The high stakes of politics in Thailand brought about the partisan election commission, while the low stakes in Indonesia made the electoral system relatively neutral. Like Thailand, the high stakes of politics in the Philippines also cause political intervention in the electoral administration.

Interstitial defects stability in FeCr alloys vs Cr concentration

Finding adequate materials to withstand the demanding conditions in the future fusion and fission reactors is a real challenge in the development of these technologies. Structural materials need to sustain high irradiation doses and temperatures that will change the microstructure over time. A better understanding of the changes produced by the irradiation will allow for a better choice of materials, ensuring a safer and reliable future power plants. High-Cr ferritic/martensitic steels head the list of structural materials due to their high resistance to swelling and corrosion. However, it is well known that these alloys present a problem of embrittlement, which could be caused by the presence of defects created by irradiation as these defects act as obstacles for dislocation motion. Therefore, the mechanical response of these materials will depend on the type of defects created during irradiation. In this work, we address a study of the effect Cr concentration has on single interstitial defect formation energies in FeCr alloys.

Clean Coal Technologies Scenario and Evaluation of Present CO2 Dwindling Initiatives to Approach Zero Emission Power Stations By Coal Combustion. Deployment Situation and Evaluation Study

In the present uncertain global context of reaching an equal social stability and steady thriving economy, power demand expected to grow and global electricity generation could nearly double from 2005 to 2030. Fossil fuels will remain a significant contribution on this energy mix up to 2050, with an expected part of around 70% of global and ca. 60% of European electricity generation. Coal will remain a key player. Hence, a direct effect on the considered CO2 emissions business-as-usual scenario is expected, forecasting three times the present CO2 concentration values up to 1,200ppm by the end of this century. Kyoto protocol was the first approach to take global responsibility onto CO2 emissions monitoring and cap targets by 2012 with reference to 1990. Some of principal CO2emitters did not ratify the reduction targets. Although USA and China spur are taking its own actions and parallel reduction measures. More efficient combustion processes comprising less fuel consuming, a significant contribution from the electricity generation sector to a CO2 dwindling concentration levels, might not be sufficient. Carbon Capture and Storage (CCS) technologies have started to gain more importance from the beginning of the decade, with research and funds coming out to drive its come in useful. After first researching projects and initial scale testing, three principal capture processes came out available today with first figures showing up to 90% CO2 removal by its standard applications in coal fired power stations. Regarding last part of CO2 reduction chain, two options could be considered worthy, reusing (EOR & EGR) and storage. The study evaluates the state of the CO2 capture technology development, availability and investment cost of the different technologies, with few operation cost analysis possible at the time. Main findings and the abatement potential for coal applications are presented. DOE, NETL, MIT, European universities and research institutions, key technology enterprises and utilities, and key technology suppliers are the main sources of this study. A vision of the technology deployment is presented.

The scenario of two-dimensional instabilities of the cylinder wake under EHD forcing: A linear stability analysis

We propose to study the stability properties of an air flow wake forced by a dielectric barrier discharge (DBD) actuator, which is a type of electrohydrodynamic (EHD) actuator. These actuators add momentum to the flow around a cylinder in regions close to the wall and, in our case, are symmetrically disposed near the boundary layer separation point. Since the forcing frequencies, typical of DBD, are much higher than the natural shedding frequency of the flow, we will be considering the forcing actuation as stationary. In the first part, the flow around a circular cylinder modified by EHD actuators will be experimentally studied by means of particle image velocimetry (PIV). In the second part, the EHD actuators have been numerically implemented as a boundary condition on the cylinder surface. Using this boundary condition, the computationally obtained base flow is then compared with the experimental one in order to relate the control parameters from both methodologies. After validating the obtained agreement, we study the Hopf bifurcation that appears once the flow starts the vortex shedding through experimental and computational approaches. For the base flow derived from experimentally obtained snapshots, we monitor the evolution of the velocity amplitude oscillations. As to the computationally obtained base flow, its stability is analyzed by solving a global eigenvalue problem obtained from the linearized Navier–Stokes equations. Finally, the critical parameters obtained from both approaches are compared.

Study of the Mechanical Strength Improvement of Wafers for EWT Solar Cells by Chemical Etching after the Drilling Process

Mechanical stability of EWT solar cells deteriorates when holes are created in the wafer. Nevertheless, the chemical etching after the hole generation process improves the mechanical strength by removing part of the damage produced in the drilling process. Several sets of wafers with alkaline baths of different duration have been prepared. The mechanical strength has been measured by the ring on ring bending test and the failure stresses have been obtained through a FE simulation of the test. This paper shows the comparison of these groups of wafers in order to obtain an optimum value of the decreased thickness produced by the chemical etching

Floating zone stability

The present state of de preparation of an experiment on floating liquid zones to be performed in the first Spacelab flight is presented. In this experiment,a liquid bridge is to be placed between two parallel coaxial disks (in the Fluid Physics Module)and subjected to very precise disturbances in order to check the theoretical predictions about its stability limits and behavior under mechanical inputs: stretching of the zone, filling or removing the liquid,axial vibration, rotation, disalignment, etc. Several aspects of the research are introduced:1) Relevance of the study. 2) Theoretical predictions of the liquid behavior regarding the floating-zone stability limits and the expected response to vibrational and rotational disturbances. 3) Ground support experiments using the Plateau technique or the small scale simulation. 4) Instrumental aspects of the experimentation: the Fluid Physics Module utilization and post-flight data analysis.5)Research program for future flights.

Comparative study of breakwater crown wall – calculation methods

An investigation was undertaken consisting of a state-of-the-art and comparative analysis of currently available methods for calculating the structural stability of wave walls in sloping breakwaters. A total of six design schemes are addressed. The conditions under which the formulations and ranges of validity are explicitly indicated by their authors, are given. The lack of definition in parameters to be used and aspects not taken into account in their investigations are discussed and the results of this analysis are given in a final table.

Study of the effect of different hole sizes on mechanical strength of wafers for back contact solar cells

Drilling process on wafers to produce EWT or MWT solar cells is a critical fabrication step, which affects on their mechanical stability. The amount of damage introduced during drilling process depends on the density of holes, their size and the chemical process applied afterwards. To quantify the relation between size of the holes and reduction of mechanical strength, several sets of wafers have been prepared, with different hole diameter. The mechanical strength of these sets has been measured by the ring on ring bending test, and the stress state in the moment of failure has been deduced by FE simulation.

Evaluation and improvement of empirical models of global solar irradiation: Case study northern Spain

This paper presents a new methodology to build parametric models to estimate global solar irradiation adjusted to specific on-site characteristics based on the evaluation of variable im- portance. Thus, those variables higly correlated to solar irradiation on a site are implemented in the model and therefore, different models might be proposed under different climates. This methodology is applied in a study case in La Rioja region (northern Spain). A new model is proposed and evaluated on stability and accuracy against a review of twenty-two already exist- ing parametric models based on temperatures and rainfall in seventeen meteorological stations in La Rioja. The methodology of model evaluation is based on bootstrapping, which leads to achieve a high level of confidence in model calibration and validation from short time series (in this case five years, from 2007 to 2011). The model proposed improves the estimates of the other twenty-two models with average mean absolute error (MAE) of 2.195 MJ/m2 day and average confidence interval width (95% C.I., n=100) of 0.261 MJ/m2 day. 41.65% of the daily residuals in the case of SIAR and 20.12% in that of SOS Rioja fall within the uncertainty tolerance of the pyranometers of the two networks (10% and 5%, respectively). Relative differences between measured and estimated irradiation on an annual cumulative basis are below 4.82%. Thus, the proposed model might be useful to estimate annual sums of global solar irradiation, reaching insignificant differences between measurements from pyranometers.

Stresses and stability of a mine paste fill

Se describe el comportamiento de los rellenos de pasta de las cámaras primarias de la mina de Aguas Teñidas y se calcula la resistencia que deben tener dichos rellenos para que no se desmoronen las paredes de los mismos que quedan expuestas al extraer las cámaras secundarias.Abstract:This article presents the study carried out at an underground mine to understand the stress distribution in the paste fills and to calculate the stability of the paste walls. The mine is operated using sublevel stopes. Three-dimensional numerical models designed with the FLAC 3D software are used to study the distribution of the vertical stresses in the paste. The numerical models have demonstrated that an arc-like effect is produced in the paste fills of the primary stopes. This effect relieves the vertical stresses and increases the stability of the exposed paste wall fill. Based on the results of the numerical models, in the 30m high secondary stopes, the arc effect starts to be evident only in paste walls with a width/height ratio lower than 0.8. 3-D calculations show that the use of Mitchell, R. J. et al. (1982) formula may be risky when estimating the fill stability in secondary stopes.