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.) ^

The wine hedonic price models in the "Old and New World": : state of the art

The basic hedonic hypothesis is that goods are valued for their utility-bearing characteristics and not for the good itself. Each attribute can be evaluated by consumers when making a purchasing decision and an implicit price can be identified for each of them. Thus, the observed price of a certain good can be analyzed as the sum of the implicit prices paid for each quality attribute. Literature has reported hedonic models estimates in the case of wines, which are excellent examples of differentiated goods worldwide.The impact of different wine attributes (intrinsic or extrinsic) on consumers’ willingness to pay has been analyzed with dissimilar results. Wines coming from "New World" producers seem to be appreciated for different attributes than wines produced in the "Old World". Moreover, "Old and New World" consumers seem to value differently the wine’s characteristics. To our knowledge, no cross country analysis has been done dealing with "New World" wines in "Old World" countries, leaving an important gap in understanding underlying attributes influencing buying decisions.

Geochemistry of pore water and sediments offshore Nicaragua and Costa Rica

Four volcanic ash-bearing marine sediment cores and one ash-free reference core were examined during research cruise RV Meteor 54/2 offshore Nicaragua and Costa Rica to investigate the chemical composition of pore waters related to volcanic ash alteration. Sediments were composed of terrigenous matter derived from the adjacent continent and contained several distinct ash layers. Biogenic opal and carbonate were only minor components. The terrigenous fraction was mainly composed of smectite and other clay minerals while the pore water composition was strongly affected by the anaerobic degradation of particulate organic matter via microbial sulphate reduction. The alteration of volcanic matter showed only a minor effect on major element concentrations in pore waters. This is in contrast to prior studies based on long sediment cores taken during the DSDP, where deep sediments always showed distinct signs of volcanic ash alteration. The missing signal of ash alteration is probably caused by low reaction rates and the high background concentration of major dissolved ions in the seawater-derived pore fluids. Dissolved silica concentrations were, however, significantly enriched in ash-bearing cores and showed no relation to the low but variable contents of biogenic opal. Hence, the data suggest that silica concentrations were enhanced by ash dissolution. Thus, the dissolved silica profile measured in one of the sediment cores was used to derive the in-situ dissolution rate of volcanic glass particles in marine sediments. A non-steady state model was run over a period of 43 kyr applying a constant pH of 7.30 and a dissolved Al concentration of 0.05 ?M. The kinetic constant (AA) was varied systematically to fit the model to the measured dissolved silica-depth profile. The best fit to the data was obtained applying AA = 1.3 * 10**-U9 mol of Si/cm**2/ s. This in-situ rate of ash dissolution at the seafloor is three orders of magnitude smaller than the rate of ash dissolution determined in previous laboratory experiments. Our results therefore imply that field investigations are necessary to accurately predict natural dissolution rates of volcanic glasses in marine sediments.

Impact of changing carbonate chemistry, temperature, and salinity on growth and test degradation of the benthic foraminifer Ammonia aomoriensis

The present study investigated the combined effects of ocean acidification, temperature, and salinity on growth and test degradation of Ammonia aomoriensis. This species is one of the dominant benthic foraminifera in near-coastal habitats of the southwestern Baltic Sea that can be particularly sensitive to changes in seawater carbonate chemistry. To assess potential responses to ocean acidification and climate change, we performed a fully crossed experiment involving three temperatures (8, 13, and 18°C), three salinities (15, 20, and 25) and four pCO2 levels (566, 1195, 2108, and 3843 µatm) for six weeks. Our results highlight a sensitive response of A. aomoriensis to undersaturated seawater with respect to calcite. The specimens continued to grow and increase their test diameter in treatments with pCO2 <1200 µatm, when Omega calc >1. Growth rates declined when pCO2 exceeded 1200 µatm (Omega calc <1). A significant reduction in test diameter and number of tests due to dissolution was observed below a critical Omega calc of 0.5. Elevated temperature (18°C) led to increased Omega calc, larger test diameter, and lower test degradation. Maximal growth was observed at 18°C. No significant relationship was observed between salinity and test growth. Lowered and undersaturated Omega calc, which results from increasing pCO2 in bottom waters, may cause a significant future decline of the population density of A. aomoriensis in its natural environment. At the same time, this effect might be partially compensated by temperature rise due to global warming.

Assessment of species diversity and state of "Stipa tenacissima" steppes

North African steppes are subjected to extreme degradation resulting in the reduction of their surface, genetic erosion of resources, and decrease in biodiversity. "Stipa tenacissima" steppes, which constitute one of the most representative vegetation types in the driest areas of the Mediterranean basin, are continuously degrading. With the aim of contributing to a better knowledge of the floristic composition and diagnosing the state of degradation of these steppes, we conducted a phytoecological analysis of 10 "S. tenacissima" sites in Tunisia. Floristic inventory compiled a systematic list of 46 vascular plant species belonging to 43 genera and 26 families. Species richness ranged from 4 to 18 species per 900 m2. Total vegetation cover was moderate and fluctuated between 22.8% and 49.9%. Our results revealed also a decreasing trend in species richness with increasing elevation (ρ = –0.585). Indeed, species richness was negatively correlated with slope (ρ = –0.19) and positively correlated with sand content (ρ = 0.262). Biological types were dominated by chamaephytes; this chamaephytization is due to the phenomenon of aridization and overgrazing. Moreover, the low species cover and the appearance of nonpalatable species highlighted the vulnerability of these steppes to degradation.

Non contact inspection of the fatigue damage state of carbon fiber reinforced polymer by optical surface roughness measurements

This work presents the evaluation of a new non-contact technique to assess the fatigue damage state of CFRP structures by measuring surface roughness parameters. Surface roughness and stiffness degradation have been measured in CFRP coupons cycled with constant amplitude loads, and a Pearson?s correlation of 0.79 was obtained between both variables. Results suggest that changes on the surface roughness measured in strategic zones of components made of the evaluated CFRP, could be indicative of the level of damage due to fatigue loads. This methodology could be useful for other FRP due to similarities in the fatigue damage process.

Optical sensing of the fatigue damage state of CFRP under realistic aeronautical load sequences

We present an optical sensing methodology to estimate the fatigue damage state of structures made of carbon fiber reinforced polymer (CFRP), by measuring variations on the surface roughness. Variable amplitude loads (VAL), which represent realistic loads during aeronautical missions of fighter aircraft (FALSTAFF) have been applied to coupons until failure. Stiffness degradation and surface roughness variations have been measured during the life of the coupons obtaining a Pearson correlation of 0.75 between both variables. The data were compared with a previous study for Constant Amplitude Load (CAL) obtaining similar results. Conclusions suggest that the surface roughness measured in strategic zones is a useful technique for structural health monitoring of CFRP structures, and that it is independent of the type of load applied. Surface roughness can be measured in the field by optical techniques such as speckle, confocal perfilometers and interferometry, among others.

A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse–chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

Chaperone rings in protein folding and degradation

Chaperone rings play a vital role in the opposing ATP-mediated processes of folding and degradation of many cellular proteins, but the mechanisms by which they assist these life and death actions are only beginning to be understood. Ring structures present an advantage to both processes, providing for compartmentalization of the substrate protein inside a central cavity in which multivalent, potentially cooperative interactions can take place between the substrate and a high local concentration of binding sites, while access of other proteins to the cavity is restricted sterically. Such restriction prevents outside interference that could lead to nonproductive fates of the substrate protein while it is present in non-native form, such as aggregation. At the step of recognition, chaperone rings recognize different motifs in their substrates, exposed hydrophobicity in the case of protein-folding chaperonins, and specific “tag” sequences in at least some cases of the proteolytic chaperones. For both folding and proteolytic complexes, ATP directs conformational changes in the chaperone rings that govern release of the bound polypeptide. In the case of chaperonins, ATP enables a released protein to pursue the native state in a sequestered hydrophilic folding chamber, and, in the case of the proteases, the released polypeptide is translocated into a degradation chamber. These divergent fates are at least partly governed by very different cooperating components that associate with the chaperone rings: that is, cochaperonin rings on one hand and proteolytic ring assemblies on the other. Here we review the structures and mechanisms of the two types of chaperone ring system.

Becoming a State: Zionist and Palestinian Movements for National Liberation

This study examines the road to statehood for the Zionist and Palestinian movements. There are three components which frame this investigation: 1. social movements and the practices in which they engage that are aimed at establishing statehood for a people; 2. distinctive configurations of the international system and the manner in which both the material and ideational foundations of that system pulls units towards conformity and predictable behavior; and finally, 3. the role of agency, that is, the way in which instrumentally rational individuals attempt to push the structure in which they are embedded towards a configuration that is better suited to their interests and objectives The most influential factor guiding these struggles for national liberation are those forces which emanate from the prevailing structure of the international system. Not only was it demonstrated that the established material and ideational preferences of existing states have strong bearing on a movement’s ideological orientation and by consequence its chosen course of struggle, but hegemonic order configurations also define political cleavages and in so doing present movement leaders with both tactical and strategic opportunities by harnessing or exploiting those cleavages. From the agency perspective, the cases showed that the leadership of each movement was highly influential in the determination of a movement’s success or failure.

Proposal of a New Parameter for the Weathering Characterization of Carbonate Flysch-Like Rock Masses: The Potential Degradation Index (PDI)

The susceptibility of clay bearing rocks to weathering (erosion and/or differential degradation) is known to influence the stability of heterogeneous slopes. However, not all of these rocks show the same behaviour, as there are considerable differences in the speed and type of weathering observed. As such, it is very important to establish relationships between behaviour quantified in a laboratory environment with that observed in the field. The slake durability test is the laboratory test most commonly used to evaluate the relationship between slaking behaviour and rock durability. However, it has a number of disadvantages; it does not account for changes in shape and size in fragments retained in the 2 mm sieve, nor does its most commonly used index (Id2) accurately reflect weathering behaviour observed in the field. The main aim of this paper is to propose a simple methodology for characterizing the weathering behaviour of carbonate lithologies that outcrop in heterogeneous rock masses (such as Flysch slopes), for use by practitioners. To this end, the Potential Degradation Index (PDI) is proposed. This is calculated using the fragment size distribution curves taken from material retained in the drum after each cycle of the slake durability test. The number of slaking cycles has also been increased to five. Through laboratory testing of 117 samples of carbonate rocks, extracted from strata in selected slopes, 6 different rock types were established based on their slaking behaviour, and corresponding to the different weathering behaviours observed in the field.

Knowledge-Bearing Elites and Industrial Performance in France and Germany. CES Germany & Europe Working Papers, No. 04.5, December 1993

Most comparative studies of public policies for competitiveness focus on the links among public agencies and industrial sectors. This paper argues that the professions---or knowledge-bearing elites-that animate these organizational links are equally significant. For public policies to promote technological advance, the visions and self-images of knowledge-bearing elites are par­ ticularly important. By examining administrative and technical elites in France and Germany in the 1980s, the paper identifies characteristics that enable these elites to implement policy in some cases, but not in others. France's "state-created" elites were well-positioned to initiate and implement large technology projects, such as digitizing the telecommunications network. Germany's state-recognized elites were, by contrast, better positioned to facilitate framework­ oriented programs that aimed at the diffusion of new technologies throughout industry. The linkages among administrative and technical elites also explain why French policymakers had difficulty adapting policy to changing circumstances over time while German policymakers managed in many cases to learn more from previous policy experiences and to adapt subsequent initiatives accordingly.

Transactions of the Medical Society of the State of New York.

Lists of members in each volume; some of these are merely partial lists, members in attendance at the annual meetings, etc.

[Documents bearing on the suit brought by the Commonwealth of Massachusetts in the Supreme Court of the U.S. to test the constitutionality of the Sheppard-Towner Act involving the principle of federal aid legislation.]

Mode of access: Internet.