Academic literature on the topic 'Salt Screen'

The objective was to evaluate the initial growth and gas exchange of yellow passion fruit seedlings irrigated with saline water under different environments. The experiment was conducted of the University of International Integration of Afro-Brazilian Lusophony, Redenção-CE. The experimental design used was entirely randomized, in factor arrangement 4 x 2, referring to four environments with different shade screens (black screen; whitescreen; red screen; all with 50% shading; and full sun) and the values of electrical conductivity of irrigationwater (0.3 and 3.0 dS m-1), with five repetitions. The following variables were evaluated: number of leaves, plant height, leaf area, photosynthesis, transpiration, stomach conductance, instantaneous water use efficiency, electrical conductivity of the saturation extract and soil pH. The environment with black screen provides greater performance in leaf area, plant height and root length and was more efficient for the production of dry mass of the aerial part and the root of passion fruit seedlings in both irrigation waters. The red screen environment stimulates greater photosynthesis, transpiration, stomach conductance and chlorophyll in passion fruit seedlings irrigated with low salinity water, while the black screen mitigated saline stress for these variables. The full sun environment provided greater instantaneous water use efficiency in passion fruit seedlings for treatment with low salinity water and the black screen environment with high salinity water.

Cholesterol-lowering strains were obtained by high throughput screening technology and ortho-phthalaldehyde method. We used oxford cup method to screen again to obtain strains of high yield bile salt hydrolase and illuminate action mechanism ofLactobacillusreducing cholesterol. Screened six strains had the ability of high yield bile salt hydrolase and good ferment ability. The results of identifying bacteria species: strain KTxKL1J1 wereLactobacillus casei, strain Tx wasStreptococcus thermophilus, strain KS4P1 wereLactococcus lactis subsp.lactis, where the last two bacteria were strain of high yield bile salt hydrolase to be few known in literature. This work showed that dissociation bile salts and cholesterol conjuncted sediments by bile salt hydrolase decomposing conjugated bile salts.

Most commercially available crystallization screens are sparse-matrix screens with a predominance of inorganic salts and polyethylene glycols (PEGs) as precipitants. It was noted that commercially available screens are largely unsatisfactory for the purpose of the crystallization of multimeric protein and protein–nucleic acid complexes. This was reasoned to be a consequence of the redundancy in screening crystallization parameter space by the predominance of PEG as a precipitant in standard screens and it was suggested that this limitation could be overcome by introducing a variety of other organic polymers. Here, a set of 288 crystallization conditions was devised based on alternative polymeric precipitants and tested against a set of 20 different proteins/complexes; finally, a screen comprising the 96 most promising conditions designed to complement PEG- and salt-based commercial screens was proposed.

Soil salinity is a major abiotic stress in Australian lentil-producing areas. It is therefore imperative to identify genetic variation for salt tolerance in order to develop lentil varieties suitable for saline soils. Conventional screening methods include the manual assessment of stress symptoms, which can be very laborious, time-consuming, and error-prone. Recent advances in image-based high-throughput phenotyping (HTP) technologies have provided unparalleled opportunities to screen plants for a range of stresses, such as salt toxicity. The current study describes the development and application of an HTP method for salt toxicity screening in lentils. In a pilot study, six lentil genotypes were evaluated to determine the optimal salt level and the growth stage for distinguishing lentil genotypes using red–green–blue (RGB) images on a LemnaTec Scanalyzer 3D phenomics platform. The optimized protocol was then applied to screen 276 accessions that were also assessed earlier in a conventional phenotypic screen. Detailed phenotypic trait assessments, including plant growth and green/non-green color pixels, were made and correlated to the conventional screen (r = 0.55; p < 0.0001). These findings demonstrated the improved efficacy of an image-based phenotyping approach that is high-throughput, efficient, and better suited to modern breeding programs.

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of themecA gene for the detection of methicillin resistance inStaphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.

We illustrate the performance of the generalized screen propagator on real seismic data for 3D zero‐offset and prestack depth imaging. We use TotalFinaElf's L7D data set from the North Sea, a 3D marine seismic survey that contained limited azimuthal coverage. The subsurface shows significant tectonic deformation, including an intrusive salt body in sedimentary sequences. A transformation to common azimuth is applied prior to the 3D prestack depth imaging procedure. We compare the performance of the generalized screen propagator with that of a hybrid phase shift plus interpolation (PSPI)/split‐step Fourier method. Three‐dimensional prestack results confirm the generalized screen method handles multipathing more accurately. Comparisons are also made with Kirchhoff migration results. The results differ mainly in the fine‐scale irregularities of the image and not in the wavefront set of the image. Using synthetic models of similar structure (the SEG/EAGE salt model), we further illustrate the importance of multipathing and multiple scattering. Overall, our results show that our wave‐equation approach produces better images than the Kirchhoff approach to prestack depth migration; we attribute this mainly to a more complete handling of wave diffraction in the generalized screen expansion, which becomes important in strongly heterogeneous and irregular velocity models such as the ones containing salt bodies.

In the context of seaside landscaping, one of the greatest challenges for practitioners and scientists is to select suitable species that are able to tolerate salt spray. This is the key aspect for a wide number of potentially suitable species. The objectives of this study were (1) to identify plant traits associated with species tolerance to salt stress and (2) to evaluate the responses of different shrub species to salt spray. For this purpose, a study was conducted to determine the effects of salt spray on twenty-four ornamental shrubs using rapid and easy-to-use methodology. The species were subjected twice a week to nebulization treatment with simulated seawater solution for 60 days. Every 20 days, net photosynthesis, stomatal conductance, transpiration rate, and chlorophyll a fluorescence were determined. Furthermore, dry biomass of the different organographic portions, leaf number, leaf area, Specific Leaf Area, chlorophyll content, and leaf damage were determined at the end of the experiment. The species exposed to seawater solution showed different physiological and morphological responses. Based on the above indices, these ornamental shrubs were screened and categorized as tolerant, moderately tolerant, or susceptible. The results suggest that Convolvulus, Ceratonia, and Ligustrum are more tolerant to salt spray than numerous other genotypes; L. langmaniae, Cascabela, and L. frutescens, conversely, are more sensitive. Among the plant traits, the morphological parameters thoroughly characterized the effects of the salt spray, but they were destructive, with the only exception being the leaf damage percentage. This last non-destructive parameter is interesting considering the aesthetic value that ornamental plants must have. The physiological parameters, and in particular photosynthesis activity, can instead be used as a non-destructive screening method to select species suitable for ornamental green spaces near the sea.

Underground salt caverns are widely used in large-scale energy storage, such as natural gas, compressed air, oil, and hydrogen. In order to quickly build large-scale natural gas reserves, an unusual building method was established. The method involves using the existing salt caverns left over from solution mining of salt to build energy storages. In 2007, it was first applied to Jintan Natural Gas Storage of China. Based on this successful project, several existing salt caverns were screened to build energy storages in China. Engineering experience indicates that the key to successful reusing is how to select the most suitable of the numerous available caverns and confirm it. This paper summarizes and reviews relevant theories and testing methods, including: (1) the primary selection principle for using existing salt caverns to build energy storage, (2) the testing method and evaluation theory of tightness of the existing salt cavern, and (3) the typical project case of using the existing salt caverns to build energy storage in China. From the practical application results, the selection principle proposed in this paper can quickly screen available existing salt caverns with energy storage potential, and the brine injection method can effectively evaluate their tightness. It provides a technical roadmap for the subsequent implementation of existing salt cavern utilization projects on a large scale.

Regelbunden fysisk aktivitet motverkar en rad fysiska och mentala sjukdomar. Hälsovinster av fysisk aktivitet är bland annat en högre funktionell muskulär- och kardiovaskulär kapacitet samt en högre livskvalitet. Stillasittande och inaktivitet kan leda till övervikt, kardiovaskulära sjukdomar, cancer, psykosociala problem och metaboliska sjukdomar. Mellan män och kvinnor finns fysiologiska skillnader som visar sig i kroppsstorlek och muskelmassa. Detta ger generellt sett kvinnor mer flexibel fysik medan män är fysiskt starkare. WHOs rekommendationer om daglig fysisk aktivitet är 150 min/vecka av moderat aerobisk träning eller 75 min mer ansträngande aerobisk träning. Styrketräning som involverar större muskelgrupper rekommenderas i åldrarna 18-64.   Till hjälp att undersöka hypotesen har två oberoende test används. En skriftlig enkät, SF-36v2 Health Survey, samt ett fysiskt test, Functional Movement Screen. I denna studie undersöks huruvida det finns ett samband mellan en ökad träningsmängd och ett högre FMS-resultat samt om en högre ålder ger lägre FMS-resultat, oavsett kön. Vi tror oss även se ett samband mellan högre FMS-poäng och ett högre uppskattat mentalt och fysiskt mående. Testpersonerna (N:30) som deltog i studien var arbetande eller studerande män (N:15) och kvinnor (N:15) i åldrarna 20-65år. FMS-resultaten visade ingen signifikant skillnad mellan kön eller stigande ålder. Däremot ser vi signifikanta skillnader mellan könen vid specifika styrke- eller rörlighetstester i FMS. Kvinnorna visar tydligt via resultaten att de generellt har en mer flexibel fysik medan männen är starkare. Som slutsats av denna studie kan vi konstatera att FMS som testmetod är könsneutralt och kan användas på blandade populationer. Detta ger testmetoden en bred användbarhet på just en blandad population. Fler studier krävs för att få fram normerande poängsättning om FMS skall användas på medelmotionären.

OBJECTIVES: Study the interdependence between stimulus duration and stimulus strength, to observe if by changing the stimulus duration, the current threshold levels will also change during pedicle screw stimulation. Then comparing the pedicle screw threshold data to 3-Dimensional imaging to confirm the effectiveness of pedicle screw stimulation. BACKGROUND: This study examined the stimulus parameters of triggered electromyography in the operating room during posterior spinal fusions with pedicle screw fixation, and how changing stimulus duration would affect the threshold value of a pedicle screw. Triggered Electromyography (t-EMG) or pedicle screw stimulation has been used for years as Gold Standard to test whether pedicle screws are properly placed or well insulated by bone in the pedicle of the vertebrae. In a triggered EMG test, a stimulus is sent to the pedicle screw, when the electrical stimulus activates nervous tissue, a compound muscle action potential is elicited, at this point a current threshold value is recorded. This threshold is compared to normative values, which determine if the screw is located intrapedicular or has breached the pedicle wall. A breach in the pedicle wall by a pedicle screw would cause a post-operative neurological deficit, such as nerve irritation. To examine the effects duration has on threshold values, the same screw was stimulated three different times with three different durations. The threshold values were statistically evaluated to see if there was a significance between each stimulus duration and its corresponding threshold value. Duration was examined because it is often overlooked as a parameter that may change the threshold value of a triggered EMG test. Having discrepancies due to duration can produce inaccurate results which could potentially harm the patient or change the surgical protocol. It may harm the patient by leaving a screw in place that has breached the pedicle wall of a vertebrae, which will most likely cause post-operative nerve irritation. Pedicle screw stimulation is an additional modality used to verify screw placement along with radiographic imaging in the operating theatre when posterior spinal fusions are taking place. METHODS: The current technique of pedicle screw stimulation using threshold numbers (in milliamperes) was evaluated against different stimulation parameters, and later the position of the screw was visually verified by a neurosurgeon with 3-dimensional imaging. Fundamentally, the imaging was used to validate the effectiveness that pedicle screw stimulation has on determining a well-positioned screw. Patients already scheduled to undergo spinal fusions with pedicle screw fixation were eligible for this study. A 3-Dimensional (3D) image of their spine was taken intraoperatively before insertion of spinal fixation instrumentation and one was taken after pedicle screws were inserted. These images were used to evaluate the position of the pedicle screw by a neurosurgeon. After screws were positioned, triggered EMG was utilized to check whether screws were properly placed. Screw measurements were taken from patients undergoing a posterior spinal fusion in either the thoracic, lumbar or sacral region. 213 screw measurements were taken in total from 40 patients. Factors like sex, age, height and weight were not considered for this study. The triggered EMG test threshold was then compared to the post screw insertion 3D image to verify the accuracy of the triggered EMG. In other words, the image was used to justify whether the triggered EMG test alone was an accurate indicator of a properly placed screw. In addition, the triggered EMG test itself was further evaluated, by stimulating the same screw three times, with three different stimulus durations, 300µsec, 200µsec, and 100µsec. The values produced by each stimulus duration were then compared to the corresponding stimulus threshold to see if there was a significant difference. Triggered EMG stimulus durations were assessed to see if stimulus parameter settings play a role in the threshold number. A change in stimulus duration, could change the triggered EMG threshold number, which when compared to normative data could possibly indicate a properly placed screw from a mal-positioned screw with a difference in threshold of as little as 1 milliampere. From all the stimulus parameters, a focus was put on the stimulus duration because this could directly affect the triggered EMG current threshold number, or the current value where a compound muscle action potential is elicited. Which depending on where the threshold value fell against pre-determined normative values, could directly affect whether a screw is interpreted as well-placed or not. To summarize, each pedicle screw was tested three times, at three different stimulus durations, then the corresponding threshold numbers were compared to currently established normative data thresholds and evaluated to see if duration could affect the results of a pedicle screw’s position. Remember, thresholds have been established that deem a properly placed screw, these have been used throughout the years, but no emphasis has been given to the stimulus parameters set for these thresholds. This study evaluated the importance of setting the proper stimulus parameters, mainly the stimulus duration, when using certain normative thresholds tested at specific durations to deem a properly positioned screw in the pedicle of a vertebrae. After stimulation, 3D imaging was taken intraoperatively to compare the triggered electromyography data to the actual placement of the screws. The threshold values were compared to the 3 D image of the same screws tested, to verify if indeed these established threshold values determined well-positioned screws. RESULTS: 213 screws were stimulated, out of the 213 screws, 2 screw measurements were excluded because stimulations were not obtained from all three durations. Thus 211 screws and 40 patients were included in this study. 211 screw measurements were taken in total from 40 patients with screws confirmed to be placed intrapedicular. The triggered EMG fell within normative data thresholds for 206 screws (98%), these screws were found to be intrapedicularly placed in the 3D imaging, which was confirmed by the neurosurgeon. Stimulation durations were found to be important in five of the screws, where there was a difference in the thresholds between the three stimulations, these were significant because the values were lying borderline on normative values, questioning if the screws were indeed well-placed or possibly causing a medial breach. These five screws were thoroughly examined by the neurosurgeon via 3-dimensional imaging and were found to be acceptable in placement, and not near nervous tissue. CONCLUSION: Intraoperative 3D imaging has shown that triggered electromyography is a reliable indicator of properly placed pedicle screws. Statistical data has also shown that stimulus duration can affect the interpretation of a properly placed screw, and threshold values do vary with different durations. Threshold values were gathered from 211 screws at three different stimulus durations, the first at 300µsec, the second at 200µsec and the third at 100µsec. At 300µsec stimulus duration, the mean threshold value was at 27.25mA (p=0.0078). At 200µsec stimulus duration, the mean threshold value was at 35.46mA (p=0.0028). At 100µsec stimulus duration, the mean threshold value was at 50.90mA (p=0.0676). These mean values were found to be statistically significant when run by the Kruskal-Wallis test, a non-parametric statistical significance test. Since, three groups of data were being compared, and thus were not normally distributed, a non-parametric significance test was used. In conclusion, the stimulus duration should be considered when using certain thresholds to interpret data. Different durations change the stimulus strength and thus, affect the results of the screw stimulation thresholds.

La adolescencia representa un período crítico durante el cual ocurren cambios físicos y psicológicos importantes. Durante este período, los problemas relacionados con la alimentación y el peso (PRAP) como la insatisfacción corporal (IC), el sedentarismo y la preocupación por el peso se vuelven predominantes. Además, durante la adolescencia se establecen elecciones y patrones de comportamiento, incluida la opción de ser físicamente activo. El objetivo de esta tesis es investigar la interrelación de la actividad física (AF) con IC, tiempo de pantalla y estado de peso en una muestra de adolescentes españoles. El Estudio1 investigó la prevalencia de AF en una muestra española y de manera longitudinal, cambios (incrementos/decrementos) en diferentes intensidades de AF (moderada-vigorosa vs. leve) a lo largo de la adolescencia temprana. En línea con las tendencias mundiales, Estudio1 mostró que la prevalencia de AF en una muestra de adolescentes españoles es baja. También se encontró que no hubo grandes caídas en los niveles de AF; de hecho, hubo pequeños incrementos para algunos grupos. Además, el Estudio1 se centró en una pregunta menos investigada: si hay cambios en la cantidad de AF realizada durante la adolescencia, ¿estos cambios son diferentes en los diferentes tipos de intensidades? Al considerar la AF como una medida global, no siempre se observaron cambios a lo largo del tiempo, pero se hicieron evidentes cuando discriminamos entre dos niveles de intensidad: actividad física moderada-a-vigorosa (AFMV) vs. actividad física leve (AFL). Específicamente, hubo incrementos significativos en AFL en chicos y en AFMV en chicas. Finalmente, al investigar potenciales cambios en las intensidades de AF considerando distintos estatus de peso, se observaron incrementos en el AFL en chicos con sobrepeso/obesidad y aumentos en AFMV y AF global en chicas con sobrepeso/obesas. Esta información es valiosa para futuras intervenciones. En particular, para comprender qué tipo de intensidades deben ser más específicas y/o cuáles pueden ser más fáciles de invitar a los adolescentes a practicar. Estudio2 es un análisis transversal que investigó si los altos niveles de IC pueden ser una barrera para participar en AFMV. Además, tuvo como objetivo investigar el impacto de tiempo de pantalla (TV /ordenador) sobre IC. El Estudio2 mostró dentro de una amplia muestra de adolescentes españoles que la IC puede funcionar como una barrera y no como un motivador para realizar AF. Es importante destacar que se encontró que el uso de ordenadores durante el tiempo de ocio se asoció negativamente con la IC de las chicas. Los hallazgos del presente estudio tienen implicancias para el desarrollo de programas destinados a prevenir el amplio espectro de PRAP con un enfoque en mejorar la satisfacción corporal y la AF simultáneamente, así como el consumo crítico de mensajes entregados a través de nuevas tecnologías. Conclusiones La presente tesis contribuye al análisis de temas que son relevantes para las intervenciones de salud pública ya que estos problemas tienen importantes consecuencias físicas y psicológicas. Además, considera una muestra de adolescentes no-anglosajona, lo que contribuye a la comprensión de este fenómeno en una población menos estudiada. Los hallazgos resaltan que sólo un porcentaje muy pequeño de jóvenes adolescentes cumple con la recomendación de la AF y que se necesitan intervenciones para aumentar su participación. Los resultados de esta tesis sugieren que intervenciones de intensidades específicas de AF, para diferentes grupos en función de su género y estatus de peso, pueden ser beneficiosas. Asimismo, a partir de esta tesis se desprende que las intervenciones relacionadas con el peso deberían mejorar la imagen corporal y la AF simultáneamente, mientras que el consumo crítico de medios masivos debería incluir un componente de redes sociales y nuevas tecnologías.
Adolescence represents a critical period of development during which important physical and psychological changes occur. During this period the eating and weight related problems, such as body dissatisfaction (BD), sedentarism and weight-concern also become quite prevalent. Also, during adolescence personal lifestyle choices and behavior patterns establish, including the choice to be physically active. The aim of this thesis is to investigate the interrelationship of physical activity (PA) in early adolescence with BD, screen-time and weight status in an under- researched sample of Spanish adolescents. Study1 investigated PA prevalence in a Spanish sample, and longitudinally investigated changes (drops/ increments) in different intensities of PA (moderate-vigorous vs. light) throughout early adolescence. In line with worldwide trends, Study1 showed that PA prevalence in a sample of Spanish adolescents is low. It was also found that there were no big drops in PA levels; in fact, there were slight increments for some groups. Furthermore, Study1 focused in a less researched question: if there are changes in the amount of PA performed during adolescence, are these changes different across different types of intensities? When considering PA as a global measure, changes overtime were not always observed, but they became clear when we discriminated between two levels of intensity: moderate-to-vigorous physical activity (MVPA) vs. light physical activity (LPA). Specifically, there were significant increments in LPA for boys, and in MVPA for girls. Finally, investigating potential changes overtime in PA intensities across different weight-statuses, increments in LPA in overweight/obese boys and increments in MVPA and global PA in overweight/obese girls were observed. This information is valuable for future interventions. In particular for understanding which kind of intensities need to be targeted more heavily and/or which ones may be easier to invite adolescents to practice. Study2 is a cross-sectional analysis that investigated whether high levels of BD may be a barrier to engage in MVPA. In addition, Study2 aimed to add to the existing literature on the impact of TV and magazines on BD, by investigating the statistical association of computer time exposure and BD. Study2 showed within a large sample of Spanish adolescents that BD can work as a barrier and not as a motivator to PA engagement. Importantly, it was found that the use of computers during leisure time was negatively associated with girls’ body image. Findings of the present study along with previous research findings have implications for the development of programs aimed at preventing the broad spectrum of weight related disorders with a focus on improving body satisfaction and physical activity simultaneously, as well as the critical consumption of messages delivered via new technologies. Conclusions The present thesis contributes to the analysis of issues that are relevant for public health interventions as these issues have important physical and psychological consequences. Moreover, it considers a sample of Spanish adolescents, which contributes to the understanding of this phenomena in the less studied non-Anglo-Saxon population. The findings of this thesis are an addition to the literature and highlight that only a very small percentage of young adolescents meet the PA recommendation and that interventions to increase participation are needed. In line with this, the results of this thesis suggest that interventions targeting specific intensities of PA for different groups based on their gender and weight status, may be beneficial. Likewise, from this thesis it was observed that BD can work as a barrier to PA adherence, and that therefore, weight-related interventions should improve body image and PA simultaneously, while critical consumption of mass-media interventions should include both an internet technology and a social network component.

Intresset för flervåningshus i trä ökar stadigt. Trä är ett lätt och mjukt material vilket är problematiskt med avseende på stomstabilisering och horisontella deformationer. Syftet har varit att undersöka förbandstyper för KL-trä och huvudfrågan var hur stommens horisontella deformationer påverkas av infästningstyperna och deras styvhet. En jämförande studie av självborrande skruvar och beräkning av deras förskjutningsmodul har utförts. En modell på 22 våningar med en stomme av KL-trä modellerades i RFEM. Analyser genomfördes för olika värden på förskjutningsmodul för att kunna jämföra de horisontella deformationerna. Resultatet visade på att deformationerna blev små och att skillnaden mellan olika förskjutningsmoduler gav en knappt märkbar skillnad i horisontell deformation, trots att variationen av värdet på förskjutningsmodulerna var stor. Detta kan förklaras av att modellen i sig är väldigt stabil, vilket främst skulle kunna bero på fasadelementens vertikala förskjutning och höjd. Andra bidragande orsaker var att modellen saknade urtag för dörrar och fönster, KL-elementens tjocklek samt att pågjutning av betong applicerades på varje våning.
The interest for multi storey buildings with timber is growing. Timber is a material that is light and ductile, characteristics that make timber a challenging material in terms of horizontal stabilization and horizontal displacements. The purpose was to examine connectors for CLT and the main question was how the horizontal displacement of the frame is affected by the connectors and their stiffness. A parametric study for self-tapping screws and calculation of their slip modulus was carried out. A model of a 22-storey building with a CLT frame was modelled in RFEM. Analyses were run for different values of the slip modulus for comparison of the horizontal displacements connected to each slip modulus. The results show that the deformations were small and that the differences in slip modulus only caused small differences in horizontal deformations even though the variation in slip modulus was wide. The reason for this could be due to the modelling; the model in itself is very stiff, possibly as a result of the façade panels’ vertical adjustment and height. Other factors contributing were the lack of openings for doors and windows in the model, the thickness of the CLT panels as well as the concrete decks on all floors.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm derived from mature B cells characterized by the presence of the t(11;14)(q13;q32) translocation that leads to the overexpression of Cyclin D1. Cyclin D1 plays a well-established role in G1/S progression, although other functions including transcription or DNA damage response (DDR) can be regulated by this cyclin. Therefore, the main goal of this thesis is the characterization of the cyclin D1 non-canonical function in MCL and lymphoid cells. Firstly, we analyzed the genomic binding of endogenous cyclin D1 in four MCL cell lines, showing widespread occupancy around the transcription start site of active promoters. Overexpressed cyclin D1 in lymphoblastic cell lines causes a global transcription downmodulation, while cyclin D1 silencing increased the RNA content. In addition, higher levels of cyclin D1 correlated with lower transcription outputs in MCL and multiple myeloma cell lines. We also found that cyclin D1 colocalized with Pol II, but its overexpression increased the RNA polymerase II pausing and decreased elongation. Cyclin D1 overexpressing cells showed higher sensitivity to transcription inhibitors, revealing a synthetic lethality interaction. As expected, MCL and multiple myeloma cell lines displaying higher levels of cyclin D1 were more sensitive to the effects of the drug. Next, we studied the capacity of cyclin D1 to induce DNA replication stress in lymphoblastic cell lines. Cyclin D1 overexpression caused increased cell proliferation, especially the mutant form cyclin D1 T286A that codifies for a more stable protein. However, cyclin D1 overexpressing cells displayed a slower progression through S-phase. The analysis of DNA fibers showed that cyclin D1 overexpression caused DNA replication stress. Indeed, constitutive overexpression of cyclin D1 led to basal DDR activation, which was detected by the induction of γH2AX and pCHK2 phosphoproteins. These results led us to wonder if MCL can display a high, constitutive hyperactivation of DDR in primary samples. Concordantly, 66% showed positive γH2AX staining and 33% of all cases showed a concomitant pCHK2expression. The activation of DDR correlated to worse survival, more chromosome abnormalities, higher proliferation and genetic alterations of genes involved in the DDR. Finally, we aimed to study the potential contribution of altered DNA methylation in the development and/or progression of MCL. To do so, we performed genome-wide methylation profiling of a large cohort of primary MCL tumors and normal lymphoid tissue samples. Primary MCL displayed a heterogeneous methylation pattern dominated by DNA hypomethylation when compared to controls. Annotation analysis of hypermethylated promoters recognized pathways related to cell proliferation. The promoters of WNT pathway inhibitors and several other tumor suppressor genes were shown frequently methylated. A substantial fraction of the genes with promoter hypermethylation showed a significant downregulation of their transcription levels. Furthermore, we identified a subset of tumors with extensive CpG methylation that had an increased proliferation signature, higher number of chromosomal alterations and poor prognosis. Our results suggest that a subset of highly proliferative MCL cases displays a dysregulation of DNA methylation characterized by the accumulation of CpG hypermethylation that may influence the clinical behavior of the tumors. Overall, we have characterized for the first time the new functions that cyclin D1 performs in transcription and replication stress in MCL. The elucidation of these mechanisms may be useful not only for a better understanding of the tumor, but also for improving diagnostic and treatment of MCL patients. A subset of aggressive cases displayed dysregulated DDR and higher methylation levels and were associated with higher proliferation. Our findings suggest that cyclin D1, in addition to its canonical role in cell cycle regulation, plays other functions that may be important for MCL lymphomagenesis.
Les neoplàsies limfoides són un grup heterogeni de tumors que, en molts casos, es caracteritzen per un esdeveniment genètic inicial i l'acumulació de canvis moleculars secundaris que condicionen la progressió tumoral. Freqüentment, les alteracions genètiques primàries són translocacions cromosòmiques que provoquen la sobreexpressió aberrant d'un oncogen. Aquest primer esdeveniment oncogènic altera la proliferació, l'apoptosi o la diferenciació normal linfoide que determinen de forma essencial la biologia del tumor. El limfoma de cèl·lules del mantell (LCM) és un subtipus de neoplàsia limfoide madura amb un curs clínic en general poc favorable i baixa supervivència. Aquest limfoma es caracteritza genèticament per la translocació t(11; 14)(q13; q32) i la conseqüent sobreexpressió de ciclina D1. De fet, l'estudi immunohistoquímic de la cicina D1 s'ha convertit en una eina imprescindible per a realitzar el diagnòstic diferencial d'aquest limfoma, atès que l'expressió d'aquest oncogen en neoplàsies limfoides es limita a la pràctica totalitat de casos de LCM i un percentatge baix de casos de mieloma múltiple (MM) i la tricoleucèmia. S'han descrit dues variants citològiques principals de LCM: clàssica i blastoide. Les formes blastoides generalment presenten una major proliferació i cariotips més complexos. La identificació en els últims anys de casos de LCM que no mostren els criteris convencionals ha complicat la classificació dels LCM fent necessari un diagnòstic basat en criteris clínics, histomorfológics, citogenètics i moleculars. Dins d'aquest grup s'enquadra un conjunt de pacients amb LCM i que presenten un curs clínic indolent sense necessitat de tractament durant un temps relativament llarg. Aquesta variant específica de LCM s’anomena limfoma de célules del mantell leucèmic no-nodal. La identificació d'aquest grup de pacients és important perquè es podrien beneficiar d'aproximacions terapèutiques més conservadores sense que es produeixi un impacte negatiu en la seva supervivència global. A més de la t(11; 14)(q13; q32) com alteració oncogènica inicial, la majoria de casos presenten, en comparació amb altres limfomes, un nombre elevat d'alteracions cromosòmiques secundàries. Els gens dianes de moltes d'aquestes alteracions cromosòmiques s’han identificat i en molts casos corresponen a gens implicats en el control del cicle cel·lular i en els mecanismes de resposta i reparació de l'ADN. El gen que codifica per a la proteïna ciclina D1, CCND1, és un dels oncogens més freqüentment amplificats en tumors humans, especialment en tumors de mama o de vies respiratòries. A més, tumors hematològics tenen mecanismes genètics que també causen la sobreexpressió d'aquest oncogen, com la translocació t(11; 14) en MCL i MM. Així mateix, ciclina D1 es troba sobrexpresado en una gran varietat de càncers diferents, com fetge, colon, melanoma o pàncrees; mitjançant altres mecanismes no genètics. Funcionalment, ciclina D1 juga un paper essencial en la transició G1 / S en el cicle cel·lular. Ciclina D1 s'expressa com a resposta a estímuls mitogènics. La seva unió amb les quinases dependents de ciclina (CDK) 4 i 6 determina l'activació d'aquestes últimes, que s'encarreguen de la fosforilació de la proteïna RB. Quan RB no està fosforilat, s'uneix i inactiva els factors de transcripció (TF) de la família E2F. No obstant això, la fosforilació de RB canvia la conformació de la proteïna i permet que les proteïnes E2F activin la transcripció de gens de fase S i que se superi el punt de restricció en el cicle cel·lular. Per això es considera que la principal funció oncogènica de ciclina D1 estaria relacionada amb el seu paper en la progressió a la fase S, que determinaria un augment de la proliferació cel·lular. No obstant això, cada vegada són més les evidències que mostren que ciclina D1 pot estar participant en altres funcions, moltes d'elles independents a la seva unió amb CDK4 / 6. S'han trobat més de 30 proteïnes que interaccionen amb ciclina D1, regulant processos com la transcripció, la reparació del ADN, apoptosi, migració i metabolisme mitocondrial. No obstant això, molts dels estudis utilitzen la sobreexpressió de ciclina D1 exògena i / o han estat únicament validats en pocs models. A més, no està clar si aquestes funcions tenen lloc tant en cèl·lules neoplàsiques com teixit normals, o fins i tot si són pròpies de determinats teixits. L'objectiu principal d'aquesta tesi doctoral és la caracterització de funcions no canòniques que ciclina D1 pogués exercir durant la linfomagènesi del LCM. En aquest projecte de tesi ens hem centrat en l'estudi del possible paper de ciclina D1 com a regulador de la transcripció (Estudi 1) i de la capacitat d'induir estrès replicatiu i dany a l'ADN (Estudi 2). L'alteració d'aquests processos, juntament amb la d'altres mecanismes ja descrits en el LCM, poden ser tant causa com conseqüència de la desregulació epigenòmica en cèl·lules tumorals. Per tot això, també és un objectiu d'aquesta tesi la caracterització dels canvis de metilació en una cohort àmplia de casos de LCM (Estudi 3). En l'estudi 1 es va analitzar el patró d'unió que ciclina D1 mostra en quatre línies cel·lulars de LCM mitjançant tècniques d'immunoprecipitació de cromatina i seqüenciació (ChIP-Seq). Inesperadament, identificarem més de 40.000 regions genòmiques que van mostrar interacció amb ciclina D1 endògena. Aquestes regions d'interacció amb ciclina D1 van mostrar estar enriquides en seqüències promotores. També analitzem diverses marques d'histones i els llocs de sensibilitat a ADNsa I en els promotors units per ciclina D1. Amb aquestes anàlisis identifiquem que ciclina D1 s'uneix d'una manera global a tots els gens amb transcripció activa. Aquesta conclusió va ser corroborada amb dades de seqüenciació de RNA (RNA-Seq). De fet, unions més fortes de ciclina D1 amb un promotor correlacionaven amb nivells més alts de transcripció del gen. Aquest patró d'unió és molt similar al que fa uns anys s'havia descrit per l'oncogen MYC, que es va definir com un amplificador transcripcional. Es va observar que Myc també s'unia especialment a una gran quantitat de promotors, correlacionava amb els nivells d'expressió i amb marques epigenètiques d'activació. La sobreexpressió de MYC causava l'amplificació del contingut global de RNA, de manera que nosaltres decidirem quantificar l'efecte de ciclina D1 sobre els nivells de RNA total cel·lular. Sorprenentment, els nivells de RNA disminuïen en models linfoblàstics quan es sobreexpressava la forma normal de ciclina D1 o la variant amb la mutació T286A, que li atorga major estabilitat i provoca majors nivells de ciclina D1 nuclear. Aquest efecte sobre la transcripció ho confirmarem mitjançant el silenciament de ciclina D1 en línies de LCM, que va causar un augment en la quantitat total de RNA. A continuació, vam comprovar en línies de LCM i MM que la concentració de ciclina D1 correlacionava amb nivells més baixos de transcripció, indicant que la ciclina D1 endògena estaria comportant-se igual que l'exògena. Mitjançant la utilització de mètodes de quantificació digital confirmarem que la sobre expressió de ciclina D1 també determinava una disminució dels nivells de RNA missatger. El nostre següent objectiu es va centrar en determinar el mecanisme pel qual ciclina D1 exerceix el seu efecte sobre la transcripció. Per a això ens centrem en l'estudi de la maquinària transcripcional i la seva relació amb la sobreexpressió de ciclina D1. Mitjançant anàlisi de ChIP-Seq de la polimerasa II (Pol II) vam determinar que els nivells de ciclina D1 en els promotors correlacionava amb els de Pol II. De fet, varem trobar que la sobreexpressió de ciclina D1 incrementava la parada de la polimerasa, especialment en aquells gens que unien més quantitat de ciclina D1 al seu promotor. Aquesta parada correlacionava amb un canvi en el patró de fosforilació de la polimerasa II, observant una disminució significativa de la fosforilació Ser5 de Pol II. Aquesta fosforilació s’associava amb l'activació de l'elongació i en conseqüència observarem un augment de l'índex de parada de la Pol II. Com la fosforilació Ser5 és depenent de CDK9, vam decidir comprovar si l'efecte de ciclina podria ser a través la unió a CDK9. Després d'observar que ciclina D1 s'unia a aquesta proteïna, vam comprovar si la sobreexpressió de ciclina D1 en models linfoblàstics augmentava la sensibilitat al 5, 6-dicloro-1-beta-D-ribofuranosylbenzimidazole (DRB) , un inhibidor específic de CDK9. La nostra hipòtesi era que podria existir una letalitat sintètica en aquells casos amb més ciclina D1 i, per tant, menors nivells de transcripció. Conseqüentment, vam comprovar que línies amb majors nivells de transcripció i nivells inferiors de ciclina D1 eren menys sensibles a l'inhibidor. Atès que la inhibició de CDK9 s'aconsegueix a dosis molt altes de DRB i, per tant, l'inhabilita per a la seva administració terapéutica, utilitzarem una droga anomenada triptolide utilitzada en assaigs clínics i que produeix una inhibició de la transcripció. Com esperàvem, en línies cel·lulars de LCM i MM, la inhibició de la transcripció és un bon candidat per a desenvolupar noves estratègies terapèutiques contra tumors de baix potencial transcripcional / alta concentració de ciclina D1. L'objectiu de l'estudi 2 va ser estudiar in vitro l'efecte de ciclina D1 sobre la replicació en el limfoma de les cèl·lules del mantell. Ciclina D1 va demostrar ser capaç d'augmentar la proporció de cèl·lules en fase S, però inhbitint la seua progressió durant la fase S. Les cèl·lules amb sobreexpressió de ciclina D1 mostraven clars defectes durant la fase S. Primer, vam comprovar que la fase S era més lenta en aquelles cèl·lules ciclina D1 positives. A més, vam detectar per primera vegada que ciclina D1 estava causant problemes en la progressió de les forquilles de replicació en models de cèl·lula B. Entre aquests problemes destaquem la disminució de la velocitat de progressió de la forquilla de replicació, un increment del nombre de nous orígens activats, la reducció del percentatge de forquilles amb elongació activa i l'augment de forquilles bloquejades. A més, també vam detectar la presència d'una població de forquilles asimètriques en el cas de la sobreexpressió de ciclina D1. També hem observat que la sobreexpressió de ciclina D1 pot comprometre la recuperació de les cèl·lules a un estrès que generi una parada de les forquilles, per exemple després del tractament amb hydroxyurea. Les cèl·lules que sobrexpressaven ciclina D1 van mostrar major apoptosi que les ciclina D1 negatives. Tot això ens va fer concloure que els nivells de ciclina D1 causen estrès replicatiu en línies cel·lulars limfoblàstiques. Tots els resultats antreriors ens fan pensar que el paper de ciclina D1 en la linfomagènesi del LCM va més enllà que el seu efecte en l'increment de la proliferació. L'estrès replicatiu pot causar inestabilitat genòmica i activació dels mecanismes de resposta al dany al ADN, per tant vam estudiar els efectes després d'una setmana d'inducció de ciclina D1, observant que s'incrementava la quantitat de proteïna H2AX i CHK2 fosforilades, marcadors d'aquesta activació del dany causada per ciclina D1. A banda, ciclina D1 augmentava significativament la proporció de cel·lules tetraploids. A continuació, donat que el LCM es caracteritza per alts nivells de ciclin D1, volguerem analitzar si els casos primaris d’aquest càncer expressaven marcadors de dany a l'ADN i dels mecanismes de resposta. Estudiarem mitjançant immunohistoquímica l'expressió de les formes fosforilades de H2AX i CHK2 en mostres primàries de MCL.24/37 (64.9%) del casos tenien activació de H2AX, mentre 14/24 (58.3%) tenien activació concomitant de CHK2. Això ens permet distingir casos amb alta activació de la resposta a dany al ADN (ambdues proteïnes fosforilades) o amb baixa / nul·la activació de la resposta a dany al ADN. El grup amb major dany a l’ADN presentava més anormalitats cromosòmiques, menor supervivència i més alteracions en gens supressors de tumors com CDKN2A o TP53. Així mateix, aquests casos també eren els més proliferatius (major Ki67). La caracterització de les alteracions epigenómicas al LCM es va desenvolupar en profunditat en l'estudi 3. Es va realitzar un estudi de metilació amb la plataforma HumanMethylation27 BeadChip de 132 casos primaris de MCL i 6 línies cel·lulars. En aquest estudi vam poder observar que el LCM és un limfoma molt heterogeni i que mostra un gran nombre d'anormalitats epigenètiques quan es compara amb un teixit normal. Curiosament, els fenòmens de hipermetilació i hipometilació de novo van mostrar diferents comportament. La hipometilació es concentrava en regions intergèniques, mentre la hipermetilació apareixia freqüentment associada a promotors. De fet, es van identificar un total de 454 gens amb promotors hipermetilats i 875 gens amb promotors hipometilats en almenys el 10% dels casos. En el nostre estudi vam observar que la hipermetilació s'associava amb una reducció de l'expressió, afectant freqüentment a gens supressors de tumors. Així, els promotors hipermetilats corresponien a gens que regulaven processos com la proliferació cel·lular i altres vies de senyalització com la via de WNT, de la qual molts dels seus inhibidors estaven de novo hipermetilats. Aquest fenomen suggeriria la inactivació oncogènica mitjançant hipermetilació de gens supressors de tumors en casos primaris de LCM. L'anàlisi dels fenòmens d’hipermetilació ens va permetre caracteritzar un subgrup de casos que presentaven un major nombre de ecanvis epigenètics, un major percentatge d'alteracions genètiques i una menor supervivència global. Especialment destacable és el fet que aquest grup de casos mostraven també una elevada signatura de proliferació. Consistent amb aquests resultats, el gen supressor de tumors CDKN2A es trobava hipermetilat i inactivat en un gran nombre de casos. Globalment, els nostres resultats suggereixen que la desregulació de l'epigenoma al LCM pot ser una conseqüència d’una proliferació descontrolada, en part per la sobreexpressió de ciclina D1, i l'adquisició de determinades epimutacions que poden participar en la progressió tumoral. Les conclusions d'aquest projecte de tesi doctoral han estat: 1) Ciclina D1 mostra un patró global d'unió al la cromatina, unint-se preferentment a promotors de gens actius i que correlaciona de manera significativa amb el nivell de transcripció del gen. 2) Ciclina D1 es comporta com un regulador negatiu global de la transcripció tant en cèl·lules de MCL com en models linfoblàstoids. 3) La sobreexpressió de ciclina D1 incrementa la parada de la Pol II al promotor i dificulta l'elongació, probablement a través de la seva unió inactivant amb CDK9. 4) Els inhibidors de la transcripció induixen apoptosis en línies cel·lulars de MCL, MM i en models linfoblàstoids de sobreexpressió de ciclina D1, suggerint que aquesta letalitat sintètica pot representar una nova estratègia terapèutica per al tractament de limfomes agressius amb nivells alts de ciclina D1. 5) Ciclina D1 augmenta la proliferació cel·lular i l'entrada a fase S quan és expressada en línies limfoblàstiques. 6) La inducció de ciclina D1 provoca un augment significatiu moderat en la proporció de cèl·lules tetraploides en línies limfoblàstiques. 7) Les cèl·lules que sobrexpressen ciclina D1 presenten defectes en fase S i signes d'estrès replicatiu, manifestant parades de les forquilles de la replicació, activació de nous orígens, ralentiment de la replicació i requereixen de més temps per completar la replicació del ADN. En conseqüència, la sobreexpressió de ciclina D1 dificulta la recuperació cel·lular després d'un estrès replicatiu. 8) Temps llargs d'inducció de ciclina D1 activen els mecanismes de resposta al dany a l'ADN, fosforilant les proteïnes CHK2 i H2AX en cèl·lules limfoides. 9) El limfoma de les cèl·lules del mantell és un càncer caracteritzat per alts nivells d'activació de marcadors de resposta al dany a l'ADN, com γH2AX i pCHK2. 10) Els casos de MCL amb alts nivells d'activació de la resposta al dany a l’ ADN tenen pitjors taxes de supervivència i s'associen amb una major inactivació de gens supressors de tumors, amb morfologies més agressives i amb un índex de proliferació més gran. 11) L'anàlisi de la metilació de casos primaris de MCL indica que la hipermetilació es dirigeix essencialment a silenciar els promotors de gens supressors de tumors relacionats amb proliferació, per exemple les inhbidors de la via de WNT. 12) Els casos de MCL que tenen majors nivells de CpGs hipermetiladas s'associen amb un pitjor pronòstic, major nombre d'anormalitats cromosòmiques i major proliferació.
Las neoplasias linfoides son un grupo heterogéneo de tumores que, en muchos casos, se caracterizan por un evento genético inicial y la acumulación de cambios moleculares secundarios que condicionan la progresión tumoral. Frecuentemente, las alteraciones genéticas primarias son translocaciones cromosómicas que provocan la sobrexpresión aberrante de un oncogen. Este primer evento oncogénico altera la proliferación, la apoptosis o la diferenciación normal linfoide que determinan de forma esencial la biología del tumor. El linfoma de células del manto (LCM) es un subtipo de neoplasia linfoide madura con un curso clínico en general poco favorable y baja supervivencia. Este linfoma se caracteriza genéticamente por la translocación t(11;14)(q13;q32) y la consecuente sobreexpresión de ciclina D1. De hecho, el estudio inmunohistoquímico de la cicina D1 se ha convertido en una herramienta imprescindible para realizar el diagnóstico diferencial de este linfoma, dado que la expresión de este oncogen en neoplasias linfoides se limita a la práctica totalidad de casos de LCM y un porcentaje bajo de casos de mieloma múltiple (MM) y tricoleucemia. Se han descrito dos variantes citológicas principales de LCM: clásica y blastoide. Las formas blastoides generalmente presentan una mayor proliferación y cariotipos más complejos. La identificación en los últimos años de casos de LCM que no muestran los criterios convencionales ha complicado la clasificación de los LCM haciendo necesario un diagnostico basado en criterios clínicos, histomorfológicos, citogenéticos y moleculares. Dentro de este grupo se encuadra un conjunto de pacientes con LCM y que presentan un curso clínico indolente sin necesidad de tratamiento durante un tiempo relativamente largo. Esta variedad específica de LCM se llama linfoma de las células del manto leucémico no-nodal. La identificación de estos pacientes es importante porque se podrían beneficiar de aproximaciones terapéuticas más conservadoras sin que se produzca un impacto negativo en su supervivencia global. Además de la t(11;14)(q13;q32) como alteración oncogénica inicial, la mayoría de casos presentan, en comparación con otros linfomas, un número elevado de alteraciones cromosómicas secundarias. Se han identificado los genes diana de muchas de estas alteraciones cromosómicas y en muchos casos corresponden a genes implicados en el control del ciclo celular y en los mecanismos de respuesta y reparación del ADN. El gen que codifica para la proteína ciclina D1, CCND1, es uno de los oncogenes más frecuentemente amplificado en tumores humanos, especialmente en tumores de mama o de vías respiratorias. Además, tumores hematológicos tienen mecanismos genéticos que también causan la sobrexpresión de este oncogen, como la translocación t(11;14) en MCL y MM. Asimismo, ciclina D1 se encuentra sobrexpresado en una gran variedad de canceres diferentes, como hígado, colón, melanoma, páncreas mediante otros mecanismos no genéticos. Funcionalmente, ciclina D1 juega un papel esencial en la transición G1/S en el ciclo celular. Ciclina D1 se expresa como respuesta a estímulos mitogénicos. Su unión con las quinasas dependientes de ciclina (CDK) 4 y 6 determina la activación de estas últimas, que se encargan de la fosforilación de la proteina RB. Cuando RB no está fosforilado, se une e inactiva los factores de transcripción (TF) de la familia E2F. Sin embargo, la fosforilacion de RB cambia la conformación de la proteína y permite que los factores E2F activen la transcripción de genes de fase S y que se supere el punto de restricción en el ciclo celular. Por ello se considera que la principal función oncogénica de ciclina D1 estaría relacionada con su papel en la progresión a la fase S, que determinaría un aumento de la proliferación celular. Sin embargo, cada vez son más las evidencias que muestran que ciclina D1 puede estar participando en otras funciones, muchas de ellas independientes a su unión con CDK4/6. Se han encontrado más de 30 proteínas que interaccionan con ciclina D1, regulando procesos como la transcripción, la reparación del ADN, apoptosis, migración y metabolismo mitocondrial. Sin embargo, muchos de los estudios utilizan la sobrexpresión de ciclina D1 exógena y/o han sido únicamente validados en pocos modelos. Además, no está claro si estas funciones tienen lugar tanto en células neoplásicas como en tejido normales, o incluso si son específicas de determinados tejidos. El objetivo principal de esta tesis doctoral es la caracterización de funciones no canónicas que ciclina D1 pudiera ejercer durante la linfomagénesis del LCM. En este proyecto de tesis nos hemos centrado en el estudio del posible papel de ciclina D1 como regulador de la transcripción (Estudio 1) y de la capacidad de inducir estrés replicativo y daño al ADN (Estudio 2). La alteración de estos procesos, junto con la de otros mecanismos ya descritos en el LCM, pueden ser tanto causa como consecuencia de la desregulación epigenómica en células tumorales. Por todo ello, también es un objetivo de esta tesis la caracterización de los cambios en metilación en una cohorte amplia de casos de LCM (Estudio 3). En el estudio 1 se analizó el patrón de unión al ADN que ciclina D1 muestra en cuatro líneas celulares de LCM mediante técnicas de inmunoprecipitación de cromatina y secuenciación (ChIP-Seq). Inesperadamente, identificamos más de 40.000 regiones genómicas que mostraron interacción con ciclina D1 endógena. Estas regiones de unión a ciclina D1 mostraron estar enriquecidas en secuencias promotoras. También analizamos varias marcas de histonas y los sitios de sensibilidad a ADNsa I en los promotores unidos por ciclina D1. Con estos análisis identificamos que ciclina D1 se unía de una manera global a todos los genes con transcripción activa. Esta conclusión fue corroborada con datos de secuenciación de RNA (RNA-Seq). De hecho, uniones más fuertes de ciclina D1 con un promotor correlacionaban con mayores niveles de transcripción del gen. Este patrón de unión es muy similar al que hace unos años se había descrito para el oncogen MYC, que se definió como un amplificador transcripcional. Se observó que Myc también se unía especialmente a una gran cantidad de promotores, correlacionaba con los niveles de expresión y con marcas epigenéticas de activación. La sobrexpresión de MYC causaba la amplificación del contenido global de RNA, por lo que nosotros decidimos cuantificar el efecto de ciclina D1 sobre la cantidad de RNA total celular. Sorprendentemente, los niveles de RNA disminuían en modelos linfoblastoides cuando se sobreexpresaba la forma normal de ciclina D1 o la variante con la mutación T286A, que le otorga mayor estabilidad y provoca mayores niveles de ciclina D1 nuclear. Este efecto sobre la transcripción lo confirmamos mediante el silenciamiento de ciclina D1 en líneas de LCM que causó un aumento en la cantidad total de RNA. A continuación, comprobamos en líneas de LCM y MM que los niveles de ciclina D1 correlacionaban con niveles más bajos de transcripción, indicando que la ciclina D1 endógena estaría comportándose igual que la exógena. Mediante la utilización de métodos de cuantificación digital confirmamos que la sobrrexpresión de ciclina D1 también determinaba una disminución de los niveles de RNA mensajero. Nuestro siguiente objetivo se dirigió a determinar el mecanismo por el que ciclina D1 ejerce su efecto sobre la transcripción. Para ello nos centramos en el estudio de la maquinaria transcripcional y su relación con la sobreexpresión de ciclina D1. Mediante análisis de ChIP-Seq de la polimerasa II (Pol II) determinamos que los niveles de ciclina D1 en los promotores correlacionaba con los niveles de Pol II. De hecho, nuestro estudió encontró que la sobrexpresión de ciclina D1 incrementaba la parada de la polimerasa, especialmente en aquellos genes que unían más cantidad de ciclina D1 a su promotor. Esta parada correlacionaba con un cambio en el patrón de fosforilación de la polimerasa II, caracterizado por una disminución significativa de la fosforilación Ser5 de Pol II. Esta fosforilación se correlaciona con la activación de la elongación y en consecuencia observamos un aumento del índice de parada de la Pol II. Como la fosforilación Ser5 es dependiente de CDK9, decidimos comprobar si el efecto de ciclina podría ser a través la unión a CDK9. Tras observar que ciclina D1 se unía a esta proteína, comprobamos si la sobrexpresión de ciclina D1 en modelos linfoblástoides aumentaba la sensibilidad al 5, 6-dicloro-1-beta-D-ribofuranosylbenzimidazole (DRB) , un inhibidor específico de CDK9. Nuestra hipótesis era que podría existir una letalidad sintética en aquellos casos con más ciclina D1 y, por tanto, menores niveles de transcripción. Consecuentemente, comprobamos que líneas con mayores niveles de transcripción y niveles inferiores de ciclina D1 eran menos sensibles al inhibidor. Dado que la inhibición de CDK9 se consigue a dosis muy altas de DRB, lo que lo inhabilita para su administración terapéutica, utilizamos una droga llamada triptolide, utilizada en ensayos clínicos y que también produce una inhibición de la transcripción. Como esperábamos, en líneas celulares de LCM y MM, la inhibición de la transcripción es un buen candidato para desarrollar nuevas estrategias terapéuticas contra tumores de bajo potencial transcripcional/ alta concentración de ciclina D1. El objetivo del estudio 2 fue estudiar in vitro los efectos de ciclina D1 sobre la replicación en el linfoma de las células del manto. Ciclina D1 demostró ser capaz de aumentar la proporción de células en fase S promoviendo la progresión a fase S. Sin embargo, las células con sobrexpresión de ciclina D1 mostraban claros defectos durante la fase S. Primero, comprobamos que la fase S era más lenta en aquellas células ciclina D1 positivas. Además, detectamos por primera vez que ciclina D1 estaba causando problemas en la progresión de las horquillas de replicación en modelos de célula B. Entre estos problemas destacamos la disminución de la velocidad de progresión de la horquilla de replicación, un incremento del número de nuevos orígenes activados, la reducción del porcentaje de horquillas con elongación activa y el aumento de horquillas bloqueadas. Además, también detectamos la presencia de una población de horquillas asimétricas en el caso de la sobrexpresión de ciclina D1. También hemos observado que la sobreexpresión de ciclina D1 puede comprometer la la recuperación de las células a un stress que genere una parada de las horquillas, por ejemplo tras el tratamiento con hydroxyurea. Las células que sobrexpresaban ciclina D1 mostraron mayor apoptosis que las ciclina D1 negativas. Todo esto nos hizo concluir que los niveles de ciclina D1 causan estrés replicativo en líneas celulares linfoblásticas. Estos resultados nos inducen a pensar que el papel de ciclina D1 en la linfomagénesis del LCM va más allá que su efecto en el incremento de la proliferación. El estrés replicativo puede causar inestabilidad genómica y activación de los mecanismos de respuesta al daño al ADN, por tanto estudiamos los efectos tras una semana de inducción de ciclina D1, observando que se incrementaba la cantidad de proteína H2AX y CHK2 fosfosriladas, marcadores de esta activación del daño causada por ciclina D1. Además, ciclina D1 aumentaba de forma significativa la proporción de células tetraploides. A continuación, dado que el LCM se caracteriza por altos niveles de ciclina D1, quisimos analizar si los casos primarios de este cáncer expresaban marcadores de daño al ADN y de los mecanismos de respuesta. Estudiamos mediante inmunohistoquímica la expresión de las formas fosforiladas de H2AX y CHK2 en muestras primarias de MCL.24/37 (64.9%) de los casos tenían activación de H2AX, mientras 14/24 (58.3%) tenían activación concomitante de CHK2. Esto nos permite distinguir tumores con alta activación de la respuesta a daño al ADN (ambas proteínas fosforiladas) o con baja/nula activación de la respuesta a daño al ADN. El grupo con mayor daño al ADN presentaba más anormalidades cromosómicas, menor supervivencia y más alteraciones en genes supresores de tumores como CDKN2A o TP53. Asimismo, estos casos también eran los más proliferativos (mayor Ki67). La caracterización de las alteraciones epigenómicas en el LCM se desarrolló en profundidad en el estudio 3. Se realizó un estudio de metilación con la plataforma HumanMethylation27 BeadChip de 132 casos primarios de MCL y 6 líneas celulares. En este estudio pudimos observar que el LCM es un linfoma muy heterogéneo y que muestra un gran número de anormalidades epigenéticas cuando se compara con un tejido normal. Curiosamente, los fenómenos de hipermetilación e hipometilación de novo mostraron diferente comportamiento. Por ejemplo, la hipometilación se concentraba en regiones intergénicas, mientras la hipermetilación de novo aparecía frecuentemente asociada a promotores. De hecho, se identificaron un total de 454 genes con promotores hipermetilados y 875 genes con promotores hipometilados en en al menos el 10% de los casos. En nuestro estudio observamos que la hipermetilacion se asociaba con una reducción de la expresión, afectando frecuentemente a genes supresores de tumores. Así, los promotores hipermetilados correspondían a genes que regulaban procesos como la proliferación celular y otras vías de señalización como la vía de WNT, de la que muchos de sus inhibidores estaban metilados. Este fenómeno sugeriría una inactivación oncogénica mediante hipermetilación de genes supresores de tumores en casos primarios de LCM. El análisis de los fenómenos de hipermetilación en LCM nos permitió caracterizar un subgrupo de casos que presentaban un mayor número de epimutaciones, un mayor porcentaje de alteraciones genéticas y una menor supervivencia global. Especialmente destacable es el hecho de que este grupo de casos mostraban también una elevada firma de proliferación. Consistente con estos resultados, el gen supresor de tumores CDKN2A se encontraba hipermetilado e inactivado en un gran número de casos. Globalmente, nuestros resultados sugieren que la desregulación del epigenoma en el LCM puede ser una consecuencia de la proliferación descontrolada mediada en parte por la sobrexpresión de ciclina D1 además de la adquisición de determinadas epimutaciones que pueden participar en la progresión tumoral. Las conclusiones de este proyecto de tesis doctoral han sido: 1) Ciclina D1 muestra un patrón global de unión a la cromatina, uniéndose preferentemente a promotores de genes activos que correlacionan de forma significativa con el nivel transcripción del gene. 2) Ciclina D1 se comporta como un regulador negativo global de la transcripción tanto en células de MCL como en modelos linfoblastoides. 3) La sobrexpresión de ciclina D1 incrementa la parada de la Pol II en el promotor y dificulta la elongación, probablemente a través de su unión inactivante con CDK9. 4) Los inhibidores de la transcripción provocan una gran respuesta apoptótica en líneas celulares de MCL, MM y en modelos linfoblastoides de sobrexpresión de ciclina D1, sugiriendo que esta letalidad sintética puede representar una nueva estrategia terapéutica para el tratamiento de linfomas agresivos con niveles altos de ciclina D1. 5) Ciclina D1 aumenta la proliferación celular y la entrada a fase S cuando es expresada en líneas linfoblásticas. 6) La inducción de ciclina D1 provoca un incremento moderado significativo en la proporción de células tetraploides en línias linfoblásticas. 7) Las células que sobrexpresan ciclina D1 presentan defectos en fase S y signos de estrés replicativo, manifestando paradas de las horquillas de la replicación, activación de nuevos orígenes, ralentización de la replicación y requieren más tiempo para completar la replicación del ADN. En consecuencia, la sobrexpresión de ciclina D1 dificulta la recuperación celular tras un estrés replicativo. 8) Tiempos largos de inducción de la ciclina D1 activan los mecanismos de respuesta al daño al ADN, fosforilando las proteínas CHK2 y H2AX en células linfoides. 9) El linfoma de las células del manto es un cáncer caracterizado por altos niveles de activación de marcadores de respuesta al daño al ADN, como γH2AX y PCHK2. 10) Los casos de MCL con altos niveles de activación de la respuesta al daño al ADN tienen peores tasas de supervivencia y se asocian con una mayor inactivación de genes supresores de tumores, con morfologías más agresivas y con un índice de proliferación mayor. 11) El análisis de la metilación de casos primarios de MCL indica que la hipermetilación se dirige esencialmente a silenciar los promotores de genes supresores de tumores relacionados con proliferación, por ejemplo la vía de WNT. 12) Los casos de MCL que tienen mayores niveles de CpGs hipermetiladas se asocian con un peor pronóstico, mayor número de anormalidades cromosómicas y mayor proliferación

Capteur électrochimique basé sur des matériaux carbonés fonctionnalisés, pour la détection de deux micropolluants faisant partie des substances prioritaires de la Directive Cadre européenne sur l’Eau (DCE2000/60/CE) : le plomb et l’anthracène. Les capteurs électrochimiques permettent d’atteindre des limites de détection et une sélectivité adéquates pour l’analyse de micropolluants dont les concentrations sont de l’ordredu μg/L, et sont simples d’utilisation pour des analyses in situ à moindre coût comparés aux appareils d’analyses conventionnels. Leur robustesse est un paramètre important afin de permettre des mesures en continu ou semi-continu dans les eaux. Cette thèse propose tout d’abord le développement du capteur pour la détection du plomb. La formulation d’une encre conductrice de carbone est étudiée pour la sérigraphie de ’électrode réceptrice, permettant ainsi de contrôler la composition de l’encre et d’étudier l’influence de la phase carbonée sur les propriétés électrocatalytiques des électrodes. La fonctionnalisation des électrodes par greffage électrochimique d’un sel de diazonium est également étudiée afin de maîtriser la sensibilité et la reproductibilité des électrodes greffées, en contrôlant l’épaisseur et la qualité des couches. Avec cet objectif, la fonctionnalisation dans un liquide ionique protique qui permet le contrôle de la monocouche en modulant la viscosité de ce milieu a été étudiée. Les électrodes greffées montrent des performances analytiques améliorées notamment en termes de répétabilité et de reproductibilité. Enfin ce travail de thèse porte également sur le développement du capteur pour la détection électrochimique de l’anthracène, molécule sans fonctions chimiques. Les électrodes sont, dans ce cas, fonctionnalisées par un polymère à empreinte moléculaire, matériau connu pour sa très grande sélectivité. Les performances de ce capteur, dont la sélectivité est basée uniquement sur le facteur de forme de la molécule, pour la détection de l’anthracène sont alors mises en évidence
Electrochemical sensor based on functionalized carbon materials, for the detection of two micropollutants, lead and anthracene, which are among of the priority substances of the European Framework Directive on Water(DCE 2000/60 / EC). Electrochemical sensors allow to achieve detection limits and selectivities for the analysis of micropollutants whose concentrations are of the order of μg/L. They are easy to use for in situ analyzes at lower costs compared to those of the conventional analysis equipment. Their robustness is an important parameter in order to allow continuous or semi-continuous measurements in water. First of all, this thesis proposes the development of a sensor for lead detection. The conductive carbon ink formulation is studied for the screen-printing of the receiving electrode, thus allowing to control the ink composition and to study the influence of the carbon phase on the electrocatalytic properties of electrodes. Functionalization of electrodes by electrochemical grafting of a diazonium salt is also studied in order to control the sensitivity and reproducibility of grafted electrodes, by controlling the thickness and the quality of the layers. With this aim it has been studied the functionalization in a protic ionic liquid in order to allow the control of the monolayer bymodulating the viscosity of this medium. The grafted electrodes show improved analytical performance especially in terms of repeatability and reproducibility. Finally, this work reports the development of a sensor for the electrochemical detection of anthracene, a molecule without chemical functions. In this case, a molecularly imprinted polymer, a material known for its very high selectivity, functionalizes the electrodes.Having a selectivity is only based on the form factor of the molecule, the performance of the sensor developed for the detection of anthracene is also highlighted

Объектами исследования служили фармацевтическая субстанция «Аргамид» и готовая лекарственная форма 1% крем «Аргамид» на основе серебряной соли сульфадимидина (Приоритетная справка на выдачу патента № 2015153574 от 14.12.2015). Данная соль и лекарственные средства на ее основе были разработаны на базе кафедры фармации УГМУ с целью обеспечения российского фармацевтического рынка эффективными антибактериальными и ранозаживляющими лекарственными препаратами отечественного производства согласно стратегии развития фармацевтической промышленности «ФАРМА-2020». Цель работы: разработка и валидация методик количественного определения серебра в исследуемых лекарственных средствах. Анализ литературных данных показал, что оптимальным методом определения серебра является метод осадительного титрования с потенциометрической индикацией конечной точки титрования. При этом, учитывая содержание серебра в анализируемых лекарственных средствах, для его определения в субстанции был выбран метод роданометрического, а в готовой лекарственной форме – йодометрического титрования. Для анализируемых лекарственных средств были подобраны оптимальные условия пробоподготовки: масса навески, концентрация растворителя; время и температура обработки пробы. В качестве индикаторного вместо дорогостоящего импортного серебряного дискового электрода использовали отечественный толстопленочный серебросодержащий электрод. Положительные результаты валидационной оценки разработанных методик, проведенной согласно требованиям Государственной Фармакопеи РФ XIII изд., позволяют считать их приемлемыми и рекомендовать для включения в проекты фармакопейных статей на исследуемые лекарственные средства. Контроль содержания серебра по разработанным методикам не требует дорогостоящего оборудования, привлечения высококвалифицированного персонала, отличается относительно коротким временем анализа, а, следовательно, может применяться испытательными лабораториями предприятия в процессе производства и центрами контроля качества во время обращения на фармацевтическом рынке исследуемых лекарственных средств.
The objects of study were pharmaceutical substance "Argamid" and finished dosage form of 1% cream "Argamid" based on silver salt sulfadimidine (Priority reference patent number 2015153574 from 14.12.2015). This salt and drugs based on it have been developed at the Department of Pharmacy UGMU to ensure that the Russian pharmaceutical market effective antibacterial and wound healing medicines domestic production according to the strategy of development of the pharmaceutical industry "Pharma 2020". Objective: Development and validation of methods of quantitative determination of silver in the investigational medicinal product. Analysis of published data shows that the best method of determining silver is the method of precipitation titration with potentiometric indication end point. However, given the content of silver in the analyzed medicaments for identification substance rodanometricheskogo method was selected, and in the final dosage form - iodometric titration. optimal conditions for sample preparation were selected to be analyzed drugs: sample weight, the concentration of solvent; Sample processing time and temperature. As an indicator instead of expensive imported silver disk electrode used domestic thick-film silver-electrode. Positive results validation assessment developed techniques conducted in accordance with the requirements of the RF State Pharmacopoeia XIII ed., Allow us to consider them acceptable and recommended for inclusion in the draft articles on the pharmacopoeia investigational medicinal products. Control of the silver content by developed techniques does not require expensive equipment, highly skilled personnel, has a relatively short analysis time, and, therefore, can be applied testing laboratories of the enterprise in the production process and quality control center during handling in the pharmaceutical market of investigational medicinal products.

Salinity is a major obstacle to global grain crop production, especially rice and wheat. The identification and improvement of salt-tolerant rice and wheat depending upon the genetic diversity and salt stress response could be a promising solution to deal with soil salinity and the increasing food demands. Plant responses to salt stress occur at the organismic, cellular, and molecular levels and the salt stress tolerance in those crop plant involving (1) regulation of ionic homeostasis, (2) maintenance of osmotic potential, (3) ROS scavenging and antioxidant enzymes activity, and (4) plant hormonal regulation. In this chapter, we summarize the recent research progress on these four aspects of plant morpho-physiological and molecular response, with particular attention to ionic, osmolytic, enzymatic, hormonal and gene expression regulation in rice and wheat plants. Moreover, epigenetic diversity could emerge as novel of phenotypic variations to enhance plant adaptation to an adverse environmental conditions and develop stable stress-resilient crops. The information summarized here will be useful for accelerating the breeding of salt-tolerant rice. This information may help in studies to reveal the mechanism of plant salt tolerance, screen high efficiency and quality salt tolerance in crops.

(−)-Lasonolide A 4, isolated from the Caribbean sponge Forcepia sp., showed remarkable anticancer activity in the NIH 60-cell screen. The central step in the syn­thesis of 4 reported (J. Am. Chem. Soc. 2014, 136, 88) by Barry M. Trost of Stanford University was the remarkably selective, convergent Ru-mediated coupling of 1 with 2 to give 3. To prepare 1, the authors took advantage of the underutilized Cu-mediated addi­tion of a Grignard reagent 6 to propargyl alcohol 5, to give 7. Coupling with the ace­tonide 8 followed by deprotection led to the phosphonium salt 9. The other half of 1 was prepared from the acetonide 10 of the commodity chemical 1,1,1-tris(hydroxymethyl)ethane. Oxidation followed by Zn-catalyzed aldol addition of the ketone 11 led to the alcohol 12. Diastereoselective reduction followed by protection gave 13. Condensation with benzaldehyde proceeded with remarkable diastereoselection, setting the quaternary center of 14. Spontaneous intramolecular Michael addition proceeded under the conditions of the subse­quent Horner-Emmons reaction, leading to the aldehyde 15. Wittig reaction with the phosphonium salt 9 followed by deprotection completed the preparation of the alkyne 1. The β-ketoester 18 prepared by the addition of 17 to 16 was prone to unwanted conjugation, and the terminal alkene was easily reduced under hydrogenation con­ditions. Enzymatic conditions were found to effect dynamic kinetic resolution and reduction, to give 19. The derived ketone 21, from coupling with 20 was reduced using the Corey organocatalyst, then hydrosilated, leading to 22. Under metathesis with 23, the product unsaturated aldehyde cyclized to 24. Homologation followed by allylation then completed the construction of 2. Acetone was the solvent of choice for the coupling of 1 with 2. This led to the acetonide 3, that was hydrolyzed and protected to give 25. Yamaguchi macrolac­tonization followed by deprotection then delivered (−)-lasonolide A 4. It is instruc­tive to compare this work to the four previous total syntheses of 4, one of which (Org. Highlights November 26, 2007) we have previously highlighted.

This chapter analyzes the circumstances of Levine’s sale of his company, Embassy, to the Avco Corporation in 1968. Because of various interrelated factors, corporations were looking to take over film companies in the mid- to late 1960s, and Levine stood to benefit greatly. Embassy was privately owned, had a huge library of films that were attractive to television, was not burdened by real estate or equipment, and had just had a huge success with The Graduate. Levine turned all of these positive points to his advantage and sold Embassy to Avco for an astonishing $40 million in the bubble created by the corporate feeding frenzy. This chapter also investigates two of Levine’s productions from this time, The Producers and The Lion in Winter, both of which stand as examples of Levine’s continued willingness to take risks.

A Small man sat in a small parlour, partitioned off from a small shop by a small screen, pasted all over with small scraps of newspapers. In company with the small man, was almost any amount of small children you may please to...

MSR (Molten salt reactor) is a reactor with the fission material dissolved in the fluoride salt as the fuel, which continuously circulates through the primary loop. The liquid fuel makes the online refueling and reprocessing be possible, consequently, more fuel cycle options would be presented due to the introduction of various refueling fuel type and reprocessing mode (continuous / batch reprocessing) comparing with that of the solid fuel reactor systems. It is important to evaluate all the possible fuel cycle options and screen out the promising ones to narrow the R&D activities of TMSR (Thorium-based Molten-Salt Reactor nuclear energy system) program. In this study, we firstly established a screening and decision-making framework, then conducted an initial evaluation work and identified the potential promising fuel cycle options. Synthesizing the goal of TMSR program and technology readiness, we proposed a “Three-steps” fuel cycle development strategy with the aim for gradually increasing the thorium resource utilization while considering the challenges of the reprocessing technology.

Electronic paper devices also known as electrochromic devices are known for their ability to change color with option to remain in certain color state even after the electrical power has been lifted. Traditional devices such as electrochromic windows used mechanisms like electrophoresis or intercalation and use special materials such as water-less electrolyte, and transparent conductive materials (TCM). As such, these devices can sometimes be relatively difficult to assemble, especially in developing countries, where such materials are harder to come by. Recently, certain improvements have been made by using alternative electrode positioning, that they do not require TCM. Presented here is a novel type of electronic paper that is recyclable, can be constructed as a monochrome or multi-color device and can be scaled accordingly to desired screen size. Presented electronic paper, is a semi-open electrochemical device, which uses pH indicator dyes and water-salt solution, combined with easily obtained sheet steel.

Fresh pig heart tissues were homogenized and deli-pidized with cold acetone. The dry acetone powder was extracted with 0.45 M potassium acetate pH 4.5, the extract was fractionated with ammonium sulfate to 60% saturation, successively followed by four chromatography steps: chromatography on CM-Sepharose CL-6B; gel filtration on Sephadex G-100; Fibrin-Sepharose affinity chromatography and chromatography on DEAE-Sepharose CL-6B. The fibrin plate method was used for the tissue plasminogen activator (t-PA) activity determination. The porcine t-PA purified was proved to be homogeneous either by SDS gel electrophoresis or by high performance liguid chromatography with a molecular weight of 67,000. Using the human melanoma t-PA antigen and its rabbit antiserum, the double immunodiffusion and immu-noelectrophoretic tests demonstrated that the porcine t-PA possessed a cross-reaction with the human t-PA. During the purification procedure, it was found that the porcine t-PA was contaminated and reversibly bound with the cell fibronectin. The binding ability depends on salt concentration. They could be separated from each other on Sepharose G-100 gel filtration at high salt concentration, this reversible binding was further confirmed by mixing these two purified components monitored on high performance liquid chromatography. The fibronectin-bound t-PA retained its ability to activate plasminogen, indicating that these two proteins may exist in a form of complex in the in vivo tissue. Some monoclonal antibodies against the porcine t-PA were obtained, being used to screen what domain of the enzyme is responsible for binding the cell fibronectin.

Several methods are being attempted to improve the performance of PEM Fuel cell electrodes so that the cost of the overall system can be brought down. The performance can be improved if the utilization of the catalyst in the electrode increases. One of the early successful method was to add a proton conducting polymer, such as NafionR to the catalyst layer. However there is a limit to the amount of NafionR that can be added as too much NafionR affect the gas diffusion. The other method is to increase the surface area of the catalyst used which has also been adequately demonstrated. Alternative methods for providing increased proton conductivity and catalyst utilization are thus of great interest, and a number of them have been investigated in the literature. One method that is being extensively investigated is to introduce the catalyst onto the polymer electrolyte membrane followed by lamination with gas diffusion electrode. In the present work, we have carried out two methods i) screen print the catalyst ink on the NafionR membrane ii) catalyze the NafionR membranes by reducing a suitable platinum salt on the membrane. Standard gas diffusion electrodes were then laminated onto this membrane. The performances of Membrane Electrode Assemblies (MEAs) prepared by these routes have been compared with the commercially available Gore catalysed membrane. It was observed that catalysed NafionR membranes show a better performance compared to the catalyst ink screen printed on the NafionR membrane and commercial Gore membrane under identical operating conditions. However MEAs with Gore membrane give a better performance in the iR region compared to the other MEAs prepared using NafionR membrane. The lesser performance with Gore membrane is probably due to the limitations in the lamination method employed.

Parabolic trough and power tower technologies provide inherent advantage of thermal energy storage and high efficiency of the Concentrating Solar Power (CSP) systems for utility scale solar plants. High efficiency CSP power generation with minimal water use is one of the SunShot goals of the US Department of Energy. The specular reflectance efficiency of the solar mirrors plays a critical role in the efficiency of power generation. The optical surface of the mirrors and the receiver must be kept clean for efficient operation of the plant. Some environmental challenges in operating the large-scale CSP plants at high reflectance efficiency arise from high concentration of atmospheric dust, wind speed and variation of relative humidity (RH) over a wide range. Deposited dust and other contaminant particles, such as soot, salt, and organic particulate matters attenuate solar radiation by scattering and absorption. Adhesion of these particles on the mirror surface depends strongly by their composition and the moisture content in the atmosphere. Presence of soluble inorganic and organic salts cause corrosion of the mirror unless the contaminants are cleaned frequently. In this paper, we briefly review (1) source of atmospheric dust and mechanisms involved in degradation of mirrors caused by salt particles, (2) loss of specular reflection efficiency as a function of particle size distribution and composition, and (3) an emerging technology for removing dust layer by using thin transparent electrodynamic screen (EDS). Feasibility of integration of EDS on the front surface of the solar collectors has been established to provide active self-cleaning properties for parabolic trough and heliostat reflectors. Prototype EDS-integrated solar collectors including second-surface glass mirrors, metallized acrylic film mirrors, and dielectric mirrors, were produced and tested in an environmental test chambers simulating desert atmospheres. The test results show that frequent removal of dust layer can maintain the specular reflectivity of the mirrors above 90% under dust deposition at a rate ranging from 0 to 10 g/m2, with particle size varying from 1 to 50 μm in diameter. The energy required for removing the dust layer from the solar was less than 10 Wh/m2 per cleaning cycle. EDS based cleaning could therefore be automated and performed as frequently as needed to maintain reflection efficiency above 90% and thus reducing water usage for cleaning mirrors in the solar field. A comparative cost analysis was performed between EDS and deluge water based cleaning that shows the EDS method is commercially viable and would meet water conservation needs.

Abstract Renewable energy sources and related conversion technologies are considered as the main solution for resolving the current issues related to global warming and environmental protection. Salinity gradient energy (SGE) is a source of renewable energy which can be defined as the Gibbs Energy of mixing when two solutions with different salinities mix together. The difference in the salinity of salt solutions is the main driving force of energy production by the SGE conversion technologies. One of the main conversion technologies of SGE is reverse electrodialysis (RED). In this technology the gradient between the concentrated and diluted salt solutions, the ions with a negative charge (anion) and positive charge (cation) pass through selective ion exchange membranes known as anion exchange membrane and cation exchange membrane. The driving force for diffusion of the ions is a function of the concentration gradient. The chemical potential of the salt solution is a function of the concentration of the salt solution and plays an important role in the Gibbs energy of mixing. The chemical potential of the salt solution is a thermodynamic property which is a function of the concentration and activity coefficient of the salt solution. The activity coefficient of the salt solution is a unique parameter which depends on the ionic strength of the solution and the type of ions in the salt solution. The salts with higher activity coefficient have a higher potential to be used in the SGE conversion process due to higher released Gibbs Energy during the mixing process. In this paper the thermodynamic model presented by Bromley [1], is used to calculate activity coefficient of 20 salts at different concentrations (0.01–6 molal). Two dimensionless parameters, Φ and Ψ, are defined as the ratio of activity coefficient and concentration between the concentrated and diluted solutions in 6 and 0.5 molal respectively. Using the dimensionless parameters, the theoretical open circuit voltage (OCV) of salt solutions in a RED cell is calculated. The salts are screened and ranked based on the activity coefficients and the theoretical open circuit voltage (OCV). The best salts are selected for use in a RED cell based on the activity coefficients and theoretical OCV. These alts could have potential for developing SGE storage systems in combination with renewable energy devices.

Abstract Saih Rawl gas is located in the South Oman Salt Basin. There are two main formations targeted for gas production; Barik & Miqrat Formations. These formations are tight and exhibit low permeability. In order to enhance gas production, these formations have to be hydraulically stimulated. The main objectives of this paper is to demonstrate the petrophysical properties of the hydraulically fractured zones. Assess gas flow contribution thru the individual zones measured by production logging and comparing with the amount of proppant placed in the formation. In addition, the paper discusses reservoir properties and characteristics obtained from logging, post stimulation operations results and post stimulation gas production. The paper discusses 20 wells; 10 from the crest and 10 from the flank. The two formations Barik and Miqrat cover approximately 17 sub reservoir units. The total overall placement ratio is 95% and 78% for the crest and flank respectively with 156 hydraulic stimulation stages. It was observed that five sub reservoir units proved to be challenging to place the desired proppant. The maximum operating pressure is reached before achieving the desired proppant concentration leading to a screen out; concentrations of 2 – 3 pounds per gallon. Petrophysical evaluation of porosity and permeability cross plots showed a linear relationship in the wells in the crest. While there was no clear relationship was seen in the flank. Radioactive tracers used are to understand if there is any proppant propergation into the higher or lower zones. Not all the five challenging sub reservoir units showed propergation to other units. The wells located in the crest showed a better production rate as compared to the flank. The paper highlights the importance of the using petrophysical evaluation to optimize hydraulic fracturing design for successful operations.

Abstract Since the field test of polymer flooding technology was carried out in the Bohai Oilfield in 2003, problems such as plugging of polymer-response wells have become increasingly worse, and conventional acidizing and plugging removal measures have had poor results. Therefore, this paper carries out research to provide a basis for effective plug removal in oil wells, to improve the productivity of polymer flooding oil wells. In this paper, the component analysis of the plug samples from the benefit injection wells in the field was carried out. The clogging mechanism was studied through X-ray diffraction, scanning electron microscopy, energy spectrum analysis, infrared spectroscopy, and chromatography, as well as through dynamic simulation evaluation of plugs and dynamic displacement experiments of long cores. Through simulation experiments, the clogging mechanism is clarified and the blockage radius range was obtained by various methods such as inverse effect of comprehensive measures, well test interpretation, and empirical formula calculation. The analysis results of the plugs show that the inorganic components of the plugs are calcium and magnesium carbonate scales, clay minerals and iron salt precipitation, and the organic components are the micelles formed by the crosslinking of trivalent metal ions. The greater the concentration of polymer produced, the greater the strength of calcium-magnesium scale aggregates and aluminum-iron-colloid elastomer, the greater the degree and depth of reservoir pore throat clogging, the larger the screen clogging area, which will even block the inlet of electric submersible pump. This will result in poor acidizing plugging effect and rapid decline in oil well productivity. Through various methods such as the inverse effect of comprehensive measures, well test interpretation, and empirical formula calculation, the blockage radius of polymer flooding response wells is greater than 4 meters. Based on this understanding, in the five wells plug removal measures, the unblocking radius and unblocking chemical agent system were adjusted and optimized. On-site application effect tracking shows that the plug removal measures have achieved good oil incremental effects, and the measures are all effective. Through the classification and comparison of oil well productivity characteristics, formation water composition, output polymer properties and other characteristics, this paper established the identification mark of plugging of polymer-response wells. In addition, an analysis method for clogging was established to clarify the composition and formation mechanism of the clogging. Finally, the plug radius calculation method was established by means of backstepping the effects of plug removal measures and well test interpretation analysis.

T-2 toxin, a toxic product belongs to the trichothecene mycotoxins, attracts major interest because of its severe detrimental effects on the health of human and farm animals. The occurrence of trichothecenes contamination is global and they are very resistant to physical or chemical detoxification techniques. Trichothecenes are absorbed in the small intestine into the blood stream. The hypothesis of this project was to develop a protecting system using probiotic bacteria that will express trichothecene 3-O-acetyltransferase (Tri101) that convert T-2 to a less toxic intermediate to reduce ingested levels in-situ. The major obstacle that we had faced during the project is the absence of stable and efficient expression vectors in probiotics. Most of the project period was invested to screen and isolate strong promoter to express high amounts of the detoxify enzyme on one hand and to stabilize the expression vector on the other hand. In order to estimate the detoxification capacity of the isolated promoters we had developed two very sensitive bioassays.The first system was based on Saccharomyces cerevisiae cells expressing the green fluorescent protein (GFP). Human liver cells proliferation was used as the second bioassay system.Using both systems we were able to prove actual detoxification on living cells by probiotic bacteria expressing Tri101. The first step was the isolation of already discovered strong promoters from lactic acid bacteria, cloning them downstream the Tri101 gene and transformed vectors to E. coli, a lactic acid bacteria strain Lactococcuslactis MG1363, and a probiotic strain of Lactobacillus casei. All plasmid constructs transformed to L. casei were unstable. The promoter designated lacA found to be the most efficient in reducing T-2 from the growth media of E. coli and L. lactis. A prompter library was generated from L. casei in order to isolate authentic probiotic promoters. Seven promoters were isolated, cloned downstream Tri101, transformed to bacteria and their detoxification capability was compared. One of those prompters, designated P201 showed a relatively high efficiency in detoxification. Sequence analysis of the promoter region of P201 and another promoter, P41, revealed the consensus region recognized by the sigma factor. We further attempted to isolate an inducible, strong promoter by comparing the protein profiles of L. casei grown in the presence of 0.3% bile salt (mimicking intestine conditions). Six spots that were consistently overexpressed in the presence of bile salts were isolated and identified. Their promoter reigns are now under investigation and characterization.

The trichothecene deoxynivalenol (DON, vomitoxin), one of the most common mycotoxin contaminants of grains, is produced by members of the Fusarium genus. DON poses a health risk to consumers and impairs livestock performance because it causes feed refusal, nausea, vomiting, diarrhea, hemolytic effects and cellular injury. The occurrence of trichothecenes contamination is global and they are very resistant to physical or chemical detoxification techniques. Trichothecenes are absorbed in the small intestine into the blood stream. The overall objective of this project was to develop a protecting system using probiotic bacteria that will express trichothecene 3-O-acetyltransferase (Tri101) that convert T-2 to a less toxic intermediate to reduce ingested levels in-situ. The major obstacle that we had faced during the project is the absence of stable and efficient expression vectors in probiotics. Most of the project period was invested to screen and isolate strong promoter to express high amounts of the detoxify enzyme on one hand and to stabilize the expression vector on the other hand. In order to estimate the detoxification capacity of the isolated promoters we had developed two very sensitive bioassays.The first system was based on Saccharomyces cerevisiae cells expressing the green fluorescent protein (GFP). Human liver cells proliferation was used as the second bioassay system.Using both systems we were able to prove actual detoxification on living cells by probiotic bacteria expressing Tri101. The first step was the isolation of already discovered strong promoters from lactic acid bacteria, cloning them downstream the Tri101 gene and transformed vectors to E. coli, a lactic acid bacteria strain Lactococcuslactis MG1363, and a probiotic strain of Lactobacillus casei. All plasmid constructs transformed to L. casei were unstable. The promoter designated lacA found to be the most efficient in reducing T-2 from the growth media of E. coli and L. lactis. A prompter library was generated from L. casei in order to isolate authentic probiotic promoters. Seven promoters were isolated, cloned downstream Tri101, transformed to bacteria and their detoxification capability was compared. One of those prompters, designated P201 showed a relatively high efficiency in detoxification. Sequence analysis of the promoter region of P201 and another promoter, P41, revealed the consensus region recognized by the sigma factor. We further attempted to isolate an inducible, strong promoter by comparing the protein profiles of L. casei grown in the presence of 0.3% bile salt (mimicking intestine conditions). Six spots that were consistently overexpressed in the presence of bile salts were isolated and identified. Their promoter reigns are now under investigation and characterization.

The objectives that were outlined in our original proposal have largely been achieved or will be so by the end of the project in February 1995 with one exception; that of mapping cold tolerance loci based on the segregation of tolerance in the BC1 progeny population. Briefly, our goals were to 1) construct a densely populated linkage map of the citrus genome: 2) map loci important in cold and/or salt stress tolerance; and 3) characterize the expression of genes responsive to cold land salt stress. As can be seen by the preceding listing of accomplishments, our original objectives A and B have been realized, objective C has been partially tested, objective D has been completed, and work on objectives E and F will be completed by the end of 1995. Although we have yet to map any loci that contribute to an ability of citrus to maintain growth when irrigated with saline water, our very encouraging results from the 1993 experiment provides us with considerable hope that 1994's much more comprehensive and better controlled experiment will yield the desired results once the data has been fully analyzed. Part of our optimism derives from the findings that loci for growth are closely linked with loci associated with foliar Cl- and Na+ accumulation patterns under non-salinization conditions. In the 1994 experiment, if ion exclusion or sequestration traits are segregating in the population, the experimental design will permit their resolution. Our fortunes with respect to cold tolerance is another situation. In three attempts to quantitatively characterize cold tolerance as an LT50, the results have been too variable and the incremental differences between sensitive and tolerant too small to use for mapping. To adequately determine the LT50 requires many plants, many more than we have been able to generate in the time and space available by making cuttings from small greenhouse-grown stock plants. As it has turned out, with citrus, to prepare enough plants needed to be successful in this objective would have required extensive facilities for both growing and testing hardiness which simply were not available at University of Florida. The large populations necessary to overcome the variability we encountered was unanticipated and unforeseeable at the project's outset. In spite of the setbacks, this project, when it is finally complete will be exceedingly successful. Listing of Accomplishments During the funded interval we have accomplished the following objectives: Developed a reasonably high density linkage map for citrus - mapped the loci for two cold responsive genes that were cloned from Poncirus - mapped the loci for csa, the salt responsive gene for glutathione peroxidase, and ccr a circadian rhythm gene from citrus - identified loci that confer parental derived specific DNA methylation patterns in the Citrus X Poncirus cross - mapped 5 loci that determine shoot vigor - mapped 2 loci that influence leaf Na+ accumulation patterns under non-saline conditions in the BC1 population - mapped 3 loci that influence leaf Na+ accumulation paterns during salt sress - mapped 2 loci that control leaf Cl- accumulation patterns under non-saline conditions - mapped a locus that controls leaf Cl- accumulation patterns during salt stress Screened the BC1 population for growth reduction during salinization (controls and salinized), and cold tolerance - determined population variation for shoot/root ratio of Na+ and Cl- - determined levels for 12 inorganic nutrient elements in an effort to examine the influence of salinization on ion content with emphasis on foliar responses - collected data on ion distribution to reveal patterns of exclusion/sequestration/ accumulation - analyzed relationships between ion content and growth Characterization of gene expression in response to salt or cold stress - cloned the gene for the salt responsive protein csa, identified it as glutathione peroxidase, determined the potential target substrate from enzymatic studies - cloned two other genes responsive to salt stress, one for the citrus homologue of a Lea5, and the other for an "oleosin" like gene - cold regulated (cor) genes belonging to five hybridization classes were isolated from Poncirus, two belonged to the group 2 Lea superfamily of stress proteins, the others show no significant homology to other known sequences - the expression of csa during cold acclimation was examined, and the expression of some of the cor genes were examined in response to salt stress - the influence of salinization on cold tolerance has been examined with seedling populations - conducted protein blot studies for expression of cold stress proteins during salt stress and vice versa

The long-term goal of this research is to understand how pollen cope with stress, and identify genes that can be manipulated in crop plants to improve reproductive success during heat stress. The specific aims were to: 1) Compare heat stress dependent changes in gene expression between wild type pollen, and mutants in which pollen are heat sensitive (cngc16) or heat tolerant (apx2-1). 2) Compare cngc16 and apx2 mutants for differences in heat-stress triggered changes in ROS, cNMP, and Ca²⁺ transients. 3) Expand a mutant screen for pollen with increased or decreased thermo-tolerance. These aims were designed to provide novel and fundamental advances to our understanding of stress tolerance in pollen reproductive development, and enable research aimed at improving crop plants to be more productive under conditions of heat stress. Background: Each year crop yields are severely impacted by a variety of stress conditions, including heat, cold, drought, hypoxia, and salt. Reproductive development in flowering plants is highly sensitive to hot or cold temperatures, with even a single hot day or cold night sometimes being fatal to reproductive success. In many plants, pollen tube development and fertilization is often the weakest link. Current speculation about global climate change is that most agricultural regions will experience more extreme environmental fluctuations. With the human food supply largely dependent on seeds, it is critical that we consider ways to improve stress tolerance during fertilization. The heat stress response (HSR) has been intensively studied in vegetative tissues, but is poorly understood during reproductive development. A general paradigm is that HS is accompanied by increased production of reactive oxygen species (ROS) and induction of ROS-scavenging enzymes to protect cells from excess oxidative damage. The activation of the HSR has been linked to cytosolic Ca²⁺ signals, and transcriptional and translational responses, including the increased expression of heat shock proteins (HSPs) and antioxidative pathways. The focus of the proposed research was on two mutations, which have been discovered in a collaboration between the Harper and Miller labs, that either increase or decrease reproductive stress tolerance in a model plant, Arabidopsis thaliana (i.e., cngc16--cyclic nucleotide gated channel 16, apx2-1--ascorbate peroxidase 2,). Major conclusions, solutions, achievements. Using RNA-seq technology, the expression profiles of cngc16 and apx2 pollen grains were independently compared to wild type under favourable conditions and following HS. In comparison to a wild type HSR, there were 2,776 differences in the transcriptome response in cngc16 pollen, consistent with a model in which this heat-sensitive mutant fails to enact or maintain a normal wild-type HSR. In a comparison with apx2 pollen, there were 900 differences in the HSR. Some portion of these 900 differences might contribute to an improved HSR in apx2 pollen. Twenty-seven and 42 transcription factor changes, in cngc16 and apx2-1, respectively, were identified that could provide unique contributions to a pollen HSR. While we found that the functional HS-dependent reprogramming of the pollen transcriptome requires specific activity of CNGC16, we identified in apx2 specific activation of flavonol-biosynthesis pathway and auxin signalling that support a role in pollen thermotolerance. Results from this study have identified metabolic pathways and candidate genes of potential use in improving HS tolerance in pollen. Additionally, we developed new FACS-based methodology that can quantify the stress response for individual pollen in a high-throughput fashion. This technology is being adapted for biological screening of crop plant’s pollen to identify novel thermotolerance traits. Implications, both scientific and agricultural. This study has provided a reference data on the pollen HSR from a model plant, and supports a model that the HSR in pollen has many differences compared to vegetative cells. This provides an important foundation for understanding and improving the pollen HSR, and therefor contributes to the long-term goal of improving productivity in crop plants subjected to temperature stress conditions. A specific hypothesis that has emerged from this study is that pollen thermotolerance can be improved by increasing flavonol accumulation before or during a stress response. Efforts to test this hypothesis have been initiated, and if successful have the potential for application with major seed crops such as maize and rice.