Negative regulation of transcription factors by Srb10 cyclin-dependent kinase

The ubiquitin-dependent proteolytic pathway plays an important role in a broad array of cellular processes, inducting cell cycle control and transcription. Biochemical analysis of the ubiquitination of Sic1, the B-type cyclin-dependent kinase (CDK) inhibitor in budding yeast helped to define a ubiquitin ligase complex named SCF^cdc4 (for Skp1, Cdc53/cullin, F-box protein). We found that besides Sic1, the CDK inhibitor Far1 and the replication initiation protein Cdc6 are also substrates of SCF^cdc4 in vitro. A common feature in the ubiquitination of the cell cycle SCF^cdc4 substrates is that they must be phosphorylated by the major cell cycle CDK, Cdc28. Gcn4, a transcription activator involved in the general control of amino acid biosynthesis, is rapidly degraded in an SCF^cdc4-dependent manner in vivo. We have focused on this substrate to investigate the generality of the SCF^cdc4 pathway. Through biochemical fractionations, we found that the Srb10 CDK phosphorylates Gcn4 and thereby marks it for recognition by SCF^cdc4 ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Furthermore, we found that at least two different CDKs, Pho85 and Srb10, conspire to promote the rapid degradation of Gcn4 in vivo. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. Our results suggest a general theme that Srb10 represses the transcription of specific genes by directly antagonizing the transcriptional activators.

Developmentally regulated transcription factors in Drosophila melanogaster

During early stages of Drosophila development the heat shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo, it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a nuclear transport protein Drosophila karyopherin-α3(dKap- α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3.Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

The pair-rule gene fushi tarazu (ftz) is a well-studied zygotic segmentation gene that is necessary for the development of the even-numbered parasegments in Drosophila melanogastor. During early embryogenesis, ftz is expressed in a characteristic pattern of seven stripes, one in each of the even-numbered parasegments. With a view to understand how ftz is transcriptionally regulated, cDNAs that encode transcription factors that bind to the zebra element of the ftz promoter have been cloned. Chapter Ill reports the cloning and characterization of the eDNA encoding zeb-1 (zebra element binding protein), a novel steroid receptor-like molecule that specifically binds to a key regulatory element of the ftz promoter. In transient transfection assays employing Drosophila tissue culture cells, it has been shown that zeb-1 as well as a truncated zeb-1 polypeptide (zeb480) that lacks the putative ligand binding domain function as sequencespecific trans-activators of the ftz gene.

The Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Chapter IV reports the eDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in-situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20-40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in fourteen stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. The data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

Factors affecting the susceptibility of salmonid fish to disease

A review article discussing the degree of susceptibility of fish to outbreaks of disease and whether, besides from changes in the physical and chemical characteristics of the environment, this susceptibility is instrumental in determining whether or not pathogenic challenge results in disease. The article summarises a decade of work on this subject at the Windermere laboratory of the Freshwater Biological Association and suggests possible directions for future research. The article covers experimental design, effects of environmental stress (including discussion on the hypothalamic-pituitary-interrenal (HPI) axis in salmonid fish), sexual maturation, research areas for future development and evolutionary considerations. There are a number of accompanying figures and images.

The promoter of cell growth- and RNA protection-associated SND1 gene is activated by endoplasmic reticulum stress in human hepatoma cells

Background: Staphyloccocal nuclease domain-containing protein 1 (SND1) is involved in the regulation of gene expression and RNA protection. While numerous studies have established that SND1 protein expression is modulated by cellular stresses associated with tumor growth, hypoxia, inflammation, heat- shock and oxidative conditions, little is known about the factors responsible for SND1 expression. Here, we have approached this question by analyzing the transcriptional response of human SND1 gene to pharmacological endoplasmic reticulum (ER) stress in liver cancer cells. Results: We provide first evidence that SND1 promoter activity is increased in human liver cancer cells upon exposure to thapsigargin or tunicamycin or by ectopic expression of ATF6, a crucial transcription factor in the unfolded protein response triggered by ER stress. Deletion analysis of the 5'-flanking region of SND1 promoter identified maximal activation in fragment (-934, +221), which contains most of the predicted ER stress response elements in proximal promoter. Quantitative real- time PCR revealed a near 3 fold increase in SND1 mRNA expression by either of the stress- inducers; whereas SND1 protein was maximally upregulated (3.4-fold) in cells exposed to tunicamycin, a protein glycosylation inhibitor. Conclusion: Promoter activity of the cell growth- and RNA-protection associated SND1 gene is up-regulated by ER stress in human hepatoma cells.

Osmotic stress does not trigger brevetoxin production in the dinoflagellate Karenia brevis

With the global proliferation of toxic Harmful Algal Bloom (HAB) species, there is a need to identify the environmental and biological factors that regulate toxin production. One such species, Karenia brevis, forms nearly annual blooms that threaten coastal regions throughout the Gulf of Mexico. This dinoflagellate produces brevetoxins, potent neurotoxins that cause neurotoxic shellfish poisoning and respiratory illness in humans, as well as massive fish kills. A recent publication reported that a rapid decrease in salinity increased cellular toxin quotas in K. brevis and hypothesized that brevetoxins serve a role in osmoregulation. This finding implied that salinity shifts could significantly alter the toxic impacts of blooms. We repeated the original experiments separately in three different laboratories and found no evidence for increased brevetoxin production in response to low-salinity stress in any of the eight K. brevis strains we tested, including three used in the original study. Thus, we find no support for an osmoregulatory function of brevetoxins. The original publication also stated that there was no known cellular function for brevetoxins. However, there is increasing evidence that brevetoxins promote survival of the dinoflagellates by deterring grazing by zooplankton. Whether they have other as yet unidentified cellular functions is currently unknown.

The effect of vitamins C and E on some of growth factors of cultured Acipenser ruthenus

The effect of vitamins C and E on some of growth factors of cultured Acipenser ruthenus was studied in this thesis. For this purpose diets supplemented with a combination of 100 and 400 mg/kg vitamin C, L-ascorbyl-2-polyphosphate and 100 and 400 mg/kg vitamin E, D-alpha-tocopherol,were each fed to sterlet in 2 replications for 15 weeks. Fifteen fish with average weight of 350.92±14.28 gr were distributed to each of 18 tanks after adaptation with experimental diet. After 5 weeks, there were significant differences in RBC, ESR, HCT and differential counting of white blood cells among the treatments (P<0.05), but there was no significant difference in the amount of WBC among the treatments (P>0.05). After 10 weeks, there were significant differences in the amounts of Monocytes, Lymphocytes and Eosinophils (P<0.05), but there was no significant differences in the amount of other hematologic factors (P>0.05). At the end of the experiment (15th week) only WBC, RBC, Monocyte, lymphocyte and neutrophil showed significant differences between the treatments (P<0.05) and other hematologic factors did not show any significant differences between the treatments (P>0.05). The results of biochemical indices analysis showed significant differences (P<0.05) among treatments for all of the parameters for 5th weeks, total protein and glucose for 10th week and only cholesterol for 15th week. The carcass analysis at the end of experiment showed that only the amount of carbohydrate, protein and ash were significantly difference between the treatments (P<0.05). The results of growth parameters at the end of 3th, 9th, 12th and 15th week showed significant differences between the treatments (P<0.05), but at the end of 6th week only GR was significantly different between the treatments (P<0.05). After concerning acute stress test including reduce water volume, cutting the aeration for 30 minutes at the end of experiment, cortisol and glucose significantly increased (P<0.05) compared with prestress period, but the lowest response to the stressor was observed in fish fed by E400 C 400 mg/kg. On the other hand the survival was 100% during the experiment and no mortality was occurred during this period. Results of this study indicate that Vitamins C and E can have remarkable effects on hematological, biochemical and growth indices in different growth periods. So regarding the effects of vitamin C and E on some growth indices, it seems that the diet containing E400 C 100 mg/kg can be considered as the optimum diet in the rearing condition for this weigth range of fish.

The study of Indian shrimp larvae feeding by enriched rotifer with vitamin C and unsaturated fatty acid (epa, dha) and its effects on growth factors, survival rate and environmental stresses

In this experiment, the feeding of Indian white shrimp larvae by unenriched rotifers (treatment 1) and enriched with highly unsaturated fatty acid (treatment 2) and highly unsaturated fatty acid along with vitamin C (treatment 3) on the growth factors, survival and resistance against salinity and formalin stress tests were studied and their differences with control treatment including newly hatched Artemia nauplii is compared. In this the study four treatments in a completely randomized design with 3 replicates per treatment were used. Farming of shrimp larvae of Zoea II to postlarvae 5 was done in 20 liter plastic bucket. Present results indicated that growth factors and survival rate of stage Zoea II to postlarvae 1 in treatments 1, 2 and 3 improve rather than control in which this case was due to optimal size rotifer rather than Artemia nauplii. Also, treatments 2 and 3 feeding with oil liver cod emulsion enriched rotifer have the highest concentration of DHA (mg/g DW) and the ratio DHA/EPA in which due to have shown the highest growth factors and a significant difference (P<0.05) with treatments 1 and control. The highest survival at stage PL1 were observed in treatment 3 that was enriched with ascorbyl palmitate in which have to the synergistic properties of vitamin C rather than treatments 2, 1 and control and showed a significant difference (P<0.05). But in stage PL5 the highest amount of growth and survival rates were related to control treatment which showed a significant difference (P<0.05) with other treatments that control has higher size rather than treatments 1, 2 and 3. Also, among experiment treatments that the two treatments 2 and 3 due to enrichment had higher growth and survival rates compared with treatment 1 in which their differences have also been significant (P<0.05). In the case of stress tests, results indicated that the highest survival rate has been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids with vitamin C. So that in stage PL1 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3. As for the second, treatment 2 showed a significant difference (P<0.05) with treatment 3. It is worth mentioning that treatment 3 showed a higher survival rate compared to treatment 2 due to the synergistic properties of vitamin C. The difference between these two treatments with treatment 1 and control was also significant. No significant difference was observed in formalin stress test 100 ppm in this stage between treatments 3 and 2 which shows the highest survival rate. But their difference with treatments 1 and control was significant (P<0.05). Also, in stage PL5 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3 which showed no significant difference (P<0.05) with control treatment. While their difference in the amount of survival rate with treatment 1 and 2 was significant (P<0.05). In this stage, the highest observed survival rate in formalin stress test 100 ppm included treatments control, 3 and 2 among which there were no significant differences (P<0.05). While the difference between these three treatments with treatment 1 was significant.

Factors affecting seismic response of submarine slopes

The response of submerged slopes on the continental shelf to seismic or storm loading has become an important element in the risk assessment for offshore structures and "local" tsunami hazards worldwide. The geological profile of these slopes typically includes normally consolidated to lightly overconsolidated soft cohesive soils with layer thickness ranging from a few meters to hundreds of meters. The factor of safety obtained from pseudo-static analyses is not always a useful measure for evaluating the slope response, since values less than one do not necessarily imply slope failure with large movements of the soil mass. This paper addresses the relative importance of different factors affecting the response of submerged slopes during seismic loading. The analyses use a dynamic finite element code which includes a constitutive law describing the anisotropic stress-strain-strength behavior of normally consolidated to lightly overconsolidated clays. The model also incorporates anisotropic hardening to describe the effect of different shear strain and stress histories as well as bounding surface principles to provide realistic descriptions of the accumulation of the plastic strains and excess pore pressure during successive loading cycles. The paper presents results from parametric site response analyses on slope geometry and layering, soil material parameters, and input ground motion characteristics. The predicted maximum shear strains, permanent deformations, displacement time histories and maximum excess pore pressure development provide insight of slope performance during a seismic event. © 2006 Author(s). This work is licensed under a Creative Commons License.

Deposition of low stress amorphous zinc tin oxide at ambient temperature using a remote plasma sputtering process suitable for delicate substrates

ZnxSnyOz thin films (<100nm thickness), deposited by remote sputtering from a metal target using a confined argon plasma and oxygen gas jet near the sample, were investigated for their material properties. No visible deformation or curl was observed when deposited on plastic. Materials were confirmed to be amorphous and range between 5 and 10 at.% Sn concentration by x-ray diffraction, x-ray photoemission spectroscopy and energydispersive x-ray spectroscopy. Factors affecting the material composition over time are discussed. Depletion of the Sn as the target ages is suspected. © The Electrochemical Society.

Genetic diversity of plankton community as depicted by PCR-DGGE fingerprinting and its relation to morphological composition and environmental factors in lake donghu

To collect information about the genetic diversity of the plankton community and to study how plankton respond to environmental conditions, plankton samples were collected from five stations representing different trophic levels in a shallow, eutrophic lake (Lake Donghu), and investigated by PCR-DGGE fingerprinting. A total of 100 bands (61 of 16S rDNA bands and 39 of 18S rDNA bands) were detected. The DGGE bands unique to any single station accounted for 38% of the total bands, whereas common bands detected at all five stations accounted for only 11%. Using UPGMA clustering and MDS ordination of DGGE fingerprints, stations I and II were found to initially group together into one cluster, which was later joined by station V. Stations III and IV were isolated into two separate groups of one station each. Some differences in grouping relationships were found when analysis was completed on the basis of chemical characteristics and morphological composition, with zooplankton composition showing the greatest variability. However, the most similar stations (I and II) were always initially grouped into one cluster. Moreover, stations that exhibited the same or similar trophic level (stations III and IV), but different concentrations of heavy metals, were further differentiated by the DGGE method. Results of the present study indicated that PCR-DGGE fingerprinting was more sensitive than the traditional methods, as other studies suggested. Additionally, PCR-DGGE appears to be more appropriate for diversity characterization of the plankton community, as it is more canonical, systematic, and effective. Most importantly, fingerprinting results are more convenient for the comparative analyses between different studies. Therefore, the use of the described fingerprinting analysis may provide an operable and sensitive biomonitoring approach to identify critical, and potentially negative, stress within an aquatic ecosystem.

Comparison and combination of several stress relief methods for cubic boron nitride films deposited by ion beam assisted deposition

Cubic boron nitride (c-BN) films were prepared by ion beam assisted deposition (IBAD) technique, and the stresses were primary estimated by measuring the frequency shifts in the infrared-absorption peaks of c-BN samples. To test the possible effects of other factors, dependencies of the c-BN transversal optical mode position on film thickness and c-BN content were investigated. Several methods for reducing the stress of c-BN films including annealing, high temperature deposition, two-stage process, and the addition of a small amount of Si were studied, in which the c-BN films with similar thickness and cubic phase content were used to evaluate the effects of the various stress relief methods. It was shown that all the methods can reduce the stress in c-BN films to various extents. Especially, the incorporation of a small amount of Si (2.3 at.%) can result in a remarkable stress relief from 8.4 to similar to 3.6 GPa whereas the c-BN content is nearly unaffected, although a slight degradation of the c-BN crystallinity is observed. The stress can be further reduced down below I GPa by combination of the addition of Si with the two-stage deposition process. (c) 2008 Elsevier B.V. All rights reserved.

Factors influencing the climatological mixed layer depth in the South China Sea: numerical simulations

The mixed layer depth (MLD) in the upper ocean is an important physical parameter for describing the upper ocean mixed layer. We analyzed several major factors influencing the climatological mixed layer depth (CMLD), and established a numerical simulation in the South China Sea (SCS) using the Regional Ocean Model System (ROMS) with a high-resolution (1/12A degrees x1/12A degrees) grid nesting method and 50 vertical layers. Several ideal numerical experiments were tested by modifying the existing sea surface boundary conditions. Especially, we analyzed the sensitivity of the results simulated for the CMLD with factors of sea surface wind stress (SSWS), sea surface net heat flux (SSNHF), and the difference between evaporation and precipitation (DEP). The result shows that of the three factors that change the depth of the CMLD, SSWS is in the first place, when ignoring the impact of SSWS, CMLD will change by 26% on average, and its effect is always to deepen the CMLD; the next comes SSNHF (13%) for deepening the CMLD in October to January and shallowing the CMLD in February to September; and the DEP comes in the third (only 2%). Moreover, we analyzed the temporal and spatial characteristics of CMLD and compared the simulation result with the ARGO observational data. The results indicate that ROMS is applicable for studying CMLD in the SCS area.

Osmotic stress tolerance mechanisms in Lactococcus lactis

The response of Lactococcus lactis subsp. cremoris NCDO 712 to low water activity (aw) was investigated, both in relation to growth following moderate reductions in the aw and in terms of survival following substantial reduction of the aw with NaCI. Lc.lactis NCDO 712 was capable of growth in the presence of ≤ 4% w/v NaCI and concentrations in excess of 4% w/v were lethal to the cells. The presence of magnesium ions significantly increased the resistance of NCDO 712 to challenge with NaCI and also to challenge with high temperature or low pH. Survival of Lc.lactis NCDO 712 exposed to high NaCI concentrations was growth phase dependent and cells were most sensitive in the early exponential phase of growth. Pre-exposure to 3% w/v NaCI induced limited protection against subsequent challenge with higher NaCI concentrations. The induction was inhibited by chloramphenicol and even when induced, the response did not protect against NaCI concentrations> 10% w/v. When growing at low aw, potassium was accumulated by Lc. lactis NCDO 712 growing at low aw, if the aw was reduced by glucose or fructose, but not by NaCI. Reducing the potassium concentration of chemically defined medium from 20 to 0.5 mM) produced a substantial reduction in the growth rate, if the aw was reduced with NaCI, but not with glucose or fructose. The reduction of the growth rate correlated strongly with a reduction in the cytoplasmic potassium concentration and in cell volume. Addition of the compatible solute glycine betaine, partially reversed the inhibition of growth rate and partially restored the cell volume. The potassium transport system was characterised in cells grown in medium at both high and low aw. It appeared that a single system was present, which was induced approximately two-fold by growth at low aw. Potassium transport was assayed in vitro using cells depleted of potassium; the assay was competitively inhibited by Na+ and by the other monovalent cations NH4+, Li+, and Cs+. There was a strong correlation between the ability of strains of Lc. lactis subsp. lactis and subsp. cremoris to grow at low aw and their ability to accumulate the compatible solute glycine betaine. The Lc. lactis subsp. cremoris strains incapable of growth at NaCI concentrations> 2% w/v did not accumulate glycine betaine when growing at low aw, whereas strains capable of growth at NaCI concentrations up to 4% w/v did. A mutant, extremely sensitive to low aw was isolated from the parent strain Lc. lactis subsp. cremoris MG 1363, a plasmid free derivative of NCDO 712. The parent strain tolerated up to 4% w/v NaCI and actively accumulated glycine betaine when challenged at low aw. The mutant had lost the ability to accumulate glycine betaine and was incapable of growth at NaCI concentrations >2% w/v or the equivalent concentration of glucose. As no other compatible solute seemed capable of substitution for glycine betaine, the data suggest that the traditional; phenotypic speciation of strains on the basis of tolerance to 4% w/v NaCI can be explained as possession or lack of a glycine betaine transport system.

Investigation of stress and burnout in Irish second-level teachers: A mixed-methods approach.

This study explores the experiences of stress and burnout in Irish second level teachers and examines the contribution of a number of individual, environmental and health factors in burnout development. As no such study has previously been carried out with this sample, a mixed-methods approach was adopted in order to comprehensively investigate the subject matter. Teaching has consistently been identified as a particularly stressful occupation and research investigating its development is of great importance in developing measures to address the problem. The first phase of study involved the use of focus groups conducted with a total of 20 second-level teachers from 11 different schools in the greater Cork city area. Findings suggest that teachers experience a variety of stressors – in class, in the staff room and outside of school. The second phase of study employed a survey to examine the factors associated with burnout. Analysis of 192 responses suggested that burnout results from a combination of demographic, personality, environmental and coping factors. Burnout was also found to be associated with a number of physical symptoms, particularly trouble sleeping and fatigue. Findings suggest that interventions designed to reduce burnout must reflect the complexity of the problem and its development. Based on the research findings, interventions that combine individual and organisational approaches should provide the optimal chance of effectively tackling burnout.

Elicitation techniques for the evaluation of speech under stress: Towards a framework for use in a controlled environment

Existing work in Computer Science and Electronic Engineering demonstrates that Digital Signal Processing techniques can effectively identify the presence of stress in the speech signal. These techniques use datasets containing real or actual stress samples i.e. real-life stress such as 911 calls and so on. Studies that use simulated or laboratory-induced stress have been less successful and inconsistent. Pervasive, ubiquitous computing is increasingly moving towards voice-activated and voice-controlled systems and devices. Speech recognition and speaker identification algorithms will have to improve and take emotional speech into account. Modelling the influence of stress on speech and voice is of interest to researchers from many different disciplines including security, telecommunications, psychology, speech science, forensics and Human Computer Interaction (HCI). The aim of this work is to assess the impact of moderate stress on the speech signal. In order to do this, a dataset of laboratory-induced stress is required. While attempting to build this dataset it became apparent that reliably inducing measurable stress in a controlled environment, when speech is a requirement, is a challenging task. This work focuses on the use of a variety of stressors to elicit a stress response during tasks that involve speech content. Biosignal analysis (commercial Brain Computer Interfaces, eye tracking and skin resistance) is used to verify and quantify the stress response, if any. This thesis explains the basis of the author’s hypotheses on the elicitation of affectively-toned speech and presents the results of several studies carried out throughout the PhD research period. These results show that the elicitation of stress, particularly the induction of affectively-toned speech, is not a simple matter and that many modulating factors influence the stress response process. A model is proposed to reflect the author’s hypothesis on the emotional response pathways relating to the elicitation of stress with a required speech content. Finally the author provides guidelines and recommendations for future research on speech under stress. Further research paths are identified and a roadmap for future research in this area is defined.