Transcriptional regulatory codes underlying Arabidopsis stress responses

Plant adaptation to stress is dependent upon the initialisation of molecular signalling networks that regulate the expression of stress-related genes. By examining high-resolution microarray datasets it has been possible to track gene expression changes over time during senescence and in response to infection by fungal pathogen Botrytis cineria in the model organism Arabidopsis thaliana. Dramatic variations in gene expression are observed at the onset of stress with different groups of genes showing different expression time-courses. This observation must, for a large part, be down to the action of different transcription factors (TFs) binding to the cis-regulatory DNA in the promoters of genes in each group and it is this regulatory code that underpins the gene regulatory networks that regulate stress responses. This thesis presents an interdisciplinary investigation of the regulatory codes that are responsible for controlling plant stress responses. Computational analysis of non-coding sequences provides a powerful approach to identify patterns within DNA that may function to regulate gene expression. This thesis covers the development of Analysis of Plant Promoter-Linked Elements (APPLES), an object-orientated software framework for the analysis of non-coding DNA. Within this environment, methods were developed to probe the regulatory codes that exist within these non-coding sequences and identify regulatory motifs that may function to regulate stress responses in Arabidopsis. APPLES methods were used to identify a novel motif that is likely to play a role in regulating drought responses in Arabidopsis, with experimental approaches providing support for this view. Using known motifs that describe previously characterised TF binding sites, it was possible to identify motifs that are associated with clusters of co-regulated genes identified from the senescence and Botrytis microarray time-course datasets. This analysis revealed cis-regulatory elements that may contribute to generating the observed expression patterns. In a contrasting approach to in silico identification of regulatory elements, the Yeast-1-Hybrid (Y1H) assay was used to experimentally identify interactions between TFs and non-coding DNA. The use of a TF library allowed the ability of approximately 1400 Arabidopsis TFs to interact with a given DNA sequence in a single assay. Using the stress-associated ANAC092 promoter as a test case, it was possible to use this highthroughput procedure to identify TFs that can bind to the promoter of this gene. This high-throughput Y1H system was then used to perform a detailed mapping of protein- DNA interactions that can occur across the core promoters of three highly related stress inducible TF-encoding genes, ANAC019, ANAC055 and ANAC072. Microarrays were used to assess the regulatory consequence of a subset of these interactions by perturbing the expression of interacting TFs and observing the effect on target gene expression during multiple stresses. This approach confirmed predicted regulatory relationships and therefore enhanced the current understanding of the transcriptional regulatory networks that operate during stress responses in Arabidopsis

Assessing Compressed Air Energy Storage (CAES) Potential in Kentucky to Augment Energy Production from Renewable Resources

Fossil fuel power plants in Kentucky have some of the highest emissions of greenhouse gasses in the United States. One potential strategy for mitigating greenhouse gasses from electric power generation is the co-installation of Compressed Air Energy Storage (CAES) and a renewable source such as photovoltaic solar electricity generation (PV solar generation). CAES with complementary co-installed PV solar generation enhances stand-alone PV solar generation because CAES power is available at night. CAES, however, requires both a site where large volumes of compressed air can be stored in the subsurface, and a heat source to prepare the stored air prior to entering the electricity-generating turbines. Co-installed PV solar electricity can provide the required thermal energy, but compressed air storage can be problematic. The two existing CAES plants, in Germany and Alabama, store compressed air in subsurface solution-mined salt caverns, however the thick salt deposits necessary to develop a compressed air storage cavern are not a part of Kentucky’s geology. Six compressed air storage models were reviewed as part of this project: acid solution-mined caverns, abandoned limestone mines, advanced energy storage in mined air storage chambers, depleted gas fields aquifer storage; and cased wellbore energy storage. Each of these models has the potential for application in Kentucky. Two issues need to be addressed in applying CAES and its variations in Kentucky: ownership of the subsurface pore space where compressed air would be stored in depleted geologic reservoirs and aquifers, and social equity of the CAES electric power generation process. Pore space ownership is addressed under both state and federal law, generally from the standpoint of natural gas storage in depleted gas fields. These storage reservoirs would require an Environmental Protection Agency (EPA) injection permit. CAES models that do not impact porosity or groundwater may require other state and federal operational permits. Because CAES is both site-flexible and easily scalable, it provides a starting point for the conversation surrounding energy equity in the U.S. CAES with co-installed PV solar electricity generation provides a path to equitable power generation for all Americans

Transcriptional regulatory codes underlying Arabidopsis stress responses

Plant adaptation to stress is dependent upon the initialisation of molecular signalling networks that regulate the expression of stress-related genes. By examining high-resolution microarray datasets it has been possible to track gene expression changes over time during senescence and in response to infection by fungal pathogen Botrytis cineria in the model organism Arabidopsis thaliana. Dramatic variations in gene expression are observed at the onset of stress with different groups of genes showing different expression time-courses. This observation must, for a large part, be down to the action of different transcription factors (TFs) binding to the cis-regulatory DNA in the promoters of genes in each group and it is this regulatory code that underpins the gene regulatory networks that regulate stress responses. This thesis presents an interdisciplinary investigation of the regulatory codes that are responsible for controlling plant stress responses. Computational analysis of non-coding sequences provides a powerful approach to identify patterns within DNA that may function to regulate gene expression. This thesis covers the development of Analysis of Plant Promoter-Linked Elements (APPLES), an object-orientated software framework for the analysis of non-coding DNA. Within this environment, methods were developed to probe the regulatory codes that exist within these non-coding sequences and identify regulatory motifs that may function to regulate stress responses in Arabidopsis. APPLES methods were used to identify a novel motif that is likely to play a role in regulating drought responses in Arabidopsis, with experimental approaches providing support for this view. Using known motifs that describe previously characterised TF binding sites, it was possible to identify motifs that are associated with clusters of co-regulated genes identified from the senescence and Botrytis microarray time-course datasets. This analysis revealed cis-regulatory elements that may contribute to generating the observed expression patterns. In a contrasting approach to in silico identification of regulatory elements, the Yeast-1-Hybrid (Y1H) assay was used to experimentally identify interactions between TFs and non-coding DNA. The use of a TF library allowed the ability of approximately 1400 Arabidopsis TFs to interact with a given DNA sequence in a single assay. Using the stress-associated ANAC092 promoter as a test case, it was possible to use this highthroughput procedure to identify TFs that can bind to the promoter of this gene. This high-throughput Y1H system was then used to perform a detailed mapping of protein- DNA interactions that can occur across the core promoters of three highly related stress inducible TF-encoding genes, ANAC019, ANAC055 and ANAC072. Microarrays were used to assess the regulatory consequence of a subset of these interactions by perturbing the expression of interacting TFs and observing the effect on target gene expression during multiple stresses. This approach confirmed predicted regulatory relationships and therefore enhanced the current understanding of the transcriptional regulatory networks that operate during stress responses in Arabidopsis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Zircon U-Pb Geochronology of Two Basement Cores (Kentucky, USA): Implications for Late Mesoproterozoic Sedimentation and Tectonics in the Eastern Midcontinent

Basement cores from two wells drilled west and east of the Grenville front consist of feldspathic litharenite and granitic orthogneiss, respectively. Detrital zircon U-Pb ages for the litharenite define a broad dominant U-Pb age mode at ca. 1115 Ma. The dominant mode matches that for the type locality of the Middle Run Formation in the Ohio subsurface and is interpreted to consist of detrital zircons sourced from East Continent Rift volcanic sources (ca. 1100 Ma) and Grenville Shawinigan granites/gneisses (1120–1180 Ma). The youngest detrital zircon ages (ca. 1020 Ma) require a maximum depositional age that is at least 70 My younger than the time of Midcontinent and East Continent rifting and magmatism. We propose that the litharenite is correlative with the Middle Run Formation in Ohio and was deposited in an evolving late Grenville rift/foreland basin adjacent to the exhuming Grenville orogen. Zircon U-Pb secondary-ion mass spectrometry ages from orthogneiss define a discordant array with intercepts of ca. 1500 and 1000 Ma. The oldest concordant dates (ca. 1450 Ma, from oscillatory-zoned cores) are interpreted as the crystallization age of the igneous protolith of the orthogneiss. Metamorphic zircon rims define a weighted mean U-Pb age of 1018 ± 19 Ma (2σ) Ma, interpreted to represent the time of high-grade metamorphism during the late Ottawan phase of the Grenville orogeny. This age pattern matches that of exposed basement in the Central Gneiss Belt of the Grenville Province (Ontario) and similar basement orthogneisses in Ohio and Kentucky that are interpreted to be of Eastern Granite-Rhyolite Province affinity. All age data are consistent with a provenance model of an actively exhuming Grenville orogen at ca. 1000 Ma producing sediment that is mixing with recycled East Continent Rift sediments

The Claims Culture: A Taxonomy of Industry Attitudes

This paper presents an analysis of a familiar aspect of construction industry culture that we have dubbed 'the claims culture'. This is a culture of contract administration that lays a strong emphasis on the planning and management of claims. The principal elements of the analysis are two sets of distinctions. The first comprises economic and occupational orders, referring to two kinds of control that are exercised over the construction process; predicated respectively on economic ownership and occupational competence. The second refers to contrasting attitudes towards relationships and problem solving within these orders: respectively 'distributive' and 'integrative'. The concepts of economic and occupational order entail further sub-categories. The various attitudes associated with these categories and sub-categories are described. They are assessed as to their consequences for change initiatives in the industry

High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation

Leaf senescence is an essential developmental process that impacts dramatically on crop yields and involves altered regulation of thousands of genes and many metabolic and signaling pathways, resulting in major changes in the leaf. The regulation of senescence is complex, and although senescence regulatory genes have been characterized, there is little information on how these function in the global control of the process. We used microarray analysis to obtain a highresolution time-course profile of gene expression during development of a single leaf over a 3-week period to senescence. A complex experimental design approach and a combination of methods were used to extract high-quality replicated data and to identify differentially expressed genes. The multiple time points enable the use of highly informative clustering to reveal distinct time points at which signaling and metabolic pathways change. Analysis of motif enrichment, as well as comparison of transcription factor (TF) families showing altered expression over the time course, identify clear groups of TFs active at different stages of leaf development and senescence. These data enable connection of metabolic processes, signaling pathways, and specific TF activity, which will underpin the development of network models to elucidate the process of senescence

Identification and Recommendations for Correction of Equipment Factors Causing Fatigue in Snowplow Operations

MnDOT No. 1001325The objective of this project was to recommend cost-effective equipment solutions to mitigate fatigue experienced by winter maintenance operators. A questionnaire collected the opinions on the relationship between equipment and fatigue from 2,011 winter maintenance operators in 23 Clear Roads states. An analysis of the existing literature and questionnaire results produced eight cost-effective equipment solutions and eight non-equipment solutions to mitigate winter maintenance operator fatigue. These 16 solutions are believed to be the most promising to mitigate fatigue at low cost (in no particular order): (1) provide dimmable interior cab lighting, (2) use light emitting diode (LED) bulbs for exterior lighting, (3) equip winter maintenance vehicles with warning lights that have a nighttime setting (i.e., dimmable), (4) install a compact disc player or satellite radio in all winter maintenance vehicles, (5) equip winter maintenance vehicles with a heated windshield, (6) install snow deflectors on front plows, (7) install LED narrow-beam bulbs on auxiliary lighting, (8) use an ergonomically designed seat with vibration dampening/air-ride technology, (9) instruct winter maintenance operators to take a 15- to 30-minute break every 4 to 5 hours, (10) provide education and training to winter maintenance operators to identify early signs of fatigue, (11) investigate reduced shift lengths, start/end times, and overtime rules/limits, (12) create an agency-wide fatigue management policy, (13) investigate methods to provide early notifications of an impending swing shift, (14) encourage heathy lifestyles, (15) encourage winter maintenance operator input in equipment purchases, and (16) provide a dedicated place for winter maintenance operators to rest at each garage/terminal

Clinically recognizable error rate after the transfer of comprehensive chromosomal screened euploid embryos is low

ObjectiveTo determine the clinically recognizable error rate with the use of quantitative polymerase chain reaction (qPCR)–based comprehensive chromosomal screening (CCS).DesignRetrospective study.SettingMultiple fertility centers.Patient(s)All patients receiving euploid designated embryos.Intervention(s)Trophectoderm biopsy for CCS.Main Outcome Measure(s)Evaluation of the pregnancy outcomes following the transfer of qPCR-designated euploid embryos. Calculation of the clinically recognizable error rate.Result(s)A total of 3,168 transfers led to 2,354 pregnancies (74.3%). Of 4,794 CCS euploid embryos transferred, 2,976 gestational sacs developed, reflecting a clinical implantation rate of 62.1%. In the cases where a miscarriage occurred and products of conception were available for analysis, ten were ultimately found to be aneuploid. Seven were identified in the products of conception following clinical losses and three in ongoing pregnancies. The clinically recognizable error rate per embryo designated as euploid was 0.21% (95% confidence interval [CI] 0.10–0.37). The clinically recognizable error rate per transfer was 0.32% (95% CI 0.16–0.56). The clinically recognizable error rate per ongoing pregnancy was 0.13% (95% CI 0.03–0.37). Three products of conception from aneuploid losses were available to the molecular laboratory for detailed examination, and all of them demonstrated fetal mosaicism.Conclusion(s)The clinically recognizable error rate with qPCR-based CCS is real but quite low. Although evaluated in only a limited number of specimens, mosaicism appears to play a prominent role in misdiagnoses. Mosaic errors present a genuine limit to the effectiveness of aneuploidy screening, because they are not attributable to technical issues in the embryology or analytic laboratories