Design and Characterization of Bifunctional Glucocorticoid Ligands Capable of Producing Novel Transcriptional Profiles.

The process of transcription underlies the expression of all gene profiles. Normal expression is caused by carefully coordinating the assembly of transcriptional proteins at specific genes, a procedure mediated through transcription factors that bind specific sequences of DNA. The glucocorticoid receptor (GR) is a transcription factor that influences the expression of genes involved in inflammation pathways. Drug-like molecules targeting GR are capable of stimulating GR to bind DNA; however, these molecules are unable to exert complete control over the members of the transcriptional complex recruited by GR. As such, we are limited in our control of GR activity. My dissertation focuses on addressing this with new GR ligands designed to recruit specific transcriptional proteins to produce novel, desired expression profiles. We first conjugated a GR molecule to a ligand of the protein FKBP. This bifunctional ligand was capable of localizing GR to DNA, recruiting FKBP-fusions to the GR-regulated gene, and producing transcriptional activity dictated by the recruitment. This was achieved with the recruitment of both a transcriptionally activating protein and a repressing protein, demonstrating the adaptability of this system to toggle the output of a gene of interest. The design of our system benefits from its inherent modularity; expanding to new targets is simply achieved through synthetic conjugation to an alternative ligand. In a first application of this strategy, we conjugated an agonistic GR ligand to a selective inhibitor of the transcriptional protein BRD4. Recruitment of BRD4 to GR resulted in the suppression of transcription at select genes, and this selectivity drives a novel profile of activated and suppressed genes. In a second application, we conjugated a GR antagonist to the BRD4 inhibitor, allowing forthe further production of novel transcriptional profiles with potential pharmacological utility. This dissertation also includes a study aimed at introducing undergraduate students to the scientific principles of modern chemical biology research. To enhance student learning, we have developed a new guided-inquiry experimental module for biochemistry laboratory courses. This has been well received, and assessment metrics indicate that the incorporation into the University of Michigan’s biochemistry course has raised student cognitive abilities in analysis and application.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113323/1/jcarolan_1.pd

Electrical and physical characterization of the Al₂O₃/ <i>p</i>-GaSb interface for 1%, 5%, 10%, and 22% (NH₄)₂S surface treatments

In this work, the impact of ammonium sulfide ((NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;S) surface treatment on the electrical passivation of the Al&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;/ &lt;i&gt;p&lt;/i&gt;-GaSb interface is studied for varying sulfide concentrations. Prior to atomic layer deposition of Al&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;, GaSb surfaces were treated in 1%, 5%, 10%, and 22% (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;S solutions for 10 min at 295 K. The smallest stretch-out and flatband voltage shifts coupled with the largest capacitance swing, as indicated by capacitance-voltage (&lt;i&gt;CV&lt;/i&gt;) measurements, were obtained for the 1% treatment. The resulting interface defect trap density (&lt;i&gt;D&lt;/i&gt;&lt;sub&gt;it&lt;/sub&gt;) distribution showed a minimum value of 4 x 10&lt;sup&gt;12&lt;/sup&gt; cm&lt;sup&gt;-2&lt;/sup&gt;eV&lt;sup&gt;-1&lt;/sup&gt; at &lt;i&gt;E&lt;/i&gt;&lt;sub&gt;v&lt;/sub&gt; + 0.27 eV. Transmission electron microscopy and atomic force microscopy examination revealed the formation of interfacial layers and increased roughness at the Al&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;/ &lt;i&gt;p&lt;/i&gt;-GaSb interface of samples treated with 10% and 22% (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;S. In combination, these effects degrade the interface quality as reflected in the &lt;i&gt;CV&lt;/i&gt; characteristics

Improving and disaggregating N2O emission factors for ruminant excreta on temperate pasture soils

pre-printCattle excreta deposited on grazed grasslands are a major source of the greenhouse gas (GHG) nitrous oxide (N2O). Currently, many countries use the IPCC default emission factor (EF) of 2% to estimate excreta-derived N2O emissions. However, emissions can vary greatly depending on the type of excreta (dung or urine), soil type and timing of application. Therefore three experiments were conducted to quantify excreta-derived N2O emissions and their associated EFs, and to assess the effect of soil type, season of application and type of excreta on the magnitude of losses. Cattle dung, urine and artificial urine treatments were applied in spring, summer and autumn to three temperate grassland sites with varying soil and weather conditions. Nitrous oxide emissions were measured from the three experiments over 12 months to generate annual N2O emission factors. The EFs from urine treated soil was greater (0.30–4.81% for real urine and 0.13–3.82% for synthetic urine) when compared with dung (− 0.02–1.48%) treatments. Nitrous oxide emissions were driven by environmental conditions and could be predicted by rainfall and temperature before, and soil moisture deficit after application; highlighting the potential for a decision support tool to reduce N2O emissions by modifying grazing management based on these parameters. Emission factors varied seasonally with the highest EFs in autumn and were also dependent on soil type, with the lowest EFs observed from well-drained and the highest from imperfectly drained soil. The EFs averaged 0.31 and 1.18% for cattle dung and urine, respectively, both of which were considerably lower than the IPCC default value of 2%. These results support both lowering and disaggregating EFs by excreta type.This research was financially supported under the National Development Plan, through the Research Stimulus Fund, administered by the Department of Agriculture, Food and the Marine (Grant numbers RSF10/RD/SC/716 and 11S138)

The Role of Selective Attention in the Positivity Offset: Evidence from Event Related Potentials

Some research suggests that positive and negative valence stimuli may be processed differently. For example, negative material may capture and hold attention more readily than equally arousing positive material. This is called the negativity bias, and it has been observed as both behavioural and electroencephalographic (EEG) effects. Consequently, it has been attributed to both automatic and elaborative processes. However, at the lowest levels of arousal, faster reaction times and stronger EEG responses to positive material have been observed. This is called the positivity offset, and the underlying cognitive mechanism is less understood. To study the role of selective attention in the positivity offset, participants completed a&nbsp;negative affective priming&nbsp;(NAP) task modified to dissociate priming for positive and negative words. The task required participants to indicate the valence of a target word, while simultaneously ignoring a distractor. In experiment 1, a behavioural facilitation effect (faster response time) was observed for positive words, in stark contrast to the original NAP task. These results were congruent with a previously reported general categorization advantage for positive material. In experiment 2, participants performed the task while EEG was recorded. In additional to replicating the behavioural results from experiment 1, positive words elicited a larger Late Positive Potential (LPP) component on ignored repetition relative to control trials. Surprisingly, negative words elicited a larger LPP than positive words on control trials. These results suggest that the positivity offset may reflect a greater sensitivity to priming effects due to a more flexible attentional set

Defect chemistry and vacancy concentration of luminescent europium doped ceria nanoparticles by solvothermal method

Pure phase and europium-doped ceria nanocrystals have been synthesized by a single step simple solvothermal process. Different spectroscopic, diffractive, and microscopic techniques were used to determine the morphology, size, crystal structure, and phase of all the samples. Electron energy loss spectroscopy (EELS) for elemental mapping confirmed that good solid solutions were formed and that the particles had a homogeneous distribution of europium. The defect chemistry was more complex than might be expected with the incorporation of each Eu3+ ion resulting in the production of an anion vacancy since the doping results in charge compensating (i.e., for Eu3+) anion vacancies as well as vacancies due to oxygen removal from the crystallite surface. Variations in nanoparticles dimension and lattice parameters were measured as a function of dopant concentrations and their variations explained. The band gap of the samples could be tailored by the doping. The doped samples were found to be luminescent due to the substitution of Ce4+ ions in the cubic symmetric lattice by the dopant ions. The thermal stability of the fluorescence properties was also investigated

Spatiotemporal Dynamics of Reward and Punishment Effects Induced By Associative Learning

While reward associative learning has been studied extensively across different species, punishment avoidance learning has received far less attention. Of particular interest is how the two types of learning change perceptual processing of the learned stimuli. We designed a task that required participants to learn the association of emotionally neutral images with reward, punishment, and no incentive value outcomes through trial-and-error. During learning, participants received monetary reward, neutral outcomes or avoided punishment by correctly identifying corresponding images. Results showed an early bias in favor of learning reward associations, in the form of higher accuracy and fewer trials needed to reach learning criterion. We subsequently assessed electrophysiological learning effects with a task in which participants viewed the stimuli with no feedback or reinforcement. Critically, we found modulation of two early event-related potential components for reward images: the frontocentral P2 (170–230 ms) and the anterior N2/Early Anterior Positivity (N2/EAP; 210–310 ms). We suggest that reward associations may change stimuli detection and incentive salience as indexed by P2 and N2/EAP. We also reported, on an exploratory basis, a late negativity with frontopolar distribution enhanced by punishment images

Temperate Grassland Yields and Nitrogen Uptake Are Influenced by Fertilizer Nitrogen Source

This research was supported under the National Development Plan through the Research Stimulus Fund administered by the Department of Agriculture, Food and the Marine (Grants RSF10-/RD/SC/716 and RSF11S138) and from the Department of Agriculture and Rural Development for Northern Ireland and by the Teagasc Walsh Fellowship Scheme.peer reviewedIn temperate grasslands, N source influences greenhouse gas emissions. Nitrification and urea hydrolysis inhibitors can reduce these losses. The objective of this study was to evaluate the impact of N source, urease inhibitors, and nitrification inhibitors on temperate grassland yields and N uptake. Experiments were conducted at three locations over 2 years (6 site-years) on the island of Ireland, covering a range of soils and climatic conditions. Results showed that calcium ammonium nitrate (CAN), urea+N-(n-butyl) thiophosphoric triamide (NBPT), urea+NBPT+dicyandiamide (DCD), and urea had equal annual dry matter yield. Urea+DCD had lower dry matter yield than CAN for 3 site-years. Calcium ammonium nitrate and urea+NBPT consistently had the same N uptake, urea+DCD had lower N uptake than CAN in 4 of 6 site-years, urea had lower N uptake than CAN in 2 site-years, and urea+NBPT+DCD had lower N uptake than CAN in 1 site-year. Urea+NBPT is a cost-effective alternative to CAN, which is consistently equal in terms of yield and N uptake in temperate grassland.Teagasc Walsh Fellowship ProgrammeDepartment of Agriculture and Rural Development for Northern IrelandDepartment of Agriculture, Food and the Marin

Size and space controlled hexagonal arrays of superparamagnetic iron oxide nanodots: magnetic studies and application

Highly dense hexagonally arranged iron oxide nanodots array were fabricated using PS-b-PEO self-assembled patterns. The copolymer molecular weight, composition and choice of annealing solvent/s allows dimensional and structural control of the nanopatterns at large scale. A mechanism is proposed to create scaffolds through degradation and/or modification of cylindrical domains. A methodology based on selective metal ion inclusion and subsequent processing was used to create iron oxide nanodots array. The nanodots have uniform size and shape and their placement mimics the original self-assembled nanopatterns. For the first time these precisely defined and size selective systems of ordered nanodots allow careful investigation of magnetic properties in dimensions from 50 nm to 10 nm, which delineate the nanodots are superparamagnetic, well-isolated and size monodispersed. This diameter/spacing controlled iron oxide nanodots systems were demonstrated as a resistant mask over silicon to fabricate densely packed, identical ordered, high aspect ratio silicon nanopillars and nanowire features

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

AbstractThe ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states

Access resistance reduction in Ge nanowires and substrates based on non-destructive gas-source dopant in-diffusion

To maintain semiconductor device scaling, in recent years industry has been forced to move from planar to non-planar device architectures. This alone has created the need to develop a radically new, non-destructive method for doping. Doping alters the electrical properties of a semiconductor, related to the access resistance. Low access resistance is necessary for high performance technology and reduced power consumption. In this work the authors reduced access resistance in top–down patterned Ge nanowires and Ge substrates by a non-destructive dopant in-diffusion process. Furthermore, an innovative electrical characterisation methodology is developed for nanowire and fin-based test structures to extract important parameters that are related to access resistance such as nanowire resistivity, sheet resistance, and active doping levels. Phosphine or arsine was flowed in a Metalorganic Vapour Phase Epitaxy reactor over heated Ge samples in the range of 650–700 °C. Dopants were incorporated and activated in this single step. No Ge growth accompanied this process. Active doping levels were determined by electrochemical capacitance–voltage free carrier profiling to be in the range of 1019 cm−3. The nanowires were patterned in an array of widths from 20–1000 nm. Cross-sectional Transmission Electron Microscopy of the doped nanowires showed minimal crystal damage. Electrical characterisation of the Ge nanowires was performed to contrast doping activation in thin-body structures with that in bulk substrates. Despite the high As dose incorporation on unpatterned samples, the nanowire analysis determined that the P-based process was the better choice for scaled features