A Colorimetric Hormone-Dependent Bacterial Biosensor for Early Drug Discovery and Environmental Toxicology

2014 William G. Lowrie Department of Chemical & Biomolecular Engineering Outstanding Undergraduate Researcher of Excellence, 2013 Undergraduate Research ScholarshipMany biosensors that have been developed to sense environmental changes or chemicals rely on change of phenotype assays. These assays often require special equipment or addition of reagents to detect fluorescence and can be both time and cost intensive. Here, a colorimetric in-vitro ligand–binding assay is proposed using β-galactosidase (β-gal, from the lacZ gene) in bacterially-expressed protein biosensors. The use of β-galactosidase-based assays (based on ONPG hydrolysis) for ligand-binding biosensors presents the advantage that β-galactosidase and the related reagents/equipment necessary are cost-effective, easily reproducible, commercially available, and much faster than traditional assays. The work to be presented was started with an established biosensor that transduces a test hormone-binding activity (estrogen) into a growth-phenotype. To obtain a colorimetric signal for binding rather than a growth phenotype, the growth phenotype reporter gene (thymidylate synthase) was replaced in the biosensor expression vector with the β-galactosidase gene. To verify our vector activity, cells were grown with and without estrogen and colorimetric assays were carried out on cell lysates to determine β-gal activity. Originally, the vector containing β-galactosidase was expressed in BL21(DE3 T7g1) recA lacZ+ strain (BLR) but the chromosomal lacZ+ was contributing background noise to the assay. To eliminate this background signal, the strain ER2566 (ΔlacZ::T7g1) was used for expression studies and three trials of β-gal assays implied higher β-gal / lacZ+ activity for cells grown with ligand than cells grown without ligand, suggesting the strain background was no longer contributing to noise. Next, cells were grown in the absence of the estrogen ligand and were lysed as estrogen was added to the lysate with the colorimetric assay reactant in-vitro while optimizing the dilution to allow for a longer assay so that the in-vitro additions of ligand would have adequate time to bind and potentially increase activity. Despite multiple attempts, no appreciable results were obtained during this step. A new experiment was devised which consisted of purifying the target protein of any proteases and cell debris through affinity chromatography involving binding to an amylose resin column. Estrogen ligand was then added to the purified protein and colorimetric assays were performed to test for increased β-gal activity. The in-vitro estrogen ligand additions to the purified protein showed no discernible changes in activity trends between the controls and samples tested. A hypothesis was formed that when cells took up the ligand in-vivo during growth, the ligand helped the ligand-binding domain fold properly during translation which produced the desired activity increase. However, it is believed that when the ligand was introduced in-vitro after lysis or purification, the ligand binding domain was improperly folded or the active site was buried which caused the suppression of changes in enzyme activity. A series of experiments were then carried out involving the in-vivo introduction of a weakly-binding stabilizing molecule (an estrogen antagonist) that would bind and help the ligand-binding domain fold properly during translation with the hope of introducing estrogen agonists in-vitro after lysis or purification that would theoretically compete for binding and cause an increase in enzyme activity. Several experimental variations had been utilized along with many trials, but in-vitro estrogen ligand additions with or without antagonist molecules introduced in-vivo failed to produce any changes in β-gal enzyme activity. An additional experiment that had been performed concurrently to those presented here involved a similar construct to the human estrogen receptor but the receptor was of the human thyroid (TRβ), which has intrinsically higher signal to noise. The addition of thyroid ligands taken up in-vivo showed an identical trend with the thyroid receptor biosensor to that of estrogen additions to the estrogen biosensor. Although increased activity was defined at this step in an analogous manner, the failed progress of the estrogen receptor biosensor led to conclusion that the thyroid biosensor would behave in a similar manner and yield inconclusive in-vitro findings. As such, in-vitro ligand additions with the thyroid biosensor were never attempted due to the comparable nature of the hypothesized ligand-binding domain folding problem and due to the fact that there are no known thyroid antagonists that could potentially bind in-vivo only to get competitively displaced with the addition of binding thyroid agonists in-vitro.No embargoAcademic Major: Chemical Engineerin

Learning the Lessons of Openness

The Open Educational Resources (OER) movement has built up a record of experience and achievements since it was formed 10 years ago as an identifiable approach to sharing online learning materials. In its initial phase, much activity was driven by ideals and interest in finding new ways to release content, with less direct research and reflection on the process. It is now important to consider the impact of OER and the types of evidence that are being generated across initiatives, organisations and individuals. Drawing on the work of OLnet (http://olnet.org) in bringing people together through fellowships, research projects and supporting collective intelligence about OER, we discuss the key challenges facing the OER movement. We go on to consider these challenges in the context of another project, Bridge to Success (http://b2s.aacc.edu), identifying the services which can support open education in the future

PDE3A signalling in blood platelets

Cyclic 3’, 5’ adenosine monophosphate (cAMP) signalling downstream of prostacyclin (PGI₂) is a key inhibitory pathway in blood platelets. This pathway is dynamically regulated by phosphodiesterase 3A (PDE3A), which hydrolyses cAMP into metabolically inactive AMP. Although PDE3A is an established drug target in anti-platelet therapies, the molecular mechanisms that underlie its function in platelets remain unclear. Therefore, the major aim of this study was to further explore PDE3A signalling in human platelets. Using a combination of cell fractionation and immunoblotting we identified two PDE3A splice variants in platelets, PDE3A1 and PDE3A2, that were differentially localised within the cell. PDE3A1 was located in the membrane fraction, whereas PDE3A2 was primarily located in the cytosolic fraction. Treatment of platelets with PGI2 induced a transient phosphorylation of PDE3A2 at Ser³¹² in a PKA dependent manner. In contrast, no phosphorylation of PDE3A1 was detected. The phosphorylation of PDE3A2 was associated with increased PDE3A enzymatic activity, which suggested that cAMP signalling activated only the cytosolic form of the enzyme. In many cells, A-kinase anchoring proteins (AKAPs) orchestrate a coordinated response between PKA and its effector proteins. The phosphorylation and activation of PDE3A2 in response to PGI2 was blunted by a cell permeable peptide inhibitor of PKA-AKAP interactions suggesting that PKA-mediated activation of PDE3A2 was dependent on an AKAP. Using a cAMP-pull down approach to enrich cAMP binding proteins combined with immunoblotting, we confirmed the presence of two AKAP7 isoforms (δ and γ) in platelets. Additionally, we found that AKAP7δ co-precipitated with PDE3A2 and possessed associated PDE3A activity. Furthermore, AKAP7 also possessed PKA activity, which was a result of its constitutive association with PKA-II. Critically, immunoprecipitated PDE3A was found to be co-associated with both PKA-II and AKAP7δ. The findings in this thesis suggest that blood platelets express multiple differentially-regulated PDE3A splice variants, of which PDE3A2 is regulated by PKA-II within a novel cytosolic AKAP7δ facilitated signalling complex. The selective inhibition of PDE3A splice variants and/or pharmacological disruption of PDE3A signalosomes may provide safer and more specific ways of controlling pathological platelet activation

States and sequences of paired subspace ideals and their relationship to patterned brain function

It is found here that the state of a network of coupled ordinary differential equations is partially localizable through a pair of contractive ideal subspaces, chosen from dual complete lattices related to the synchrony and synchronization of cells within the network. The first lattice is comprised of polydiagonal subspaces, corresponding to synchronous activity patterns that arise from functional equivalences of cell receptive fields. This lattice is dual to a transdiagonal subspace lattice ordering subspaces transverse to these network-compatible synchronies. Combinatorial consideration of contracting polydiagonal and transdiagonal subspace pairs yields a rich array of dynamical possibilities for structured networks. After proving that contraction commutes with the lattice ordering, it is shown that subpopulations of cells are left at fixed potentials when pairs of contracting subspaces span the cells' local coordinates - a phenomenon named glyph formation here. Treatment of mappings between paired states then leads to a theory of network-compatible sequence generation. The theory's utility is illustrated with examples ranging from the construction of a minimal circuit for encoding a simple phoneme to a model of the primary visual cortex including high-dimensional environmental inputs, laminar speficicity, spiking discontinuities, and time delays. In this model, glyph formation and dissolution provide one account for an unexplained anomaly in electroencephalographic recordings under periodic flicker, where stimulus frequencies differing by as little as 1 Hz generate responses varying by an order of magnitude in alpha-band spectral power. Further links between coupled-cell systems and neural dynamics are drawn through a review of synchronization in the brain and its relationship to aggregate observables, focusing again on electroencephalography. Given previous theoretical work relating the geometry of visual hallucinations to symmetries in visual cortex, periodic perturbation of the visual system along a putative symmetry axis is hypothesized to lead to a greater concentration of harmonic spectral energy than asymmetric perturbations; preliminary experimental evidence affirms this hypothesis. To conclude, connections drawn between dynamics, sensation, and behavior are distilled to seven hypotheses, and the potential medical uses of the theory are illustrated with a lattice depiction of ketamine xylazine anaesthesia and a reinterpretation of hemifield neglect

Investigating the role of language in children's early educational outcomes

Most children develop speech and language skills effortlessly, but some are slow to develop these skills and then go on to struggle with literacy and academic skills throughout their schooling. It is the first few years of life that are critical to their subsequent performance.\ud This project looks at what we know about the early communication environment in a child’s first two years of life, and the role this plays in preparing children for school using data from a large longitudinal survey of young people (ALSPAC - the Avon Longitudinal Study of Parents and Children).\ud It examines the characteristics of the environment in which children learn to communicate (such as activities undertaken with children, the mother’s attitude towards her baby, and the wider support available to the family) and the extent to which this affects a child’s readiness for school entry (defined as their early language, reading, writing, and maths skills that they need in school).\ud \ud Key Findings:\ud •\ud There is a strong association between a child’s social background and their readiness for school as measured by their scores on school entry assessments covering language, reading, maths and writing.\ud •\ud Language development at the age of 2 years predicts children’s performance on entry to primary school. Children’s understanding and use of vocabulary and their use of two or three word sentences at 2 years is very strongly associated with their performance on entering primary school.\ud •\ud The children’s communication environment influences language development. The number of books available to the child, the frequency of visits to the library, parents teaching a range of activities, the number of toys available, and attendance at pre-school, are all important predictors of the child’s expressive vocabulary at 2 years. The amount of television on in the home is also a predictor; as this time increased, so the child’s score at school entry decreased.\ud •\ud The communication environment is a more dominant predictor of early language than social background. In the early stages of language development, it is the particular aspects of a child’s communication environment that are associated with language acquisition rather than the broader socio-economic context of the family.\ud •\ud The child’s language and their communication environment influence the child’s performance at school entry in addition to their social background. Children’s success at school is governed not only by their social background; the child’s communication environment\ud before their second birthday and their language at the age of two years also have a strong influence

Estates, Trusts, and Wills

Estates, Trusts, and Will

Pathological affections and relations of false membranes

n/

Observations on the exhibition of mercury in minute doses

n/