Acetylation of BMAL1 by TIP60 controls BRD4-P-TEFb recruitment to circadian promoters.

Many physiological processes exhibit circadian rhythms driven by cellular clocks composed of interlinked activating and repressing elements. To investigate temporal regulation in this molecular oscillator, we combined mouse genetic approaches and analyses of interactions of key circadian proteins with each other and with clock gene promoters. We show that transcriptional activators control BRD4-PTEFb recruitment to E-box-containing circadian promoters. During the activating phase of the circadian cycle, the lysine acetyltransferase TIP60 acetylates the transcriptional activator BMAL1 leading to recruitment of BRD4 and the pause release factor P-TEFb, followed by productive elongation of circadian transcripts. We propose that the control of BRD4-P-TEFb recruitment is a novel temporal checkpoint in the circadian clock cycle

Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting

Amphiphilic molecules emerged as versatile building blocks for the generation of nano-sized architectures in water, as they can be programmed to self-assemble into a wide range of different topologies. In this thesis the generation of auto-fluorescent heterovalent nano-sized structures was explored using two types of amphiphilic scaffolds: disc-shaped and linear amphiphiles self-assembling in water into columnar polymers or into amorphous spherical nanoparticles, respectively. Numerous applications for self-assembling nanostructures were reported in literature based on amphiphilic molecules, ranging from imaging to diagnostics, and from drug delivery to tissue engineering. Many of these applications require the capability of the supramolecular system to actively target specific cell surface receptors. This is typically achieved through decoration with bioactive epitopes such as small molecules, peptides, and proteins. As discussed in chapter 1, the bioactive epitopes can either already be part of the monomeric supramolecular building blocks (pre-functionalization) or introduced after self-assembly via covalent attachment to appending reactive groups (post-functionalization). Selective and multivalent binding of disc-shaped amphiphiles to bacterial receptors was previously shown through the introduction of three functional groups at the periphery of the ethylene oxide tails and subsequent functionalization with bioactive ligands. Here, to expand the library of scaffolds, amphiphiles containing either nine amine functionalities or a single amine, azide and propargyl group were synthesized. Their decoration with bioactive ligands such as peptides, carbohydrates, small molecules and fluorescent dyes using both amide coupling and copper-catalyzed azide-alkyne cycloaddition is described in chapter 2. The orthogonality of the copper-catalyzed azide-alkyne cycloaddition allowed the functionalization with unprotected ligands. Whereas the functionalization of discotics with a carbohydrate was quantitative, the coupling of peptides proceeded with at best 40% conversion. This was probably due to steric crowding of peripheral functionalities in the self-assembly inducing solvent, which is required for solubility of unprotected ligands. In contrast, discotics bearing a single amine emerged as a versatile non-sterically hindered scaffold for ligand attachment as they were rapidly and quantitatively functionalized with a range of peptidic- and non-peptidic ligands using both NHS ester and HBTU activation techniques under non-assembling solvent conditions. The ability to fine-tune the density and display of bioactive epitopes and thereby creating more complex dynamic and heterovalent structures without interfering with the self-assembling process is a key prerequisite for the development of a platform technology for targeting. A versatile and non-sterically hindered scaffold for ligand attachment, such as the presented discotic bearing a single amine, might constitute the basis for such a technology. The functionalization of this discotic leads to monovalent ligand functionalized discotics. The display of multiple ligands, which is important for enhanced binding affinities, will be accomplished upon self-assembly into columnar stacks. This so-called multivalency upon self-assembly has been probed with a number of monovalent ligand-functionalized discotics in chapter 3. Enzyme-linked lectin assay revealed a three-fold increase in binding activity compared with the non self-assembling counterpart. The self-assembly into a columnar stack and the accompanied display of multiple ligands was as well confirmed studying the binding of monovalent streptavidin to discotics functionalized with a single biotin using Förster resonance energy transfer and SDS-PAGE. The formation of heterovalent supramolecular polymers through dynamic intermixing of different functionalized building blocks was shown using mixtures of biotin and fluorescein functionalized discotics incubated with streptavidin coated magnetic beads. Thus the self-assembly into supramolecular polymers not only generates a multivalent, but as well a heterovalent system. The possibility to generate heterovalent supramolecular polymers via simple intermixing of discotics has a great potential in view of advanced biological applications, for example in the field of targeted imaging. To gain further inside into the dynamics of this intermixing process, discotics bearing a single O6 benzylguanine moiety were covalently post-functionalized with two FRET-pairing fluorescent proteins. Firstly, the covalent post-functionalization with proteins, ligands which are incompatible with the pre-functionalization strategy, was confirmed with several analytical techniques such as SDS-PAGE and LC-MS in chapter 4. The covalent protein conjugation at the same time leads to Förster resonance energy transfer from the auto-fluorescent discotic scaffold to the yellow fluorescent protein and allows on-line monitoring of the conjugation. At the same time the protein conjugation does not interfere with the self-assembling process, leading to a multivalent protein display on a supramolecular wire, as visualized via energy transfer from the cyan to the yellow fluorescent protein. Secondly, the system maintains its intermixing dynamics, which allows the formation of hetero-functionalized supramolecular protein-conjugated polymers through exchange of the protein-functionalized discotics over time. The supramolecular wires act as dynamic framework on which the two proteins can assemble and exchange in a dynamic manner, leading to effective protein interactions, as observed by energy transfer. The cellular uptake of amine-decorated discotics and the dependence of cellular uptake on the peripheral amine density were explored in chapter 5. Using the auto-fluorescence of the discotic scaffolds, their internalization was studied using live cell multiphoton fluorescence microscopy Discotics bearing three or nine amine groups at their periphery efficiently translocated through the plasma membrane via endocytosis. Additionally, the knowledge about the formation of intermixed supramolecular polymers obtained in chapter 3 and 4 was applied to generate multi-functional supramolecular polymers consisting of up to three different cell-permeable and non cell-permeable discotic monomers. Through intermixing with cell-permeable discotic monomers in the supramolecular polymer, the cellular uptake of non-cell permeable discotics was induced and each of the components could be individually visualized, demonstrating the potential of dynamic multi-component supramolecular polymers. The functionalization of self-assembling p-conjugated nanoparticles with bioactive epitopes, a prerequisite for applications in targeted multimodal imaging, was investigated in the last chapter. Upon microinjection into water, these linear and auto-fluorescent amphiphiles self-assemble into highly-fluorescent amorphous nanoparticles of 80-100 nm. Azide and mannose groups were introduced at the periphery of the ethylene glycol chains of the amphiphile and did not interfere with the self-assembly process. The binding of mannose functionalized nanoparticles to proteins and bacteria confirmed the accessibility of the introduced ligand. Co-assembly of different amphiphiles enabled the fine-tuning of ligand density, which was confirmed with Förster resonance energy transfer. Additionally, using copper catalyzed azide-alkyne cycloaddition reaction, azide bearing nanoparticles were post-functionalized with different ligands. Successful combination of both functionalization strategies via intermixing of mannose and azide bearing amphiphiles and subsequent copper catalyzed azide-alkyne cycloaddition led to heterovalent nanoparticles. Nano-sized columnar and spherical supramolecular assemblies were functionalized with a wide range of ligands such as carbohydrates, peptides, and proteins using both pre- and post-functionalization strategies. This allowed for expanding the ligand diversity at two independent stages in the fabrication process of these bioactive nano-structures. Supramolecular synthesis enabled the facile generation of complex heterovalent bioactive assemblies; in the case of nanoparticles via co-assembly of different amphiphiles and in the case of discotics via dynamic intermixing of building blocks between the supramolecular stacks. With this knowledge in hand advanced applications of complex multitargeting and multimodal supramolecular nano-sized structures in imaging can be envisioned; carrying for example several targeting ligands as well as an alternative imaging probe. The ability to tune the optical properties in the case of the nanoparticles should additionally enable multi-color imaging. At the same time, the self-assembling nature of these nanoparticles allows the incorporation of hydrophobic (drug) molecules and functionalized lipids, expanding the scope of functionalization strategies and with it of possible applications. The absence of unspecific adsorption of the bare scaffolds of both the disc-shaped and linear amphiphiles proves their broad potential as selective biological targeting tools

Non-parametric inference on calibration of predicted risks

Moderate calibration, the expected event probability among observations with predicted probability z being equal to z, is a desired property of risk prediction models. Current graphical and numerical techniques for evaluating moderate calibration of risk prediction models are mostly based on smoothing or grouping the data. As well, there is no widely accepted inferential method for the null hypothesis that a model is moderately calibrated. In this work, we discuss recently-developed, and propose novel, methods for the assessment of moderate calibration for binary responses. The methods are based on the limiting distributions of functions of standardized partial sums of prediction errors converging to the corresponding laws of Brownian motion. The novel method relies on well-known properties of the Brownian bridge which enables joint inference on mean and moderate calibration, leading to a unified 'bridge' test for detecting miscalibration. Simulation studies indicate that the bridge test is more powerful, often substantially, than the alternative test. As a case study we consider a prediction model for short-term mortality after a heart attack, where we provide suggestions on graphical presentation and the interpretation of results. Moderate calibration can be assessed without requiring arbitrary grouping of data or using methods that require tuning of parameters

end of magnolia season

20 pagesDrawing is a balancing act of reality and fiction. Line and form are markers, representative of space and experience, a record of things tangible and ephemeral. There is the capacity for a linear trajectory but it can be elusive: coalescing and cycling through states at once concrete but also synthetic. Shifts in actuality or factuality occur from the inevitable slips and lapses of translation as a line and form is drawn and repeatedly redrawn, altered a little bit in each iteration. An indeterminate looping of degradative and generative processes, a never ending cycle

Evaluating zircon strain chronometry of a Grenville Front deformation fabric through microstructural analysis and quartz piezometry

Zircon strain chronometry can allow correlation of isotopic ages with microstructural fabrics to place an absolute age on the timing of deformation. However, the general applicability of zircon strain chronometry to problems in regional continental tectonics is still being tested and this study is the first effort to apply the approach to the ~1 Ga (billion year old) Grenville Province of the Precambrian Canadian Shield. Samples were analyzed from the 1747 +6/-5 Ma Wanapitei Complex, a poly-deformed mafic metaplutonic body that preserves fabric evidence for several stages in the tectonic history of the Grenville Front. This study is the first to combine in situ strain measurements of zircon with independent measurements of past differential stress using quartz grain size piezometry. Zircon grains in Wanapitei Complex samples exhibiting the latest, D3, ductile deformation were analyzed using electron backscatter diffraction whereas grain size piezometry was completed on a cross-cutting dyke and a marginal gneiss. It was determined that the stress conditions in the Grenville Front near the Wanapitei Complex were not sufficient to deform the generally small (\u3c 50 microns) zircon grains seen in thin sections and result in discordance that would date deformation. Future work targeting significantly larger zircon grains as well as other accessory phases such as baddeleyite and monazite would more thoroughly test the viability of U-Pb strain chronometry in the Grenville Front region