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Selective measurement of proteins from single cells is an important and challenging task in the study of cell biology. Protein signaling underlies many important cellular processes such as stem cell differentiation and tumor metastasis. However, due to the diversity of proteins and the subtlety of the chemical modifications that transduce the signals, it is challenging to selectively measure a specific protein from single cells. Further, determining the localization of proteins in individual cells is challenging due to the artifacts introduced by the chemical fixation required by current tools. To address each of these analytical gaps, we introduce two new single cell protein analysis platforms and two supporting technologies.First, to eliminate fixation-induced artifacts in single-cell protein localization measurements, we developed the subcellular western blotting platform. We designed a set of buffers that enabled independent separations of the cytoplasmic and nuclear compartments of the same single cell. To enable buffer exchange without disturbing the intact cellular components, we developed a hydrogel-based lid for the delivery of lysis reagents purely by diffusion. We characterized the selectivity of the fractionation by assaying a panel of proteins with known localization and demonstrated the platform with an NF-$\kappa$B translocation assay.Next, to enhance the selectivity of single-cell protein analysis, introduced an assay for performing single-cell isoelectric focusing. Building upon the hydrogel lid based reagent delivery system, we developed a microfluidic device that integrates sample preparation, analyte separation, and detection. We characterized the theoretical performance of the device using a fluorescent ladder and purified proteins. Further, we used the single-cell isoelectric focusing assay to separate isoforms and TurboGFP and $\beta$-tubulin from single cells. Additionally, we extended the platform by designing a lid system that generates a r

Joan Flasch Artists' Book Collection

Developing selective compounds for ligand-gated ion channel receptors is a difficult task because of the conserved binding sites in the multitude of receptor subtypes, especially with nicotinic acetylcholine receptors (nAChR). Subtypes of neuronal nAChRs are widely distributed in brain tissue and integrate into various neuronal circuits mediating cognitive processes such as learning, reward, and memory. The objective of the dissertation was to identify important determinants involved in [alpha]7-nAChR selectivity and to apply these principles to develop selective leads. My first approach in determining [alpha]7-nAChR selectivity was utilizing our large congeneric library of triazoles, optimized for binding to the acetylcholine binding proteins (AChBP), for screening and characterizing functional receptor responses of [alpha]7-nAChRs, [alpha]4[beta]2-nAChRs, and the 5-HT₃A receptors to identify selective chemical moieties. The second approach utilized in situ click-chemistry with an [alpha]7 mutated form of AChBP to generate triazoles with increased [alpha]7-nAChR selectivity. The third approach elucidated the protein determinants of the receptors and AChBPs governing activation and binding. This was accomplished with protein-sequence alignments and x-ray crystallographic analyses of common nicotinic ligands and triazoles with the AChBPs. Pharmacological characterization and rank ordering, found that potent agonists at the [alpha]7-nAChR subtype consistently contained (1,4)-anti triazoles of quaternary aza-bicyclics with rigid aromatic ring systems. Additional in situ reactions were performed with a mutated-AChBP template towards the [alpha]7-nAChR subtype to gain selectivity. Both characterization studies elucidated distinct functional responses for the three receptors. [alpha]7- nAChR compounds identified were all agonists, preferring those containing quaternary amines with aromatic rigid moieties. [alpha]4[beta]2-nAChR compounds identified were all antagonists and

The International Workshop on X-Ray Mirror Design, Fabrication, and Metrology (IWXM), Osaka, Japan, was held as a satellite meeting of the Conference on Synchrotron Radiation Instrumentation (SRI) 2009, Melbourne, Australia, in October, 2009. The workshop was organized by a collaboration of scientists from a number of leading synchrotron institutions and universities around the World, such as Osaka University, SPring-8, KEK (Japan), ALS, APS and NSLS (USA), ELETTRA (Italy), ESRF, Synchrotron SOLEIL (France), BESSY (Germany), Diamond (UK), SSRF (China), NSRRC (Taiwan) and PAL (Korea). The workshop followed a series of parallel workshops focused on metrology (1st, 2nd and 3rd International Workshop on Metrology for X-ray and Neutron Optics) and on active X-ray optics (1st and 2nd X-ray and XUV Active Optics Workshop, ACTOP06 and ACTOP08) and included the 3rd workshop on X-ray and EUV active optics (ACTOP09). The workshop brought together more than 100 participants: manufacturers, optical and mechanical engineers, designers, and users of X-ray optics; allowing for free exchange of ideas, highlighting of existing problems and challenges, and searching for ways to improve existing instrumentation for sub-microradian and sub-nanometer accuracy. A visit to the Osaka University mirror fabrication laboratory, SPring-8, and the X-ray free electron laser (XFEL) facility was included in the workshop.

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.

Time-resolved EPR spectra were observed by means of the transient EPR technique for two kinds of fullerene dimers C120 and C120O in the lowest excited triplet (T1) states and compared with those of mono-adducts of fullerene (C60). It was found that zero-field splitting (zfs) parameters of both dimers are nearly the same as those of the monomers, indicating that the excitation is almost completely localized at one of the two fullerene units in the T1 state. © 2002 Elsevier Science B.V. All rights reserved.

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