Rossukon Thongwichian

Thammasat University, Krung Thep, Bangkok, Thailand

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Publications (6)34.3 Total impact

  • Thomas Schendel · Rossukon Thongwichian
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    ABSTRACT: A common approach to estimate extreme flood events is the annual block maxima approach, where for each year the peak streamflow is determined and a distribution (usually the generalized extreme value distribution (GEV)) is fitted to this series of maxima. Eventually this distribution is used to estimate the return level for a defined return period. However, due to the finite sample size, the estimated return levels are associated with a range of uncertainity, usually expressed via confidence intervals. Previous publications have shown that existing bootstrapping methods for estimating the confidence intervals of the GEV yield too narrow estimates of these uncertainty ranges. Therefore, we present in this article a novel approach based on the less known test inversion bootstrapping, which we adapted especially for complex quantities like the return level. The reliability of this approach is studied and its performance is compared to other bootstrapping methods as well as the Profile Likelihood technique. It is shown that the new approach improves significantly the coverage of confidence intervals compared to other bootstrapping methods and for small sample sizes should even be favoured over the Profile Likelihood.
    No preview · Article · May 2015 · Advances in Water Resources
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    ABSTRACT: Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their indi-vidual activities in a semi-continuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.
    No preview · Article · May 2015 · Journal of the American Chemical Society
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    ABSTRACT: We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the (15)N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D (1)H-(15)N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.
    Preview · Article · Jun 2013 · Nature Protocol
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    ABSTRACT: We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments.
    No preview · Article · Jan 2013 · Biochimica et Biophysica Acta
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    ABSTRACT: Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.
    Full-text · Article · Sep 2012 · Journal of Biomolecular NMR
  • Rossukon Thongwichian · Philipp Selenko
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    ABSTRACT: For the purpose of studying IDPs inside cells of higher organisms, several eukaryotic in-cell NMR systems have been developed over the past years. In this chapter we will focus on high-resolution in-cell NMR applications in Xenopus laevis oocytes, the first eukaryotic cellular model system to be established. In contrast to prokaryotic in-cell NMR samples, eukaryotic in-cell NMR specimens are prepared by cytoplasmic delivery of an exogenously produced, isotope-labeled protein into the non-isotope-labeled environment of the respective "host" cell. In-cell NMR applications in Xenopus oocytes rely on intracellular sample deposition by direct microinjection into the oocyte cytoplasm. Here, we describe the preparation of oocyte in-cell NMR samples for IDP studies in this cellular model environment.
    No preview · Article · Jul 2012 · Methods in molecular biology (Clifton, N.J.)