Development of Chemical Proteomic Methods to Quantify Cysteine Oxidation and Reactivity

Protein oxidation is not a fully-understood concept in biology, where the identity of proteins containing oxidative post-translational modifications, their specific sites of modification, and the extent of changing oxidation under stress remain largely undefined. A major contributor to the cellular oxidative response is reversible oxidation of the sulfur atom on cysteine residues (i.e., protein thiols), which can modulate the structure and/or function of modified proteins and ultimately perturb metabolic and/or signaling pathways. Measuring the in vivo oxidation status of protein thiols has traditionally been challenging because these modifications are labile, prone to artifactual oxidation during sample preparation, and low in abundance. The fundamental goals of this dissertation are to develop chemical strategies to enrich for proteins thiols (Chapter 2) and determine their abundance using liquid chromatography-tandem mass spectrometry and label-free quantification (Chapter 3). Using the developed techniques, the proteome of the unicellular green alga Chlamydomonas reinhardtii was surveyed for proteins with reversibly oxidized cysteines and also phosphorylation sites to reveal a more complete understanding of PTM crosstalk, which highlights the reciprocity of signaling cascades across biological pathways (Chapter 4). The cysteine reactivity in the Chlamydomonas proteome was additionally measured to empirically determine sentinel proteins and define a characteristic fingerprint of biological response to oxidative stress (Chapter 5). As demonstrated, this dissertation provides novel tools to identify intracellular pathways targeted by protein thiol oxidation and are widely applicable in future proteomic studies using cells/tissues from disparate organisms. This work ultimately helps to measure cellular fitness by identifying novel markers of protein oxidation and provides targetable hypotheses to better understand tolerance mechanisms toward stress and disease.Doctor of Philosoph

Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets

Recuperado de: https://www.biorxiv.org/content/10.1101/310102v1Target of rapamycin (TOR) kinase is a conserved regulator of cell growth whose activity is modulated in response to nutrients, energy and stress. Key proteins involved in the pathway are conserved in the model photosynthetic microalga Chlamydomonas reinhardtii, but the substrates of TOR kinase and downstream signaling network have not been elucidated. Our study provides a new resource for investigating the phosphorylation networks governed by the TOR kinase pathway in Chlamydomonas. We used quantitative phosphoproteomics to investigate the effects of inhibiting Chlamydomonas TOR kinase on dynamic protein phosphorylation. Wild-type and AZD-insensitive Chlamydomonas strains were treated with TOR-specific chemical inhibitors (rapamycin, AZD8055 and Torin1), after which differentially affected phosphosites were identified. Our quantitative phosphoproteomic dataset comprised 2547 unique phosphosites from 1432 different proteins. Inhibition of TOR kinase caused significant quantitative changes in phosphorylation at 258 phosphosites, from 219 unique phosphopeptides. Our results include Chlamydomonas homologs of TOR signaling-related proteins, including a site on RPS6 with a decrease in phosphorylation. Additionally, phosphosites on proteins involved in translation and carotenoid biosynthesis were identified. Follow-up experiments guided by these phosphoproteomic findings in lycopene beta/epsilon cyclase showed that carotenoid levels are affected by TORC1 inhibition and carotenoid production is under TOR control in algae.National Science Foundation CAREER MCB-155252

Limits: Essays on the Limitations of Science and Religion

The work begins with an investigation of the mind-body problem by Arden Baxter, who argues that the mind is not reducible to the brain in practice even if it may be in theory. Evan Rapone opines that a new and concise definition of science is required, and then provides one. In an analysis of Kant’s Critique of Pure Reason, Madeleine Scott suggests that “the unthinkable will become thinkable” should we be able to transcend the limits of cognitive science by combining it with philosophical investigation. Morgan McConnell investigates the relationship between metaphors and models and concludes that the limits of science are intimately connected to the limits of language. Finally, Sara Nelson brings a Christian perspective to the work and proposes that the nature of the divine ensures that humans can never answer some of the most fundamental questions

The Saharan heat low and moisture transport pathways in the central Sahara-multiaircraft observations and Africa-LAM evaluation

We present a characterization of the Saharan heat low (SHL) based on dropsonde observations made on 22 June 2011 by two simultaneously flying aircraft during the Fennec project. The observations are used to identify moisture transport pathways and to validate the UK Met Office limited area model for northern Africa (Africa-LAM). The observations capture the SHL, harmattan, and monsoon surge. The SHL has a northeast-southwest orientated elongated shape centered over northern Mauritania. The SHL core is associated with a 950 hPa temperature minimum (36.4°C) in the morning caused by the monsoon surge and a maximum (42.6°C) in the afternoon. The monsoon surge east of the SHL core splits into two transport pathways: (a) curving around the SHL core in the north, especially pronounced in a morning near-surface layer, and (b) northeastward transport within the ~2km deep monsoon surge (afternoon observations only). In the morning the model forecasts the harmattan, monsoon surge, and the SHL geographic location and northeast-southwest orientation well but the model represents the SHL flatter and more spatially extended and overestimates the convective boundary layer (CBL) by up to ~0.3 km. The simulated afternoon SHL location appears shifted westward by up to ~1°. The model overestimates the shallow afternoon monsoon surge CBL depth of ~1.8km by >2kmresulting in southwestward transport of vertically mixed moisture above ~2.5km contrasting observed northeastward-only transport at lower levels. This moisture distribution model error is likely to have consequences for simulations of Saharan thermodynamics and dust emissions caused by convection-driven cold pools

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Sleep deprivation reduces the dextran radial distribution and 125I-apoE inflow from CSF into brain. A-B) Representative images of cascade blue dextran (CB) in mice on normal sleep cycle (A) and in mice during sleep deprivation (SD) (B). Cascade blue dextran (10 kDa) was injected into cisterna magna and the mice perfusion fixed (PFA) at 15 min. The vasculature was outline by lectin (green). Scale bars 100 μm (A-B). C) 125I-ApoE2 (yellow column), 125I-apoE3 (red column) and 125I-apoE4 (orange column) inflow into brain from the CSF were reduced in SD mice. D) 14C-inulin inflow into brain from the CSF was reduced with SD and not affected by apoE isoforms. 125I-ApoE (10 nM) and 14C-inulin were intracisternally injected and the brain analyzed for radioactivity. Values are mean ± SEM. N = 6 mice per group. (EPS 15099 kb

Extensive Liquid Meltwater Storage in Firn Within the Greenland Ice Sheet

The accelerating loss of mass from the Greenland ice sheet is a major contribution to current sea level rise. Increased melt water runoff is responsible for half of Greenlands mass loss increase. Surface melt has been increasing in extent and intensity, setting a record for surface area melt and runoff in 2012. The mechanisms and timescales involved in allowing surface melt water to reach the ocean where it can contribute to sea level rise are poorly understood. The potential capacity to store this water in liquid or frozen form in the firn (multi-year snow layer) is significant, and could delay its sea-level contribution. Here we describe direct observation of water within a perennial firn aquifer persisting throughout the winter in the southern ice sheet,where snow accumulation and melt rates are high. This represents a previously unknown storagemode for water within the ice sheet. Ice cores, groundairborne radar and a regional climatemodel are used to estimate aquifer area (70 plue or minus 10 x 10(exp 3) square kilometers ) and water table depth (5-50 m). The perennial firn aquifer represents a new glacier facies to be considered 29 in future ice sheet mass 30 and energy budget calculations

The MassiveBlack-II simulation: the evolution of haloes and galaxies to z 0

(Abridged for arXiv)We investigate the properties of halos, galaxies and blackholes to z=0 in the high resolution hydrodynamical simulation MassiveBlack-II (MBII) which evolves a LCDM cosmology in a comoving volume Vbox=100(Mpc/h)^3. MBII is the highest resolution simulation of this size which includes a self-consistent model for star formation, black hole accretion and associated feedback. We provide a simulation browser web application which enables interactive search and tagging of halos, subhalos and their properties and publicly release our galaxy catalogs. Our analysis of the halo mass function (MF) in MBII reveals that baryons have strong effects, with changes in the halo abundance of 20-35% below the knee of the MF (Mhalo < 10^13.2 Msun/h at z=0) when compared to fits based on dark matter only simulations. We provide a fitting function for the halo MF out to redshift z=11 and discuss how the onset of non-universality in the MF limits the accuracy of our fit. We study the halo occupation distribution and clustering of galaxies, in particular the evolution and scale dependence of stochasticity and bias finding reasonable agreement with observational data. The shape of the cosmic spectral energy distribution predicted by MBII is consistent with observations, but lower in amplitude. The Galaxy Stellar Mass Function (GSMF) function is broadly consistent with observations at z>=2. At z<2, the population of passive low mass (for M*<10^9 Msun) galaxies in MBII makes the GSMF too steep compared to observations whereas at the high mass end (M*>10^11 Msun) galaxies hosting bright AGNs make significant contributions to the GSMF. The quasar bolometric luminosity function is also largely consistent with observations. We note however that more efficient AGN feedback (beyond simple thermal coupling used here) is likely necessary for the largest, rarest objects/clusters at low redshifts.Comment: 26 pages, 25 figures. Submitted to MNRAS. High-resolution version and MBII galaxy catalogs can be found at http://mbii.phys.cmu.edu/data