20 research outputs found
Multivariate methods for the statistical analysis of hyperdimensional high-content screening data
No full textThesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2014.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.In the post-genomic era, greater emphasis has been placed on understanding the function of genes at the systems level. To meet these needs, biologists are creating larger, and increasingly complex datasets. In recent years, high-content screening (HCS) using RNA interference (RNAi) or other perturbation techniques in combination with automated microscopy has emerged as a promising investigative tool to explore intricate biological processes. Image-based HC screens produce massive hyperdimensional data sets. To identify novel components of the DNA damage response (DDR) after ionizing radiation, we recently performed an image-based HC RNAi screen in an osteosarcoma cell line. Robust univariate hit identication methods and manual network analysis identied an isoform of BRD4, a bromodomain and extra-terminal domain family member, as an endogenous inhibitor of DDR signaling. However, despite the plethora of data generated from our and other HC screens, little progress has been made in analyzing HC data using multivariate computational methods that exploit the full richness of hyperdimensional data and identify more than just the most salient knockdown phenotypes to gain a detailed understanding of how gene products cooperate to regulate complex cellular processes. We developed a novel multivariate method using logistic regression models and least absolute shrinkage and selection operator regularization for analyzing hyperdimensional HC data. We applied this method to our HC screen to identify genes that exhibit subtle but consistent phenotypic changes upon knockdown that would have been missed by conventional univariate hit identication approaches. Our method automatically selects the most predictive features at the most predictive time points to facilitate the more ecient design of follow-up experiments and puts the identied hits in a network context using the Prize-Collecting Steiner Tree algorithm. This method offers superior performance over the current gold standard for the analysis of HC RNAi screens. A surprising finding from our analysis is that training sets of genes involved in complex biological phenomena used to train predictive models must be broken down into functionally coherent subsets in order to enhance new gene discovery. Additionally, we found that in the case of RNAi screening, statistical cell-to-cell variation in phenotypic responses in a well of cells targeted by a single shRNA is an important predictor of gene dependent events.by Jonathan Rameseder.Ph. D
Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer
No full textDespite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80–90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.American Society for Radiation Oncology (Junior Faculty Career Research Training Award)Harvard University. Joint Center for Radiation Therapy (Foundation Grant)Dana-Farber/Harvard Cancer Center (SPORE Developmental Research Project Award in Lung Cancer Research)National Cancer Institute (U.S.) (Award K08CA172354
The Proteomics Identifications database: 2010 update
No full textThe Proteomics Identifications database (PRIDE, http://www.ebi.ac.uk/pride) at the European Bioinformatics Institute has become one of the main repositories of mass spectrometry-derived proteomics data. For the last 2 years, PRIDE data holdings have grown substantially, comprising 60 different species, more than 2.5 million protein identifications, 11.5 million peptides and over 50 million spectra by September 2009. We here describe several new and improved features in PRIDE, including the revised submission process, which now includes direct submission of fragment ion annotations. Correspondingly, it is now possible to visualize spectrum fragmentation annotations on tandem mass spectra, a key feature for compliance with journal data submission requirements. We also describe recent developments in the PRIDE BioMart interface, which now allows integrative queries that can join PRIDE data to a growing number of biological resources such as Reactome, Ensembl, InterPro and UniProt. This ability to perform extremely powerful across-domain queries will certainly be a cornerstone of future bioinformatics analyses. Finally, we highlight the importance of data sharing in the proteomics field, and the corresponding integration of PRIDE with other databases in the ProteomExchange consortium.European Union (ProDaC grant LSHG-CT-2006-036814)Burroughs Wellcome Fund (Grant WT085949MA
The Applicability of Functional Data in Multi-Unit Auctions
No full textThe present thesis aims to contribute to the literature on functional data anal-
ysis (FDA), in particular the prediction challenge in multi-unit auctions with pay-as-bid pricing. It demonstrates that FDA provides a beneficial toolkit
for empirical questions in relation to auctions by applying and extending FDA
methods. All the chapters of this work deal with real auction data collected and
preprocessed by the author. While analyzing the data, fundamental problems
related to the applicability and interpretability are generalized and investigated.
In this context and always motivated by real data, the thesis makes empirical
but also methodological contributions
Pride-asap: automatic fragment ion annotation of identified PRIDE spectra
No full textAbstractWe present an open source software application and library written in Java that provides a uniform annotation of identified spectra stored in the PRIDE database. Pride-asap can be ran in a command line mode for automated processing of multiple PRIDE experiments, but also has a graphical user interface that allows end users to annotate the spectra in PRIDE experiments and to inspect the results in detail. Pride-asap binaries, source code and additional information can be downloaded from http://pride-asa-pipeline.googlecode.com.This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics
The mTOR-Regulated Phosphoproteome Reveals a Mechanism of mTORC1-Mediated Inhibition of Growth Factor Signaling
No full textSeptember 21 Author ManuscriptThe mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.National Institutes of Health (U.S.) (Grant CA103866)National Institutes of Health (U.S.) (Grant AI47389)United States. Department of Defense (W81XWH-07-0448)W. M. Keck FoundationLAM FoundationAmerican Cancer SocietyHoward Hughes Medical Institute (Investigator
Molecular brakes regulating mTORC1 activation in skeletal muscle following synergist ablation
No full textThe goal of the current work was to profile positive (mTORC1 activation, autocrine/paracrine growth factors) and negative [AMPK, unfolded protein response (UPR)] pathways that might regulate overload-induced mTORC1 activation with the hypothesis that a number of negative regulators of mTORC1 will be engaged during a supra-physiological model of hypertrophy. To achieve this, mTORC1-IRS1/2 signaling, BiP/CHOP/IRE1a, and AMPK activation were determined in rat plantaris muscle following synergist ablation (SA). SA resulted in significant increases in muscle mass of ~4% per day throughout the 21 days of the experiment. The expression of the insulin-like growth factors were high throughout the 21d of overload. However, IGF signaling was limited since IRS1 and 2 were undetectable in the overloaded muscle from day 3 to day 9. The decreases in IRS1/2 protein were paralleled by increases in GRB10(Ser501/503) and S6K1(Thr389) phosphorylation, two mTORC1 targets that can destabilize IRS proteins. PKB(Ser473) phosphorylation was higher from 3-6 days and this was associated with increased TSC2(Thr939) phosphorylation. The phosphorylation of TSC2(Thr1345) (an AMPK site) was also elevated whereas phosphorylation at the other PKB site, Thr(1462), was unchanged at 6d. In agreement with the phosphorylation of Thr(1345), synergist ablation led to activation of a1-AMPK during the initial growth phase, lasting the first 9 days before returning to baseline by day 12. The UPR markers CHOP and BiP were elevated over the first 12 days following ablation, whereas IRE1a levels decreased. These data suggest that during supra-physiological muscle loading, at least three potential molecular brakes engage to down-regulate mTORC1
The bromodomain protein Brd4 insulates chromatin from DNA damage signalling
No full textDNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.David H. Koch Institute for Integrative Cancer Research at MITNational Institutes of Health (U.S.) (National Institute of Environmental Health Sciences Core Grant P30CA14051)National Institutes of Health (U.S.) (National Institute of Environmental Health Sciences Core Grant ES-002109)R01-ES153391-U54-CA112967-04R21-NS063917SPARC GrantAmerican Board of Radiology (Holman Pathway Research Resident Seed Grant)American Society for Radiation Oncology (Junior Faculty Research Training Award
Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome
No full textSystematic phosphorylation analysis of human mitotic protein complexes.
No full textProgression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1
