Integrated methodologies to study human transcriptional regulation from functional genomics data

Abstract : The human body is composed of a huge number of cells acting together in a concerted manner. The current understanding is that proteins perform most of the necessary activities in keeping a cell alive. The DNA, on the other hand, stores the information on how to produce the different proteins in the genome. Regulating gene transcription is the first important step that can thus affect the life of a cell, modify its functions and its responses to the environment. Regulation is a complex operation that involves specialized proteins, the transcription factors. Transcription factors (TFs) can bind to DNA and activate the processes leading to the expression of genes into new proteins. Errors in this process may lead to diseases. In particular, some transcription factors have been associated with a lethal pathological state, commonly known as cancer, associated with uncontrolled cellular proliferation, invasiveness of healthy tissues and abnormal responses to stimuli. Understanding cancer-related regulatory programs is a difficult task, often involving several TFs interacting together and influencing each other's activity. This Thesis presents new computational methodologies to study gene regulation. In addition we present applications of our methods to the understanding of cancer-related regulatory programs. The understanding of transcriptional regulation is a major challenge. We address this difficult question combining computational approaches with large collections of heterogeneous experimental data. In detail, we design signal processing tools to recover transcription factors binding sites on the DNA from genome-wide surveys like chromatin immunoprecipitation assays on tiling arrays (ChIP-chip). We then use the localization about the binding of TFs to explain expression levels of regulated genes. In this way we identify a regulatory synergy between two TFs, the oncogene C-MYC and SP1. C-MYC and SP1 bind preferentially at promoters and when SP1 binds next to C-NIYC on the DNA, the nearby gene is strongly expressed. The association between the two TFs at promoters is reflected by the binding sites conservation across mammals, by the permissive underlying chromatin states 'it represents an important control mechanism involved in cellular proliferation, thereby involved in cancer. Secondly, we identify the characteristics of TF estrogen receptor alpha (hERa) target genes and we study the influence of hERa in regulating transcription. hERa, upon hormone estrogen signaling, binds to DNA to regulate transcription of its targets in concert with its co-factors. To overcome the scarce experimental data about the binding sites of other TFs that may interact with hERa, we conduct in silico analysis of the sequences underlying the ChIP sites using the collection of position weight matrices (PWMs) of hERa partners, TFs FOXA1 and SP1. We combine ChIP-chip and ChIP-paired-end-diTags (ChIP-pet) data about hERa binding on DNA with the sequence information to explain gene expression levels in a large collection of cancer tissue samples and also on studies about the response of cells to estrogen. We confirm that hERa binding sites are distributed anywhere on the genome. However, we distinguish between binding sites near promoters and binding sites along the transcripts. The first group shows weak binding of hERa and high occurrence of SP1 motifs, in particular near estrogen responsive genes. The second group shows strong binding of hERa and significant correlation between the number of binding sites along a gene and the strength of gene induction in presence of estrogen. Some binding sites of the second group also show presence of FOXA1, but the role of this TF still needs to be investigated. Different mechanisms have been proposed to explain hERa-mediated induction of gene expression. Our work supports the model of hERa activating gene expression from distal binding sites by interacting with promoter bound TFs, like SP1. hERa has been associated with survival rates of breast cancer patients, though explanatory models are still incomplete: this result is important to better understand how hERa can control gene expression. Thirdly, we address the difficult question of regulatory network inference. We tackle this problem analyzing time-series of biological measurements such as quantification of mRNA levels or protein concentrations. Our approach uses the well-established penalized linear regression models where we impose sparseness on the connectivity of the regulatory network. We extend this method enforcing the coherence of the regulatory dependencies: a TF must coherently behave as an activator, or a repressor on all its targets. This requirement is implemented as constraints on the signs of the regressed coefficients in the penalized linear regression model. Our approach is better at reconstructing meaningful biological networks than previous methods based on penalized regression. The method is tested on the DREAM2 challenge of reconstructing a five-genes/TFs regulatory network obtaining the best performance in the "undirected signed excitatory" category. Thus, these bioinformatics methods, which are reliable, interpretable and fast enough to cover large biological dataset, have enabled us to better understand gene regulation in humans.

The development and pilot-testing of a training curriculum in adolescent medicine and health.

PURPOSE: To select and propose a set of knowledge, attitudes, and skills essential for the care of adolescents; to encourage the development of adolescent health multidisciplinary networks; and to set up training programs in as many European countries as possible. METHODS: The curriculum was developed by 16 physicians from 11 European countries with various professional specializations. In line with modern guidelines in medical education, it is a modular, flexible instrument which covers the main teaching areas in the field, such as basic skills (i.e. setting, rights and confidentiality, gender and cultural issues) as well as specific themes (i.e. sexual and reproductive health, eating disorders, chronic conditions). It consists of 17 thematic modules, each containing detailed objectives, learning approaches, examples, and evaluation methods. RESULT: Two international one-week summer schools were used to assess the feasibility and appropriateness of the curriculum. The overall evaluation was good, with most of the items surpassing three on a four-point Likert scale. However, it pointed to several aspects (process and content) which will need to be refined in the future, such as an increase in interactive sessions (role playing), and a better mix of clinical and public health issues.

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing.

Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.

Angelo Panebiancos institutionaliseringshypotes: testing i en nordisk kontext

Abstract: Angelo Panebianco's institutionalizationhypothesis : testing in nordic context

Integrated Ladinian bio-chronostratigraphy and geochrononology of Monte San Giorgio (Southern Alps, Switzerland)

New biostratigraphic data significantly improve the age assignment of the Ladinian succession of Monte San Giorgio (UNESCO World Heritage List site, Southern Alps, Switzerland), whose world-famous fossil marine vertebrate faunas are now dated to the substage and zone levels. High-resolution single-zircon U-Pb dating was performed using ID-TIMS and chemical abrasion (CA) pre-treatment technique on volcanic ash layers intercalated in the biostratigraphically-defined intervals of the Meride Limestone. It yielded ages of 241.07 +/- 0.13 Ma (Cava superiore beds, P. gredleri Zone), 240.63 +/- 0.13 Ma (Cassina beds, P gredleri/P. archelaus transition Zone) and 239.51 +/- 0.15 Ma (Lower Kalkschieferzone, P. archelaus Zone). Our results suggest that the time interval including the vertebrate-bearing Middle Triassic section spans around 4 Myr and is thus significantly shorter than so far assumed. The San Giorgio Dolomite and the Meride Limestone correlate with intervals of the Buchenstein Formation and the Wengen Formation in the reference section at Bagolino, where the Global boundary Stratotype Section and Point (GSSP) for the base of the Ladinian was defined. The new radio-isotopic ages of the Meride Limestone are up to 2 Myr older than those published for the biostratigraphically-equivalent intervals at Bagolino but they are consistent with the recent re-dating of the underlying Besano Formation, also performed using the CA technique. Average sedimentation rates at Monte San Giorgio are by more than an order of magnitude higher compared to those assumed for the Buchenstein Formation, which formed under sediment-starved pelagic conditions, and reflect prevailing high subsidence and high carbonate mud supply from the adjoining Salvatore/Esino platforms. Finally, the high-resolution U-Pb ages allow a correlation of the vertebrate faunas of the Cava superiore/Cava inferiore beds with the marine vertebrate record of the Prosanto Formation (Upper Austroalpine), so far precluded by the poor biostratigraphic control of the latter.

SARM-S4 and metabolites detection in sports drug testing: a case report.

Recently, pharmaceutical industry developed a new class of therapeutics called Selective Androgen Receptor Modulator (SARM) to substitute the synthetic anabolic drugs used in medical treatments. Since the beginning of the anti-doping testing in sports in the 1970s, steroids have been the most frequently detected drugs mainly used for their anabolic properties. The major advantage of SARMs is the reduced androgenic activities which are the main source of side effects following anabolic agents' administration. In 2010, the Swiss laboratory for doping analyses reported the first case of SARMs abuse during in-competition testing. The analytical steps leading to this finding are described in this paper. Screening and confirmation results were obtained based on liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. Additional information regarding the SARM S-4 metabolism was investigated by ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS).

Final report on the Portable Weather Station, March 2012

This station was required to have air temperature, relative humidity, wind speed and direction, and pavement temperature sensors of similar quality to the traditional RWIS sensors, have an integrated solar powered battery system, and be trailer-mounted for ease of transport. The station was tested by the Iowa DOT for basic reliability and sensor performance for a month and a half in Ames, Iowa before being moved to near Osceola, Iowa in early February 2010 for further field testing and evaluation. DOT field maintenance staff was able to successfully set up the station with minimal instruction and found the station to be relatively intuitive in its installation. Air temperature, wind speed, and wind direction observations from the station were compared to a nearby RWIS station and had good agreement. Pavement temperature readings were compared to sites within 40 miles of the station, and the readings correlated. The station has had good reliability.

Development of a Database for Drilled Shaft Foundation Testing (DSHAFT), June 2012

Drilled shafts have been used in the US for more than 100 years in bridges and buildings as a deep foundation alternative. For many of these applications, the drilled shafts were designed using the Working Stress Design (WSD) approach. Even though WSD has been used successfully in the past, a move toward Load Resistance Factor Design (LRFD) for foundation applications began when the Federal Highway Administration (FHWA) issued a policy memorandum on June 28, 2000.The policy memorandum requires all new bridges initiated after October 1, 2007, to be designed according to the LRFD approach. This ensures compatibility between the superstructure and substructure designs, and provides a means of consistently incorporating sources of uncertainty into each load and resistance component. Regionally-calibrated LRFD resistance factors are permitted by the American Association of State Highway and Transportation Officials (AASHTO) to improve the economy and competitiveness of drilled shafts. To achieve this goal, a database for Drilled SHAft Foundation Testing (DSHAFT) has been developed. DSHAFT is aimed at assimilating high quality drilled shaft test data from Iowa and the surrounding regions, and identifying the need for further tests in suitable soil profiles. This report introduces DSHAFT and demonstrates its features and capabilities, such as an easy-to-use storage and sharing tool for providing access to key information (e.g., soil classification details and cross-hole sonic logging reports). DSHAFT embodies a model for effective, regional LRFD calibration procedures consistent with PIle LOad Test (PILOT) database, which contains driven pile load tests accumulated from the state of Iowa. PILOT is now available for broader use at the project website: http://srg.cce.iastate.edu/lrfd/. DSHAFT, available in electronic form at http://srg.cce.iastate.edu/dshaft/, is currently comprised of 32 separate load tests provided by Illinois, Iowa, Minnesota, Missouri and Nebraska state departments of transportation and/or department of roads. In addition to serving as a manual for DSHAFT and providing a summary of the available data, this report provides a preliminary analysis of the load test data from Iowa, and will open up opportunities for others to share their data through this quality–assured process, thereby providing a platform to improve LRFD approach to drilled shafts, especially in the Midwest region.

A novel in vitro metabolomics approach for neurotoxicity testing, proof of principle for methyl mercury chloride and caffeine

There is a need for more efficient methods giving insight into the complex mechanisms of neurotoxicity. Testing strategies including in vitro methods have been proposed to comply with this requirement. With the present study we aimed to develop a novel in vitro approach which mimics in vivo complexity, detects neurotoxicity comprehensively, and provides mechanistic insight. For this purpose we combined rat primary re-aggregating brain cell cultures with a mass spectrometry (MS)-based metabolomics approach. For the proof of principle we treated developing re-aggregating brain cell cultures for 48h with the neurotoxicant methyl mercury chloride (0.1-100muM) and the brain stimulant caffeine (1-100muM) and acquired cellular metabolic profiles. To detect toxicant-induced metabolic alterations the profiles were analysed using commercial software which revealed patterns in the multi-parametric dataset by principal component analyses (PCA), and recognised the most significantly altered metabolites. PCA revealed concentration-dependent cluster formations for methyl mercury chloride (0.1-1muM), and treatment-dependent cluster formations for caffeine (1-100muM) at sub-cytotoxic concentrations. Four relevant metabolites responsible for the concentration-dependent alterations following methyl mercury chloride treatment could be identified using MS-MS fragmentation analysis. These were gamma-aminobutyric acid, choline, glutamine, creatine and spermine. Their respective mass ion intensities demonstrated metabolic alterations in line with the literature and suggest that the metabolites could be biomarkers for mechanisms of neurotoxicity or neuroprotection. In addition, we evaluated whether the approach could identify neurotoxic potential by testing eight compounds which have target organ toxicity in the liver, kidney or brain at sub-cytotoxic concentrations. PCA revealed cluster formations largely dependent on target organ toxicity indicating possible potential for the development of a neurotoxicity prediction model. With such results it could be useful to perform a validation study to determine the reliability, relevance and applicability of this approach to neurotoxicity screening. Thus, for the first time we show the benefits and utility of in vitro metabolomics to comprehensively detect neurotoxicity and to discover new biomarkers.

Report on a review of selected general and application controls over the Iowa Department of Administrative Service’s Human Resource Information System (HRIS), Payroll System and Integrated Information for Iowa System (I/3) for the period March 26, 2012 through April 27, 2012

Report on a review of selected general and application controls over the Iowa Department of Administrative Service’s Human Resource Information System (HRIS), Payroll System and Integrated Information for Iowa System (I/3) for the period March 26, 2012 through April 27, 2012

Demonstration of Load Rating Capabilities through Physical Load Testing: Sioux County Bridge Case Study, RB32-013, 2013

The objective of this work, Pilot Project - Demonstration of Capabilities and Benefits of Bridge Load Rating through Physical Testing, was to demonstrate the capabilities for load testing and rating bridges in Iowa, study the economic benefit of performing such testing, and perform outreach to local, state, and national engineers on the topic of bridge load testing and rating. This report documents one of three bridges inspected, load tested, and load rated as part of the project, the Sioux County Bridge (FHWA #308730), including testing procedures and performance of the bridge under static loading along with the calculated load rating from the field-calibrated analytical model. Two parallel reports document the testing and load rating of the Ida County Bridge (FHWA #186070) and the Johnson County Bridge (FHWA #205750). A tech brief provides overall information about the project.

Demonstration of Load Rating Capabilities through Physical Load Testing: Ida County Bridge Case Study, RB32-013, 2013

The objective of this work, Pilot Project - Demonstration of Capabilities and Benefits of Bridge Load Rating through Physical Testing, was to demonstrate the capabilities for load testing and rating bridges in Iowa, study the economic benefit of performing such testing, and perform outreach to local, state, and national engineers on the topic of bridge load testing and rating. This report documents one of three bridges inspected, load tested, and load rated as part of the project, the Ida County Bridge (FHWA #186070), including testing procedures and performance of the bridge under static loading along with the calculated load rating from the field-calibrated analytical model. Two parallel reports document the testing and load rating of the Sioux County Bridge (FHWA #308730) and the Johnson County Bridge (FHWA #205750). A tech brief provides overall information about the project.

Demonstration of Load Rating Capabilities through Physical Load Testing: Johnson County Bridge Case Study, RB32-013, 2013

The objective of this work, Pilot Project - Demonstration of Capabilities and Benefits of Bridge Load Rating through Physical Testing, was to demonstrate the capabilities for load testing and rating bridges in Iowa, study the economic benefit of performing such testing, and perform outreach to local, state, and national engineers on the topic of bridge load testing and rating. This report documents one of three bridges inspected, load tested, and load rated as part of the project, the Johnson County Bridge (FHWA #205750), including testing procedures and performance of the bridge under static loading along with the calculated load rating from the field-calibrated analytical model. Two parallel reports document the testing and load rating of the Sioux County Bridge (FHWA #308730) and the Ida County Bridge (FHWA #186070). A tech brief provides overall information about the project.

Pilot Project - Demonstration of Load Rating Capabilities through Physical Load Testing: Tech Transfer Summary, RB32-013, 2013

This project demonstrated the capabilities for load testing bridges in Iowa, developed and presented a webinar to local and state engineers, and produced a spreadsheet and benefit evaluation matrix that others can use to preliminarily assess where bridge testing may be economically feasible given truck traffic and detour lengths.