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.

Hepatic hemangiomas: Factors associated with T2 shine-through effect on diffusion-weighted MR sequences.

PURPOSE: To determine the frequency and factors associated with the presence of T2 shine-through effect in hepatic hemangiomas on diffusion-weighted (DW) magnetic resonance (MR) sequences. MATERIALS AND METHODS: This retrospective study was approved by institutional review board with waiver of informed consent. One hundred forty-nine consecutive patients with 388 hepatic hemangiomas who underwent a liver MR between January 2010 and November 2011 were included. MR analysis evaluated the lesion characteristics (signal intensities and enhancement patterns (classical, rapidly filling, delayed filling)), the presence of T2 shine-through effect on DW sequences (b values of 0, 150, and 600s/mm(2)), and apparent diffusion coefficient (ADC) values. Multivariate analysis was performed to study the factors associated with the T2 shine-through effect. RESULTS: T2 shine-through effect was observed in 204/388 (52.6%) of hepatic hemangiomas and in 100 (67.1%) patients. Mean ADC value of hemangiomas with T2 shine-through effect was significantly lower than hemangiomas without (2.0±0.48 vs 2.38±0.45, P<.0001). On multivariate analysis, high signal intensity on fat-suppressed T2-weighted fast spin-echo images, hemangiomas with classical or delayed enhancement, and the ADC of the liver were the only significant factors associated with T2 shine-through effect. CONCLUSION: T2 shine-through effect is commonly observed in hepatic hemangiomas and is related to hemangiomas characteristics. Radiologists should be aware of this phenomenon which could lead to misdiagnosis. Its presence should not question the diagnosis of hemangiomas when typical MR findings are found.

Implementation of raltegravir in routine clinical practice: selection criteria for choosing this drug, virologic response rates, and characteristics of failures.

BACKGROUND: Raltegravir (RAL) achieved remarkable virologic suppression rates in randomized-clinical trials, but today efficacy data and factors for treatment failures in a routine clinical care setting are limited. METHODS: First, factors associated with a switch to RAL were identified with a logistic regression including patients from the Swiss HIV Cohort Study with a history of 3 class failure (n = 423). Second, predictors for virologic outcome were identified in an intent-to-treat analysis including all patients who received RAL. Last observation carried forward imputation was used to determine week 24 response rate (HIV-1 RNA >or= 50 copies/mL). RESULTS: The predominant factor associated with a switch to RAL in patients with suppressed baseline RNA was a regimen containing enfuvirtide [odds ratio 41.9 (95% confidence interval: 11.6-151.6)]. Efficacy analysis showed an overall response rate of 80.9% (152/188), whereas 71.8% (84/117) and 95.8% (68/71) showed viral suppression when stratified for detectable and undetectable RNA at baseline, respectively. Overall CD4 cell counts increased significantly by 42 cells/microL (P < 0.001). Characteristics of failures were a genotypic sensitivity score of the background regimen <or=1, very low RAL plasma concentrations, poor adherence, and high viral load at baseline. CONCLUSIONS: Virologic suppression rates in our routine clinical care setting were promising and comparable with data from previously published randomized-controlled trials.

IL-17F co-expression improves cell growth characteristics and enhances recombinant protein production during CHO cell line engineering.

The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame.

Novel acid phosphatase in Candida glabrata suggests selective pressure and niche specialization in the phosphate signal transduction pathway.

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including Saccharomyces cerevisiae. However, C. glabrata still had phosphate starvation-inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three-gene family that contains a phosphomutase-like domain. This small-scale gene duplication event could allow for sub- or neofunctionalization. On the basis of phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate starvation-inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

Antecedents, psychiatric characteristics and follow-up of adolescents hospitalized for suicide attempt of overwhelming suicidal ideation.

OBJECTIVES: To evaluate the socio-demographic as well as the health and psychiatric profiles of adolescents hospitalised for suicide attempt or overwhelming suicide ideation and to assess repetition of suicide attempt over a period of 18 months. PATIENTS AND METHODS: Between April 2000 and September 2001, all patients aged 16 to 21 years admitted to the University Hospitals of Geneva and Lausanne for suicide attempt or ideation were included in the study. At this time (T0) semi-structured face to face interviews were conducted to identify socio-demographic data, mental health and antecedents regarding suicidal conducts. Current psychiatric status was assessed with the MINI (Mini International Neuropsychiatric Instrument). At T1 and T2, reassessments included psychiatric status (MINI) as well as lifestyles, socio-professional situation and suicidal behaviours. RESULTS: At T0, 269 subjects met the study criteria, among whom 83 subjects (56 girls and 27 boys) left the hospital too quickly to be involved or refused to participate in the study (final sample at T0: 149 girls; 37 boys). The participation rate at T1 and T2 was respectively 66% and 62% of the original sample. The percentage of adolescents meeting the criteria for psychiatric diagnoses (91%) was high: affective disorder (78%); anxiety disorder (64%); substance use disorder (39%); eating disorder (9%); psychotic disorder (11%); antisocial personality (7%) with most subjects (85%) having more than one disorder. Around 90% of the subjects interviewed at T1, and/or T2, had received follow-up care after their hospitalisation, either by a primary care physician or a psychotherapist or both. Two subjects died of violent death and 18% made a further suicide attempt. CONCLUSION: Most adolescents hospitalised for suicidal episodes suffer from psychiatric problems which should be addressed by a careful psychiatric assessment, followed up if needed by a structured after care plan.

Relationship of hyperactivity/inattention with adiposity and lifestyle characteristics in preschool children.

We performed a cross-sectional study in 450 nonreferred preschool children aged 4 to 6 years to assess the association between hyperactivity/inattention with adiposity and lifestyle characteristics. Measurements included scores of hyperactivity/inattention, adiposity, objectively measured physical activity, television viewing, and eating habits. Higher scores of hyperactivity/inattention were associated with lower percentage body fat, higher levels of physical activity, and less time spent in sedentary activity (all P ≤ .01). However, higher scores of hyperactivity/inattention were also associated with more television viewing and less healthy eating habits (all P ≤ .04). Except for some selected eating habits (P ≥ .07), those relationships remained significant after adjustment for age, sex, and sociodemographic confounders. To conclude, higher scores of hyperactivity/inattention are linked to different lifestyle characteristics that may in part contribute to a future development of overweight/obesity. Precise mechanisms explaining these associations and possible preventive approaches should be further investigated.

Proximate basis of the covariation between a melanin-based female ornament and offspring quality.

In contradiction to sexual selection theory, several studies showed that although the expression of melanin-based ornaments is usually under strong genetic control and weakly sensitive to the environment and body condition, they can signal individual quality. Covariation between a melanin-based ornament and phenotypic quality may result from pleiotropic effects of genes involved in the production of melanin pigments. Two categories of genes responsible for variation in melanin production may be relevant, namely those that trigger melanin production (yes or no response) and those that determine the amount of pigments produced. To investigate which of these two hypotheses is the most likely, I reanalysed data collected from barn owls ( Tyto alba). The underparts of this bird vary from immaculate to heavily marked with black spots of varying size. Published cross-fostering experiments have shown that the proportion of the plumage surface covered with black spots, a eumelanin composite trait so-called "plumage spottiness", in females positively covaries with offspring humoral immunocompetence, and negatively with offspring parasite resistance (i.show $132#e. the ability to reduce fecundity of ectoparasites) and fluctuating asymmetry of wing feathers. However, it is unclear which component of plumage spottiness causes these relationships, namely genes responsible for variation in number of spots or in spot diameter. Number of spots reflects variation in the expression of genes triggering the switch from no eumelanin production to production, whereas spot diameter reflects variation in the expression of genes determining the amount of eumelanin produced per spot. In the present study, multiple regression analyses, performed on the same data sets, showed that humoral immunocompetence, parasite resistance and wing fluctuating asymmetry of cross-fostered offspring covary with spot diameter measured in their genetic mother, but not with number of spots. This suggests that genes responsible for variation in the quantity of eumelanin produced per spot are responsible for covariation between a melanin ornament and individual attributes. In contrast, genes responsible for variation in number of black spots may not play a significant role. Covariation between a eumelanin female trait and offspring quality may therefore be due to an indirect effect of melanin production.

Investigation of the Reproductibility of Third-Level Characteristics

The process of comparing a fingermark recovered from a crime scene with the fingerprint taken from a known individual involves the characterization and comparison of different ridge details on both the mark and the print. Fingerprints examiners commonly classify these characteristics into three different groups, depending on their level of discriminating power. It is commonly considered that the general pattern of the ridge flow constitutes first-level detail, specific ridge flow and minutiaes (e.g. ending ridges, bifurcations) constitutes second-level detail, and fine ridge details (e. g. pore positions and shapes) are described as third-level details.In this study, the reproducibility of a selection of third-level characteristics is investigated. The reproducibility of these features is examined on serveral recordings of a same finger, first acquired using only optical visualization techniques and second on impressions developed using common firngermark development techniques. Prior to the evaluation of the reproducibility of the considered characteristics, digital images of the fingerprints were recorded at two different resolutions (1000 and 2000 ppi). This allowed the study to also examine the influence of higher resolution on the considered characteristics. It was observed that the increase in the resolution did not result in better feature detection or comparison between images.The examination of the reproducibility of a selection of third-level characteristics showed that the most reproducible features observed were minutiae shapes and pore positions along the ridges.

Improvement of cardiovascular risk prediction: time to review current knowledge, debates, and fundamentals on how to assess test characteristics.

Cardiovascular risk assessment might be improved with the addition of emerging, new tests derived from atherosclerosis imaging, laboratory tests or functional tests. This article reviews relative risk, odds ratios, receiver-operating curves, posttest risk calculations based on likelihood ratios, the net reclassification improvement and integrated discrimination. This serves to determine whether a new test has an added clinical value on top of conventional risk testing and how this can be verified statistically. Two clinically meaningful examples serve to illustrate novel approaches. This work serves as a review and basic work for the development of new guidelines on cardiovascular risk prediction, taking into account emerging tests, to be proposed by members of the 'Taskforce on Vascular Risk Prediction' under the auspices of the Working Group 'Swiss Atherosclerosis' of the Swiss Society of Cardiology in the future.

Estimation of the noisy component of anatomical backgrounds.

The knowledge of the relationship that links radiation dose and image quality is a prerequisite to any optimization of medical diagnostic radiology. Image quality depends, on the one hand, on the physical parameters such as contrast, resolution, and noise, and on the other hand, on characteristics of the observer that assesses the image. While the role of contrast and resolution is precisely defined and recognized, the influence of image noise is not yet fully understood. Its measurement is often based on imaging uniform test objects, even though real images contain anatomical backgrounds whose statistical nature is much different from test objects used to assess system noise. The goal of this study was to demonstrate the importance of variations in background anatomy by quantifying its effect on a series of detection tasks. Several types of mammographic backgrounds and signals were examined by psychophysical experiments in a two-alternative forced-choice detection task. According to hypotheses concerning the strategy used by the human observers, their signal to noise ratio was determined. This variable was also computed for a mathematical model based on the statistical decision theory. By comparing theoretical model and experimental results, the way that anatomical structure is perceived has been analyzed. Experiments showed that the observer's behavior was highly dependent upon both system noise and the anatomical background. The anatomy partly acts as a signal recognizable as such and partly as a pure noise that disturbs the detection process. This dual nature of the anatomy is quantified. It is shown that its effect varies according to its amplitude and the profile of the object being detected. The importance of the noisy part of the anatomy is, in some situations, much greater than the system noise. Hence, reducing the system noise by increasing the dose will not improve task performance. This observation indicates that the tradeoff between dose and image quality might be optimized by accepting a higher system noise. This could lead to a better resolution, more contrast, or less dose.

Characteristics and early and long-term outcome in patients with acute ischemic stroke and low ejection fraction.

OBJECTIVES: We assessed the clinical characteristics of patients with acute ischemic stroke (AIS) with left ventricular ejection fraction (EF) ≤ 35% and investigated the association of low EF with early and long-term outcome. METHODS: A total of 2439 patients of the Acute Stroke Registry and Analysis of Lausanne (ASTRAL) were selected. Demographics, risk factors, pre-stroke treatment, and clinical, radiological and metabolic variables in patients with and without low EF were compared. Functional independence (modified Rankin Score ≤ 2) and mortality were recorded 1 week up to 12 months from admission. RESULTS: Low EF patients (n=119) were more commonly men, older, had higher rates of coronary artery disease and atrial fibrillation (AF), and more frequent pretreatment with anticoagulants, antiplatelets and antihypertensive agents. On admission, they presented with higher stroke severity and had lower values of systolic blood pressure, higher heart rate, and worse estimated glomerular filtration rate. Stroke-related disability and death rates were higher in low EF patients during follow-up (19.5% vs. 7.8% at 1 week, and 36.1% vs. 16.5% at 12 months). Increasing age, stroke severity, and AF were independent predictors of one-year mortality in these patients while prior use of statins had a favorable effect on early mortality. CONCLUSIONS: AIS in patients with low EF is associated with older age, cardiac comorbidities, and more severe clinical presentation. Low EF can identify a subset of AIS patients at high risk of early and long-term functional disability and mortality.

Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.

Acute opiate overdose: characteristics of 190 consecutive cases.

AIMS: To characterize the population of drug users consulting the Emergency Room (ER) of a university hospital with acute opiate overdose (AOO) and to assess rate of referral to specialized treatment programme. DESIGN: Survey of a 12-month sample of AOO patients. MEASUREMENTS: Medical and psychosocial features of the drug users, details of emergency treatment and referral by a mobile resuscitation team (SMUR) and the ER of our hospital (CHUV-Lausanne, Switzerland). In addition fatal AOO cases were collected by the Institute of Forensic Medicine (IFM) during the same period. FINDINGS: One hundred and eighty-four cases of AOO (134 patients) were treated. The files of the IFM detailed six additional deceased cases. This population of drug users was characterized by an over-representation of men (73%), by young age (27.4 years), by a high rate of multi-drugs use (90%) and by a high rate of multiple previous overdoses (2.6). Average length of stay was 20.1 hours but 41% of cases stayed less than 8 hours. Only one patient was readmitted within an 8-hour period. When discharged, 78% returned home. Unexpectedly, 67% of patients were not referred to any therapeutic programme for drug addiction. CONCLUSION: This study shows the low mortality of AOO when treated but also demonstrates the need to improve psychosocial evaluation and referral of drug addicts admitted with AOO.