Evolution of mRNA expression levels of autosomal and sex-linked genes in amniotes

In addition to differences in protein-coding gene sequences, changes in expression resulting from mutations in regulatory sequences have long been hypothesized to be responsible for phenotypic differences between species. However, unlike comparison of genome sequences, few studies, generally restricted to pairwise comparisons of closely related mammalian species, have assessed between-species differences at the transcriptome level. They reported that gene expression evolves at different rates in various organs and in a pattern that is overall consistent with neutral models of evolution. In the first part of my thesis, I investigated the evolution of gene expression in therian mammals (i.e.7 placental and marsupials), based on microarray data from human, mouse and the gray short-tailed opossum (Monodelphis domestica). In addition to autosomal genes, a special focus was given to the evolution of X-linked genes. The therian X chromosome was recently shown to be younger than previously thought and to harbor a specific gene content (e.g., genes involved in brain or reproductive functions) that is thought to have been shaped by specific sex-related evolutionary forces. Sex chromosomes derive from ordinary autosomes and their differentiation led to the degeneration of the Y chromosome (in mammals) or W chromosome (in birds). Consequently, X- or Z-linked genes differ in gene dose between males and females such that the heterogametic sex has half the X/Z gene dose compared to the ancestral state. To cope with this dosage imbalance, mammals have been reported to have evolved mechanisms of dosage compensation.¦In the first project, I could first show that transcriptomes evolve at different rates in different organs. Out of the five tissues I investigated, the testis is the most rapidly evolving organ at the gene expression level while the brain has the most conserved transcriptome. Second, my analyses revealed that mammalian gene expression evolution is compatible with a neutral model, where the rates of change in gene expression levels is linked to the efficiency of purifying selection in a given lineage, which, in turn, is determined by the long-term effective population size in that lineage. Thus, the rate of DNA sequence evolution, which could be expected to determine the rate of regulatory sequence change, does not seem to be a major determinant of the rate of gene expression evolution. Thus, most gene expression changes seem to be (slightly) deleterious. Finally, X-linked genes seem to have experienced elevated rates of gene expression change during the early stage of X evolution. To further investigate the evolution of mammalian gene expression, we generated an extensive RNA-Seq gene expression dataset for nine mammalian species and a bird. The analyses of this dataset confirmed the patterns previously observed with microarrays and helped to significantly deepen our view on gene expression evolution.¦In a specific project based on these data, I sought to assess in detail patterns of evolution of dosage compensation in amniotes. My analyses revealed the absence of male to female dosage compensation in monotremes and its presence in marsupials and, in addition, confirmed patterns previously described for placental mammals and birds. I then assessed the global level of expression of X/Z chromosomes and contrasted this with its ancestral gene expression levels estimated from orthologous autosomal genes in species with non-homologous sex chromosomes. This analysis revealed a lack of up-regulation for placental mammals, the level of expression of X-linked genes being proportional to gene dose. Interestingly, the ancestral gene expression level was at least partially restored in marsupials as well as in the heterogametic sex of monotremes and birds. Finally, I investigated alternative mechanisms of dosage compensation and found that gene duplication did not seem to be a widespread mechanism to restore the ancestral gene dose. However, I could show that placental mammals have preferentially down-regulated autosomal genes interacting with X-linked genes which underwent gene expression decrease, and thus identified a novel alternative mechanism of dosage compensation.

Growth decrease in Alpine whitefish: investigating the relative contribution of fishing induced selection and environmental change and its implication for conservation measures

La pression exercée par les activités humaines menace pratiquement tous les écosystèmes aquatiques du globe. Ainsi, sous l'effet de divers facteurs tels que la pollution, le réchauffement climatique ou encore la pêche industrielle, de nombreuses populations de poissons ont vu leurs effectifs chuter et divers changements morphologiques ont été observés. Dans cette étude, nous nous sommes intéressés à une menace particulière: la sélection induite par la pêche sur la croissance des poissons. En effet, la génétique des populations prédit que la soustraction régulière des individus les plus gros peut entraîner des modifications rapides de certains traits physiques comme la croissance individuelle. Cela a par ailleurs été observé dans de nombreuses populations marines ou lacustres, dont les populations de féras, bondelles et autres corégones des lacs suisses. Toutefois, malgré un nombre croissant d'études décrivant ce phénomène, peu de plans de gestion en tiennent compte, car l'importance des effets génétiques liés à la pêche est le plus souvent négligée par rapport à l'impact des changements environnementaux. Le but premier de cette étude a donc été de quantifier l'importance des facteurs génétiques et environnementaux. Dans le premier chapitre, nous avons étudié la population de palée du lac de Joux (Coregonus palaea). Nous avons déterminé les différentiels de sélection dus à la pêche, c'est-à-dire l'intensité de la sélection sur le taux de croissance, ainsi que les changements nets de croissance au cours du temps. Nous avons observé une baisse marquée de croissance et un différentiel de sélection important indiquant qu'au moins 30% de la diminution de croissance observée était due à la pression de sélection induite par la pêche. Dans le deuxième chapitre, nous avons effectué les mêmes analyses sur deux espèces proches du lac de Brienz (C. albellus et C. fatioi) et avons observé des effets similaires dont l'intensité était spécifique à chaque espèce. Dans le troisième chapitre, nous avons analysé deux autres espèces : C. palaea et C. confusus du lac de Bienne, et avons constaté que le lien entre la pression de sélection et la diminution de croissance était influencé par des facteurs environnementaux. Finalement, dans le dernier chapitre, nous avons étudié les effets potentiels de différentes modifications de la taille des mailles des filets utilisés pour la pêche à l'aide de modèles mathématiques. Nous concluons que la pêche a un effet génétique non négligeable (et donc peu réversible) sur la croissance individuelle dans les populations observée, que cet effet est lié à la compétition pour la nourriture et à la qualité de l'environnement, et que certaines modifications simples de la taille des mailles des filets de pêche pourraient nettement diminuer l'effet de sélection et ainsi ralentir, voir même renverser la diminution de croissance observée.

Fast quantification of ten psychotropic drugs and metabolites in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A sensitive and selective ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was developed for the fast quantification of ten psychotropic drugs and metabolites in human plasma for the needs of our laboratory (amisulpride, asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, norquetiapine, olanzapine, paliperidone, quetiapine and risperidone). Stable isotope-labeled internal standards were used for all analytes, to compensate for the global method variability, including extraction and ionization variations. Sample preparation was performed by generic protein precipitation with acetonitrile. Chromatographic separation was achieved in less than 3.0min on an Acquity UPLC BEH Shield RP18 column (2.1mm×50mm; 1.7μm), using a gradient elution of 10mM ammonium formate buffer pH 3.0 and acetonitrile at a flow rate of 0.4ml/min. The compounds were quantified on a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The method was fully validated according to the latest recommendations of international guidelines. Eight point calibration curves were used to cover a large concentration range 0.5-200ng/ml for asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, olanzapine, paliperidone and risperidone, and 1-1500ng/ml for amisulpride, norquetiapine and quetiapine. Good quantitative performances were achieved in terms of trueness (93.1-111.2%), repeatability (1.3-8.6%) and intermediate precision (1.8-11.5%). Internal standard-normalized matrix effects ranged between 95 and 105%, with a variability never exceeding 6%. The accuracy profiles (total error) were included in the acceptance limits of ±30% for biological samples. This method is therefore suitable for both therapeutic drug monitoring and pharmacokinetic studies.

Reproducibility and relative validity of a food-frequency questionnaire for French-speaking Swiss adults.

BACKGROUND: Due to the distinct cultural and language differences that exist in Switzerland, there is little information on the dietary intake among the general Swiss population. Adequately assessing dietary intake is thus paramount if nutritional epidemiological studies are to be conducted. OBJECTIVE: To assess the reproducibility and validity of a food-frequency questionnaire (FFQ) developed for French-speaking Swiss adults. DESIGN: A total of 23 men and 17 women (43.1+/-2.0 years) filled out one FFQ and completed one 24-hour dietary recall at baseline and 1 month afterward. RESULTS: Crude Pearson's correlation coefficients between the first and the second FFQ ranged from 0.58 to 0.90, intraclass correlation coefficient (ICC) ranged between 0.53 and 0.92. Lin's concordance coefficients ranged between 0.55 and 0.87. Over 80% of participants were classified in the same or adjacent tertile using each FFQ. Macronutrient intakes estimated by both FFQs were significantly higher than those estimated from the 24-hour recall for protein and water, while no significant differences were found for energy, carbohydrate, fats (five groups), and alcohol. De-attenuated Pearson's correlation coefficients between the 24-hour recall and the first FFQ ranged between 0.31 and 0.49, while for the second FFQ the values ranged between 0.38 and 0.59. Over 40 and 95% of participants fell into the same or the adjacent energy and nutrient tertiles, respectively, using the FFQs and the 24-hour recall. CONCLUSIONS: This FFQ shows good reproducibility and can be used determining macronutrient intake in a French-speaking Swiss population in an epidemiological setting.

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients.

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC-MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-(13) C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3-6.3%) and accurate (3.8-7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, -20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service. Copyright © 2013 John Wiley & Sons, Ltd.

Temporal quantification of MAPK induced expression in single yeast cells.

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Discordances in the application of different criteria for quantification of paediatric abdominal obesity: an analysis of two Swiss studies.

Several definitions of paediatric abdominal obesity have been proposed but it is unclear whether they lead to similar results. We assessed the prevalence of abdominal obesity using five different waist circumference-based definitions and their agreement with total body fat (TBF) and abdominal fat (AF). Data from 190 girls and 162 boys (Ballabeina), and from 134 girls and 113 boys (Kinder-Sportstudie, KISS) aged 5-11 years were used. TBF was assessed by bioimpedance (Ballabeina) or dual energy X-ray absorption (KISS). On the basis of the definition used, the prevalence of abdominal obesity varied between 3.1 and 49.4% in boys, and 4.7 and 55.5% in girls (Ballabeina), and between 1.8 and 36.3% in boys and 4.5 and 37.3% in girls (KISS). Among children considered as abdominally obese by at least one definition, 32.0 (Ballabeina) and 44.7% (KISS) were considered as such by at least two (out of five possible) definitions. Using excess TBF or AF as reference, the areas under the receiver operating curve varied between 0.577 and 0.762 (Ballabeina), and 0.583 and 0.818 (KISS). We conclude that current definitions of abdominal obesity in children lead to wide prevalence estimates and should not be used until a standard definition can be proposed.

Biological exposure indicators: quantification of biological variability using toxicokinetic modeling

Compartmental and physiologically based toxicokinetic modeling coupled with Monte Carlo simulation were used to quantify the impact of biological variability (physiological, biochemical, and anatomic parameters) on the values of a series of bio-indicators of metal and organic industrial chemical exposures. A variability extent index and the main parameters affecting biological indicators were identified. Results show a large diversity in interindividual variability for the different categories of biological indicators examined. Measurement of the unchanged substance in blood, alveolar air, or urine is much less variable than the measurement of metabolites, both in blood and urine. In most cases, the alveolar flow and cardiac output were identified as the prime parameters determining biological variability, thus suggesting the importance of workload intensity on absorbed dose for inhaled chemicals.

Quantification of thermotolerant campylobacter in Swiss water treatment plants, by real-time quantitative polymerase chain reaction

A total of 49 wastewater samples from 23 different wastewater treatment plants (WWTPs) were analyzed using real-time quantitative polymerase chain reaction for the presence and quantity of thermotolerant campylobacters. Thermotolerant campylobacters were detected in 87.5% (21/24) and 64% (16/25) of untreated and treated wastewater samples, respectively. Their concentration was sufficiently high to be quantified in 20.4% (10/49) of the samples. In these samples, the concentration ranged from 68 000 to 2292 000 cells/L in untreated wastewater and from 10 800 to 28 000 cells/L in treated water. We conclude that thermotolerant campylobacters present a health hazard for workers at WWTPs in Switzerland. [Authors]

Quantification of magmatic and hydrothermal processes in a peralkaline syenite-alkali granite complex based on textures, phase equilibria, and stable and radiogenic isotopes

The Puklen complex of the Mid-Proterozoic Gardar Province, South Greenland, consists of various silica-saturated to quartz-bearing syenites, which are intruded by a peralkaline granite. The primary mafic minerals in the syenites are augite +/- olivine + Fe-Ti oxide + amphibole. Ternary feldspar thermometry and phase equilibria among mafic silicates yield T = 950-750degreesC, a(SiO2) = 0.7-1 and an f(O2) of 1-3 log units below the fayalite-magnetite-quartz (FMQ) buffer at 1 kbar. In the granites, the primary mafic minerals are ilmenite and Li-bearing arfvedsonite, which crystallized at temperatures below 750degreesC and at f(O2) values around the FMQ buffer. In both rock types, a secondary post-magmatic assemblage overprints the primary magmatic phases. In syenites, primary Ca-bearing minerals are replaced by Na-rich minerals such as aegirine-augite and albite, resulting in the release of Ca. Accordingly, secondary minerals include ferro-actinolite, (calcite-siderite)(ss), titanite and andradite in equilibrium with the Na-rich minerals. Phase equilibria indicate that formation of these minerals took place over a long temperature interval from near-magmatic temperatures down to similar to300degreesC. In the course of this cooling, oxygen fugacity rose in most samples. For example, late-stage aegirine in granites formed at the expense of arfvedsonite at temperatures below 300degreesC and at an oxygen fugacity above the haematite-magnetite (HM) buffer. The calculated delta(18)O(melt) value for the syenites (+5.9 to +6.3parts per thousand) implies a mantle origin, whereas the inferred delta(18)O(melt) value of <+5.1parts per thousand for the granitic melts is significantly lower. Thus, the granites require an additional low-delta(18)O contaminant, which was not involved in the genesis of the syenites. Rb/Sr data for minerals of both rock types indicate open-system behaviour for Rb and Sr during post-magmatic metasomatism. Neodymium isotope compositions (epsilonNd(1170 Ma) = -3.8 to -6.4) of primary minerals in syenites are highly variable, and suggest that assimilation of crustal rocks occurred to variable extents. Homogeneous epsilon(Nd) values of -5.9 and -6.0 for magmatic amphibole in the granites lie within the range of the syenites. Because of the very similar neodymium isotopic compositions of magmatic and late- to post-magmatic minerals from the same syenite samples a principally closed-system behaviour during cooling is implied. In contrast, for the granites an externally derived fluid phase is required to explain the extremely low epsilon(Nd) values of about -10 and low delta(18)O between +2.0 and +0.5parts per thousand for late-stage aegirine, indicating an open system in the late-stage history. In this study we show that the combination of phase equilibria constraints with stable and radiogenic isotope data on mineral separates can provide much better constraints on magma evolution during emplacement and crystallization than conventional whole-rock studies.

Continuous monitoring and quantification of multiple parameters of daily physical activity in ambulatory Duchenne muscular dystrophy patients.

Multiple motor function and strength assessment tools exist for the evaluation of neuromuscular diseases, but most do not directly assess functional ability in the patients' daily physical activity in their home environment. In this study our aim was to assess: 1) the feasibility and accuracy of physical activity monitoring during two days in a home environment of five DMD patients using a non-commercialized monitor containing a 3D accelerometer and a gyroscope, 2) if a difference in the physical activity parameters could be measured before and one month after starting prednisolone. We reliably quantified the time spend sitting, standing, lying, walking, the number of steps taken, the cadence, the number of walking episodes and their duration as well as how these were distributed over the day. Parameters possibly reflecting endurance, such as the duration of the walking episodes or the succession of two or three walking episodes lasting more than 30 s were the most improved after prednisolone treatment. This degree of detailed determination of physical activity in a home environment has not been previously reported in neuromuscular disorders to our knowledge and some of the reported parameters are potential new outcome measures in clinical trials.

Quantification of Myocardial Perfusion Using 13N-ammonia PET: a Cross-Comparison Study on Analysis Software Packages - Carimas, PMOD and FlowQuant

Aim. Several software packages (SWP) and models have been released for quantification of myocardial perfusion (MP). Although they all are validated against something, the question remains how well their values agree. The present analysis focused on cross-comparison of three SWP for MP quantification of 13N-ammonia PET studies. Materials & Methods. 48 rest and stress MP 13N-ammonia PET studies of hypertrophic cardiomyopathy (HCM) patients (Sciagrà et al., 2009) were analysed with three SW packages - Carimas, PMOD, and FlowQuant - by three observers blinded to the results of each other. All SWP implement the one-tissue-compartment model (1TCM, DeGrado et al. 1996), and first two - the two-tissue-compartment model (2TCM, Hutchins et al. 1990) as well. Linear mixed model for the repeated measures was fitted to the data. Where appropriate we used Bland-Altman plots as well. The reproducibility was assessed on global, regional and segmental levels. Intraclass correlation coefficients (ICC), differences between the SWPs and between models were obtained. ICC≥0.75 indicated excellent reproducibility, 0.4≤ICC<0.75 indicated fair to good reproducibility, ICC<0.4 - poor reproducibility (Rosner, 2010). Results. When 1TCM MP values were compared, the SW agreement on global and regional levels was excellent, except for Carimas vs. PMOD at RCA: ICC=0.715 and for PMOD vs. FlowQuant at LCX:ICC=0.745 which were good. In segmental analysis in five segments: 7,12,13, 16, and 17 the agreement between all SWP was excellent; in the remaining 12 segments the agreement varied between the compared SWP. Carimas showed excellent agreement with FlowQuant in 13 segments and good in four - 1, 5, 6, 11: 0.687≤ICCs≤0.73; Carimas had excellent agreement with PMOD in 11 segments, good in five_4, 9, 10, 14, 15: 0.682≤ICCs≤0.737, and poor in segment 3: ICC=0.341. PMOD had excellent agreement with FlowQuant in eight segments and substantial-to-good in nine_1, 2, 3, 5, 6,8-11: 0.585≤ICCs≤0.738. Agreement between Carimas and PMOD for 2TCM was good at a global level: ICC=0.745, excellent at LCX (0.780) and RCA (0.774), good at LAD (0.662); agreement was excellent for ten segments, fair-to-substantial for segments 2, 3, 8, 14, 15 (0.431≤ICCs≤0.681), poor for segments 4 (0.384) and 17 (0.278). Conclusions. The three SWP used by different operators to analyse 13N-ammonia PET MP studies provide results that agree well at a global level, regional levels, and mostly well even at a segmental level. Agreement is better for 1TCM. Poor agreement at segments 4 and 17 for 2TCM needs further clarification.