Health behaviours, socioeconomic status, and mortality: further analyses of the British Whitehall II and the French GAZEL prospective cohorts

BACKGROUND: Differences in morbidity and mortality between socioeconomic groups constitute one of the most consistent findings of epidemiologic research. However, research on social inequalities in health has yet to provide a comprehensive understanding of the mechanisms underlying this association. In recent analysis, we showed health behaviours, assessed longitudinally over the follow-up, to explain a major proportion of the association of socioeconomic status (SES) with mortality in the British Whitehall II study. However, whether health behaviours are equally important mediators of the SES-mortality association in different cultural settings remains unknown. In the present paper, we examine this issue in Whitehall II and another prospective European cohort, the French GAZEL study. METHODS AND FINDINGS: We included 9,771 participants from the Whitehall II study and 17,760 from the GAZEL study. Over the follow-up (mean 19.5 y in Whitehall II and 16.5 y in GAZEL), health behaviours (smoking, alcohol consumption, diet, and physical activity), were assessed longitudinally. Occupation (in the main analysis), education, and income (supplementary analysis) were the markers of SES. The socioeconomic gradient in smoking was greater (p<0.001) in Whitehall II (odds ratio [OR] = 3.68, 95% confidence interval [CI] 3.11-4.36) than in GAZEL (OR = 1.33, 95% CI 1.18-1.49); this was also true for unhealthy diet (OR = 7.42, 95% CI 5.19-10.60 in Whitehall II and OR = 1.31, 95% CI 1.15-1.49 in GAZEL, p<0.001). Socioeconomic differences in mortality were similar in the two cohorts, a hazard ratio of 1.62 (95% CI 1.28-2.05) in Whitehall II and 1.94 in GAZEL (95% CI 1.58-2.39) for lowest versus highest occupational position. Health behaviours attenuated the association of SES with mortality by 75% (95% CI 44%-149%) in Whitehall II but only by 19% (95% CI 13%-29%) in GAZEL. Analysis using education and income yielded similar results. CONCLUSIONS: Health behaviours were strong predictors of mortality in both cohorts but their association with SES was remarkably different. Thus, health behaviours are likely to be major contributors of socioeconomic differences in health only in contexts with a marked social characterisation of health behaviours. Please see later in the article for the Editors' Summary.

Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome.

AIMS: Brugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit Na(v)1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel BrS SCN5A mutation. METHODS AND RESULTS: SCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. Sodium currents (I(Na)) were analysed using the whole-cell patch-clamp technique at 37 degrees C. The electrophysiological effects of the mutation were simulated using the Luo-Rudy model, into which the transient outward current (I(to)) was incorporated. A new mutation (C1850S) was identified in the Na(v)1.5 C-terminal domain. In HEK293 cells, mutant I(Na) density was decreased by 62% at -20 mV. Inactivation of mutant I(Na) was accelerated in a voltage-dependent manner and the steady-state inactivation curve was shifted by 11.6 mV towards negative potentials. No change was observed regarding activation characteristics. Altogether, these biophysical alterations decreased the availability of I(Na). In the simulations, the I(to) density necessary to precipitate repolarization differed minimally between the two genotypes. In contrast, the mutation greatly affected conduction across a structural heterogeneity and precipitated conduction block. CONCLUSION: Our data confirm that mutations of the C-terminal domain of Na(v)1.5 alter the inactivation of the channel and support the notion that conduction alterations may play a significant role in the pathogenesis of BrS.

The future key role of LC-high-resolution-MS analyses in clinical laboratories: a focus on quantification.

For the last decade, high-resolution (HR)-MS has been associated with qualitative analyses while triple quadrupole MS has been associated with routine quantitative analyses. However, a shift of this paradigm is taking place: quantitative and qualitative analyses will be increasingly performed by HR-MS, and it will become the common 'language' for most mass spectrometrists. Most analyses will be performed by full-scan acquisitions recording 'all' ions entering the HR-MS with subsequent construction of narrow-width extracted-ion chromatograms. Ions will be available for absolute quantification, profiling and data mining. In parallel to quantification, metabotyping will be the next step in clinical LC-MS analyses because it should help in personalized medicine. This article is aimed to help analytical chemists who perform targeted quantitative acquisitions with triple quadrupole MS make the transition to quantitative and qualitative analyses using HR-MS. Guidelines for the acceptance criteria of mass accuracy and for the determination of mass extraction windows in quantitative analyses are proposed.

Event-related potential analyses via single-subject topographic classification.

Introduction: Responses to external stimuli are typically investigated by averaging peri-stimulus electroencephalography (EEG) epochs in order to derive event-related potentials (ERPs) across the electrode montage, under the assumption that signals that are related to the external stimulus are fixed in time across trials. We demonstrate the applicability of a single-trial model based on patterns of scalp topographies (De Lucia et al, 2007) that can be used for ERP analysis at the single-subject level. The model is able to classify new trials (or groups of trials) with minimal a priori hypotheses, using information derived from a training dataset. The features used for the classification (the topography of responses and their latency) can be neurophysiologically interpreted, because a difference in scalp topography indicates a different configuration of brain generators. An above chance classification accuracy on test datasets implicitly demonstrates the suitability of this model for EEG data. Methods: The data analyzed in this study were acquired from two separate visual evoked potential (VEP) experiments. The first entailed passive presentation of checkerboard stimuli to each of the four visual quadrants (hereafter, "Checkerboard Experiment") (Plomp et al, submitted). The second entailed active discrimination of novel versus repeated line drawings of common objects (hereafter, "Priming Experiment") (Murray et al, 2004). Four subjects per experiment were analyzed, using approx. 200 trials per experimental condition. These trials were randomly separated in training (90%) and testing (10%) datasets in 10 independent shuffles. In order to perform the ERP analysis we estimated the statistical distribution of voltage topographies by a Mixture of Gaussians (MofGs), which reduces our original dataset to a small number of representative voltage topographies. We then evaluated statistically the degree of presence of these template maps across trials and whether and when this was different across experimental conditions. Based on these differences, single-trials or sets of a few single-trials were classified as belonging to one or the other experimental condition. Classification performance was assessed using the Receiver Operating Characteristic (ROC) curve. Results: For the Checkerboard Experiment contrasts entailed left vs. right visual field presentations for upper and lower quadrants, separately. The average posterior probabilities, indicating the presence of the computed template maps in time and across trials revealed significant differences starting at ~60-70 ms post-stimulus. The average ROC curve area across all four subjects was 0.80 and 0.85 for upper and lower quadrants, respectively and was in all cases significantly higher than chance (unpaired t-test, p<0.0001). In the Priming Experiment, we contrasted initial versus repeated presentations of visual object stimuli. Their posterior probabilities revealed significant differences, which started at 250ms post-stimulus onset. The classification accuracy rates with single-trial test data were at chance level. We therefore considered sub-averages based on five single trials. We found that for three out of four subjects' classification rates were significantly above chance level (unpaired t-test, p<0.0001). Conclusions: The main advantage of the present approach is that it is based on topographic features that are readily interpretable along neurophysiologic lines. As these maps were previously normalized by the overall strength of the field potential on the scalp, a change in their presence across trials and between conditions forcibly reflects a change in the underlying generator configurations. The temporal periods of statistical difference between conditions were estimated for each training dataset for ten shuffles of the data. Across the ten shuffles and in both experiments, we observed a high level of consistency in the temporal periods over which the two conditions differed. With this method we are able to analyze ERPs at the single-subject level providing a novel tool to compare normal electrophysiological responses versus single cases that cannot be considered part of any cohort of subjects. This aspect promises to have a strong impact on both basic and clinical research.

Characterization of cocoa butter and cocoa butter equivalents by bulk and molecular carbon isotope analyses: Implications for vegetable fat quantification in chocolate

The fatty acids from cocoa butters of different origins, varieties, and suppliers and a number of cocoa butter equivalents (Illexao 30-61, Illexao 30-71, Illexao 30-96, Choclin, Coberine, Chocosine-Illipe, Chocosine-Shea, Shokao, Akomax, Akonord, and Ertina) were investigated by bulk stable carbon isotope analysis and compound specific isotope analysis. The interpretation is based on principal component analysis combining the fatty acid concentrations and the bulk and molecular isotopic data. The scatterplot of the two first principal components allowed detection of the addition of vegetable fats to cocoa butters. Enrichment in heavy carbon isotope (C-13) of the bulk cocoa butter and of the individual fatty acids is related to mixing with other vegetable fats and possibly to thermally or oxidatively induced degradation during processing (e.g., drying and roasting of the cocoa beans or deodorization of the pressed fat) or storage. The feasibility of the analytical approach for authenticity assessment is discussed.

Analyses critiques de ľ expression génétique

La question du filtrage de ľinformation génétique dans la cellule est fondamentale. Comment la cellule sélectionne-t-elle, avant de les transformer en RNA puis en protéines, certaines parties bien déterminées de son information génétique? Il ne sera probablement pas possible de donner une explication cohérente du développement embryonnaire, de la différentiation cellulaire et du maintien de ľétat différencie tant que nous n'aurons pas repondu de manière satis-faisante à cette question. Dans un premier chapitre, quelques notions de base concernant ľexpression génétique sont préséntées. Le dogme de flux de ľinformation génétique dans la cellule, DNARNA protéine est valable à la fois pour les procaryotes et les eucaryotes malgré des différences significatives au niveau de la structure et de la régulation des gènes. Contrairement aux génes procaryotes, la plu-part des gènes eucaryotes sont morcelés. Le DNA codant pour une protéine est interrompu par des régions non-codantes dont les transcrits sont éliminés par excision pendant la maturation du RNA messager ultérieurement traduit en protéine. Une grande variété de mécanismes interviennent dans la régulation de ľactivité de ces gènes. Le pouvoir et les limites des méthodes modernes de ľanalyse structurale et fonctionnelle des génes sont discutés dans la deuxième partie de ľarticle. Ľhybridation moléculaire reposant sur la complémentarité des bases azotées des acides nucléiques joue un rôle déterminant dans ľétude de la complexity des génomes et de leur expression. Récemment, ľapplication de la technologie du DNA recombinant et des techniques annexes a permis ľisolement ainsi que la caractérisation de plusieurs génes eucaryotes. La question de ľexpression différentielle de ces génes est actuellement intensément étudiée dans plusieurs systèmes de transcription ayant chacun ses points forts et ses faiblesses. En guise de conclusion, quelques implications de ľessor prodigieux que connaît la génétique moléculaire sont discutées.

An investigation of the potential of DIP-STR markers for DNA mixture analyses

The genetic characterization of unbalanced mixed stains remains an important area where improvement is imperative. In fact, using the standard tools of forensic DNA profiling (i.e., STR markers), the profile of the minor contributor in mixed DNA stains cannot be successfully detected if its quantitative share of DNA is less than 10% of the mixed trace. This is due to the fact that the major contributor's profile "masks" that of the minor contributor. Besides known remedies to this problem, such as Y-STR analysis, a new compound genetic marker that consists of a Deletion/Insertion Polymorphism (DIP) linked to a Short Tandem Repeat (STR) polymorphism, has recently been developed and proposed [1]. These novel markers are called DIP-STR markers. This paper compares, from a statistical and forensic perspective, the potential usefulness of these novel DIP-STR markers (i) with traditional STR markers in cases of moderately unbalanced mixtures, and (ii) with Y-STR markers in cases of female-male mixtures. This is done through a comparison of the distribution of 100,000 likelihood ratio values obtained using each method on simulated mixtures. This procedure is performed assuming, in turn, the prosecution's and the defence's point of view.

Clinical, polysomnographic and genome-wide association analyses of narcolepsy with cataplexy: a European Narcolepsy Network study.

The aim of this study was to describe the clinical and PSG characteristics of narcolepsy with cataplexy and their genetic predisposition by using the retrospective patient database of the European Narcolepsy Network (EU-NN). We have analysed retrospective data of 1099 patients with narcolepsy diagnosed according to International Classification of Sleep Disorders-2. Demographic and clinical characteristics, polysomnography and multiple sleep latency test data, hypocretin-1 levels, and genome-wide genotypes were available. We found a significantly lower age at sleepiness onset (men versus women: 23.74 ± 12.43 versus 21.49 ± 11.83, P = 0.003) and longer diagnostic delay in women (men versus women: 13.82 ± 13.79 versus 15.62 ± 14.94, P = 0.044). The mean diagnostic delay was 14.63 ± 14.31 years, and longer delay was associated with higher body mass index. The best predictors of short diagnostic delay were young age at diagnosis, cataplexy as the first symptom and higher frequency of cataplexy attacks. The mean multiple sleep latency negatively correlated with Epworth Sleepiness Scale (ESS) and with the number of sleep-onset rapid eye movement periods (SOREMPs), but none of the polysomnographic variables was associated with subjective or objective measures of sleepiness. Variant rs2859998 in UBXN2B gene showed a strong association (P = 1.28E-07) with the age at onset of excessive daytime sleepiness, and rs12425451 near the transcription factor TEAD4 (P = 1.97E-07) with the age at onset of cataplexy. Altogether, our results indicate that the diagnostic delay remains extremely long, age and gender substantially affect symptoms, and that a genetic predisposition affects the age at onset of symptoms.

Leptin and smoking cessation: secondary analyses of a randomized controlled trial assessing physical activity as an aid for smoking cessation.

BACKGROUND: Smokers have a lower body weight compared to non-smokers. Smoking cessation is associated with weight gain in most cases. A hormonal mechanism of action might be implicated in weight variations related to smoking, and leptin might be implicated. We made secondary analyses of an RCT, with a hypothesis-free exploratory approach to study the dynamic of leptin following smoking cessation. METHODS: We measured serum leptin levels among 271 sedentary smokers willing to quit who participated in a randomized controlled trial assessing a 9-week moderate-intensity physical activity intervention as an aid for smoking cessation. We adjusted leptin for body fat levels. We performed linear regressions to test for an association between leptin levels and the study group over time. RESULTS: One year after smoking cessation, the mean serum leptin change was +3.23 mg/l (SD 4.89) in the control group and +1.25 mg/l (SD 4.86) in the intervention group (p of the difference < 0.05). When adjusted for body fat levels, leptin was higher in the control group than in the intervention group (p of the difference < 0.01). The mean weight gain was +2.91 (SD 6.66) Kg in the intervention and +3.33 (SD 4.47) Kg in the control groups, respectively (p not significant). CONCLUSIONS: Serum leptin levels significantly increased after smoking cessation, in spite of substantial weight gain. The leptin dynamic might be different in chronic tobacco users who quit smoking, and physical activity might impact the dynamic of leptin in such a situation. CLINICAL TRIAL REGISTRATION NUMBER: NCT00521391.

Évaluation statistique des résultats des analyses ADN de 2005 à 2011 et recommandations stratégiques au sein de la section d'Identité judiciaire de la Police cantonale vaudoise

Notre étude a eu pour but d'analyser les résultats des analyses ADN effectuées sur les traces relevées sur des scènes de crimes ou sur des objets s'y apparentant. Cette analyse nous a permis d'en identifier les points forts et les points faibles. Les données concernant les résultats ADN des années 2005 à 2008 dans le canton de Vaud ont montré une augmentation significative du nombre de traces envoyées, avec en parallèle un déclin de la qualité des résultats. Suite à cette observation, des solutions ayant pour but de réduire le nombre de ces analyses négatives ont dû être adoptées. Des changements opérationnels ont alors été mis en place dès 2009. Il s'agissait alors, d'une part, d'agir au niveau des traces de contact en tenant compte du type de support sur lequel la trace était prélevée, et, d'autre part, d'agir sur les traces riches (sang, salive, sperme) en mettant en place l'analyse systématique de la nature de la trace à l'aide de tests indicatifs. L'évaluation des conséquences de la mise en place de ces procédures a été possible à l'aide d'une nouvelle analyse statistique. Les résultats montrent qu'un meilleur tri des traces permet une amélioration des résultats.

Genome-wide association analyses identify 18 new loci associated with serum urate concentrations.

Elevated serum urate concentrations can cause gout, a prevalent and painful inflammatory arthritis. By combining data from >140,000 individuals of European ancestry within the Global Urate Genetics Consortium (GUGC), we identified and replicated 28 genome-wide significant loci in association with serum urate concentrations (18 new regions in or near TRIM46, INHBB, SFMBT1, TMEM171, VEGFA, BAZ1B, PRKAG2, STC1, HNF4G, A1CF, ATXN2, UBE2Q2, IGF1R, NFAT5, MAF, HLF, ACVR1B-ACVRL1 and B3GNT4). Associations for many of the loci were of similar magnitude in individuals of non-European ancestry. We further characterized these loci for associations with gout, transcript expression and the fractional excretion of urate. Network analyses implicate the inhibins-activins signaling pathways and glucose metabolism in systemic urate control. New candidate genes for serum urate concentration highlight the importance of metabolic control of urate production and excretion, which may have implications for the treatment and prevention of gout.

Methods for integration site distribution analyses in animal cell genomes.

The question of where retroviral DNA becomes integrated in chromosomes is important for understanding (i) the mechanisms of viral growth, (ii) devising new anti-retroviral therapy, (iii) understanding how genomes evolve, and (iv) developing safer methods for gene therapy. With the completion of genome sequences for many organisms, it has become possible to study integration targeting by cloning and sequencing large numbers of host-virus DNA junctions, then mapping the host DNA segments back onto the genomic sequence. This allows statistical analysis of the distribution of integration sites relative to the myriad types of genomic features that are also being mapped onto the sequence scaffold. Here we present methods for recovering and analyzing integration site sequences.

Phylogenetic analyses of C4 photosynthesis evolution in grasses: genetics of convergence and convergence of genes

Summary : During the evolutionary diversification of organisms, similar ecological constraints led to the recurrent appearances of the same traits (phenotypes) in distant lineages, a phenomenon called convergence. In most cases, the genetic origins of the convergent traits remain unknown, but recent studies traced the convergent phenotypes to recurrent alterations of the same gene or, in a few cases, to identical genetic changes. However, these cases remain anecdotal and there is a need for a study system that evolved several times independently and whose genetic determinism is well resolved and straightforward, such as C4 photosynthesis. This adaptation to warm environments, possibly driven by past atmospheric CO2 decreases, consists in a CO2-concentrating pump, created by numerous morphological and biochemical novelties. All genes encoding C4 enzymes already existed in C3 ancestors, and are supposed to have been recruited through gene duplication followed by neo-functionalization, to acquire the cell specific expression pattern and altered kinetic properties that characterize Ca-specific enzymes. These predictions have so far been tested only in species-poor and ecologically marginal C4 dicots. The monocots, and especially the grass family (Poaceae), the most important C4 family in terms of species number, ecological dominance and economical importance, have been largely under-considered as suitable study systems. This thesis aimed at understanding the evolution of the C4 trait in grasses at a molecular level and to use the genetics of C4 photosynthesis to infer the evolutionary history of the C4 phenotype and its driving selective pressures. A molecular phylogeny of grasses and affiliated monocots identified 17 to 18 independent acquisitions of the C4 pathway in the grass family. A relaxed molecular clock was used to date these events and the first C4 evolution was estimated in the Chloridoideae subfamily, between 32-25 million years ago, at a period when atmospheric CO2 abruptly declined. Likelihood models showed that after the COZ decline the probability of evolving the C4 pathway strongly increased, confirming low CO2 as a likely driver of C4 photosynthesis evolution. In order to depict the genetic changes linked to the numerous C4 origins, genes encoding phopshoenolpyruvate carboxylase (PEPC), the key-enzyme responsible for the initial fixation of atmospheric CO2 in the C4 pathway, were isolated from a large sample of C3 and C4 grasses. Phylogenetic analyses were used to reconstruct the evolutionary history of the PEPC multigene family and showed that the evolution of C4-specific PEPC had been driven by positive selection on 21 codons simultaneously in up to eight C4 lineages. These selective pressures led to numerous convergent genetic changes in many different C4 clades, highlighting the repeatability of some evolutionary processes, even at the molecular level. PEPC C4-adaptive changes were traced and used to show multiple appearances of the C, pathway in clades where species tree inferences were unable to differentiate multiple C4 appearances and a single appearance followed by C4 to C3 reversion. Further investigations of genes involved in some of the C4 subtypes only (genes encoding decarboxylating enzymes NADP-malic enzyme and phosphoenolpyruvate carboxykinase) showed that these C4-enzymes also evolved through strong positive selection and underwent parallel genetic changes during the different Ca origins. The adaptive changes on these subtype-specific C4 genes were used to retrace the history of the C4-subtypes phenotypes, which revealed that the evolution of C4-PEPC and C4-decarboxylating enzymes was in several cases disconnected, emphasizing the multiplicity of the C4 trait and the gradual acquisition of the features that create the CO2-pump. Finally, phylogenetic analyses of a gene encoding the Rubisco (the enzyme responsible for the fixation of CO2 into organic compounds in all photosynthetic organisms) showed that C4 evolution switched the selective pressures on this gene. Five codons were recurrently mutated to adapt the enzyme kinetics to the high CO2 concentrations of C4 photosynthetic cells. This knowledge could be used to introgress C4-like Rubisco in C3 crops, which could lead to an increased yield under predicted future high CO2 atmosphere. Globally, the phylogenetic framework adopted during this thesis demonstrated the widespread occurrence of genetic convergence on C4-related enzymes. The genetic traces of C4 photosynthesis evolution allowed reconstructing events that happened during the last 30 million years and proved the usefulness of studying genes directly responsible for phenotype variations when inferring evolutionary history of a given trait. Résumé Durant la diversification évolutive des organismes, des pressions écologiques similaires ont amené à l'apparition récurrente de certains traits (phénotypes) dans des lignées distantes, un phénomène appelé évolution convergente. Dans la plupart des cas, l'origine génétique des traits convergents reste inconnue mais des études récentes ont montré qu'ils étaient dus dans certains cas à des changements répétés du même gène ou, dans de rares cas, à des changements génétiques identiques. Malgré tout, ces cas restent anecdotiques et il y a un réel besoin d'un système d'étude qui ait évolué indépendamment de nombreuses fois et dont le déterminisme génétique soit clairement identifié. La photosynthèse dite en Ça répond à ces critères. Cette adaptation aux environnements chauds, dont l'évolution a pu être encouragé par des baisses passées de la concentration atmosphérique en CO2, est constituée de nombreuses nouveautés morphologiques et biochimiques qui créent une pompe à CO2. La totalité des gènes codant les enzymes Ç4 étaient déjà présents dans les ancêtres C3. Leur recrutement pour la photosynthèse Ç4 est supposé s'être fait par le biais de duplications géniques suivies par une néo-fonctionnalisation pour leur conférer l'expression cellule-spécifique et les propriétés cinétiques qui caractérisent les enzymes C4. Ces prédictions n'ont jusqu'à présent été testées que dans des familles C4 contenant peu d'espèces et ayant un rôle écologique marginal. Les graminées (Poaceae), qui sont la famille C4 la plus importante, tant en termes de nombre d'espèces que de dominance écologique et d'importance économique, ont toujours été considérés comme un système d'étude peu adapté et ont fait le sujet de peu d'investigations évolutives. Le but de cette thèse était de comprendre l'évolution de la photosynthèse en C4 chez les graminées au niveau génétique et d'utiliser les gènes pour inférer l'évolution du phénotype C4 ainsi que les pressions de sélection responsables de son évolution. Une phylogénie moléculaire de la famille des graminées et des monocotylédones apparentés a identifié 17 à 18 acquisitions indépendantes de la photosynthèse chez les graminées. Grâce à une méthode d'horloge moléculaire relâchée, ces évènements ont été datés et la première apparition C4 a été estimée dans la sous-famille des Chloridoideae, il y a 32 à 25 millions d'années, à une période où les concentrations atmosphériques de CO2 ont décliné abruptement. Des modèles de maximum de vraisemblance ont montré qu'à la suite du déclin de CO2, la probabilité d'évoluer la photosynthèse C4 a fortement augmenté, confirmant ainsi qu'une faible concentration de CO2 est une cause potentielle de l'évolution de la photosynthèse C4. Afin d'identifier les mécanismes génétiques responsables des évolutions répétées de la photosynthèse C4, un segment des gènes codant pour la phosphoénolpyruvate carboxylase (PEPC), l'enzyme responsable de la fixation initiale du CO2 atmosphérique chez les plantes C4, ont été séquencés dans une centaine de graminées C3 et C4. Des analyses phylogénétiques ont permis de reconstituer l'histoire évolutive de la famille multigénique des PEPC et ont montré que l'évolution de PEPC spécifiques à la photosynthèse Ça a été causée par de la sélection positive agissant sur 21 codons, et ce simultanément dans huit lignées C4 différentes. Cette sélection positive a conduit à un grand nombre de changements génétiques convergents dans de nombreux clades différents, ce qui illustre la répétabilité de certains phénomènes évolutifs, et ce même au niveau génétique. Les changements sur la PEPC liés au C4 ont été utilisés pour confirmer des évolutions indépendantes du phénotype C4 dans des clades où l'arbre des espèces était incapable de différencier des apparitions indépendantes d'une seule apparition suivie par une réversion de C4 en C3. En considérant des gènes codant des protéines impliquées uniquement dans certains sous-types C4 (deux décarboxylases, l'enzyme malique à NADP et la phosphoénolpyruvate carboxykinase), des études ultérieures ont montré que ces enzymes C4 avaient elles-aussi évolué sous forte sélection positive et subi des changements génétiques parallèles lors des différentes origines de la photosynthèse C4. Les changements adaptatifs sur ces gènes liés seulement à certains sous-types C4 ont été utilisés pour retracer l'histoire des phénotypes de sous-types C4, ce qui a révélé que les caractères formant le trait C4 ont, dans certains cas, évolué de manière déconnectée. Ceci souligne la multiplicité du trait C4 et l'acquisition graduelle de composants participant à la pompe à CO2 qu'est la photosynthèse C4. Finalement, des analyses phylogénétiques des gènes codant pour la Rubisco (l'enzyme responsable de la fixation du CO2 en carbones organiques dans tous les organismes photosynthétiques) ont montré que l'évolution de la photosynthèse Ça a changé les pressions de sélection sur ce gène. Cinq codons ont été mutés de façon répétée afin d'adapter les propriétés cinétiques de la Rubisco aux fortes concentrations de CO2 présentes dans les cellules photosynthétiques des plantes C4. Globalement, l'approche phylogénétique adoptée durant cette thèse de doctorat a permis de démontré des phénomène fréquents de convergence génétique sur les enzymes liées à la photosynthèse C4. Les traces génétiques de l'évolution de la photosynthèse C4 ont permis de reconstituer des évènements qui se sont produits durant les derniers 30 millions d'années et ont prouvé l'utilité d'étudier des gènes directement responsables des variations phénotypiques pour inférer l'histoire évolutive d'un trait donné.