Postmortem biochemistry performed on vitreous humor after postmortem CT-angiography.

Postmortem angiography is becoming increasingly essential in forensic pathology as an adjunct to conventional autopsy. Despite the numerous advantages of this technique, some questions have been raised regarding the influence of the contrast agent injected on the results of toxicological and biochemical analyses. The aim of this study was to investigate the effect of the injection of the contrast agent Angiofil®, mixed with paraffin oil, on the results of postmortem biochemical investigations performed on vitreous humor. Postmortem biochemical investigations were performed on vitreous samples collected from bodies that had undergone postmortem angiography (n=50) and from a control group (n=50). Two vitreous samples were analyzed for each group and the results compared. Glucose, urea, creatinine, 3-β-hydroxybutyrate, sodium and chloride were tested. Different values were observed between the first and second samples in each group. However, these differences were not clinically relevant, suggesting that the injection of this contrast agent mixture does not modify the concentration of the analyzed substances in the vitreous humor.

Postmortem chemistry update part I.

Postmortem chemistry is becoming increasingly essential in the forensic pathology routine and considerable progress has been made over the past years. Biochemical analyses of vitreous humor, cerebrospinal fluid, blood and urine may provide significant information in determining the cause of death or in elucidating forensic cases. Postmortem chemistry may essentially contribute in the determination of the cause of death when the pathophysiological changes involved in the death process cannot be detected by morphological methods (e.g. diabetes mellitus, alcoholic ketoacidosis and electrolytic disorders). It can also provide significant information and useful support in other forensic situations, including anaphylaxis, hypothermia, sepsis and hormonal disturbances. In this article, we present a review of the literature that covers this vast topic and we report the results of our observations. We have focused our attention on glucose metabolism, renal function and electrolytic disorders.

Phylogenetic structures of the Holarctic Sorex araneus group and its relationships with S-samniticus, as inferred from mtDNA sequences

The shrews of the Sorex araneus group, characterized by the sexual chromosome complex XY1, Y2 have been intensively studied by morphological, karyotypical, and biochemical analyses. Nevertheless, the phylogenetic relationships among the species belonging to the araneus complex are still under debate, as different approaches gave often contradictory results. In this paper, partial nucleotide sequences of the mitochondrial DNA cytochrome b gene (1011 bp) were determined for 6 species of the araneus group from Eurasia and North America. We also included in the data set the sequences of Sorex samniticus, whose relationships with the araneus group remain controversial. Three other species representing two major karyological groups were also examined. Both parsimony and distance trees strongly support the monophyly of the araneus group. Sorex sumniticus is significantly more closely related to the araneus complex than to the other species included in the analysis. Based on the branching pattern within the araneus group, an attempt has been made to reconstruct the colonization history of the Holarctic region.

Maternal effect on female caste determination in a social insect.

Caste differentiation and division of labor are the hallmarks of social insect colonies [1, 2]. The current dogma for female caste differentiation is that female eggs are totipotent, with morphological and physiological differences between queens and workers stemming from a developmental switch during the larval stage controlled by nutritional and other environmental factors (e.g., [3-8]). In this study, we tested whether maternal effects influence caste differentiation in Pogonomyrmex harvester ants. By conducting crossfostering experiments we identified two key factors in the process of caste determination. New queens were produced only from eggs laid by queens exposed to cold. Moreover, there was a strong age effect, with development into queens occurring only in eggs laid by queens that were at least two years old. Biochemical analyses further revealed that the level of ecdysteroids was significantly lower in eggs developing into queens than workers. By contrast, we found no significant effect of colony size or worker exposure to cold, suggesting that the trigger for caste differentiation may be independent of the quantity and quality of resources provided to larvae. Altogether these data demonstrate that the developmental fate of female brood is strongly influenced by maternal effects in ants of the genus Pogonomyrmex.

Value of sTREM-1, procalcitonin and CRP as laboratory parameters for postmortem diagnosis of sepsis.

OBJECTIVES: Triggering receptor expressed on myeloid cells-1 (TREM-1) was reported to be up-regulated in various inflammatory diseases as well as in bacterial sepsis. Increased cell-surface TREM-1 expression was also shown to result in marked plasma elevation of the soluble form of this molecule (sTREM-1) in patients with bacterial infections. In this study, we investigated sTREM-1, procalcitonin and C-reactive protein in postmortem serum in a series of sepsis-related fatalities and control individuals who underwent medico-legal investigations. sTREM-1 was also measured in pericardial fluid and urine. METHODS: Two study groups were prospectively formed, a sepsis-related fatalities group and a control group. The sepsis-related fatalities group consisted of sixteen forensic autopsy cases. Eight of these had a documented clinical diagnosis of sepsis in vivo. The control group consisted of sixteen forensic autopsy cases with various causes of death. RESULTS: Postmortem serum sTREM-1 concentrations were higher in the sepsis group with a mean value of 173.6 pg/ml in septic cases and 79.2 pg/ml in control individuals. The cutoff value of 90 pg/ml provided the best sensitivity and specificity. Pericardial fluid sTREM-1 values were higher in the septic group, with a mean value of 296.7 pg/ml in septic cases and 100.9 pg/ml in control individuals. The cutoff value of 135 pg/ml provided the best sensitivity and specificity. Mean urine sTREM-1 concentration was 102.9 pg/ml in septic cases and 89.3 pg/ml in control individuals. CONCLUSIONS: Postmortem serum sTREM-1, individually considered, did not provide better sensitivity and specificity than procalcitonin in detecting sepsis. However, simultaneous assessment of procalcitonin and sTREM-1 in postmortem serum can be of help in clarifying contradictory postmortem findings. sTREM-1 determination in pericardial fluid can be an alternative to postmortem serum in those situations in which biochemical analyses are required and blood collected during autopsy proves insufficient.

Postmortem biochemistry: mith or reality?

Postmortem biochemistry is becoming increasingly essential in the forensic casework and considerable progress has been made over the past years. Biochemical analyses of blood, postmortem serum, pericardial fluid, vitreous humor and urine may indeed provide significant information in identifying the cause of death as well as characterizing the precipitating conditions and the predisposing disorders. Postmortem biochemical analyses are decisive in determining the cause of death when the pathophysiological changes involved in the process cannot be otherwise ascertained by morphological investigations (mainly diabetic and alcoholic ketoacidosis). It can also provide significant information and useful support in numerous situations on forensic interest, including cold exposure-related deaths, fatal anaphylaxis, bacterial infection-related fatalities and hormonal disturbances. In this report we present an overview pertaining to the diagnostic performance of classical and novel biochemical markers in the forensic setting.

Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover.

Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3' to 5' exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3-GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

Phosphorylation of phosphatidylinositol-3-kinase-protein kinase B and extracellular signal-regulated kinases 1/2 mediate reoxygenation-induced cardioprotection during hypoxia.

In vivo exposure to chronic hypoxia (CH) depresses myocardial performance and tolerance to ischemia, but daily reoxyenation during CH (CHR) confers cardioprotection. To elucidate the underlying mechanism, we tested the role of phosphatidylinositol-3-kinase-protein kinase B (Akt) and p42/p44 extracellular signal-regulated kinases (ERK1/2), which are known to be associated with protection against ischemia/reperfusion (I/R). Male Sprague-Dawley rats were maintained for two weeks under CH (10% O(2)) or CHR (as CH but with one-hour daily exposure to room air). Then, hearts were either frozen for biochemical analyses or Langendorff-perfused to determine performance (intraventricular balloon) and tolerance to 30-min global ischemia and 45-min reperfusion, assessed as recovery of performance after I/R and infarct size (tetrazolium staining). Additional hearts were perfused in the presence of 15 micromol/L LY-294002 (inhibitor of Akt), 10 micromol/L UO-126 (inhibitor of ERK1/2) or 10 micromol/L PD-98059 (less-specific inhibitor of ERK1/2) given 15 min before ischemia and throughout the first 20 min of reperfusion. Whereas total Akt and ERK1/2 were unaffected by CH and CHR in vivo, in CHR hearts the phosphorylation of both proteins was higher than in CH hearts. This was accompanied by better performance after I/R (heart rate x developed pressure), lower end-diastolic pressure and reduced infarct size. Whereas the treatment with LY-294002 decreased the phosphorylation of Akt only, the treatment with UO-126 decreased ERK1/2, and that with PD-98059 decreased both Akt and ERK1/2. In all cases, the cardioprotective effect led by CHR was lost. In conclusion, in vivo daily reoxygenation during CH enhances Akt and ERK1/2 signaling. This response was accompanied by a complex phenotype consisting in improved resistance to stress, better myocardial performance and lower infarct size after I/R. Selective inhibition of Akt and ERK1/2 phosphorylation abolishes the beneficial effects of the reoxygenation. Therefore, Akt and ERK1/2 have an important role to mediate cardioprotection by reoxygenation during CH in vivo.

Postmortem diagnosis of diabetes mellitus and its complications.

Diabetes mellitus has become a major cause of death worldwide and diabetic ketoacidosis is the most common cause of death in children and adolescents with type 1 diabetes. Acute complications of diabetes mellitus as causes of death may be difficult to diagnose due to missing characteristic macroscopic and microscopic findings. Biochemical analyses, including vitreous glucose, blood (or alternative specimen) beta-hydroxybutyrate, and blood glycated hemoglobin determination, may complement postmortem investigations and provide useful information for determining the cause of death even in corpses with advanced decompositional changes. In this article, we performed a review of the literature pertaining to the diagnostic performance of classical and novel biochemical parameters that may be used in the forensic casework to identify disorders in glucose metabolism. We also present a review focusing on the usefulness of traditional and alternative specimens that can be sampled and subsequently analyzed to diagnose acute complications of diabetes mellitus as causes of death.

Taxonomic status of Hylomys parvus and Hylomys suillus (Insectivora: Erinaceidae): biochemical and morphological analyses

The genus Hylomys was thought to be represented by a single widespread species. Biochemical and morphometric analyses of several Southeast Asian populations reveal that Sumatra is inhabited by two distinct species, the dwarf gymnure (H. parvus) and the lesser gymnure (H. suillus). The absence of interbreeding between these two groups along with their relatively ancient common origins are documented by several diagnostic loci and a large Nei's genetic distance (D = 0.353 +/- 0.035). The dwarf gymnure has been reported only from the slopes of the Mt. Kerinci volcano in Sumatra, where the species lives at higher elevations than its potential competitor, the lesser gymnure. Other populations of Hylomys from Java, Borneo, and Malaysia are more closely related to the Sumatran sample of H. suillus, but they exhibit strong interpopulational genetic differentiation (D = 0.165 +/- 0.040) that may be accounted for by their isolated montane habitat. In addition, a principal-components analysis based on 16 measurements of the skull clearly separates adult specimens of both species. There is little overlap in the measurements between H. suillus (which is larger) and H. parvus. On Sumatra where both species may be sympatric, the notched space between premaxillary tips, soft texture of the fur, and more delicate skull and dentition are diagnostic of H. parvus.

Use of new biochemical plasmatic markers, plasma free and total metanephrines, for the diagnosis and follow-up of neuroblastoma in children with clinical correlation

Background: Neuroblastoma is a paediatrictumour derived from the neural crest. Biochemical diagnosis and follow up rely on quantitation of urinary catecholamines (dopamine and noradrenaline) and their metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA) (gold-standard). When combined, these analyses have a sensitivity of 95%. However, they are clearly limited by inaccuracy of urine collection in young children and normalisation of catecholamine concentrations by creatininuria. Recent development in biochemical diagnosis of pheochromocytoma, another neural crest tumour found in adults, shows that plasmatic measurement of methoxylated catecholamines called metanephrines are more sensitive and specific than other biomarkers. Moreover, a study to determine the reference intervals for metanephrines in a pediatric population has recently been completed. The aim of this work is to describe the role of metanephrines monitoring in the follow up of neuroblastoma. Method: This retrospective study included patients with neuroblastoma in whom the following parameters were determined: plasma free and total metanephrines, plasma catecholamines, 24h urinary catecholamines and metanephrines in absolute value and corrected by creatinine, VMA and HVA at the diagnosis and during treatment at the University Hospital of Lausanne (Switzerland). Eleven patients aged between the first day of life and 7 years old were followed between 2005 and 2012. Clinical outcome and biochemical concentrations of the analytes were correlated. Results: At diagnosis, plasma free and total normetanephrines and methoxytyramine have a sensitivity of 100% compared to 85% for the actual gold standard. Metanephrine remain below the upper reference limit as expected since these tumours do not produce adrenaline. The relationship between biochemical markers and clinical outcome is illustrated graphically. Plasma or urinary normetanephrine and methoxytyramine correlate better with the history of the patient than VMA and HVA, as evaluated by ordinal logistic regression. Concentrations of analytes in urine show a better correlation with clinical events when the results are corrected by creatininuria. Conclusion: Normetanephrine and methoxytyramine reflect disease history in neuroblastoma patients and could play a significant role in the follow up of this type of tumour. Formal studies in a sufficient number of patients are needed to confirm this preliminary observation.

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.

Atypical EPO profiles in anti-doping analyses

Résumé : Erythropoietin (EPO) is a glycoprotein hormone endogenously produced by the kidney, whose main physiological role is the stimulation of erythropoiesis. Since the beginning of the nineties, recombinant human EPO (rhEPO), a potent anti-anaemia treatment drug, has been manufactured by pharmaceutical industries. However, the erythropoiesis stimulating power of rhEPO was rapidly misused by unscrupulous athletes in order to improve their performances in endurance sports. Endogenous EPO has the same amino-acid backbone as most of recombinant forms; the molecules however differ through their respective glycosylation patterns. This difference constitutes the basis of the usual EPO screening test (IEF) developed in 2000 and still currently used in all anti-doping laboratories of the world. Nowadays, 3 EPO generations have been commercialized. The fight against EPO abuse is a continuous challenge for anti-doping laboratories. The diversity of recombinant EPO forms and the continuous development of new ones considerably confuse the identification of EPO doping. Several facets of this fight were investigated in this work. One of the limiting aspects of doping agents screening is the availability of positive samples. Therefore, 2nd and 3rd generation EPOS, namely NESP and C.E.R.A., were injected to healthy subjects in the frame of pilot clinical studies. These latter allowed to review the current EPO identification criteria defined by the World Anti-Doping Agency (WADA) in the case of NESP and to validate and implement a new assay targeting C.E.R.A. in human serum. Both studies resulted in the determination of the respective detection windows of NESP and C.E.R.A. in biological fluids. Following that, Dynepo, a 1st generation EPO presenting similarities with the endogenous form, was also in the centre of a similar clinical study. Our work aimed to overcome the actual identification criteria, which are not adapted to Dynpeo, and to propose an alternative pattern classification method based on the discriminant analysis of IEF EPO profiles. This method might be validated for other EPO forms in the future. The detection window of this molecule was also determined. Under particular conditions, confounding effects can complicate the identification of EPO in biological matrices. For example, athletes having performed a strenuous physical effort can excrete modified isoforms of endogenous EPO, making it very similar to some recombinant forms. Such phenomena, called effort urines, were reproduced under controlled conditions and, after characterization of effort EPO, an urinary biochemical marker was proposed to unequivocally identify effort urines. It also happens that EPO analyses fail to detect endogenous levels of EPO. Such profiles were thoroughly investigated and potential causes identified. Natural reasons relying on urine properties and test specificity were underlined, but the possible addition of adulterant agents in urine samples was also considered. Therefore, a simple biochemical assay targeting the suspected substances was set up. Our work was based on the characterization of atypical EPO profiles from different origins. Therefore, 3 EPO molecules representing the 3 generations of the drug and 2 confounding effects confusing the results interpretation were studied. These studies resulted in tangible applications for the laboratory, the best example of which being the C.E.R.A. assay, but also in scientific findings allowing to improve our comprehension of EPO doping in sport.

A genetic and biochemical investigation of malondialdehyde production and turnover in Arabidopsis

Malondialdehyde (MDA) is a small reactive molecule which occurs ubiqui¬tous among eukaryotes. Interest in this molecule stems from the fact that it can be highly reactive. In green tissues of plants it is apparently formed pre¬dominantly by reactive oxygen species (ROS)-mediated non-enzymatic oxi¬dation (nLPO) of triunsaturated fatty acids (TFAs). MDA which is formed by nLPO is widely used as a disease marker and is regarded to be a cel-lular toxin. Surprisingly, sites of ROS production like mitochondria and chloroplasts possess membranes which are enriched in nLPO-prone polyun¬saturated fatty acids (PUFAs). In this work we showed that chloroplasts are the major site of MDA production in leaves of adult Arabidopsis thaliana plants, whereas analyses in seedlings revealed accumulation in meristematic tissues like the root tip, lateral roots and the apical meristem region. Char-acterizing the MDA pools in more detail, we could show that MDA in plants was predominantly present in a free, non-reactive enolate form. This might explain why it is tolerated in sites where its protonated form could poten¬tially damage the genome and proteome. Analyzing the biological fate of MDA in leaves using labeled MDA-isotopes. we were able to show that MDA is metabolized and used to assemble lipids. The major end-point metabolite was identified as 18:3-16:3-monqgalactosyldiacylglycerol (MGDG), which is the most abundant lipid in chloroplasts. We hypothesize that PUFAs in sites of ROS production, like at PS II in chloroplasts, might act as buffers pre¬venting damage of proteins, thereby generating molecules such as MDA. The MDA produced in this way appears predominantly in a non-reactive enolate form in the cell until it fulfills a biological function or until it is metabo¬lized in order to assemble polyunsaturated MGDGs. Additionally, nLPO has been reported to increase in pathogenesis and we challenged seedlings and adult plants with necrotrophic fungi. Monitoring MDA during the in¬fections, we found MDA pools in seedlings were highly inducible although they were tightly controlled in the leaves of adult plants. - Malondialdehyde (MDA) est une petite molecule réactive présente de manière ubiquitaire dans les eucaryotes. L'intérêt de cette molécule vient du fait que celle-ci pourrait être très réactive. Dans les tissus verts des plantes, la majorité du MDA est apparement formée par l'oxydation non-enzymatique (nLPO) des acides gras polyinsaturés (PUFAs) transmis par des espèces ac¬tives d'oxygène (ROS). Le MDA formé par nLPO est souvent utilisé comme marqueur de maladies et il est considéré comme une toxine cellulaire. Etonnament, les sites de production comme les mitochondries et les chloro- plastes sont riches en PUFAs qui sont sensibles à la nLPO. Dans cette thèse nous montrons que les chloroplastes répresentent le site de production de MDA dans les feuilles adultes d'Arabidopsis thaliana. Les analyses de MDA dans les plantules ont révélé que le MDA s'accumule dans les tissus meris- tematiques comme celles de la pointe de la racine, des racines latéralles et du meristème apical. Par la caractérisation du MDA présent nous avons pu montrer que la majorité du MDA était présent sous la forme d'un énolate non-réactif. Ceci pourrait expliquer pourquoi le MDA est toléré dans les sites où il pourrait casser le genome ou le protéome s'il est présent sous sa forme protonée. Les analyses du devenir du MDA dans les feuilles par des isotopes du MDA ont montré que celui-ci est metabolisé et utilisé pour assembler des lipides. Le lipide majoritairement métabolisé a été identifié comme étant le 18:3-16:3-monogalactosyldiacylglycerole (MGDG); le lipide le plus abondant dans les chloroplastes. Nous supposons que la présence des PUFAs dans les sites de production du ROS, tout comme le PS II dans les chloroplastes, pourrait jouer un rôle de tampon pour prevenir les protéines de différentes dégradations et ainsi générer des molécules telle que le MDA. La majorité du MDA produit par cette réaction est présente dans la cellule sous la forme d'énolate non-réactif, jusqu'au moment de son utilisation ou lorsqu'il serra metabolisé pour produire des MGDGs polyinsaturés. De plus, il a été décrit que nLPO pourait augmenter dans la pathogenèse, et nous avons testé des plantes adultes et des plantules en présence de champignons nécrotrophiques. L'observation du MDA pendant les infections a montré que les concentrations en MDA sont fortement induites dans les plantules mais contrôlées dans les plantes adultes.

Official Positions for FRAX® clinical regarding biochemical markers from Joint Official Positions Development Conference of the International Society for Clinical Densitometry and International Osteoporosis Foundation on FRAX®.

The best indirect evidence that increased bone turnover contributes to fracture risk is the fact that most of the proven therapies for osteoporosis are inhibitors of bone turnover. The evidence base that we can use biochemical markers of bone turnover in the assessment of fracture risk is somewhat less convincing. This relates to natural variability in the markers, problems with the assays, disparity in the statistical analyses of relevant studies and the independence of their contribution to fracture risk. More research is clearly required to address these deficiencies before biochemical markers might contribute a useful independent risk factor for inclusion in FRAX(®).