Exposure models in Switzerland: an overview of the present situation

A survey was undertaken among Swiss occupational health and safety specialists in 2004 to identify uses, difficulties, and possible developments of exposure models. Occupational hygienists (121), occupational physicians (169), and safety specialists (95) were surveyed with an in depth questionnaire. Results obtained indicate that models are not used very much in practice in Switzerland and are reserved to research groups focusing on specific topics. However, various determinants of exposure are often considered important by professionals (emission rate, work activity), and in some cases recorded and used (room parameters, operator activity). These parameters cannot be directly included in present models. Nevertheless, more than half of the occupational hygienists think that it is important to develop quantitative exposure models. Looking at research institutions, there is, however, a big interest in the use of models to solve problems which are difficult to address with direct measurements; i. e. retrospective exposure assessment for specific clinical cases and prospective evaluation for new situations or estimation of the effect of selected parameters. In a recent study about cases of acutepulmonary toxicity following water proofing spray exposure, exposure models have been used to reconstruct exposure of a group of patients. Finally, in the context of exposure prediction, it is also important to report about a measurement database existing in Switzerland since 1991. [Authors]

Indexes and boundaries for "quantitative significance" in statistical decisions.

Boundaries for delta, representing a "quantitatively significant" or "substantively impressive" distinction, have not been established, analogous to the boundary of alpha, usually set at 0.05, for the stochastic or probabilistic component of "statistical significance". To determine what boundaries are being used for the "quantitative" decisions, we reviewed pertinent articles in three general medical journals. For each contrast of two means, contrast of two rates, or correlation coefficient, we noted the investigators' decisions about stochastic significance, stated in P values or confidence intervals, and about quantitative significance, indicated by interpretive comments. The boundaries between impressive and unimpressive distinctions were best formed by a ratio of greater than or equal to 1.2 for the smaller to the larger mean in 546 comparisons, by a standardized increment of greater than or equal to 0.28 and odds ratio of greater than or equal to 2.2 in 392 comparisons of two rates; and by an r value of greater than or equal to 0.32 in 154 correlation coefficients. Additional boundaries were also identified for "substantially" and "highly" significant quantitative distinctions. Although the proposed boundaries should be kept flexible, indexes and boundaries for decisions about "quantitative significance" are particularly useful when a value of delta must be chosen for calculating sample size before the research is done, and when the "statistical significance" of completed research is appraised for its quantitative as well as stochastic components.

Nutritional status using the Mini Nutritional Assessment questionnaire and its relationship with bone quality in a population of institutionalized elderly women.

Malnutrition, a risk factor for osteoporotic fractures, is frequent in elderly people and, is underdiagnosed and undertreated. There are only few studies on the nutritional status of elderly people in Europe. The Mini Nutritional Assessment (MNA) is a non invasive and validated questionnaire to evaluate nutritional status in elderly people, classified in three groups: 1 degree score < 17: malnourished, 2 degrees score >17 and < 24: at risk of malnutrition, 3 degrees score >24: well-nourished, with a maximum of 30 points. Quantitative ultrasound of bone (QUS) is a method for assessing quality of bone which can be easily performed in nursing homes. Therefore, these two tests allowed to study the relationships between nutritional status and ultrasonic parameters of bone in 78 institutionalized women aged 86 +/- 6 years, living in 11 nursing homes around Lausanne (Switzerland). All were assessed by the MNA, had a measurement of the tricipital skin fold and of the grip strength. Functional status was evaluated by the scale "Activity of Daily Living" (ADL), and serum albumin level was measured when permitted. All had QUS of the calcaneus (with an Achilles, GE Lunar). The measured parameters are the Broadband Ultrasound Attenuation (BUA), attenuation of a band of ultrasonic frequencies through the medium, expressed in dB/MHz, and the Speed of Sound (SOS), speed of the ultrasounds through the medium, expressed in m/s. A third parameter, the stiffness index (SI), expressed as a percentage of the values obtained by the manufacturer in a young population and derived from BUA and SOS, was calculated automatically : SI = (0.67xBUA) + (0.28xSOS) - 420, expressed in percent compared to a young adult population (%YA). Fifteen percent of the women were undernourished and 58% were at risk of malnutrition. As expected, compared with the well-nourished minority, undernourished subjects had significant lower body mass index (BMI), tricipital skin fold (TSF), ADL score and albumin level (p < 0,01). The subjects "at risk of malnutrition" had significant lower BMI, ADL score (p < 0.01), tricipital skin fold and serum albumin (p < 0.05). Ultrasound parameters were low independently of the nutritional status. MNA score correlated significantly with tricipital skin fold (r = 0.508, p < 0.01), ADL (r = 0.538, p < 0.01) and albumin serum level (r = 0.409, p = 0.01). There was a trend for a correlation between the MNA and the ultrasound parameter BUA (r = 0.207, p = 0.07), whereas no correlation was found with SOS and SI. A multivariate analysis showed that tricipital skin fold and ADL explained 61% of the variance of the MNA. In conclusion, using simple and non invasive methods, this study showed that malnutrition and osteoporosis are frequent in institutionalized elderly persons in our country, and the ultrasound parameters are influenced by many others factors in addition to nutrition, especially at this age and in elderly residents of nursing homes.

A multilevel approach to understanding circadian rhythmicity : social and sleep-dependent effects in ants and mice

Résumé françaisLa majorité des organismes vivants sont soumis à l'alternance du jour et de la nuit, conséquence de la rotation de la terre autour de son axe. Ils ont développé un système interne de mesure du temps, appelé horloge circadienne, leur permettant de s'adapter et de synchroniser leur comportement et leur physiologie aux cycles de lumière. Cette dernière est considérée comme étant le signal majeur entraînant l'horloge interne et. par conséquent, les rythmes journaliers d'éveil et de sommeil. Outre sa régulation circadienne, le sommeil est contrôlé par un processus homéostatique qui détermine son besoin. La contribution de ces deux processus dans le fonctionnement cellulaire du cerveau n'a pas encore été investiguée. La mesure de l'amplitude ainsi que de la prévalence des ondes delta de l'EEG (activité delta) constitue un index très fiable du besoin de sommeil. Il a été démontré que cette activité est génétiquement déterminée et associée à un locus de trait quantitatif situé sur le chromosome 13 de la souris.Grâce à des expériences de privation de sommeil et d'analyses de transcriptome du cerveau dans trois souches de souris présentant diverses réponses à la privation de sommeil, nous avons trouvé que Homerla, localisé dans la région d'intérêt du chromosome 13, est le meilleur marqueur du besoin de sommeil. Homerla est impliqué dans la récupération de l'hyperactivité neuronale induite par le glutamate, grâce à son effet tampon sur le calcium intracellulaire. Une fonction fondamentale du sommeil pourrait donc être de protéger le cerveau et de lui permettre de récupérer après une hyperactivité neuronale imposée par une veille prolongée.De plus, nous avons montré que 2032 transcrits sont exprimés rythmiqueraent dans le cerveau de la souris, parmi lesquels seulement 391 le restent après que les animaux aient été privés de sommeil à différents moments au cours des 24 heures. Cette observation montre clairement que la plupart des changements rythmiques au niveau du transcriptome dépendent du sommeil et non de l'horloge circadienne et souligne ainsi l'importance du sommeil dans la physiologie des mammifères.La plupart des expériences concernant les rythmes circadiens ont été réalisées sur des individus isolés en négligeant l'effet du contexte social sur les comportements circadiens. Les espèces sociales, telles que les fourmis, se caractérisent par une division du travail où une répartition des tâches s'effectue entre ses membres. De plus, certaines d'entre elles doivent être pratiquées en continu comme les soins au couvain tandis que d'autres requièrent une activité rythmique comme le fourragement. Ainsi la fourmi est un excellent modèle pour l'étude de 1 influence du contexte social sur les rythmes circadiens.A ces fins, nous avons décidé d'étudier les rythmes circadiens chez une espèce de fourmi Camponotus fellah et de caractériser au niveau moléculaire son horloge circadienne. Nous avons ainsi développé un système vidéo permettant de suivre l'activité locomotrice de tous les individus d'une colonie. Nos résultats montrent que, bien que la plupart des fourmis soient arythmiques à l'intérieur de la colonie, elles développent d'amples rythmes d'activité en isolation. De plus, ces rythmes disparaissent presque aussitôt que la fourmi est réintroduite dans la colonie. Cette rythmicité observée en isolation semble être générée par l'horloge circadienne car elle persiste en condition constante (obscurité totale). Nous avons ensuite regardé si cette apparente arythmie observée dans la colonie résultait d'un effet masquant des interactions sociales sur les rythmes circadiens d'activité. Nos résultats suggèrent que l'horloge interne est fonctionnelle dans la colonie mais que l'expression de ses rythmes au niveau comportemental est inhibée par les interactions sociales. Les analyses moléculaires du statut de l'horloge dans différents contextes sociaux sont actuellement en cours. Le contexte social semble donc un déterminant majeur du comportement circadien chez la fourmi.AbstractAlmost all living organisms on earth are subjected to the alternance of day and night re-sulting from the rotation of the earth around its axis. They have evolved with an internal timing system, termed the circadian clock, enabling them to adapt and synchronize their behavior and physiology to the daily changes in light and related environmental parame¬ters. Light is thought to be the major cue entraining the circadian clock and consequently the rhythms of rest/activity. In addition to its circadian dependent timing, sleep is reg¬ulated by a homeostatic process that determines its need. The contribution of these two processes in the cellular functioning of the brain has not yet been considered. A highly reliable index of the homeostatic process of sleep is the measure of the amplitude and prevalence of the EEG delta waves (delta activity). It has been shown that sleep need, measured by delta activity, is genetically determined and associated with a Quantitative Trait Locus (QTL) located on the mouse chromosome 13. By using sleep deprivation and brain transcriptome profiling in three inbred mouse strains showing different responses to sleep loss, we found that Homerla, localized within this QTL region is the best transcrip¬tional marker of sleep need. Interestingly Homerla is primarily involved in the recovery from glutamate-induced neuronal hyperactivity by its buffering effect on intracellular cal¬cium. A fundamental function of sleep may therefore reside in the protection and recovery of the brain from a neuronal hyperactivity imposed by prolonged wakefulness.Moreover, time course gene expression experiments showed that 2032 brain tran¬scripts present a rhythmic variation, but only 391 of those remain rhythmic when mice are sleep deprived at four time points around the clock. This finding clearly suggests that most changes in gene transcription over the day are sleep-wake dependent rather than clock dependent and underlines the importance of sleep in mammalian physiology.In the second part of this PhD, I was interested in the social influence on circadian behavior. Most experiments done in the circadian field have been performed on isolated individuals and have therefore ignored the effect of the social context on circadian behav-ior. Eusocial insect species such as ants are characterized by a division of labor: colony tasks are distributed among individuals, some of them requiring continuous activity such as nursing or rhythmic ones such as foraging. Thus ants represent a suitable model to study the influence of the social context on the circadian clock and its output rhythms.The aim of this part was to address the effect of social context on circadian rhythms in the ant species Camponotus fellah and to characterize its circadian clock at the molecu¬lar level. We therefore developed a video tracking system to follow the locomotor activity of all individuals in a colony. Our results show that most ants are arrhythmic within the colony, but develop, when subjected to social isolation, strong rhythms of activity that intriguingly disappear when individuals are reintroduced into the colony. The rhythmicity observed in isolated ants seems to be driven by the circadian clock as it persists under constant conditions (complete darkness). We then tested whether the apparent arrhyth- micity in the colony stemmed from a masking effect of social interactions on circadian rhythms. Indeed, we found that circadian clocks of ants in the colony are functional but their expression at the behavioral level is inhibited by social interactions. The molecular assessment of the circadian clock functional state in the different social context is still under investigation. Our results suggest that social context is a major determinant of circadian behavior in ants.

Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome.

Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this "IPEX-like" syndrome presently remains unclear. To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3(+) T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively. TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3(+) and CD25(+)FOXP3(+) T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.

Methods for testing association between uncertain genotypes and quantitative traits.

Interpretability and power of genome-wide association studies can be increased by imputing unobserved genotypes, using a reference panel of individuals genotyped at higher marker density. For many markers, genotypes cannot be imputed with complete certainty, and the uncertainty needs to be taken into account when testing for association with a given phenotype. In this paper, we compare currently available methods for testing association between uncertain genotypes and quantitative traits. We show that some previously described methods offer poor control of the false-positive rate (FPR), and that satisfactory performance of these methods is obtained only by using ad hoc filtering rules or by using a harsh transformation of the trait under study. We propose new methods that are based on exact maximum likelihood estimation and use a mixture model to accommodate nonnormal trait distributions when necessary. The new methods adequately control the FPR and also have equal or better power compared to all previously described methods. We provide a fast software implementation of all the methods studied here; our new method requires computation time of less than one computer-day for a typical genome-wide scan, with 2.5 M single nucleotide polymorphisms and 5000 individuals.

Validation and reproducibility of a semi-quantitative Food Frequency Questionnaire for use in elderly Swiss women.

OBJECTIVE: The principal aim of this study was to develop a Swiss Food Frequency Questionnaire (FFQ) for the elderly population for use in a study to investigate the influence of nutritional factors on bone health. The secondary aim was to assess its validity and both short-term and long-term reproducibility. DESIGN: A 4-day weighed record (4 d WR) was applied to 51 randomly selected women of a mean age of 80.3 years. Subsequently, a detailed FFQ was developed, cross-validated against a further 44 4-d WR, and the short- (1 month, n = 15) and long-term (12 months, n = 14) reproducibility examined. SETTING: French speaking part of Switzerland. SUBJECTS: The subjects were randomly selected women recruited from the Swiss Evaluation of the Methods of Measurement of Osteoporotic Fracture cohort study. RESULTS: Mean energy intakes by 4-d WR and FFQ showed no significant difference [1564.9 kcal (SD 351.1); 1641.3 kcal (SD 523.2) respectively]. Mean crude nutrient intakes were also similar (with nonsignifcant P-values examining the differences in intake) and ranged from 0.13 (potassium) to 0.48 (magnesium). Similar results were found in the reproducibility studies. CONCLUSION: These findings provide evidence that this FFQ adequately estimates nutrient intakes and can be used to rank individuals within distributions of intake in specific populations.

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

A quantitative genetic signature of senescence in a short-lived perennial plant.

The evolution of senescence (the physiological decline of organisms with age) poses an apparent paradox because it represents a failure of natural selection to increase the survival and reproductive performance of organisms. The paradox can be resolved if natural selection becomes less effective with age, because the death of postreproductive individuals should have diminished effects on Darwinian fitness [1, 2]. A substantial body of empirical work is consistent with this prediction for animals, which transmit their genes to progeny via an immortal germline. However, such evidence is still lacking in plants, which lack a germline and whose reproduction is diffuse and modular across the soma. Here, we provide experimental evidence for a genetic basis of senescence in the short-lived perennial plant Silene latifolia. Our pedigree-based analysis revealed a marked increase with age in the additive genetic variance of traits closely associated with fitness. This result thus extends to plants the quantitative genetic support for the evolutionary theory of senescence.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of ribosomal RNA

RÉSUMÉ Le but d'un traitement antimicrobien est d'éradiquer une infection bactérienne. Cependant, il est souvent difficile d'en évaluer rapidement l'efficacité en utilisant les techniques standard. L'estimation de la viabilité bactérienne par marqueurs moléculaires permettrait d'accélérer le processus. Ce travail étudie donc la possibilité d'utiliser le RNA ribosomal (rRNA) à cet effet. Des cultures de Streptococcus gordonii sensibles (parent Wt) et tolérants (mutant Tol 1) à l'action bactéricide de la pénicilline ont été exposées à différents antibiotiques. La survie bactérienne au cours du temps a été déterminée en comparant deux méthodes. La méthode de référence par compte viable a été comparée à une méthode moléculaire consistant à amplifier par PCR quantitative en temps réel une partie du génome bactérien. La cible choisie devait refléter la viabilité cellulaire et par conséquent être synthétisée de manière constitutive lors de la vie de la bactérie et être détruite rapidement lors de la mort cellulaire. Le choix s'est porté sur un fragment du gène 16S-rRNA. Ce travail a permis de valider ce choix en corrélant ce marqueur moléculaire à la viabilité bactérienne au cours d'un traitement antibiotique bactéricide. De manière attendue, les S. gordonii sensibles à la pénicilline ont perdu ≥ 4 log10 CFU/ml après 48 heures de traitement par pénicilline alors que le mutant tolérant Tol1 en a perdu ≥ 1 log10 CFU/ml. De manière intéressant, la quantité de marqueur a augmenté proportionnellement au compte viable durant la phase de croissance bactérienne. Après administration du traitement antibiotique, l'évolution du marqueur dépendait de la capacité de la bactérie à survivre à l'action de l'antibiotique. Stable lors du traitement des souches tolérantes, la quantité de marqueur détectée diminuait de manière proportionnelle au compte viable lors du traitement des souches sensibles. Cette corrélation s'est confirmée lors de l'utilisation d'autres antibiotiques bactéricides. En conclusion, l'amplification par PCR du RNA ribosomal 16S permet d'évaluer rapidement la viabilité bactérienne au cours d'un traitement antibiotique en évitant le recours à la mise en culture dont les résultats ne sont obtenus qu'après plus de 24 heures. Cette méthode offre donc au clinicien une évaluation rapide de l'efficacité du traitement, particulièrement dans les situations, comme le choc septique, où l'initiation sans délai d'un traitement efficace est une des conditions essentielles du succès thérapeutique. ABSTRACT Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether ribosomal RNA (rRNA) could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts, and compared to quantitative real-time PCR amplification of either the 16S-rRNA genes (rDNA) or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost ≥ 4 log10 CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Toll mutant lost ≤ 1 log10 CFU/ml. Amplification of a 427-base fragment of 16S rDNA yielded amplicons that increased proportionally to viable counts during bacterial growth, but did not decrease during drug-induced killing. In contrast, the same 427-base fragment amplified from 16S rDNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Toll mutant (≥4 log10 CFU/ml and ≤1 lo10 CFU/ml, respectively), and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments the experiments were repeated by amplifying a 119-base region internal to the origina1 427-base fragment. The amount of 119-base amplicons increased proportionally to viability during growth, but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical to differentiate between live and dead bacteria.

Neurofibromatosis of von Recklinghausen: a quantitative study of the epidermal keratinocyte and melanocyte populations.

The numerical keratinocyte to melanocyte relation was studied in café au lait spots and adjacent normally pigmented skin of 9 patients with classical neurofibromatosis. Compared to normal skin of healthy individuals, the keratinocyte:melanocyte ratio distributions obtained in neurofibromatosis indicated a shift to lower values in the biopsies of café au lait spots and normally pigmented skin. These results are evidence in favor of an impaired tissue organization of the epidermis in neurofibromatosis with regard to the keratinocyte-melanocyte interrelation.

Controls on ostracod valve geochemistry, Part 1: Variations of environmental parameters in ostracod (micro-)habitats

The variations of environmental conditions (T°, pH, δ13CDIC, [DIC], δ18O, Mg/Ca, and Sr/Ca) of ostracod habitats were examined to determine the controls of environmental parameters on the chemical and isotopic composition of ostracod valves. Results of a one-year monitoring of environmental parameters at five sites, with depths of between 2 and 70 m, in Lake Geneva indicate that in littoral to sub-littoral zones (2, 5, and 13 m), the chemical composition of bottom water varies seasonally in concert with changes in temperature and photosynthetic activity. An increase of temperature and photosynthetic activity leads to an increase in δ13C values of DIC and to precipitation of authigenic calcite, which results in a concomitant increase of Mg/Ca and Sr/Ca ratios of water. In deeper sites (33 and 70 m), the composition of bottom water remains constant throughout the year and isotopic values and trace element contents are similar to those of deep water within the lake. The chemical composition of interstitial pore water also does not reflect seasonal variations but is controlled by calcite dissolution, aerobic respiration, anaerobic respiration with reduction of sulphate and/or nitrate, and methanogenesis that may occur in the sediment pores. Relative influence of each of these factors on the pore water geochemistry depends on sediment thickness and texture, oxygen content in bottom as well as pore water. Variations of chemical compositions of the ostracod valves of this study vary according to the specific ecology of the ostracod species analysed, that is its life-cycle and its (micro-)habitat. Littoral species have compositions that are related to the seasonal variations of temperature, δ13C values of DIC, and of Mg/Ca and Sr/Ca ratios of water. In contrast, the compositions of profundal species are largely controlled by variations of pore fluids along sediment depth profiles according to the specific depth preference of the species. The control on the geochemistry of sub-littoral species is a combination of controls for the littoral and profundal species as well as the specific ecology of the species.

Automated quantitative histology reveals vascular morphodynamics during Arabidopsis hypocotyl secondary growth.

Among various advantages, their small size makes model organisms preferred subjects of investigation. Yet, even in model systems detailed analysis of numerous developmental processes at cellular level is severely hampered by their scale. For instance, secondary growth of Arabidopsis hypocotyls creates a radial pattern of highly specialized tissues that comprises several thousand cells starting from a few dozen. This dynamic process is difficult to follow because of its scale and because it can only be investigated invasively, precluding comprehensive understanding of the cell proliferation, differentiation, and patterning events involved. To overcome such limitation, we established an automated quantitative histology approach. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with automated cell type recognition through machine learning, we could establish a cellular resolution atlas that reveals vascular morphodynamics during secondary growth, for example equidistant phloem pole formation. DOI: http://dx.doi.org/10.7554/eLife.01567.001.

New human kallikrein 14: enzymatic characterization and inhibitors development.

RESUME DESTINE A UN LARGE PUBLIC En biologie, si une découverte permet de répondre à quelques questions, en général elle en engendre beaucoup d'autres. C'est ce qui s'est produit récemment dans le monde des kallicréines. De la famille des protéases, protéines ayant la faculté de couper plus ou moins spécifiquement d'autres protéines pour exercer un rôle biologique, la famille des kallicréines humaines n'était composée que de 3 membres lors du siècle dernier. Parmi eux, une kallicréine mondialement utilisée pour détecter le cancer de la prostate, le PSA. En 2000, un chercheur de l'hôpital universitaire Mont Sinaï à Toronto, le Professeur Eleftherios Diamandis, a découvert la présence de 12 nouveaux gènes appartenant à cette famille, situés sur le même chromosome que les 3 premières kallicréines. Cette découverte majeure a placé les spécialistes des kallicréines face à une montagne d'interrogations car les fonctions de ces nouvelles protéases étaient totalement inconnues. La kallicréine humaine 14 (hK14) présente un intérêt particulier, car elle se retrouve associée à différents cancers, notamment les carcinomes ovariens et mammaires. Cette association ne répond cependant pas à la fonction de cette protéase. L'objectif de ce travail de thèse était donc de découvrir, dans un premier temps, la spécificité de cette nouvelle kallicréine, c'est-à-dire le type de coupure qu'elle engendre au niveau des protéines qu'elle cible. Utilisant une technologie de pointe qui exploite la propriété des bactériophages à se répliquer dans les bactéries à l'infini, des dizaines de millions de combinaisons protéiques aléatoires ont été présentées à hK14, qui a pu sélectionner celles qui lui étaient favorables pour la coupure. Cette technique qualitative porte le nom de Phage Display Substrate. Une fois la sélection réalisée, il fallait transférer ces séquences coupées ou substrats dans un système permettant de donner une valeur quantitative à l'efficacité de coupure. Pour cela nous avons développé une technologie qui permet d'évaluer cette efficacité en utilisant des protéines fluorescentes de méduse, modifiées génétiquement, dont l'excitation de la première (CFP : cyan fluorescent protein) par la lumière à une certaine longue d'onde permet le transfert d'énergie à la seconde (YFP : yellow fluorescent protein), via un substrat qui les lie. Pour que ce transfert d'énergie se produise, il faut que les deux protéines fluorescentes soient proches, comme c'est le cas lorsqu'elles sont liées par un substrat. La coupure de ce lien provoque un changement de transfert d'énergie qui est quantifiable en utilisant un spectrofluoromètre. Cette technologie permet donc de suivre la réaction d'hydrolyse (coupure) des protéases. Afin de poursuivre certaines expériences permettant de mieux comprendre la fonction biologique d'hK14 ainsi que son éventuelle implication dans le cancer, nous avons développé des inhibiteurs spécifiques d'hK14. Les séquences qui on été le plus efficacement coupées par hK14 ont été utilisées pour transformer deux types d'inhibiteurs classiques, qui circulent dans notre sang, en inhibiteurs d'hK14 hautement efficaces et spécifiques. Selon les résultats obtenus in vitro, ils pourront être évalués in vivo en tant que traitement potentiel contre le cancer. RESUME Les protéases sont des enzymes impliquées dans des processus physiologiques mais aussi parfois pathologiques. La famille des kallicréines tissulaires humaines représente le plus grand groupe de protéases humaines, dont plusieurs pourraient participer au développement de certaines maladies. D'autre part, ces protéases sont apparues comme des marqueurs de pathogénicité potentiels, notamment dans les cas de cancers hormono-dépendants. La kallicréine humaine 14 a été récemment découverte et son implication dans quelques maladies, particulièrement dans le cas de tumeurs, semble probable. En effet, son expression génique est augmentée au niveau des tissus cancéreux de la prostate et du sein et son expression protéique s'est révélée plus élevée dans le sérum de patientes atteintes d'un cancer du sein ou des ovaires. Cependant, comme c'est le cas pour la plupart des kallicréines, sa fonction est encore inconnue. Afin de mieux connaître son rôle biologique et/ou pathologique, nous avons décidé de caractériser son activité enzymatique. Nous avons tout d'abord mis au point un système de substrats entièrement biologique permettant d'étudier in vitro l'activité des protéases. Ce système est basé sur le phénomène de FRET, à savoir le transfert d'énergie de résonance fluorescente qui intervient entre deux molécules fluorescentes voisines si le spectre d'émission de la protéine donneuse chevauche le spectre d'excitation de la protéine receveuse. Nous avons fusionné de manière covalente une protéine fluorescente bleue (CFP) et une jaune (YFP) en les liant avec diverses séquences. Par clivage de la séquence de liaison, une perte du transfert d'énergie peut être mesurée par un spectrofluoromètre. Cette technologie représente un moyen facile de suivre la réaction d'hydrolyse des protéases. Les conditions optimales de production de ces substrats CFP-YFP ont été déterminées, de même que les paramètres pouvant éventuellement influencer le FRET. Ce système possède une grande résistance à la protéolyse non spécifique et est applicable à un grand nombre de protéase. Contrairement aux substrats fluorogéniques, il permet d'étudier les acides aminés se trouvant des deux côtés du site de clivage. Ce système étant entièrement biologique, il est le reflet des interactions protéine-protéine et représente un outil biologique facile, bon marché et rapide pour caractériser les protéases. Dans un premier temps, hK14 a été mise en présence d' une banque de haute diversité de pentapeptides aléatoires présentée à la surface de phages afin d'identifier des substrats spécifiques. Ensuite, le système CFP-YFP a été employé pour trier les peptides sélectionnés afin d'identifier les séquences de substrats les plus sensibles et spécifiques pour hK14. Nous avons montré, qu'en plus de sa prévisible activité de type trypsine, hK14 possède aussi une très surprenante activité de type chymotrypsine. Les séquences les plus sensibles ont été choisies pour cribler la banque de donnée Swissprot, permettant ainsi l'identification de 6 substrats protéiques humains potentiels pour hK14. Trois d'entre eux, la laminine α-5, le collagène IV et la matriline-4, qui sont des composants de la matrice extracellulaire, ont démontré une grande susceptibilité à l'hydrolyse par hK14. De plus, la séparation éléctrophorétique a montré que la dégradation de la laminine α-5 et de la matriline-4 par hK14 devait se produire aux sites identifiés par la technologie du phage display. Pour terminer, nous avons transformé, par mutagenèse dirigée, deux serpines (inhibiteurs de protéases de type sérine) connues, AAT et ACT (alpha anti-trypsine et alpha anti-chymotrypsine), qui inhibent un vaste éventail d'enzymes humaines en inhibiteurs d'hK14 hautement efficaces et spécifiques. Ces inhibiteurs pourront être utilisés d'une part pour poursuivre certaines expériences permettant de mieux comprendre l'implication d'hK14 dans des voies physiologiques ou dans le cancer et d'autre part pour les évaluer in vivo en tant que traitement potentiel contre le cancer. SUMMARY Proteases consist of enzymes involved in physiological events, but also, in case of dysregulation, in pathogenicity. The human tissue kallikrein family represents the largest human protease cluster and includes several members that either could participate in the course of certain diseases or emerged as potential biological markers, especially in hormone dependent cancers. The human kallikrein 14 has been recently discovered and suggested implications in some disorders, particularly in tumors since its gene expression is up-regulated in prostate and breast cancer tissues and its protein expression increased in the serum of patients with breast and ovarian cancers. However, like most kallikreins, its function remains unknown. To better understand hK14 biological and/or pathological role, we decided to characterize its enzymatic activity. First of all, we developped a biological system suitable for in vitro study of protease activity. This system is based on the so-called FRET phenomenon, that is the Fluorescence Resonance Energy Transfer that occurs between two nearby fluorescent proteins if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. We fused covalently a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) with diverses sequences. Upon cleavage of the linker sequence by protease, the loss of energy transfer can be measured by a spectrofluorometer allowing an easy following of hydrolysis reaction. The optimal conditions to produce in bacterial system these CFP-YFP substrates were determined as well as the parameters that could eventually influence the FRET. This system demonstrated a high degree of resistance to non-specific proteolysis and applicability to various conditions corresponding to a great number of existing proteases. Other avantages are the possibility to study the amino acids located both sides of the cleavage site as well as the interest to work in a full biological system reflecting protein-protein interaction. A phage substrate library with exhaustive diversity was used prior to CFP-substrate-YFP system to isolate specific human kallikrein 14 substrates. After that the CFP-YFP system was used to sort peptides and identify highly sensitive and specific substrate sequences for hK14. We showed that besides its predictable trypsin-like activity, hK14 also possesses a surprising chymotrypsin-like activity. The screening of the Swissprot database was achieved with the most sensitive sequences and allowed the identification of 6 potential human protein substrates for hK14. Three of them, laminin α-5, collagen IV and matrilin-4, which are components of the extracellular matrix were incubated with hK14, by which they were efficiently hydrolyzed. Moreover, electrophoretic separation revealed that degradation of laminin α-5 and matrilin-4 by hK14 generated fragments with identical molecular size than the predicted N-terminal fragments that would result from hK14 specific cleavage, proving the value of phage display substrate to identify potential substrates. Finally, with site-directed mutagenesis, we transformed two well-known serpins (serine protease inhibitors), AAT and ACT (alpha anti-trypsin and alpha anti-chymotrypsin), which inhibit a vast spectrum of human enzymes into highly efficient and specific hK14 inhibitors. These inhibitors will be used to pursue experiments that could help understand hK14 implication in physiological pathways as well as in cancer biology and also to perform their in vivo evalution as potential cancer treatment.