Cyril Tissot

Mathias Rouan

Etienne Neethling

Hervé Quénol

David Brosset

Hervé Quenol

Modeling of vine agronomic practices in the context of climate change

  L'archive ouverte pluridisciplinaire

INTRODUCTION

Global climate change affects regional climates and hold implications for viticulture worldwide. Many studies have addressed the issue of the impact of climate change on viticulture in most wine regions worldwide, yet few studies are devoted to observing and simulating both climate and climate change at the "terroir" scale (local scale). However, phenological variations as well as difference in grapes/wine quality are often observed over short distances in a wine-region, which are related to local characteristics (slope, soil, seasonal climate . . . ).

A wide range of the adaptation solutions suggested to reduce the impacts of climate change on viticulture have resulted from studies carried out at regional scales using computer simulations. However, results from models hold significant uncertainties e.g. difficulty to predict future climate due to uncertainties related to emissions scenarios as well as model parameterizations and integrations and, finally, regional scales are not the most appropriate scale for winegrowers. Climate variability at local scales explains more clearly the characteristics of wines, and winemakers have always been adapting their practices based on it. In the context of climate change, the development in knowledge of climate and impacts on grapevine performance and resulting wine characteristics allow to identify methods of adaption to climate change a cyril.tissot@univ-brest.fr b herve.quenol@uhb.fr c etienneneethling@gmail.com d david.brosset@ecole-navale.fr based on constant evolution of practices rather than on radical change of practices such as shifting of viticultural regions or change of cultivars.

The aim of this contribution is to conceptualize a generic model (a site specific viticultural activity) including strategies of production (modeling by decision trees) to reproduce the impact of spatial and temporal variability of climatic conditions on grapevine growth and berry maturity using an approach that combines technologies derived from GIS, agent-based models and spatial and temporal databases. The objective is to specify a systemic and integrated modeling environment for simulating grapevine growth and berry ripening under different conditions and constraints (slope, aspect, soil type, climate variability . . . ) and to integrate production strategies and corresponding adaptation rules according to the evolution of these constraints.

Modeling approach

The approach aimed to link in situ observation about phenology and agronomic action [START_REF] Bonnefoy | Analyse multi-scalaire des températures dans le vignoble du Val de Loire[END_REF] to issues in terms of seeking optimal vine adaptation to wineproducing soils' specificity [START_REF] Barbeau | Role of soil physical properties, climate and harvest period on must composition, wine type and flavour (Vitis vinifera L, cv chenin), Coteaux du Layon, France[END_REF][START_REF] Asselin | Les terroirs viticoles : du concept au produit[END_REF]. It draws on the DAHU multi-agent simulator.

The DAHU platform provides a simulation environment that can integrate multi-source and multi-scale spatiotemporal constraints as forcing variables within constrained agent models (Fig. 1).

The DAHU platform provides a simulation environment that can integrate multi-source and multi-scale spatiotemporal constraints as forcing variables within constrained agent models (Fig. 1). This approach meets the requirement of explicitly formalising spatiotemporal relationships between spatial entities, environmental processes and human activities. The aim is to set up a methodological framework that can run without a complete system specification, i.e. considering the modelling environment as the result of a set of heterogeneous constraints in scalar terms.

This approach meets the requirement of explicitly formalising spatiotemporal relationships between spatial entities, environmental processes and human activities. The aim is to set up a methodological framework that can run without a complete system specification, i.e. considering the modelling environment as the result of a set of heterogeneous constraints in scalar terms. The specificity of the DAHU platform is that the entire model is based on the technical description of human activities (activity modeling in Fig. 1). Each activity is modelized by an agent 1 .

Agents are selected based on the detailed typology of the various activities and their associated practices (Le [START_REF] Tixerant | Modelling of human activity development in coastal sea areas[END_REF][START_REF] Tissot | Modeling interaction between human activities and coastal zone environment[END_REF]). In the DAHU platform, the environment consists of a set of spatialised data. In particular, it includes constraints that influence the development of an activity. These constraints directly or indirectly influence the decisions and/or actions of agents.

Formalisation of agents

The integration of this model in the DAHU simulator aimed to optimise the connections between the supervising, operating and production agents.

-the "Supervisor" Agent plays a supervising role in the model. It sets the specifications of the various wine designations and imposes specific production methods. It is directlyrelated to the "WineGrowers" Agents who provide information regarding the quality of grapes produced by their vines. According to this information, the "Supervisor" agent may modify wine specifications;

-"Viticulteurs" Agents aim to produce wine that meets precise specifications according to the target designation. This action involves growing the grapevine in optimal conditions given the agronomic specificities of the grapevine-grower's plots;

-"Vignes" Agents are grape production entities. They generally represent a plot or an entity deemed homogeneous in terms of agronomic features (definition based on soil units; [START_REF] Bodin | Characterization of viticultural terroirs using a simple field model based on soil depth. I. Validation of the water supply regime, phenology, and vine vigour, in the Anjou vineyard (France)[END_REF]. The role of these agents is to reproduce vine growth dynamics according to climate conditions.

The relationships between these three types of agents determine the production strategies adopted by the grapevine growers. The Operating Agent group was thereby enhanced with decision-making regulations established based on regulations applied under climate constraints.

Modeling sequence

In order to relate vine management to environmental constraints, a significant knowledge base informing all biophysical and agronomic parameters of the vine as been developed. This database allows simulating the growth cycle of the vine by taking into account climate variability and agronomic practices.

In order to model the growth of the vine simple algorithms based on bioclimatic indices are used. Theses indices allows to study plant response to climate variability according to grape variety (Morlat, 2010;[START_REF] Tonietto | A multicriteria climatic classification system for grape-growing regions worldwide[END_REF][START_REF] Huglin | Biologie et écologie de la vigne[END_REF].

Temperature data are also used to calculate Huglin solar thermal index [START_REF] Huglin | Nouveau mode d'évaluation des possibilités héliothermiques d'un milieu viticole[END_REF], which is used by the 01015-p.2 The value of the Index Huglin for a station is the cumulative value of the index at the date of September 30, the accumulation start since April 1 (northern hemisphere to the southern hemisphere, index is calculated for the period from September 30 to April 1) using the following formula: The architecture of the model is to formalize a relationship between vine growth cycle, grape variety and production method used.

The aim of the simulations (the model is under development) is to relate grapevine phenological stages to climate conditions and crop management methods in a context of global change (Fig. 2). The choice of agronomic actions also depends on the production strategy winemaker. All these elements determine agronomic practices that are adjusted in real time in the model. Therefore the actions taken at time step t can influence the choices made at t + n.

Simulation

The purpose of the first tests carried out in the Coteaux du Layon region (France) is to assess the capacity of grapevine growers to adapt to the considerable rise in temperature and produce sweet wine [START_REF] Bonnefoy | Analyse multi-scalaire des températures dans le vignoble du Val de Loire[END_REF]. Three production profiles were identified: conventional (traditional vine-growing), integrated (plant protection product and fertiliser restrictions), organic (precise specifications severely limiting the use of plant protection products). An extensive survey of grapevine growers helped associate calendars of specific practices with each production profile. These calendars facilitated the development of decision trees under agroclimatic constraints. Temperature and precipitation data were used to define which days were agronomically feasible for carrying out vineyard practices. According to these data, the production profile and the agroclimate features (type of soil, slope, orientation), each WineGrowers Agent makes crop management choices.

In Argentina the Bodega AltaVista prototype is devoted to assess the control of plant water stress by optimising agronomic action and the use of irrigation in Mendoza country. The bodega has greatly contributed to the development of this model by providing bioclimatic data and information on agricultural practices The set of parameters and all the agronomic action can be controlled via the GUI model. The NetLogo platform used to support the development provide visualization tools such as maps and graphics probes. Agents choices and actions can be viewed at each time step, at daily scale for the prototype actually developed (Fig. 7). Probes are also used to visualize graphically many parameters as bioclimatic indexes for example.

GUI model is used to display relevant results and to interact with winemakers. However it is difficult to draw specific conclusions about the data without further analysis. This first stage, currently being tested, requires validation because decision-making is a complex chain of relationships to model. Two methods are currently under development. The first method focuses on adapting the production strategy from a knowledge base consulted by the Viticulteur Agent at each time step. The second, more complex method draws on iterative stochastic algorithms to estimate of the best grapevine management solution.

Conclusion

The modeling approach presented in this chapter offers a comprehensive approach to simulate the interactions between viticulture and environmental constraints. Through the development of a multi-agent model the issue of the impact of climate variability on agronomic practices and production strategies are specifically targeted. DAHU modeling platform allows simulation of vine growth from bioclimatic indices and secondly simulation of agronomic practices in the context of climate change. This step is crucial for the analysis of production strategies.

After this first development step many prospects are considered. They relate mainly to improvements in computer code of DAHU-Vine.

That needs to be expanded to assess the implications of agronomic actions on the dynamics of the vine. From a technical point of view it will be necessary to introduce feedback loops within the model to simulate the consequences of some agronomic choices on vine characteristics (vigor, earliness, resistance to pathogens . . . ). Cooperation with vine-growers also raise the issue of ownership of such a modeling approach in a context where the skills and experience are the best tools to adapt to changing climate conditions. Discussions with wine-growers are essential to validate and improve the model and also to build tangible scenarios of practical adaptation to climate change. In this context the work sessions are planned to construct co-scripted and prospective simulations.

Figure 1 .

 1 Figure 1. The DAHU simulation platform.

Figure 2 .

 2 Figure 2. Modelling vine management in the context of climate change.

Figure 3 .

 3 Figure 3. Screen capture of DAHU-Vigne model.

  I H = [(T m -10) + (T x -10)/2] * k where Tm = average temperature, Tx = maximum temperature and k = coefficient of day length ranging from 1.02 to 1.06 (depending on the latitude of the area of interest).

An agent is an entity capable of acting on itself or its environment, which reacts to its changes and has a partial representation of its environment (Wooldridge, M.

2009).


Oštir Krištof

Nuninger Laure

Paleorelief detection and modellinga case of study in eastern Languedoc (France)

Introduction

The main objective of the paper is paleo digital elevation model (paleoDEM) detection and modelling, a multidisciplinary approach involving geodesy, archaeology, and paleoenvironmental studies. The digital elevation model (DEM) is an important data layer to understand settlement pattern and socioenvironmental contexts and evolution that archaeologists are using together with GIS analysis. It can, for example help to analyze viewsheds, to define travel time between places, to simulate ancient paths, etc. Considering a period of several millennia, some topographical areas can be considered without fundamental change. Within littoral zones or fluvial areas, however, the situation is different. Sedimentation and erosion change the relief permanently, due to alluvial and sea level dynamic. A nowadays-flat area could be hilly in prehistoric times and the application of present DEM for the analysis is therefore unsuitable, since it would distort the expected result completely. Our case of study area is situated in Languedoc (France), on the littoral plain of Mauguio between the cities of Montpellier to the west and Nîmes to the north (Fig. 1). This area covers around 250 km 2 . From an archaeological point of view, the zone is very well explored with almost 350 sites inhabited from Bronze Age to Middle Ages, showing also land division evidences obtained from aerial photo interpretation. In addition, in 1990s several geoarchaeological surveys were done in the main deltas of Lez and Vidourle rivers. Unfortunately the geo-archaeological coring, performed to understand paleochannel profiles in the Vidourle delta, is concentrated in three zones only. Considering a regional scale, only three average points could be used in further analyses. The geoarchaeological approach in itself brings indices about paleorelief but since the measurements are represented as points they are not adequate to produce a proper historical DEM. Additionally the approach is slow and relatively expensive. We have therefore used remote sensing, optical image processing and radar inter-ferometry, in combination with spatial analysis to detect the paleorelief.

The first step was to create an accurate present DEM with a resolution suitable for analyses. Afterwards, the DEM, as well as SPOT and Landsat satellite images, was processed with several filtering techniques to detect "paleofeatures" -evidences of paleorelief. Features were digitalised and their spatial distribution was statistically tested according to archaeological data distribution. At last, we compared and tried to qualify the paleofeatures with the results obtained by photo-interpretation, classification and simulation.

Paleorelief interpretation

Existing technology does not allow a direct detection of paleorelief. Nevertheless, satellite imagery and current digital elevation models can be used to detect the "paleofeatures" and to relate them to past relief.

The digital elevation model of the study area has been made as a combination (weighted sum) of all available sources [START_REF] Podobnikar | Data integration for the DTM production[END_REF]. Firstly, a DEM has been produced with radar interferometry (InSAR, [START_REF] Oštir | Producing digital elevation models with radar interferometry[END_REF]) from two ERS radar images, acquired on 1996-04-04 and 1996-04-05. The images were acquired in tandem mode by ERS-1 and ERS-2 with a time difference of only one day. The coherence of the image pair is very high in the whole study area, with exception of water bodies (sea, rivers, channels, etc.). Interferometric processing produced a digital elevation model with a resolution of 25 m. The vertical accuracy of the relief is approximately 8 m and the positional accuracy is almost 5 m (one fifth of pixel size). The model has been only moderately filtered and prepared for combination with other DEMs.

Additionally the following DEMs were used: IGN DEM (with the resolution of 50 m, provided by IGN), Aster DEM (30 m, made from Aster stereo satellite images), SRTM DEM (90 m, produced with radar interferometry from the Shuttle Radar Topography Mission data). These models have different resolution and different coverage of the area: from ~60% for Aster to almost 100% for InSAR and SRTM (both are produced with interferometry and contain some areas with missing data) and complete for IGN. Standard deviations of all DEMs for the study area are less than 10 m (Table 1); Aster and InSAR are systematically higher because they include vegetation cover.

The final (combined) model is a weighted sum of partial models [START_REF] Podobnikar | Data integration for the DTM production[END_REF]. It takes the rough shape of IGN DEM and adds details of others, particularly InSAR. Its resolution is 25 m and it is very close to the control points (-0.2 m, standard deviation 3.7 m, Table 1, Fig. 1). The model is rather homogenous and could be used for further analysis and paleorelief interpretation. Optical satellite images were an additional important interpretation source. A panchromatic image acquired by SPOT 4 in 1999-04-08 and a Landsat 7 ETM+ image from 2001-08-13 were used. Optical images (Landsat and SPOT) were enhanced prior interpretation. We have been trying to observe paleofeatures indirectly through detection of edges, particularly related to humidity and vegetation anomalies. Landsat with its seven bands gives much better spectral information than SPOT while SPOT has a better spatial resolution (SPOT 4 pan with 10 m resolution has been used only).

Simple and advanced edge detection has been performed, including Sobel and high-pass filtering supplemented with edge thresholding. Special attention has been given to filtering in the direction of the Sun to expose small variations in the direction of incidence radiation. The position of the Sun has been computed from the image acquisition time and the filter has been oriented in the selected direction.

To increase the spatial and preserve spectral resolution of Landsat resolution merge of panchromatic and selected multispectral channels has been performed [START_REF] Švab | High-resolution image fusion: methods to preserve spectral and spatial resolution[END_REF]. A similar procedure has been repeated with panchromatic SPOT and multispectral Landsat data. It has to be mentioned the huge difference between 15 m resolution of panchromatic Landsat and 10 m of SPOT. The latter is much sharper, what is probably the influence of different sensor types, simplifying the observation of anomalies.

The pre-processed DEM and satellite imagery enabled paleofeature delineation. Manual on screen digitalization has been performed and all possible features have been included. The following data has been mainly used to digitalize potential features (Fig. 2):

• panchromatic and edge filtered SPOT,

• multispectral and pan-sharpened Landsat, edge filtered panchromatic Landsat, and • shaded and edge filtered combined DEM. All possible anomalies have been considered and the features have been over-digitalized and later cleaned (Fig. 3). In the iterative procedure different images were used to digitize and recognize features. Maps in the scale 1:25.000 were used to verify all the vectors by eliminating existent relief characteristics (roads, buildings, etc.) and water courses (rivers, channels, etc.). SPOT and shaded combined DEM were most useful in feature delineation and all other layers were used only to update or check the results. Much better results are expected with a digital elevation model produced from lidar imaging [START_REF] Challis | Airborne laser altimetry in alluviated landscapes[END_REF]) that is scheduled for December 2006.

Thematical identification

To test if the detected paleofeatures can be linked to real paleorelief, an analysis of archaeological sites in the area has been performed (Fig. 3). The distribution of sites in relation to the features (buffer zones, site proximity analysis) has been observed for Prehistory, Roman period and medieval period. The sites were compared to random point distribution. For all known sites the distance from digitalized paleofeatures has been determined. Only sites that are less than certain distance (2.5 km) from the features have been considered. It has been statistically proven that the average distance of sites for all periods, especially for Prehistory, is smaller than for random points (Fig. 4a). For Prehistory the average distance is only approximately 450 m, while for random points it is more than 800 m, almost twice as large. One can also observe that the majority of the sites lie less then 500 m from the detected features (Fig. 4b). Despite of the connection between evidence of paleofeatures and archaeological sites, we do not know their exact nature. Three different approaches were performed to define types of paleofeatures. First, the digitized layer was overlaid with geology, pedology, cadastre with contour lines at 1 m interval, and historical aerial photography (from allied missions in 1944). By observation of small areas chosen as test sites, several paleofeatures were identified as paleochannels, edges of natural paleodepressions, ancient roads, terraces, dried valleys etc. Nevertheless, it was noticed that some of the features are still related to the artefacts observed on the IGN DEM (stages caused by contour line interpolation) and some features remain unresolved.

The second approach was related to paleocoastline or sea border (Fig. 3). To ascertain if any of the features can be interpreted as coastline, we used simulations taking into account geoarchaeological information. A small number of cores with an acceptable spatial distribution permitted the generation of a very basic model with an approximated subsiding of the alluvial floor [START_REF] Nuninger | Contribution à la modélisation des paléo-reliefs de la plaine littorale de l'étang de Mauguio (Languedoc, France): premières approches par télédétection. In Temps et espaces de l'Homme en société. Analyses et modèles spatiaux en archéologie[END_REF]. The result shows a rather good general connection and by adding archaeological sites distribution, we were able to notice a relation between chronology and local variation. In fact, reconstitution of paleo-shore during antiquity suggests that the sea-level was approximately 1 m above the present level. However, the archaeological site locations suggest less elevated paleo-shore which is better connected to detected paleofeatures. Even if the scale of layers is different, we have to consider the local variation of sea level, due to hydro sedimentary dynamics. Thus, features detected (Fig. 5) show two steps of shore evolution from Bronze Age to Antiquity. In this case, a shore seems to have moved back until Antiquity and Bronze Age settlements were probably under water during that time.

Fig. 5. Evolution of paleo sea border from Bronze Age to Antiquity

At last, in order to automatically classify the detected evidence of paleorelief, a multivariate analysis (factor analysis with classification 1 ) was processed [START_REF] Coulon | Contribution à la recherche des paléoreliefs dans la plaine littorale de Mauguio : analyse spatiale et statistique des données pour l'élaboration d'une typologie des indices de paléoreliefs[END_REF]. First, the detected features were rasterised and filtered to delete each cells corresponding to DEM artificial stages and thus to avoid noise due to IGN DEM artefacts. Remaining cells were grouped as new entities (1020) according to neighbourhood and their thematic homogeneity (mostly soils). Then, each entity was described by several criteria:

• Archaeological density was calculated as kernel density taking into account duration of occupation (settlements). E.g. 1 settlement occupied during period 100 to 199 AD and period 200 to 299 AD counts as 2 occupations for density calculation. The factor analysis results grouped the detected paleofeatures in 7 classes for 1020 entities. We will focus to the most significant only. In spite of containing dip 6% of all entities, class 1 is very interesting. It groups features with a strong archaeological density, a very strong compactness, a principal aspect to the east and a concentration on the oldest soils (61% "fersiallitiques").

Features from this class can be interpreted as micro-knoll with a high probability. Class 7 and 8 (14% and 6%) can be interpreted as class 1 with lower probability. On the contrary within class 6 (12%), one can observe a weak archaeological density (78% from null to mean value), a weak compactness (88%), principal aspect to north or north-east (67%) and most recent soils (70%). Features from this class are probably in connection with alluvial terrace or dried valley. At last, the class 5 (10%) is represented mostly by no archaeological occupation (82%), a very strong compactness (70%) and most recent soil (85%), especially soils with hydromorphological characteristics (41%). This class should bring together small depressions and paleomeanders.

The multivariate analysis is a first stage to sort automatically the detected paleofeatures. Nevertheless, such assumptions should be verified by control on the original data (IGN map, cadastre, aerial photograph) and in the field. Then, if the classification can be validated, criteria should be integrated directly within the process of detection.

Conclusions

With the case study we have proven that remote sensing can help in the detection of paleorelief. While it is not possible to detect past environment directly, several data layers -in our case most successfully the enhanced panchromatic SPOT 4 and the combined DEM -can be used to observe paleofeatures and to relate them to relief. The described approach will not replace precise work of archaeological and paleoenvironmental investigation, but can simplify the interpretation.

The results obtained present a first step and should be improved with further processing and additional data. The project will continue with advanced processing of high resolution satellite imagery and aerial photography. Furthermore, lidar scanning is scheduled to provide better elevation data in the flat areas.

Fig. 1 .

 1 Fig. 1. Location of the study area.

Fig. 2 .

 2 Fig. 2. Enhanced SPOT (a) and Landsat (b) satellite images were used together with DEM (c) to detect paleofeatures.

Fig. 3 .

 3 Fig. 3. Digitized and cleaned paleofetures are shown in relation to archaeological sites.

Fig. 4 .

 4 Fig. 4. Average distance to of archaeological sites to paleofeatures for different periods (a) and percentage of the sites within certain distance (b) compared with random points.

Table 1 .

 1 Statistical comparison of available DEMs and the final combined DEM

		Control	IGN	Aster	SRTM	InSAR	Combined
		points	DEM 50	DEM	DEM	DEM	DEM
	Number	110	110	67	108	105	110
	Percentage	100%	100%	61%	98%	95%	100%
	Average	81.4	80.9	82.4	81.6	90.0	81.2
	Min Diff		-13	-13	-25	-15	-15
	M a x D i f f		8	2 4	1 1	2 9	8
	Average Diff		-0.5	9.1	-1.2	4.7	-0.2
	StdDev Diff		3.7	8.4	5.0	7.8	3.7

The factor analysis and classification used are an « Analyse Factorielle des Correspondances -AFC » with a « Classification Ascendante Hiérarchique -CAH »

Acknowledgments

The project was supported by CNRS within the framework of a four months associate researcher position for Krištof Oštir (Laboratory ThéMA, UMR 6049, Besançon, France). We would like to thank Jean-François Berger, Cécile Jung and M. Coulon for their collaboration and access to their data. Ziga Kokalj helped with figures and text corrections.


est destinée au dépôt

The dual beam spectropluviometer

Discrete view of rain :

Various studies allowed the modeling and the simulation of rainfall as a raindrop point process. Lavergnat andGolé, 1998 et 2006. The DBS also allowed some investigations on the DSD.

BLOC DIAGRAM

Successfully compared with other optical disdrometers.

LATMOS/IPSL/UVSQ/UPMC/CNRS

The Dual Beam spectropluviometer (DBS) measures the equivalent diameter, the fall velocity and the time of arrival of raindrops. Its main advantage over previous optical disdrometers is its ability to capture the whole measurement range of atmospheric precipitating particles (down to 0.1mm raindrops). Room for improvement and rejuvenating:

A 2014 IPSL funding is asked for in order to modernize the DBS (both hardware and software components).

The processing algorithm as well as the calibration process are under study to improve the quality of the measurements (and among others to reduce the noise for the small drops).

Intercomparisons with other instruments at Sirta is in progress.

RELATED WORKS

Power spectra of the time series collected from july 2008 to july 2009 at the Sirta. This picture display the various scaling of the rain measurements

  Nawal Akrour, Laurent Barthès, Aymeric Chazottes, Jean-Yves Delahaye, Peter Golé, Jacques Lavergnat, Jean-Paul Vinson To cite this version: Nawal Akrour, Laurent Barthès, Aymeric Chazottes, Jean-Yves Delahaye, Peter Golé, et al.. The dual beam spectropluviometer. Journée Scientifique SIRTA 2014, Jun 2014, Palaiseau, France. 2014. hal-01108783

  Continuous view of rain :At the Latmos, various studies on the multifractal properties of rain are based on measurements of the DBS.

	De Montera at al., 2009 & 2010
	Verrier et al., 2011 & 2013.

  . Akrour, L. Barthès, A. Chazottes, J.Y. Delahaye,, P. Golé, J. Lavergnat and J.P. Vinson

		THE DUAL BEAM SPECTROPLUVIOMETER
	TECHNICAL CHARACTERISTICS	FIELD EVALUATION : (DEVEX experiment)
	2D homogeneity of beams and noise
	Up Low	Up Low				Up Low	Up Low
	2 mm		2 mm			2 mm	2 mm
		Ribs	A	B	C	D	Slots
			Ø 69		250 mm	Ø 69
	Emitter		Measurement area	Receiver

Krajewski et al., DEVEX-disdrometer evaluation experiments : Basic results and implications for hydrologic studies, 2005, Advances in Water Resources.

N


Michel Jourdan

Maïlys Cren

N Robert

Karine Bolloré

Thierry Fest

Christophe Duperray

Fabien Guilloton

Dirk Hose

Karin Tarte

Bernard Klein
email: bernard.klein@inserm.fr

à la diffusion de documents scientifiques de niveau

Keywords: Plasma cell, B cell, differentiation, microarray

recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors.

INTRODUCTION

Mature memory plasma cells (PCs), termed long lived plasma cells (LLPCs), are located in the bone marrow (BM) or mucosa and may survive for years, insuring longterm immune memory. [START_REF] Radbruch | Competence and competition: the challenge of becoming a long-lived plasma cell[END_REF] LLPCs are rare cells (0.25 % of BM cells) [START_REF] Hideshima | A proto-oncogene BCL6 is up-regulated in the bone marrow microenvironment in multiple myeloma cells[END_REF][START_REF] Terstappen | Identification and characterization of plasma cells in normal bone marrow by high resolution flow cytometry[END_REF] making their study difficult in humans. Recent knowledge about their generation and maintenance was obtained with murine models mainly. After selection of antigen (Ag) specific B cells in the germinal center, involving mutations in immunoglobulin (Ig) variable genes and isotype switching, centrocytes differentiate into memory B cells (MBCs) or plasmablasts (PBs). These PBs migrate to medullary cords, exit into the lymph through a sphingosine phosphate gradient and get to the peripheral blood. [START_REF] Tangye | Staying alive: regulation of plasma cell survival[END_REF] Circulating PBs have to find a specific niche in the BM or mucosa that will provide them with the factors to survive and fully differentiate. The restricted number of suitable PC niches is supposed to be the main limiting factor explaining LLPC rarity. [START_REF] Tangye | Staying alive: regulation of plasma cell survival[END_REF] In mice, Tokoyoda et al. have reported the PC niche to be a VCAM1 + SDF-1 + stromal cell that could be shared by hematopoietic progenitors and pre-pro B cells. [START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF] Several hematopoietic cell subsets have been described to be involve in PC retention, maturation, and maintenance in the BM, including macrophages, eosinophils, and megakaryocytes. [START_REF] Chu | The establishment of the plasma cell survival niche in the bone marrow[END_REF] At least 3 growth factors and chemokines, produced by the exquisite PC niche, are recognized to control PC survival: SDF-1, APRIL/BAFF, and IL-6. [START_REF] Tangye | Staying alive: regulation of plasma cell survival[END_REF] SDF-1, produced by SCs is essential to recruit mouse PBs into the BM. [START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF][START_REF] Hargreaves | A coordinated change in chemokine responsiveness guides plasma cell movements[END_REF] BAFF and APRIL are produced by hematopoietic cells and are critical to support PC survival. [START_REF] Chu | The establishment of the plasma cell survival niche in the bone marrow[END_REF] BAFF binds to 3 receptors, BAFF receptor, BCMA and TACI, whereas APRIL binds to BCMA and TACI in vivo. [START_REF] Vincent | The BAFF/APRIL system: Emerging functions beyond B cell biology and autoimmunity[END_REF] Although both APRIL and BAFF can support murine LLPC survival, [START_REF] Benson | Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL[END_REF] APRIL appears to be more efficient than BAFF to promote LLPC survival, at least in mice. First, APRIL binds to BCMA with a higher affinity than BAFF, [START_REF] Vincent | The BAFF/APRIL system: Emerging functions beyond B cell biology and autoimmunity[END_REF] and BCMA is highly expressed in PCs. [START_REF] Jourdan | An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization[END_REF] Secondly, APRIL and its receptor TACI bind to heparan sulfate chains, in particular to the proteoglycan syndecan-1, [START_REF] Huard | APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa[END_REF][START_REF] Moreaux | APRIL and TACI interact with syndecan-1 on the surface of multiple myeloma cells to form an essential survival loop[END_REF] which is a hallmark of mature PCs. [START_REF] Wijdenes | A plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1[END_REF] Thirdly, the long-term survival of transferred LLPCs is impaired in APRIL -/-mice, but unaffected in BAFF -/-ones. [START_REF] Belnoue | Homing and Adhesion Patterns Determine the Cellular Composition of the Bone Marrow Plasma Cell Niche[END_REF] IL-6, produced by SCs or dendritic cells, is essential for the generation of PBs, and its role in supporting LLPC survival is controversial in mice. The generation of Agspecific PCs is dramatically decreased in IL-6 -/-mice [START_REF] Kopf | Impaired immune and acute-phase responses in interleukin-6-deficient mice[END_REF] and the ability of BM SCs to support the survival of purified BM LLPCs in vitro is inhibited by an anti-IL-6 monoclonal antibody (mAb) or lost using IL-6 -/-mice SCs. [START_REF] Wols | The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity[END_REF][START_REF] Cassese | Plasma Cell Survival Is Mediated by Synergistic Effects of Cytokines and Adhesion-Dependent Signals[END_REF] Similarly, the survival of LLPCs in the culture of intestine biopsies is impaired by an anti-IL-6 mAb or an APRIL/BAFF inhibitor. [START_REF] Mesin | Long-lived plasma cells from human small intestine biopsies secrete immunoglobulins for many weeks in vitro[END_REF] But the persistence of LLPCs transferred into IL-6 -/-mice is unaffected. [START_REF] Belnoue | Homing and Adhesion Patterns Determine the Cellular Composition of the Bone Marrow Plasma Cell Niche[END_REF] In humans, the factors that promote the differentiation of PBs into LLPCs are poorly identified in vivo, due to the rarity of bone marrow PCs (BMPCs) and the ethical difficulty to harvest the BM. The majority of the studies dealing with PC survival and growth were done with malignant PCs. [START_REF] Mahtouk | Growth factors in multiple myeloma: a comprehensive analysis of their expression in tumor cells and bone marrow environment using Affymetrix microarrays[END_REF][START_REF] Anderson | Pathogenesis of myeloma[END_REF] Using in vitro models of differentiation of B cells into PCs, we and others have shown that in vitro generated PCs are early PCs with a phenotype close to that of circulating PBs and PCs in healthy individuals. [START_REF] Jourdan | An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization[END_REF][START_REF] Mei | Blood-borne human plasma cells in steady state are derived from mucosal immune responses[END_REF][START_REF] Caraux | Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138-and CD138+ plasma cells[END_REF] These in vitro generated PCs expressed highly CD38 and CD31 and lack B cell antigens (CD20, CD22, CD24) except CD19. They express CD138 but at lower level than BMPCs, they express CD62L unlike BMPCs, and fail to express CD9, VCAM1, and CCR2. [START_REF] Jourdan | An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization[END_REF] Recent studies have proposed a role for osteoclasts [START_REF] Geffroy-Luseau | Osteoclasts support the survival of human plasma cells in vitro[END_REF] or SCs to support human PC survival in vitro. [START_REF] Cocco | In Vitro Generation of Long-lived Human Plasma Cells[END_REF] In this last study, blood B cells could be differentiated into Ig-secreting LLPCs in contact with soluble factors produced by a mouse SC line.

However, the molecular mechanisms of this supportive activity remain unknown. [START_REF] Cocco | In Vitro Generation of Long-lived Human Plasma Cells[END_REF] In the current study, we show that IL-6 is mandatory for the in vitro survival of LLPCs in combination with either APRIL or BAFF or APRIL/BAFF-unrelated SC soluble factors. These LLPCs are non-cell cycling, survive for months in vitro, while producing Igs continuously.

MATERIALS AND METHODS

Reagents

Cell samples

Peripheral blood cells from healthy volunteers were purchased from the French Blood Center (Toulouse, France) and CD19 + CD27 + MBCs purified ( 95% purity) as described. [START_REF] Jourdan | An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization[END_REF] When indicated, D10 early PCs (CD20 -CD138 + ) were FACS-sorted using FITC-conjugated anti-CD20 mAb and PE-or APC-conjugated anti-CD138 mAb. The purity of FACS-sorted cell populations was ≥ 95% as assayed by cytometry. Resto-6 stromal cells were used as a source of SCs. These SCs were obtained from a 15%/25% Percoll interface of dissociated human tonsil cells as previously described. [START_REF] Amé-Thomas | Human mesenchymal stem cells isolated from bone marrow and lymphoid organs support tumor B-cell growth: role of stromal cells in follicular lymphoma pathogenesis[END_REF] Plastic-adherent cells were selected and expanded in RPMI 1640 culture medium and 10% fetal calf serum (FCS) yielding to the Resto-6 SCs after 8 passages. Resto-6 SCs express usual mesenchymal stromal cell markers (CD90, CD73, and CD105) and can acquire properties of fibroblastic reticular cells (FRC) including expression of high levels of adhesion molecules and gp38/podoplanin, production of a dense meshwork of transglutaminase, and production of inflammatory and lymphoid chemokines upon stimulation by TNFα and Lymphotoxin-α1β2. [START_REF] Amé-Thomas | Human mesenchymal stem cells isolated from bone marrow and lymphoid organs support tumor B-cell growth: role of stromal cells in follicular lymphoma pathogenesis[END_REF] Resto-6 SCs support efficiently the growth and survival of normal B and T cells and of malignant lymphoma B cells, in particular after FRCcommitment. They were used between passages 8 and 15.

Cell cultures

PCs were generated through a four-step culture.

Assay for cell viability and cell growth

Cell concentration and viability were assessed using trypan blue dye exclusion test.

The number of metabolic active cells was also determined using intracellular ATP quantitation with a Cell Titer Glo Luminescent Assay (Promega Corporation, Madison, WI).

Cell cycle analysis, immunophenotypic analysis, and cytology

The cell cycle was assessed using DAPI staining (Sigma-Aldrich) and cells in the S phase using incubation with bromodeoxyuridine (BrdU) for 1 hour and labelling with an anti-BrdU antibody (APC BrdU flow kit, BD Biosciences) according to manufacturer's instructions. Cells were stained with a combination of 4 to 7 mAbs conjugated to different fluorochromes. The Cytofix/Cytoperm kit (BD Biosciences) was used for intracellular staining of IgM, IgA, IgG or Ki67 antigen. [START_REF] Jourdan | An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization[END_REF] Flow cytometry analysis was performed with a FACSAria cytometer using FACSDiva 6.1 (Becton Dickinson, San Jose, CA) and with a Cyan ADP cytometer driven by the Summit software (Beckman Coulter). Kaluza software (Beckman Coulter) was used for data analysis. The fluorescence intensity of the cell populations was quantified using the stain index (SI) formula: [mean fluorescence intensity (MFI) obtained from a given mAb minus MFI obtained with a control mAb]/[2 times the standard deviation of the MFI obtained with the same control mAb]. [START_REF] Jourdan | An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization[END_REF] Cytospin smears of cell-sorted CD20 - CD138 + D30 PCs were stained with May-Grünwald-Giemsa.

Analysis of Ig secretion

ELISA. Flow cytometry sorted PCs were cultured at 10 6 cells/ml for 24 hours and culture supernatants harvested. IgM, IgA, or IgG concentrations were assessed by ELISA using human IgM, IgA, and IgG ELISA kits from Bethyl Laboratories (Montgomery, TX), according to the manufacturer's recommendations.

ELISPOT. The number of IgM, IgA, or IgG secreting PCs was evaluated with the ELISPOT assay, [START_REF] Jourdan | Characterization of a transitional preplasmablast population in the process of human B cell to plasma cell differentiation[END_REF] seeding 500 PCs by well in ELISPOT plates and culturing cells for 18 hours. The number and size of IgM, IgA, and IgG elispots were assessed using the Biosys Bioreader 5000 apparatus (Biosys, Miami, FL).

Microarray hybridization and bioinformatics analysis

RNA was extracted and hybridized to human genome U133 Plus 2.0 GeneChip microarrays, according to the manufacturer's instructions (Affymetrix, Santa Clara, CA). Gene expression data are deposited in the ArrayExpress public database (http://www.ebi.ac.uk/microarray-as/ae/). The accession numbers are E-MEXP-3034

for prePBs, E-MEXP-2360 for PBs and BMPCs, E-MEXP-3945 for day 10 (D10) early PCs and D30 PCs, and E-MTAB-2118 for Resto-6 SCs. Gene expression data were analyzed with our bioinformatics platforms (RAGE, http://rage.montp.inserm.fr/) [START_REF] Reme | A new method for class prediction based on signed-rank algorithms applied to Affymetrix microarray experiments[END_REF] and Amazonia (http://amazonia.transcriptome.eu/). [START_REF] Carrour | Amazonia!: An Online Resource to Google and Visualize Public Human whole Genome Expression Data[END_REF] Genes differentially expressed between cell populations were determined with the SAM statistical microarray analysis software. [START_REF] Tusher | Significance analysis of microarrays applied to the ionizing radiation response[END_REF] The clustering was performed and visualized with the Cluster and TreeView softwares. [START_REF] Eisen | Cluster analysis and display of genome-wide expression patterns[END_REF] Gene annotation and networks were generated with the Reactome Functional Interaction Cytoscape plugin (http://www.cytoscape.org/).

Statistical analysis

Statistical comparisons were made with the non-parametric Mann-Whitney test, unpaired or paired Student's t-test using SPSS software. P-values ≤ .05 were considered as significant.

RESULTS

IL-6 in combination with APRIL or BAFF or APRIL/BAFF-unrelated stromal-cell soluble factors supports the generation and survival of PCs in vitro

Starting from MBCs, early PCs can be generated within 10 days using a 3-step culture. These early PCs could survive poorly in presence of IL-6 (Figure 1A). Adding human tonsil SCs could promote PC survival with 28% surviving CD138 PCs at day 30 (Figure 1A). In the presence of SCs, the CD138 fluorescence staining index at day 14 was increased 3 fold (P = .006) compared to day 14 PCs generated with IL-6 alone (Figure 1B and1C). It progressively increased 2 fold from day 14 to day 30 (P = .032) and then was stable up to day 60 (Figures 1B and1C). Coculturing early PCs separated from SCs in transwell culture plates promoted PC survival at the same extent as coculture of PCs with SCs at day 14, 17, 24 and 30, showing a contact between PCs and SCs is not mandatory (Figure 2A). In agreement, coculture supernatant of PCs with SCs (PC/SC-CM) or of SCs alone (SC-CM) also promoted PC survival at day 30 (Figure 2A). Various growth recombinant factors -APRIL, BAFF, SDF-1, IGF-1 -known to sustain malignant PC survival [START_REF] Mahtouk | Growth factors in multiple myeloma: a comprehensive analysis of their expression in tumor cells and bone marrow environment using Affymetrix microarrays[END_REF] did not induce PC survival when used alone (Figure 2B). IL-6 alone supported PC survival but at a 76% lower level than that induced by the SC-CM (P = .003). Adding APRIL together with IL-6 increased significantly 6.5 fold (P = .02) the IL-6 PC survival activity (Figure 2B).

Adding BAFF also increased the PC survival induced by IL-6 (P = .02) and SDF-1 and/or IGF-1 did not increase IL-6 activity (Figure 2B). The PC survival induced by the SC-CM was fully abrogated by an anti-IL-6 mAb showing the critical role of IL-6 produced by SCs, but was unaffected by an APRIL/BAFF inhibitor, TACI-Fc (Figure 2C). The TACI-Fc APRIL/BAFF inhibitor fully blocked the ability of APRIL to increase 2.2 fold the PC generation induced by the SC-CM (Figure 2C). It also inhibited the APRIL-induced growth of the XG1 myeloma cell line (supplementary Figure 1). [START_REF] Sprynski | The role of IGF-1 as a major growth factor for myeloma cell lines and the prognostic relevance of the expression of its receptor[END_REF] As APRIL/BAFF activate NF-κB pathways, [START_REF] Bossen | BAFF, APRIL and their receptors: structure, function and signaling[END_REF] we investigated whether these pathways could be involved in PC generation by SCs. An inhibitor of both canonical and alternative NF-κB pathways (IKK16) did not affect the generation of PCs supported by SCs, whereas it impeded the additive effect of APRIL in PC generation promoted by SCs (Figure 3). Thus, SCs produce communication signals, which cooperate with IL-6 to promote PC survival and are not inhibited by a NF-κB inhibitor. This is in agreement with the low expression of NF-κB-induced genes [START_REF] Annunziata | Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma[END_REF] in PCs generated with SCs in vitro contrarily to BMPCs (supplementary Figure 2). These communication signals are not SDF-1 or IGF-1 since the SC-CM activity was unaffected by their specific inhibitors (Figure 2C). Adding these 3 inhibitors together (APRIL/BAFF, SDF-1 and IGF-1 inhibitors) yielded to an apparent decrease in SC-CM induced PC generation, which did not reach statistical significance (Figure 2C).

Of note, PCs could survive up to 120 days in these culture conditions adding fresh IL-6, APRIL and culture medium weekly and cultures were stopped after 120 days for convenience but not due to a decline in PC survival. These PCs were thus termed LLPCs. These results indicate that IL-6 is mandatory to induce the long-term survival of LLPCs in combination with either APRIL or BAFF or with APRIL/BAFF-unrelated soluble factors produced by SCs.

Characterization of D30 PCs

D30 PCs had a more mature PC phenotype than D10 early PCs with a progressive 7.4-fold increase in CD138 density (Figure 1C, P .001), a 2.2-fold increase in CD54 staining index, a 1.5-fold increase in CD9 + PCs, and a decrease in HLA-DR, CD45, CD62L, CCR10 expressing PCs and staining indexes (P .05, Figure 4 and supplementary Figure 3). D30 PCs did not proliferate (0.04% cells incorporating BrdU), whereas a low fraction of D10 early PCs was in the S phase of the cell cycle (4% BrdU+) (Figure 5A and5B). D30 PCs displayed the cytology of mature PCs and produced cytoplasmic kappa or lambda Ig light chains (supplementary Figure 4). Day 60 PCs comprise IgG PCs only, with a quick disappearance of IgM PCs (no more detectable at day 30) and a progressive one of IgA PCs as assayed by FACS, ELISA and ELISPOT assays (Figures 6 A-F). Of note, the rate of IgG production was similar between day 30 and day 60 PCs (Figure 6F). The number of IgG and IgM producing cells and the Ig isotype produced were not significantly different starting from the same number of LLPCs generated with either SC coculture, IL-6+APRIL, SC-CM or APRIL+SC-CM (Figure 6G). There was a trend in increasing IgA secreting cells in the two experiments adding APRIL compared to the culture groups without APRIL (P < .1). Combining data of these two experiments show APRIL increased 3.4 fold the number of IgA secreting cells (47 vs. 14 IgA secreting cells/500 PCs, P < .001, Figure 6G).

B and PC transcription factors

D30 PCs had a higher expression of genes coding for IRF4 and BLIMP1 PC transcription factors than D10 early PCs and failed to express PAX5 (P .02, Figure 7). D30 PCs expressed 3-fold more the spliced form of XBP1 mRNA than D10 early PCs and 2-fold more the unspliced mRNA form than early PCs, resulting in an increased XBP1s/XBP1u mRNA ratio (P .009, Figure 7). Of note, BCL6 gene expression in D30 PCs was low compared to that in B lymphocytes but significantly higher than that in early PCs (P = .04, Figure 7).

Gene expression profile (GEP) of D30 PCs

In vitro generated D4 prePBs, D7 PBs, D10 early PCs, D30 PCs, and BMPCs purified from healthy individuals were profiled using Affymetrix U133 plus 2.0 microarrays. D30 PCs were generated using cocultures with SCs. The 5 populations are classified into 2 major clusters, a PC cluster comprising D10 early PCs, D30 PCs, and BMPCs and a PB cluster comprising prePBs and PBs (Figure 8). To look for genes indicator of LLPCs, we ran a SAM supervised analysis comparing D10 early PCs to D30PCs+BMPCs starting from the 5000 genes with the highest variance. 160 probe sets (141 unique genes) were overexpressed in D30PCs+BMPCs compared to D10 early PCs (Wilcoxon statistic, fold change 2, FDR 1%) and 490 (427 unique genes) in D10 early PCs versus D30PCs+BMPCs (supplementary Table 1). Genes coding for translation, focal adhesion, IL-6 signalling, and integrin signalling pathways were enriched in D30PC+BMPC genes (supplementary Table 2), and genes coding for DNA replication and mitosis in D10 early PCs (supplementary Table 3). The gene expression profiles of LLPCs harvested from the spleen of patients with primary immune thrombocytopenia treated with rituximab anti-CD20 mAb was recently documented and compared to that of PBs harvested from the spleen of untreated patients. [START_REF] Mahévas | B cell depletion in immune thrombocytopenia reveals splenic long-lived plasma cells[END_REF] Similarly to the current in vitro generated LLPCs, LLPCs from these patients overexpress genes coding for PC transcription factors (JUN, FOS, EGR1),

negative

DISCUSSION

This study shows i) the feasibility to generate human mature PCs in vitro, ii) the longterm survival of these PCs does not require a contact with niche cells, but only cell communication factors, in particular IL-6 and APRIL.

These PCs are called long-lived PCs because they are non-cycling PCs surviving and producing Igs for months in vitro as their counterpart in vivo. [START_REF] Radbruch | Competence and competition: the challenge of becoming a long-lived plasma cell[END_REF][START_REF] Slifka | Humoral immunity due to long-lived plasma cells[END_REF][START_REF] Manz | MAINTENANCE OF SERUM ANTIBODY LEVELS[END_REF] In addition, they have a phenotype similar to that of LLPCs in vivo: high expression of CD138, increased expression of CD9, weak expression of CD62L, CD45 and HLA-DR compared to PBs and early PCs. [START_REF] Caraux | Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138-and CD138+ plasma cells[END_REF][START_REF] Medina | The heterogeneity shown by human plasma cells from tonsil, blood, and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression[END_REF] These in vitro generated LLPCs expressed IRF4 and PRDM1 genes coding for PC transcription factors at a higher level than early PCs. Murine LLPCs also highly express Blimp1 compared to early PCs in the BM. [START_REF] Kallies | Plasma Cell Ontogeny Defined by Quantitative Changes in Blimp-1 Expression[END_REF] XBP1 is a master regulator of unfold protein response critical to protect PCs from stress induced by high Ig production. [START_REF] Iwakoshi | Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1[END_REF] XBP1 mRNA has to be spliced to encode for an active protein and, in agreement, we found that LLPCs had an increased ratio of spliced to unspliced XBP1 mRNAs compared to early PCs. LLPCs expressed weakly but significantly BCL6 gene compared to early PCs. This deserves further study since Bcl6 is also inducible in malignant PCs in response to SC-derived factors, conferring a survival advantage on them. [START_REF] Hideshima | A proto-oncogene BCL6 is up-regulated in the bone marrow microenvironment in multiple myeloma cells[END_REF] A second major finding is that the generation and survival of human LLPCs do not require a contact with niche cells in vitro, but can be obtained with 2 recombinant growth factors only, IL-6 and APRIL. APRIL can be replaced by BAFF, which activates the same receptors. In addition, APRIL can be replaced by APRIL/BAFFunrelated soluble factors produced by SCs. Of note, an inhibitor of both the canonical and alternative NF-κB pathways did not affect the generation of LLPCs supported by SCs, whereas it abrogated the additive effect of APRIL in getting LLPCs with SCs. In addition, a set of genes, whose expression is induced by NF-κB pathway activation in malignant PCs, [START_REF] Annunziata | Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma[END_REF] is poorly expressed in LLPCs generated with SCs in vitro compared to BMPCs as previously mentioned by Cocco et al. [START_REF] Cocco | In Vitro Generation of Long-lived Human Plasma Cells[END_REF] Thus the activation of the NF-κB pathway is not mandatory to generate LLPCs in vitro, but could enhance it. The current data are in line with recent findings showing that a combination of APRIL, BAFF, IGF-1, SDF-1 and VEGF can support modestly the in vitro 14-day survival of human PCs harvested from the BM, whereas STAT3 activating cytokines, in particular IL-6, are critical. [START_REF] Rodríguez-Bayona | STAT-3 Activation by Differential Cytokines Is Critical for Human -Generated Plasma Cell Survival and Ig Secretion[END_REF] Using more complex culture conditions, including IL-6, IL-21, IFN-α and SC-CM, a recent study has shown human LLPCs can be generated in vitro also. [START_REF] Cocco | In Vitro Generation of Long-lived Human Plasma Cells[END_REF] That the in vitro long-term survival of human PCs can be supported by 2 growth factors (IL-6 and APRIL) only is quite surprising regarding the current view of the complexity of the PC niche, comprising SCs and various hematopoietic cells (eosinophils, dendritic cells, megakaryocytes, neutrophils, basophils). [START_REF] Chu | The establishment of the plasma cell survival niche in the bone marrow[END_REF][START_REF] Belnoue | Homing and Adhesion Patterns Determine the Cellular Composition of the Bone Marrow Plasma Cell Niche[END_REF][START_REF] Winter | Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow[END_REF] SCs are thought to serve as docking cells bringing close together PCs and hematopoietic cells, [START_REF] Belnoue | Homing and Adhesion Patterns Determine the Cellular Composition of the Bone Marrow Plasma Cell Niche[END_REF] but also producing soluble factors promoting PC survival, in particular IL-6 and galectin. [START_REF] Tsai | Galectin-1 promotes immunoglobulin production during plasma cell differentiation[END_REF][START_REF] Anginot | Galectin 1 Modulates Plasma Cell Homeostasis and Regulates the Humoral Immune Response[END_REF] The ability of hematopoietic cells to sustain PC survival is due mainly by their ability to produce APRIL. [START_REF] Benson | Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL[END_REF][START_REF] Huard | APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa[END_REF] The fact that soluble growth factors can replace niche cells for the generation and survival of LLPCs in vitro suggests it could be the case in vivo and questions about the regulation of LLPC count in vivo. It is generally assumed that the tiny PC count in the BM is regulated by the rarity of BM niche cells [START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF] , new PCs being in competition with old ones for the availability of niche cells. 44 The current finding suggests the PC niche is mainly a liquid niche comprising a life-sustaining mixture and concentration of chemokines and growth factors, which is likely the case close to the docking SCs attracting both PCs and hematopoietic cells. [START_REF] Belnoue | Homing and Adhesion Patterns Determine the Cellular Composition of the Bone Marrow Plasma Cell Niche[END_REF] When entering the BM, if a PC cannot migrate close to a SC, it will not encounter the life-sustaining concentrations of soluble factors and die. But in case of deregulated production of these cell communication signals such as in inflammatory conditions, one can expect many PCs may survive in vivo. This may explain the accumulation of LLPCs in the spleen of patients with primary immune thrombocytopenia treated with Rituximab anti-CD20 mAb, in association with a 2-fold increase in BAFF concentration in the spleen compared to Rituximab untreated patients. [START_REF] Mahévas | B cell depletion in immune thrombocytopenia reveals splenic long-lived plasma cells[END_REF] Besides genomic abnormalities, this could also explain the progressive accumulation of premalignant PCs and then malignant PCs in patients with malignant PC disorders who display increased plasma concentrations of IL-6, [START_REF] Bataille | Serum levels of interleukin 6, a potent myeloma cell growth factor, as a reflect of disease severity in plasma cell dyscrasias[END_REF] APRIL or BAFF. [START_REF] Moreaux | BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone[END_REF] The current finding of a mandatory role of IL-6 to promote the survival of human LLPCs in vitro questions the role of IL-6 for LLPC maintenance. In mice, whereas IL-6 produced by SCs is mandatory to get the survival of BMPCs in vitro, [START_REF] Wols | The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity[END_REF][START_REF] Cassese | Plasma Cell Survival Is Mediated by Synergistic Effects of Cytokines and Adhesion-Dependent Signals[END_REF] the survival of transferred LLPCs is not impaired in IL-6 -/-mice unlike APRIL -/-ones. [START_REF] Belnoue | Homing and Adhesion Patterns Determine the Cellular Composition of the Bone Marrow Plasma Cell Niche[END_REF] This is likely due to bias in the murine or human models used. In particular, whereas LLPCs can not be transferred in APRIL -/-mice 14 , Ag-specific PCs can be generated in APRIL -/-mice using repeated Ag boosts indicating additional factors can replace APRIL. [START_REF] Belnoue | Homing and Adhesion Patterns Determine the Cellular Composition of the Bone Marrow Plasma Cell Niche[END_REF] In IL-6 -/-mice, a role of the other cytokines able to trigger gp130 IL-6 transducer chain and/or STAT3 activation and to supplement for a deficit in IL-6 induced signalling to support PC survival has not been evaluated. This is the case in humans since a recent study has emphasized that inhibition of STAT3 activation by small compounds can fully block the in vitro 14-day survival of human PCs harvested from the peripheral blood or BM of healthy individuals. STAT3 activation in these PCs could be driven by either IL-6, IL-10 or IL-21. [START_REF] Rodríguez-Bayona | STAT-3 Activation by Differential Cytokines Is Critical for Human -Generated Plasma Cell Survival and Ig Secretion[END_REF] These in vitro models to get human PCs likely introduce some bias, in particular by the method to activate B cells (through BCR, CD40, or TLR), the combination of cytokines used to generate prePBs, PBs, early PCs and then LLPCs, the origin of stromal cells and the culture conditions influencing PC metabolism (nutrients, glucose, O 2 concentration). [START_REF] Jourdan | An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization[END_REF][START_REF] Mei | Blood-borne human plasma cells in steady state are derived from mucosal immune responses[END_REF][START_REF] Cocco | In Vitro Generation of Long-lived Human Plasma Cells[END_REF][START_REF] Jourdan | Characterization of a transitional preplasmablast population in the process of human B cell to plasma cell differentiation[END_REF] In the current model, we used a stromal cell line obtained from tonsils because it grow easily until confluence and at confluence, can survive for several months without proliferating but providing a continuous SC support. In initial experiments, similar data were obtained in term of phenotype and long-term survival with BM SCs. But it is of major interest to investigate further whether the use of SCs from different tissue origins could change the phenotype and gene expression profiling of LLPCs, in particular their proximity with BMPCs. For example, a progressive loss of IgA secreting PCs occurred in cultures with tonsil SCs or SC-CM likely due to the lack of a critical survival factor for IgA PC survival in vitro.

Adding APRIL can revert this loss, increasing 3.4 fold the survival of IgA secreting PCs and this could eventually also occur with BM SCs. The ease of the current model to get PCs in vitro will make possible further identification of these possible biases.

All prePBs and PBs generated in this model express CD19 whereas Chaidos et al. [START_REF] Chaidos | Clinical drug resistance linked to interconvertible phenotypic and functional states of tumor-propagating cells in multiple myeloma[END_REF] reported recently the existence of CD19 -CD38 + CD138 -plasmablasts (called Pre-PCs) in healthy individuals, together with the known CD19 + CD38 + CD138 -PBs and CD19 + CD38 + CD138 + PCs. [START_REF] Caraux | Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138-and CD138+ plasma cells[END_REF] The malignant counterpart of these Pre-PCs are found in patients with MM. Running a supervised analysis of gene expression profiling of malignant Pre-PCs and PCs, Chaidos et al. found enrichment of genes coding for epigenetic pathways. [START_REF] Chaidos | Clinical drug resistance linked to interconvertible phenotypic and functional states of tumor-propagating cells in multiple myeloma[END_REF] The in vitro model we used likely failed to generate these CD19 -CD38 + CD138 -since all prePBs generated at day 4, PBs at day 7 and PCs at day 10 express CD19. [START_REF] Jourdan | An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization[END_REF][START_REF] Jourdan | Characterization of a transitional preplasmablast population in the process of human B cell to plasma cell differentiation[END_REF] In addition, the epigenetic genes differentially expressed between malignant Pre-PCs and PCs could not classify the current in vitro generated PBs, early PCs and LLPCs (data not shown).

Besides its interest for understanding the fine pathways controlling PC generation and survival in humans, the current model should be promising to study the mechanisms involved in malignant PC disorders and controlling the activity of drugs used to treat patients with these disorders. As several genes whose expression or abnormalities are associated with disease activity have been identified, [START_REF] Walker | A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value[END_REF][START_REF] Morgan | The genetic architecture ofmultiple myeloma[END_REF][START_REF] Kassambara | Genes with a spike expression are clustered in chromosome (sub)bands and spike (sub)bands have a powerful prognostic value in patients with multiple myeloma[END_REF][START_REF] Kassambara | Identification of pluripotent and adult stem cell genes unrelated to cell cycle and associated with poor prognosis in multiple myeloma[END_REF][START_REF] Kassambara | Inhibition of DEPDC1A, a Bad Prognostic Marker in Multiple Myeloma, Delays Growth and Induces Mature Plasma Cell Markers in Malignant Plasma Cells[END_REF][START_REF] Schoenhals | Kruppel-like factor 4 blocks tumor cell proliferation and promotes drug resistance in multiple myeloma[END_REF][START_REF] Moreaux | A highrisk signature for patients with multiple myeloma established from the molecular classification of human myeloma cell lines[END_REF][START_REF] Hose | Induction of angiogenesis by normal and malignant plasma cells[END_REF] their modulation throughout the different stages of PC generation (prePBs, PBs, early PCs, LLPCs) could help to understand better their function. Of note, it is now feasible to force or repress the expression of a given gene in these PCs using measles envelop pseudotyped lentiviral delivery. [START_REF] Schoenhals | Efficient transduction of healthy and malignant plasma cells by lentiviral vectors pseudotyped with measles virus glycoproteins[END_REF] The same holds true for drugs used to treat patients with MM, in particular to identify if these drugs could target a specific PB or PC stage and the underlying mechanism.

Human recombinant IL- 2 ,

 2 TACI-Fc, and APRIL were purchased from R&D Systems (Minneapolis, MN), IFN-α (IntronA) from Merck Canada Inc. (Kirckland, Canada), IL-6 and IL-15 from AbCys SA (Paris, France), BAFF, SDF-1α, IGF-1, and IL-10, from Peprotech (Rocky Hill, NJ, USA), the B-E8 anti-IL-6 mouse mAb from Diaclone (Besançon, France), the SDF-1 inhibitor AMD3100 from Sigma (Sigma-Aldrich, St Louis, MO), the IGF-1R inhibitor from Novartis Pharma (Basel, Switzerland), and IKK16, a selective inhibitor targeting both IKK1 and IKK2, from R&D Systems.

Figure 1 .

 1 Figure 1. Generation and survival of mature CD138 bright PCs in vitro. (A) PCs (CD38 + CD138 + cells) were generated with or without stromal cells (SCs) in the presence of IL-6 (10 ng/ml) and cell count and viability assayed at day (D) 10, 14, 24, 30, and 60 using trypan blue dye exclusion. Data are the mean concentration of viable cells ± SD determined in six separate experiments. *The mean value is significantly higher than that in culture without SCs at the same culture day using a paired t-test (P .05). (B) Expression of CD138. The histograms show FACS labelling with an anti-CD138 (black) or an isotype-matched control mAb (white) of one experiment representative of four. The percentages of CD138 positive cells and CD138 staining index (SI) are indicated in the panels. (C) CD138 fluorescence staining index. Data are the mean CD138 staining index ± SD of four separate experiments. Statistical analysis was done using a paired t-test. ns = not significant.

Figure 2 .

 2 Figure 2. IL-6 in combination with APRIL or BAFF or SC-CM supports the generation and survival of PCs in vitro.(A) D10 early PCs were FACS sorted and cultured from D10 to D30 either in contact with SCs (culture with SCs), or with SCs placed in a lower chamber of a transwell culture plate (SCs+PCs in transwells), or with supernatant of PC and SC coculture (PC/SC-CM), or culture supernatant of SCs (SC-CM). Results are the mean of viable cell counts ± SD of 3-paired experiments. ns: the mean count of PCs at a given culture day is not significantly different from that in the SC group at the same culture day using a paired t-test (B) FACS-sorted early PCs were cultured from D10 to D30 with culture medium and 10% FCS (Co) or in the presence of recombinant cytokines and/or the SC-CM. The concentrations of cytokines used were 10 ng/ml for IL-6 and IGF-1 and 200 ng/ml for APRIL, BAFF, and SDF-1. Counts of metabolic active cells

Figure 3 .

 3 Figure 3. A NF-κB inhibitor does not affect the generation of PCs by SCs but

Figure 4 .

 4 Figure 4. D30 PCs have a more mature phenotype than D10 early PCs.

Figure 5 .

 5 Figure 5. D30 PCs do not cell cycle. The cell cycle was assessed using DAPI staining and quantification of cells in the S phase using bromodeoxyuridine (BrdU) incorporation and labelling with an anti-BrdU antibody. (A) Dot plots show a representative experiment out of three. The percentages of cells in the G0/G1, S, and G2/M phases are indicated. (B) Results are the mean percentage ± SD of cells in the S phase of three separate experiments.

Figure 6 .

 6 Figure 6. D30 and D60 PCs are functional PCs that produce Igs continuously. FACS-sorted D10 early PCs were cultured with SCs (A-F) or growth factors as indicated (G). (A-C) Cytoplasmic (cy) Igs (IgG, IgA, and IgM) (A), cy-light chains (B), and surface (s) Igs (C) were assessed by flow cytometry. Results are the mean percentage ± SD of positive cells from 5 separate experiments. (D) IgG, IgA, and IgM production was assessed by ELISA. Results are the mean ± SD of Ig production in pg per cell and per day determined in 3 separate experiments. (E) The number of IgG-, IgA-, and IgM-secreting cells was assessed by ELISPOT. Results are the mean Ig-secreting cell number ± SD from 4 separate experiments. (F) ELISPOTs from a representative experiment are shown. (G) FACS sorted D10 early PCs were cultured with SCs or with a combination of cytokines and/or SC-CM as indicated. The number of Ig-secreting cells was determined by ELISPOT at D30 of culture. Results are the mean Ig-secreting cell number ± SD from 3 separate experiments. *The mean value is significantly different from that in D10 PCs using a paired t-test.

Figure 7 .

 7 Figure 7. Gene expression of transcription factors involved in PC differentiation. Naive B cells (BCs), D10 early PCs, and D30 PCs were FACS sorted. Gene expression of IRF4, PRDM1, XBP1u, XBP1s, PAX5, and BCL6 assayed by real-time RT-PCR. The mRNA level in the different cell populations was compared assigning the arbitrary value 1 to gene expression in BCs. Data are the mean value ± SD of gene expression determined in 5 separate experiments. The ratio XBP1s/XBP1u in D10 early PCs and D30 PCs is shown. *The mean value is significantly different from that in D10 PCs using a paired t-test (P .05).

Figure 8 .

 8 Figure 8. Unsupervised clustering of gene expression profile of purified D4 prePBs, D7 PBs, D10 early PCs, D30 PCs, and BMPCs. D4 prePBs, D7 PBs, D10 early PCs, D30 PCs and BMPCs were profiled using Affymetrix U133 plus 2.0 microarray and an unsupervised hierarchical clustering was run with the 5000 probe sets with the highest variance (log transform, center genes and arrays, uncentered correlation and average linkage). The 5 populations are classified into 2 major clusters, a PC cluster comprising D10 early PCs, D30 PCs and BMPCs (r = 0.11) and a plasmablast cluster comprising prePBs and PBs (r = 0.20).The horizontal lines represent the normalized and centered expression of each of the 5000 genes in the samples and are depicted according to the color scale shown at the bottom (-1.5 to 1.5 on a log base 2 scale).

  Figure 1

  Figure 5

Table 4 )

 4 . Genes coding for protein metabolism, translation, antigen processing and presentation, and CXCR4 signalling were enriched in BMPC genes (supplementary

	generated D10 early PCs, patients' PBs overexpress genes coding for positive cell
	cycle regulators (CCND2, BUB1B, BUB1, TIMELESS, CENPF, MAD2L1, BIRC5,
	ZWINT, MKI67, MCM4, CCNB2) and surface/cytokine receptors (ITGB1, TNFSF10)
	(supplementary Table 1). Comparing gene expression between BMPCs and D30
	PCs, 198 unique genes were overexpressed in BMPCs and 555 in D30 PCs (SAM
	supervised analysis, Wilcoxon statistic, fold change 2, FDR 1%, supplementary

regulators of the cell cycle (KLF4, KLF2, PPP1R15A) and cell surface/cytokine receptors (CD9, SDC1, FCRL5). Conversely, similarly to in vitro-

Table 5 )

 5 . Genes coding for glypican pathway, TGFβ receptor and Smad2/3

	signalling, protein export and proteasome were enriched in D30 PC genes
	(supplementary Table 6). GEP were done using D30 PCs generated with SCs and
	IL-6. It could be of interest to investigate further whether adding APRIL together with
	IL-6 or SC-CM could change the gene expression profiling, making it closer to that of
	BMPCs.

ACKNOWLEDGEMENTS

This work was supported by grants from ARC (SL220110603450, Paris France), from ANR (2012-109/087437), and the European Community (FP7-OVERMYR). We thank Mrs Pantesco from the Microarray Core Facility of IRB (http://irb.montp.inserm.fr/en/index.php?page=Plateau&IdEquipe=6), Dr Duperray from the cytometry platform of IRB (http://irb.montp.inserm.fr/en/index.php?page=Plateau&IdEquipe=3, Montpellier Rio Imaging) and Dr Reme from the IRB Bioinformatics platform (http://irb.montp.inserm.fr/en/index.php?page=Plateau&IdEquipe=18).

AUTHOR CONTRIBUTIONS

MJ designed research, performed the experiments and wrote the paper. KT, TF and FG, provided stromal cells and corrected the paper. MC, NR, and KB performed the experiments.

CD provided assistance for cytometry experiments.

DH provided data of gene expression profiling of BMPCs.

BK is the senior investigator who designed research and wrote the paper.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare. Supplementary information is available at Leukemia's website.


Hubert Luce

Richard Wilson

F Dalaudier

F Richard Wilson

F Truchi Truchi

Hiroyuki Hashiguchi

Masayuki K Yamamoto

Mamoru Yamamoto

Lakshmi Lakshmi

Kantha Kantha

Prof. Shoichiro Thorpe Fukao

Estimating length scales for tropospheric turbulence from MU rad Estimating length scales for tropospheric turbulence from MU radar and balloon data ar and balloon data

Introduction Instrumental set-up

The Middle and Upper Atmosphere radar (MUR) was operated in range imaging (FII) mode (5 frequencies) from 01 November 2013 16:00 LT to 09 November 2013 08:00 LT at a time resolution of 6.14 s (e.g. Luce et al. 2006). The radar beam was steered into 3 directions (vertical, and 10°off zenith toward North and East). The range sampling was performed from 1.32 km to 20.37 km. The FII mode was used for imaging the turbulent and stable layers. The Doppler spectra and moments were estimated at the range resolution of 150 m.

MUR observations

Figures 1(a)-1(b): Height-time cross-section of vertical MUR echo power in range imaging mode for all the campaign. The passage of frontal zones are clearly visible. The smoothed appearance of the radar echoes in clouds and precipitations strongly differ with the appearance of the thin stratified layers in dry air. The detailed analysis of the synoptic conditions will be performed in a subsequent work.

Conclusions ε

Estimating atmospheric turbulence parameters from ST radar measurements is an important issue. The methods used in the literature are based on the hypothesis that radar echoes at oblique incidence result from isotropic turbulence within the inertial subrange (e.g. Naström and Eaton, 2005). In such a case, the width of Doppler spectrum can be related to the turbulent energy dissipation rate . When the outer scale of turbulence is smaller than the dimensions of the radar volume, depends on the background stability N 2 (e.g., Hocking, EPS, 1999). The latter parameter is usually estimated from standard balloon measurements.

Our studies aim at estimating turbulence parameters by taking advantage of the high time resolution of radiosondes (1 sec) and concurrent radar observations. Wilson et al. (JAOT, 2011) showed that detecting turbulence from PTU measurements is possible by using Thorpe analysis (Thorpe, DSR, 1977) despite the instrumental measurement noise. The method was applied by Clayson and Kantha (JAOT, 2008) but without considering the noise effects. Turbulent layers can be directly identified through the detection of overturns produced by mixing and billows in dry or moist potential temperature profiles. The instrumental noise can lead to the detection of spurious layers especially in weakly stratified regions. Wilson et al. (JAOT, 2010) proposed objective selection criteria and procedures for minimizing noise effects. Wilson et al. (AMT, 2013) considered the air saturation effects. The Thorpe analysis also provides an important length-scale, the so-called Thorpe length LT defined as the root mean square of the Thorpe displacements in a turbulent layer. This parameter is very frequently used for characterizing turbulence in oceans: most studies revealed equivalence between LT and outer scales of turbulence (Ozmidov or buoyancy scales), for fully developed turbulence with an inertial domain (e.g. Smyth and Moum, JPO, 2000). From an intensive MU radar-balloon campaign at Shigaraki MU observatory performed in 2011, Luce et al. (RS, 2014) showed that the deepest turbulent layers identified in the potential temperature profiles (typically 50-1000 m in depth) are generally associated with nearly isotropic radar echoes, consistent with the detection of turbulence by the radar. This result gives credence to the fact that it is relevant to combine radar and balloon observations for estimating turbulence parameters, at least in a statistical sense. , 2010, , AMT, 2011, , 2013)). The labels indicate the deepest layers for which turbulence parameters have been estimated (Table 1). The deepest layers are detected in the regions where turbulence was detected by the radar and was suggested by the Ri* profiles. In particular, the most significant layers, observed on both sides of the cloud base (horizontal lines), associated with SNR and variance enhancements are detected in the temperature profiles by the Thorpe analysis for the 6 consecutive flights. Some others (deeper than 1000 m above 10 km) can be seen near the cloud top in M08 and V13 and coincide with recognizable features in Figures 4 and5. On the contrary, the selected turbulent layers below 6.0 km do not appear from one profile to another and may not be significant (except perhaps below the first radar gate, around 1.2 km).

LB1

The purpose of our investigations is to infer key parameters of atmospheric turbulence from the combination of (MU) radar and balloon data. Our approach is first based on the direct identification of turbulent layers in potential temperature profiles from Thorpe analysis with careful selection procedures. Results from two types of radiosondes (Vaisala and Meisei) were obtained with similar performances. The present studies extend the results described by Wilson et al. (JASTP 2014) obtained for a few cases only.

-For the first time, a statistical comparison between the Thorpe length and the buoyancy scale is presented for turbulent events observed from six consecutive balloons near an upper level front and within cloud could be made and for selected cases from previous campaigns. A clear relationship between LT and LB was found (i.e. LB ~LT despite an important dispersion to be more thoroughly interpreted, see Figure 6b). It is consistent with studies conducted for oceanic turbulence. This fundamental result may have dramatic impacts on the characterization of turbulence in the troposphere and may justify the estimation of energetic parameters from standard balloon data alone (Clayson and Kantha, JAOT 2008).

-Estimation of energy dissipation rates in clear air and upper level clouds could also be obtained. Preliminary results show an overall consistency of the inferred turbulence parameters. (Smyth and Moum, JPO 2000) Questions or comments? Corresponding authors: hubert.luce@univ-tln.fr richard.wilson@upmc.fr doi.org/10.1016/j.jastp.2014.01.005i)

Figure 2 :

 2 Figure 2: Close up of Figure (1a) between 01-NOV 20:00 LT and 02-NOV 05:20 LT . The nearly straight lines show the balloon altitude vs time (red: Vaisala, Black: Meisei). V02/M03, V04/M05 and V06/M07 were launched almost simultaneously but separately. The profiles are the moist potential temperature profiles used for Thorpe sorting. They were estimated from the numerical integration of dry N 2 when relative humidity was below the saturation thresholds defined by Zhang et al. (2010) for Vaisala sondes (and also used for Meisei sondes here) or moist N (Lalas and Einaudi, JAS, 1974) when above. Also shown are the vertical profiles of at a vertical resolution of 50 m. (The vertical dashed line correspond to N* 2 =0). The Meisei and Vaisala profiles are very similar. The N* 2 maxima generally coincide with radar echo enhancements.

Figure 3 :Figure 4 :(

 34 Figure 3: Close up of Figure (1a) between 02-NOV 20:00 LT and 03-NOV 05:05 LT. The black curves show the moist N* 2 and the vertical dashed lines show N* 2 =0. Deep regions of weak stability can be seen in the height range 6.0-11.5 km and coincide with a smooth appearance of the radar echoes. These echoes are not aspect sensitive (see Figure 5). Their structure contrasts with the thin layering below 6.0 km and above 11.5 km where profiles of N* 2 show a succession of narrow peaks. A frontal surface (strong temperature and humidity increases) and the cloud base are indicated by the dotted blue line (and coincide with the strong peak of N* 2 . Deep (up to ~1 km) turbulent layers can be seen on both sides of the cloud base and within the clouds. The nearly vertical striations of the echoes underneath cloud can be the signature of KH billows or convective rolls as already reported from MUR observations in FII mode for such atmospheric conditions (e.g. Luce et al, MWR, 2010).

  Figure6(a) Preliminary comparisons between buoyancy and Thorpe lengths for 18 selected cases from balloon and MUR data collected in 2011 (black dots) and 2012 (red dots) using similar radar configurations. (b) Systematic comparison for the layers exceeding 100 m in depth and detected above 5.8 km (just below and within the cloud). The blue dots corresponds to the deepest layers shown in Figure6and described in Table1. The ratio LT/LB is less than a factor 2 to 3 and correlation coefficient is 0.56. A linear regression indicates LT ~LB. (p~1)

  potential energy, TKE: Turbulent Kinetic Energy, Lo: Ozmidov scale : potential energy dissipiation rates p Energetic parameters estimated for the deepest layers (labeled in Figure 6) below cloud (LB), above cloud (LA) and inside cloud or near cloud top (LT). The values for cloudy air are emphasized by gray rectangles. Note that kinetic energy dissipation rates inside clouds are always smaller than values underneath clouds.

  et al. JASTP, 2014 for more details on the mathematical derivations http://dx.


J Delanoë

J.-P Vinson

W Brec

F Bertrand

C Caudoux

A Protat

M Haeffelin

J.-C Dupont

J Parent Du Chatelet

BASTA : a 95GHz FM-CW Cloud radar

Keywords:

As we do not have pulse, the frequency is modulated to be able to determine the echo distance. Low power amplifier (1W vs 1-2kW for pulsed radar) => cheaper!

  R) for each chirp (Radial) Once you have R you are back to a pulse radar: We use PPP (Pulse Pair Processing) to derive Doppler velocity (phase) and reflecWvity (module) for n high energy for a short period, we emit conWnuously less energy. => At the end the same amount of energy is sent! A chirp modulates the frequency of the wave in order to code the informaWon conWnuous observaWon of non-precipitaWng clouds and fog (SIRTA). ! Scien7fic aims : -Improve representaWon of clouds and associated processes in climate and forecast models. -InformaWon at different Wme scales on dynamical cloud processes, microphysics and radiaWve processes. -BeCer understanding of fog processes -RefracWvity studies ! Opera7onal aims : -ContribuWon to the development of cloud observaWon staWons for a future assimilaWon of cloud products. -ValidaWon of spaceborne acWve remote sensing instruments, cloud radars and lidars (A-Train : CALIPSO/ CloudSat, EarthCARE in 2016). ! Industrial aim : development of a low cost radar for large scale deployment. VerWcally poinWng, bistaWc, no polarisaWon.What does FM-CW stand for?FM-CW (Frequency Modulated -ConWnuous Wave)! Principle: Compensate the lack of power by emifng conWnuous signal.