Nessrine Essid
email: essid.nessrinegc@gmail.com

Anissa Eddhahak-Ouni
email: anissaeddhahak@yahoo.fr

Jamel Neji

Experimental and Numerical Thermal Properties Investigation of Cement-Based Materials Modified with PCM for Building Construction Use

Keywords: Phase-change materials (PCM), Cement-based material, Thermal conductivity, Heat storage capacity, Experimental setup, Optimization, Numerical simulation, Ansys CFX

Due to their latent heat storage capacity, phase-change materials (PCM) incorporated in wallboards are an effective solution to reduce energy consumption inside buildings. This is achieved by incorporating PCM in construction elements made of cement-based materials. The purpose of this research is to evaluate both the thermal conductivity and the heat storage capacity of mortars and concretes with different amounts of PCM in order to evaluate their thermal performance. Therefore, a laboratory-developed transient plane source experimental setup was used to measure these properties. First, several mortar and concrete specimens including different amounts of PCM (0%, 4.5%, 9%, and 13% by total mass of cement) were manufactured. Then, the experimental setup was used to measure the temperature development on PCM-concretes and PCM-mortars for a period of 1,000 s. The collected data were analyzed to back-calculate the thermal characteristics using a numerical optimization procedure. Numerical findings using the finite-element method show that the testing procedure efficiently provides accurate estimations of the thermal properties of the tested specimens. It was found that cement-based materials incorporating PCM have lower thermal conductivity and higher heat storage capacity, which indicates the improvement of their thermal behavior.

Introduction and Literature Review

In developing countries with high growth rates and improved standards of living, energy consumption and greenhouse gas emissions have significantly increased in recent decades (IEA 2017;[START_REF] Solomon | Contribution of working group I to the fourth assessment report of the intergovernmental panel on climate change[END_REF]). Thus, nowadays, saving energy and reducing gas emissions are becoming crucial issues and great challenges to industrial and developed countries for the future (IEA 2017).

Approximately 40% of the world energy was consumed by the construction sector in 2010 (IEA 2017). Indeed, buildings with low thermal mass are subject to overheating and temperature fluctuations.

To solve this problem and provide thermal comfort indoors, the use of air conditioning during the summers and heating systems during the winters is necessary [START_REF] Solomon | Contribution of working group I to the fourth assessment report of the intergovernmental panel on climate change[END_REF]Climate Initiative 2005). However, these systems consume huge quantities of energy. Therefore, researchers in the construction sector are working on improving the thermal performance of building materials in order to reduce the use of electric systems. In fact, developing new efficient alternatives based on environmentally friendly construction materials is needed. Among these alternatives, storing heat and cold for later usage has become a key technology and an attractive solution for building professionals to improve energy efficiency in the building sector [START_REF] Zalba | Review on thermal energy storage with phase change material: Heat transfer analysis and applications[END_REF][START_REF] Cabeza | Use of microencapsulated PCM in concrete walls for energy savings[END_REF].

In order to reach smart and light wallboard structures, classic construction materials, such as plaster and concrete, can be combined with those having a high latent heat. The latter have the advantage of absorbing large amounts of energy in a comparatively very small volume [START_REF] Pasupathy | Experimental investigation and numerical simulation analysis on the thermal performance of a building roof incorporating phase change material (PCM) for thermal management[END_REF][START_REF] Sharma | Review on thermal energy storage with phase change materials and applications[END_REF]). Phase-change materials (PCM), preferably those with solid-liquid transition, have a high latent heat, and therefore a high volumetric storage capacity at low temperature [START_REF] Kuznik | A review on phase change materials integrated in building walls[END_REF][START_REF] Banu | Evaluation of thermal storage as latent heat in phase change material wallboard by differential scanning calorimetry and large scale thermal testing[END_REF]. This kind of PCM presents melting temperatures within ambient temperature range. Consequently, their integration in construction materials is likely to improve the thermal storage capacity of the building and ultimately contributes to keeping the inside temperature relatively constant with low variations [START_REF] Cabeza | Materials used as PCM in thermal energy storage in buildings: A review[END_REF][START_REF] Devaux | Benefits of PCM underfloor heating with PCM wallboards for space heating in winter[END_REF]. For instance, incorporation of PCM in a 1.5-cm-thick plaster wall resulted in the same thermal storage capacity as a concrete wall that is 9-cm-thick or a brick wall that is 12-cm-thick (Fig. 1).

It is known that the addition of any substance to cement-based materials changes their properties. [START_REF] Wang | Effects of nanomaterials on the hydration kinetics and rheology of portland cement pastes[END_REF] investigated the effects of nanomaterials on the hydration kinetics and rheology of ordinary portland cement pastes. The experimental results indicated that the addition of nanolimestone and nanosilica accelerated early cement hydration. More specifically, the maximum heat flow increased by nearly 2 mW/g when the nanomaterials increased by 1.5%, whereas the time to reach the heat-flow peaks decreased. Toufigh et al. studied polymer concrete (PC) as a potential replacement for concrete based on ordinary portland cement (OPC) [START_REF] Toufigh | Strength evaluation and energy-dissipation behavior of fiber-reinforced polymer concrete[END_REF]). The experimental results indicate that the PC and fiber-reinforced polymer concrete (FRPC) specimens exhibit significantly higher compressive and tensile strength compared to OPC. They are also tougher than OPC specimens. FRPC specimens have higher tensile strength and toughness relative to PC specimens, but both sets are comparable in compressive strength [START_REF] Toufigh | Strength evaluation and energy-dissipation behavior of fiber-reinforced polymer concrete[END_REF].

In our case, there are inherent disadvantages of PCM application in buildings. Indeed, low thermal conductivities of many PCM have posed a continuous challenge. Furthermore, despite the existence of many different phase-change transitions-from solid to solid, from solid to gas, from liquid to gas, or from solid to liquid-the one mainly used is the solid-to-liquid change. Some limitations are related to the other phase-change transitions such as:

• In the case of liquid-to-gas phase change, the thermal storage requires large volumes to store the heat.

• In the case of solid-to-solid phase change, the transformation is too slow and has a low transformation heat [START_REF] Leppers | Development of smart microencapsulated organic phase-change materials for enhancing heat storage capacities of concrete. WP4: Design and construction of concrete (concrete compositions)[END_REF][START_REF] Hawes | The stability of phase change materials in concrete[END_REF]). The use of PCM in construction applications is also limited by the appearance of some phenomena, such as hysteresis, phase segregation, and supercooling problems [START_REF] Sharma | Review on thermal energy storage with phase change materials and applications[END_REF][START_REF] Kuznik | A review on phase change materials integrated in building walls[END_REF]. Because the use of PCM is still experimental, they are quite expensive.

The use of PCM in buildings has been one of the greatest research topics of the last few decades. PCM are currently classified into three groups: organic PCM, inorganic PCM, and eutectic PCM [START_REF] Cabeza | Materials used as PCM in thermal energy storage in buildings: A review[END_REF].

In 2008, Castellon studied PCM effects on air-conditioning usage. He proved that 5% of consumed energy is saved during the summer period when using only 5% of PCM in practice.

Despite the numerous advantages of PCM from a thermal point of view, a major mechanical disadvantage is emphasized, mainly the compressive resistance of the construction material decreases due to the increased porosity with addition of PCM. This drawback was noticed in many experimental studies [START_REF] Hunger | The behavior of self-compacting concrete containing microencapsulated phase change materials[END_REF]Meshgin and Xi 2012).

Thanks to their important advantages, several types of organic PCM with a large range of melting points have been combined with different construction materials. The main results about their effect on the material properties have been published in many journals [START_REF] Sharma | Review on thermal energy storage with phase change materials and applications[END_REF][START_REF] Kuznik | A review on phase change materials integrated in building walls[END_REF]. However, the behavior of PCM is complex, and their properties are often not easy to measure. For example, the thermal behavior can be affected by the shape, the geometry, the dimensions, and especially the heat transfer mechanism (Meshgin and Xi 2012).

Therefore, studying the amounts of exchanged heat between the material and the surrounding environment is important because it allows planning an optimal control of the thermal energy storage and release. In addition, the measurement of the heat flux and the temperature development is necessary for the design of structures with PCM-cementitious materials. To the authors' best knowledge, very few research works have been devoted to the problem of temperature and heat-flux measurements using representative specimens of PCM-cement-based materials, and much less to large-size materials such as PCM walls. The thermal methodologies most commonly used have often been based on the study of PCM specimens of a few milligrams by thermogravimetric analysis (TGA) or differential scanning calorimetry (DSC) techniques. This can raise questions about the specimens used for measurement with respect to their homogeneity and representativeness. In addition, there is an incompatibility between the needed TGA and DSC specimens' size and the aggregates' dimensions when studying concrete. Hence, this study was carried out in the framework of a wider research program of the authors that deals with a thermal characterization method using a laboratory-developed setup. The experimental procedure allows the determination of the thermophysical properties (thermal conductivity and heat storage capacity) of materials based on simultaneous measurements of heat flux and temperature elevation.

This paper is structured as follows: In the following section, "Materials and Methods," the studied materials, the experimental setup, and the theoretical background of a mathematical approach aiming to measure the thermal conductivity and the specific heat capacity of PCM-cementitious materials are described. The "Results and Discussions" section is devoted to the numerical simulation of the testing setup and compares its results with those found experimentally. Then, in the "Conclusions" section, the obtained results are discussed followed by the main conclusions of the study.

Materials and Methods

Materials Description

Phase-Change Materials

The PCM used in this study are commercial products named Micronal DS 5038 (BASF The Chemical Company, Ludwigshafen, Germany) in capsule powder form, with a solid-liquid phase transition. These PCM were chosen in this study because of their organic properties. In fact, unlike inorganic PCM characterized by the presence of supercooling and incongruent melting phenomena, organic PCM have shown good stability in the concrete mixes [START_REF] Kuznik | A review on phase change materials integrated in building walls[END_REF].

These PCM in their capsule powder form are made with a paraffin core microencapsulated in PMMA polymer shell, forming a thin protective insulating film. The scanning electron microscope (SEM) observations of these PCM, illustrated in Fig. 2, and highlighted in previous studies [START_REF] Eddhahak-Ouni | Experimental and multi-scale analysis of the thermal properties of portland cement concretes embedded with microencapsulated phase change materials (PCMs)[END_REF][START_REF] Drissi | Thermal analysis by DSC of phase change materials, study of the damage effect[END_REF][START_REF] Drissi | Development of new concrete 'energy accumulator': Experimental, probabilistic and numerical study of its thermal behavior[END_REF], revealed that they generally have a spherical shape with a diameter ranging between 100 and 300 µm. In reality, these capsules represent the agglomeration of many thousands of microcapsules ranging between 2 and 20 µm bounded together. This phenomenon could be explained by the spray drying of the initial PCM solution, which results in the creation of bigger secondary particles for dust prevention and better handling. Some damaged capsules were observed before their incorporation in the concrete/mortar mix. According to the technical data sheets of the PCM supplier (BASF 2012), their melting point and phase-change enthalpy are 25°C and 110 kJ/kg, respectively. These values were also checked using DSC technique during the study by [START_REF] Drissi | Thermal analysis by DSC of phase change materials, study of the damage effect[END_REF]. In addition, measurements by laser flash analysis (LFA) were carried out, showing that PCM present low thermal conductivities with an average value of about 0.155 W/m/K in the temperature range -10°C to 60°C.

The first dynamic scan was performed by cooling the sample from 20°C to -10°C, and then an isothermal segment of 5 min was programmed so that the sample could reach an isothermal equilibrium state. Next, a heating dynamic segment was carried out from -10°C to 55°C followed by an isothermal step before cooling the sample from 55°C to -10°C. This last cooling segment was arranged so that the DSC signal reversibility could be checked. To finish, a heating segment was performed from -10°C to 20°C. The temperature range of the DSC program was selected to mirror the expected natural climate temperatures. The DSC tests were performed using samples with the same mass (10 mg). For every scanning rate, the test was repeated at least three times, using virgin samples each time. In addition, the DSC experiment was also used to measure the specific heat capacity of PCM.

The DSC results showed that PCM exhibit comparable thermal behavior during heating and cooling processes with an increase of the sensible and latent heats in the exothermic signal. For more details, see the study by [START_REF] Drissi | Thermal analysis by DSC of phase change materials, study of the damage effect[END_REF].

Mortar and Concrete Confection

Different samples of PCM-concretes and PCM-mortars were fabricated in this study. Concrete and mortar specimens with no addition of PCM were also produced to serve as controls. All prepared specimens have a square cross section (4 × 4 cm 2 ). Two different thicknesses of specimen were considered: e = 2 cm and e = 4 cm. In order to avoid capsule damage during the mixing process, PCM were incorporated with the mixture at the last stage.

The mortar and concrete formulations considered in the present study are those optimized by the authors in previous works [START_REF] Drissi | Development of new concrete 'energy accumulator': Experimental, probabilistic and numerical study of its thermal behavior[END_REF]. Tables 1 and2 summarize the quantities for the different ingredients used to manufacture concretes and mortars, respectively.

Portland cement (CEM II/A-L 32.5 N), sand (with a particle size distribution between 0 and 250 µm), and a water-to-cement ratio equal to 0.5 were used in this study. Different PCM amounts (0%, 4.5%, 9%, and 13%) by total mass of the cement were used to manufacture the PCM-concretes and PCM-mortars. It can be noticed that the cement volume fractions in the PCM-mortars are respectively the same as the ones in the PCM-concretes.

After casting, the samples were placed in a room at a temperature of 22°C and a relative humidity of 50%. The specimens were dried inside the laboratory for 28 days and then used for the experiments. Note that the mass of the specimens was monitored before and after the thermal tests, and no significant change was found, thereby showing stable water content during the tests.

Experimental Setup Description

The classical DSC technique often used for the identification of thermal dynamic properties of materials is not adaptable for the case of PCM-cement-based materials because of the incompatibility of the specimen sizes with the crucible sizes [START_REF] Devaux | Benefits of PCM underfloor heating with PCM wallboards for space heating in winter[END_REF]. In order to characterize the thermal properties of PCM-concretes and PCM-mortars using specimens that represent their geometry and microstructure, an original laboratory-developed experimental setup was used (Fig. 3).

The proposed DEsProTherm device designed by EPSILON-FAHRENHEIT (certified ISO 9001 version 2008) looks like hotplate setups commonly used for thermal conductivity measurement (Mathis [START_REF] Instruments | Effect of phase-change materials on properties of concrete[END_REF].

In order to minimize the heat transfer dissipation through the lateral sides of the tested material, a 4-cm thickness of insulation layer with known thermal properties was placed in contact with the lateral surfaces of the specimens. Whereas the lower and upper surfaces of the specimens were put in contact with the two isothermal heat exchange plates of aluminum linked to a heat source (Fig. 4). Heat flux was measured by a thin tangential gradient fluxmeter placed at the interface between the heating element and the specimen. The temperature development was measured by thermocouples placed on the lower and upper surfaces of the specimen. The DEsProTherm setup measured the temperature elevation when the material was subject to transient heat flux. Then, analysis of the measured temperature over time on the upper and lower surfaces of the specimen allowed the calculation of the volumetric heat storage capacity and the thermal conductivity of the material. The measurement period was set to 1,000 s. All testing was performed at a temperature (Troom ≈ 22°C) below the PCM temperature so that PCM were in solid state at the start of the testing.

In the next subsection, the theoretical development used for solving the heat transfer problem in transient regime is presented.

Theoretical Background

Because the material was insulated on the lateral edges, it is safe to assume a one-dimensional (1D) heat transfer direction into the specimen. Therefore, the mathematical model is based on the transient unidirectional heat conduction phenomenon in a multilayer wall, which can be written as follows (Mathis Instruments 2001):

∂T (z, t) ∂t = λ ρC p ∂ 2 T (z, t) ∂ 2 z (1)
where T(z, t) = measured temperature; λ = thermal conductivity; and ρ = volumetric heat capacity of the considered material.

To solve Eq. ( 1), it is necessary to specify initial and boundary conditions. The initial condition is

T (z, t = 0) = T 0 for 0 < z < e (2)
The boundary conditions at the sample lower and upper surfaces are

-λ ∂T (z = 0, t) ∂z = φ (3) λ ∂T(z = e, t) ∂z = 0 (4)
where φ = imposed heat flux to the material. Solving Eq. ( 1) using the conditions [Eqs. ( 2)-( 4)] yields to the analytical temperature expression

T (z, t) = T 0 + φe 3λ + z 2λe (z -2e)φ + φt ρC p e - 2φe λ +∞ p=1 exp (-(λ/ρC p )(Pπ/e) 2 t) (pπ) 2 cos pπ e z (5)
Then, one can derive the evolutions versus time of the sample temperature at z = 0 and z = e, respectively given by Eqs. ( 6) and ( 7 where N = number of measurements.

The determination of the material thermal conductivity and volumetric heat capacity is obtained by solving the system of Eqs. ( 6) and ( 7), in order to find the best fit for the measured temperature development denoted T exp . For this purpose, an objective function J is given by the following equation and minimized using the leastsquares method:

J = k=N k=1 T exp (e, t k ) -T (e, t k ) 2 (8)

Numerical Simulation of the Samples' Thermal Behavior and Comparison with the Experimental Results

The specimens' thermal behavior was simulated using the Ansys CFX 15 software. Ansys is a transient system simulation program that simulates the thermal behavior of physical systems. The heat equation is then solved by a finite volumes method that makes it possible to calculate the temperature at each node. Before developing the equations that govern the heat transfer process occurring in the system, some assumptions were made in order to simplify its solving process. These assumptions are that

• Thermal properties are constant with respect to temperature.

• End effects are neglected.

• Interfacial resistances are negligible. • Heat transfer through the wall is assumed 1D.

• Inducted buoyancy flow in the melted PCM is neglected. Fig. 5 shows the physical system studied in the present work. It consists of a geometry made with a specific material (mortar, PCM-mortar, concrete, PCM-concrete) with homogenous properties. The total thickness of the geometry is either 2 cm or 4 cm. Its corresponding properties are the results given by the mathematical model studied in the "Materials and Methods" section. The sample was initially at a uniform temperature close to the thermal comfort requirement, T(z, 0) = T0 = 22°C. The boundary conditions on the outer surfaces are due to the flux variation. The lateral surfaces are adiabatic.

This problem is mathematically expressed by the following equations:

∂T (z, t) ∂t = λ ρC p ∂ 2 T(z, t) ∂z 2 ; z ∈ [0, e], t > 0 (9) -λ ∂T ∂z = φ; z = 0, t > 0 (10) λ ∂T ∂z = 0; z = e, t > 0 (11) T (z, 0) = T 0 ; z ∈ [0, e] (12)
After optimizing the finite mesh and the time step and after validating the model, the different studied cases were simulated using Ansys software. The total time was 1,000 s, and the time step was 0.1 s. For samples with 2 cm thickness, the flow at z = 0 was taken around 265 W/m 2 , while for samples with 4 cm thickness, it was about 1,180 W/m 2 .

Results and Discussions

This section presents the main results obtained by the DEsPro-Therm experimental setup, as well as the results of the optimization procedure used for the thermal properties calculation. The sampling frequency of readings was 0.1 s, and the readings were recorded continuously.

Experimental Results (Thermograms) Fig. 6 presents the measured temperature over time on the lower (z = 0) and upper (z = e) surfaces of the control and PCM-concrete specimens with a thickness of 2 cm. Similarly, Fig. 7 shows the collected data for the control and PCM-mortar specimens with a thickness of 2 cm.

Thermophysical Properties

The objective function minimization given by Eq. ( 8) allows the computation of the thermophysical properties (thermal conductivity and volumetric heat storage capacity) of PCM at T = 22°C. The results are summarized in Figs. 8 and9 for the 2-cm-and 4-cm-thick concrete specimens, respectively. The symbols at the top of each bar in Figs. 8-11 are indicators of the data. These figures show that addition of PCM in concrete and mortar results in a decrease of thermal conductivity and an increase of the energy storage capacity. These results are in agreement with those found in the literature [START_REF] Leppers | Development of smart microencapsulated organic phase-change materials for enhancing heat storage capacities of concrete. WP4: Design and construction of concrete (concrete compositions)[END_REF]Meshgin and Xi 2012).

It is also noticed that the decrease of the thermal conductivity is more important for the cubic PCM-concrete specimens with 4-cm thickness, than for the thinner specimens (2-cm thickness). This phenomenon can probably be attributed to the lower thermal diffusivity of cubic samples, in comparison with the thinner concrete ones.

Moreover, an increase of about 15% was noticed in the volumetric heat storage capacity with 13% of PCM for a specimen thickness of 2 cm, whereas, an enhancement of only 9% was found for the specimen thickness of 4 cm. This difference could be explained by many causes including the presence of more damaged PCM particles and consequently more pores found in the 4-cm-hick specimen.

For the control concrete specimens, the specific heat was slightly lower for the thicker one. This small difference could be attributed to the margin of error of the experimental measurements. For the concrete with 9% PCM specimens, the results seem to be irregular, which could also be attributed to the uncertainties of the experimental measurements. Fig. 10 plots the results of the thermal conductivity and the volumetric heat storage capacity for the PCM-mortar specimens with a thickness of 2 cm. The results of PCM-mortar specimens with a thickness of 4 cm are represented in Fig. 11.

For the mortar specimens, a significant decrease in the thermal conductivity and a good improvement of the specific heat capacity are noted. Moreover, the effect of the PCM addition seems to be higher in the mortar case than in the concrete case. In the case of the cubic specimen (e = 4 cm), the decrease of thermal conductivity reaches 37% for PCM-mortars, whereas it is about 30% for PCM-concretes. However, the increase of the heat capacity was estimated at 13% for PCM-mortars versus 9% for PCM-concretes. This result could be explained by the presence of gravel in concrete, which may have influenced the heat transfer process and created more pores in the microstructure of the concrete compared with the mortar. In addition, during the mixture phase, the contact between the PCM and the gravel damaged the PCM-microcapsules; thus, their effect on the thermal properties of concrete was lower than in the mortar. Accordingly, the different aspects linked to the PCM damage and its effect on the long-term energy efficiency of the PCM-concrete final product merit further study in the future in order to improve our understanding and knowledge of the sustainability of PCM-concrete technology.

Discussion of PCM Effect

As presented previously, a numerical investigation of the thermal behavior of both concrete and PCM-concrete specimens was performed. In addition, the heat equations were solved analytically using MATLAB software package (MathWorks, Natick, MA). This section presents a comparison between the measured temperature development and those predicted using the Ansys model and analytically. For example, Fig. 12 shows the temperature development at the upper surface (z = e) as obtained with the three methods (measured, Ansys model, and analytical). A perfect agreement is achieved among the cases.

The temperature difference (ΔT ) between the upper and lower surfaces of the specimens is an important indicator of the performance of PCM. In order to evaluate this parameter correctly, a normalization was performed on the temperature data. Fig. 13 shows the results from the measured temperature and from the numerically predicted ones for the four treated PCM rates (0%, 4.5%, 9%, and 13%) in the case of concrete specimens with a thickness of e = 2 cm. It can be seen that the temperature rise on the upper surface, z = e, decreases as the PCM is incorporated. Indeed, the PCM is considered a condenser for the temperature propagation. Thus, about 15% reduction is noticed for 13% of PCM in the case of e = 2 cm. These results are coherent with the expected thermal conductivity reduction with the PCM addition. Moreover, the numerical results are very close to the experimental ones (maximum deviation of 2.5% and minimum deviation of 0.3% as compared with the experiment).

Conclusions

In this work, an experimental investigation of smart cement-based materials incorporating PCM using an original laboratory setup are presented. The purpose of the study was to evaluate both the thermal conductivity and the heat storage capacity of mortars and concretes, including different PCM proportions. Based on the transient plane source theory, the experimental setup enabled monitoring of the temperature development on the material specimen subjected to a unidirectional heat flux signal.

Using the measured temperature development on the specimen faces and the analytical temperature solution of the heat transfer problem, thermal conductivity, and heat storage capacity of the tested material are identified. The research results highlight the effects of PCM on the thermal behavior of cement-based material. More specifically, a decrease of the thermal conductivity and an improvement of the heat storage capacity were noticed in both mortar and concrete cases with the addition of PCM. For cubic specimens, the maximal loss of thermal conductivity was evaluated at 30% with PCM-concretes versus 37% for the case of PCM-mortars. On the other hand, the maximal gain in the specific heat for PCM-concrete was about 2% versus 13% for PCM-mortar. Although the same PCM volume proportions were considered for mortars and concretes, the effect of PCM in the mortar case was greater than that in the concrete case. This finding can be explained by the presence of aggregates in the concrete case. In fact, the concrete specimens are more porous because of the presence of interfacial transition zones around the gravel particles. This can be attributed to the progressive departure of water during the drying process and therefore the creation of voids. Further study is necessary in order to check the variation of the porosity of the concrete with the addition of PCM.

These findings successfully demonstrate not only the efficiency of the developed test bench to evaluate the thermal conductivity and specific heat of materials using relatively large specimens, but also the effect of PCM addition to provide important advantages for thermal storage systems and heat transfer applications.

In the future, it would be interesting to make use of the obtained experimental results in this study to validate a numerical finiteelement model that has been already developed in a previous study [START_REF] Drissi | Thermal analysis by DSC of phase change materials, study of the damage effect[END_REF]. The numerical simulation allows a fast prediction of the PCM-cement-based material's thermal response at a macroscale when subjected to complex scenarios of extreme ambient temperatures. Moreover, in order to determine the effect of those materials on the thermal comfort of a building, it is necessary to study numerically and experimentally other wall thicknesses and other amounts of PCM to incorporate with the cement-based material. The arrangement (number, positions, etc.) of the PCM walls in the building could thus be judiciously determined. The construction of a test cell would also help to understand the actual contribution of these walls.

Fig. 1 .

 1 Fig. 1. Comparison of thickness between brickwork, concrete wall, and plaster with PCM wall having the same thermal heat storage.

Fig. 2 .

 2 Fig. 2. SEM observations of PCM capsules: (a) overview; (b) micro-PCM agglomeration; and (c) PCM damage.

  ): θ(0, λ, ρC p , t) = T 0 + φe 3λ

Fig. 3 .

 3 Fig. 3. Experimental setup: material testing. (Image by Nessrine Essid.)

Fig. 5 .

 5 Fig. 5. The studied system.

Fig. 6 .

 6 Fig. 6. Temperature evolutions recorded by the experimental setup (PCM-concretes, e = 2 cm): (a) 0% PCM; (b) 4.5% PCM, (c) 9% PCM; and (d) 13% PCM.

Fig. 7 .Fig. 8 .

 78 Fig. 7. Temperature evolutions recorded by the experimental setup (PCM-mortars, e = 2 cm): (a) 0% PCM; (b) 4.5% PCM; (c) 9% PCM; and (d) 13% PCM.

Fig. 9 .Fig. 10 .

 910 Fig. 9. Thermal properties of PCM-concretes (e = 4 cm): (a) thermal conductivity; and (b) volumetric heat capacity.

Fig. 11 .

 11 Fig. 11. Thermal properties of PCM-mortars (e = 4 cm): (a) thermal conductivity; and (b) volumetric heat capacity.

Fig. 13 .

 13 Fig. 13. Temperature rise of the upper face for PCM-concrete (e = 2 cm).

Table 1 .

 1 Reference concrete formulation for making 1 m 3 of concrete

	Component	Value
	Cement (kg)	360
	W:C	0.5
	Sand (kg)	748
	Gravel (kg)	1,076

Note: W:C = water-to-cement ratio.

Table 2 .

 2 Reference mortar formulation

	Component	Value
	Cement (g)	500
	W:C	0.5
	Sand (g)	1,038

Note: W:C = water-to-cement ratio.

Acknowledgments

The authors gratefully acknowledge the scientific collaboration of the I2M team from ENSAM-Bordeaux and especially Mr. Denis Bruneau and Mr. Awarou Waste Aregba.

Notation

The following symbols are used in this paper:


domain: sdv info

text:
Type of presentation: N/A Chromatin spatial organization and transcription regulation during early embryonic development : the example of ribosomal genes

Acknowledgement: This work was supported by the REVIVE Labex and by an INRA bottom-up grant. We acknowledge the MIMA2 Platform.


Amira Gabsi
email: amira.gabsi@gmail.com

Xavier Heim

Akram Dlala

Asma Gati

Haifa Sakhri

Ahmed Abidi

Sonia Amri

Bilel Neili

Aurelie S Leroyer

Alexandrine Bertaud

Monia Smiti Khanfir

Fatma Said

Mohamed Habib Houman

Brigitte Granel

Marcel Blot-Chabaud

Nathalie Bardin

& Raja

TH17 cells expressing CD146 are significantly increased in patients with Systemic sclerosis

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

differentiation and for production of cytokines which participates in protective immunity at boundary tissues [START_REF] Castro | RORγt and RORα signature genes in human TH17 cells[END_REF] . IL17A primarily binds to IL-17RA [START_REF] Gu | IL-17 family: Cytokines, receptors and signaling[END_REF] and initiates a pro-inflammatory signal, which is strongly involved in the progression of many autoimmune diseases in humans [START_REF] Bălănescu | IL-17 and TH17 cells in systemic sclerosis: a comprehensive review[END_REF][START_REF] Amatya | IL-17 Signaling: The Yin and the Yang[END_REF] .

In SSc, the mRNA and protein expression of IL17A was found to be positively correlated to an outcome index, the modified Rodnan skin score [START_REF] Bălănescu | IL-17 and TH17 cells in systemic sclerosis: a comprehensive review[END_REF][START_REF] Zhou | The elevated expression of TH17-related cytokines and receptors is associated with skin lesion severity in early systemic sclerosis[END_REF] .

In view of the role played by CD146 in SSc, we hypothesized a preponderant role of CD146 positive TH17 cells in SSc. To investigate this hypothesis, we explored in the present study, the concentrations of sCD146 and IL17A in serum of patients with SSc as compared to healthy controls. In parallel, we evaluated the proportion of TH17 cells expressing CD146 (CD146+ TH17) over total TH17 cells and analyzed the potential correlations with clinical events. At last, effect of sCD146 was tested on the proportion of CD146+ TH17 among total TH17cells of patients with SSc.

Materials and Methods

Ethics statement. All patients gave informed consent and all methods were performed in accordance with the relevant Guidelines and regulations. The Medical Bioethics Committee at the Pasteur Institute of Tunis approved This study under the reference 2015/04/E/FST. Blood samples. Peripheral blood samples were obtained from 50 patients with SSc admitted to internal medicine departments of university hospital La RABTA Tunis Tunisia; all patients fulfilled the ACR/EULAR Classification Criteria and were then sub classified according to Leroy et al. criteria [START_REF] Van Den Hoogen | Classification Criteria for Systemic Sclerosis: An American College of Rheumatology/European League Against Rheumatism Collaborative Initiative: ACR/EULAR Classification Criteria for SSc[END_REF] . The cohort is explained in Table 1. Fifty sera of age and sex matched blood controls (median age 61 (57-70) and sex ratio 10 male/40 female).

Separation of blood cells on a FICOLL density gradient. Peripheral blood mononuclear cells (PBMCs) were prepared by centrifugation over a Ficoll-Hypaque Gradient (Histopaque; Sigma Diagnostics, St Louis, MO) and cryopreseved in 10% dimethyl sulphoxide (DMSO) in fetal bovine serum (FBS). Sera were also collected and stocked in -20° until use.

Soluble CD146 detection.

We measured serum sCD146 concentration using a capture enzyme-linked immunosorbent assay kit (CYQUANT ELISA, Biocytex Marseille, France) according to the manufacturer's protocol. The ELISA assay was validated previously [START_REF] Kaspi | Identification of CD146 as a novel molecular actor involved in systemic sclerosis[END_REF] . A plastic support coated with specific mouse monoclonal anti human CD146 F(ab') 2 fragments binds to the sCD146 to be measured.

Next the mouse monoclonal antibody anti-CD146 coupled with peroxidase binds to a remaining free antigenic determinant of the CD146. The bound enzyme peroxidase is then revealed by its activity in a predetermined time on the TMB substrate. After stopping the reaction, the intensity of the signal is directly related to the concentration of sCD146 initially contained in the sample.

Quantification of IL17A.

IL17A was quantified in serum of healthy controls and patients with SSc, using commercially available ELISA kit (Quantikinie ELISA human IL-17A immunoassay R&D Systems France). All the amount of cytokine present in diluted serum will be captured. After several washes to remove the excess, a polyclonal antibody bound to an enzyme specific for human IL17A is added. After washes, the reaction is revealed with a substrate (TMB), giving a color whose intensity is proportional to the amount of IL17A bound and the plates are read at 450 nm and optical density were converted to concentration using a four parametric logistic regression. mRNA quantification. RNA isolation and cDNA synthesis. Total cellular RNA was extracted from cryopreseved PBMCs using the acid guanidine-phenol-chloroform technique with Trizol (GIBCO, BRL, Bethesda, MD), according to the manufacturer's instruction. After incubation for 10 min at 65 °C, RNA samples were stored Samples were run in duplicate, and relative quantification of mRNA level was performed using β-actin as an endogenous reference. We obtained the primer sequences (Table 2) from literature and checked through PCR in silico. We used three primers: RORγT, IL17A and CD146. qPCR data was analyzed by calculating the fold difference individually for each gene. Cycle threshold (Ct) is defined as the number of PCR cycles at which the fluorescence signal rises above the threshold value and is inversely proportional to the amount of template present in the reaction. Ct values of genes and control samples were compared and the fold difference calculated like: Fold CHANGE = 2-ΔCt where ΔCt = CT sample-CT endogenous control. The Ct values were plotted, assuming the same threshold for all genes evaluated. Ct values < 40 were only used for calculation of the PCR efficiency. (This was explained in Step-one-plus book).

Flow cytometry analysis.

PBMCs were prepared by centrifugation over a Ficoll technique and cryopserved in 10% SVF-DMSO. Thawed cells were routinely tested for viability by trypan blue exclusion. For cytokine secretion assays, PBMCs were stimulated with complete cell culture medium (Eurobio France) with 1/1500-inonomycine. -1/2000 PMA -1/3000 Golgi Stop overnight. Then cells were washed twice with 5% fetal bovine serum (FBS)-phosphate-buffered saline (PBS) prior to staining. First of all, cells were incubated for ten minutes with F c blocking (Human TrueStain FcX TM Ozyme FRANCE) as an isotype control. Then, PBMCs were stained with up to five antibodies for 30 min at room temperature. After staining, the cells were fixed with 2% formaldehyde-PBS prior to analysis on a BD canto flow cytometry (BD Bioscience, San Diego, CA, USA). Lymphocytes were gated based on forward-scatter and side-scatter properties; at least 10,000-gated events were analyzed using the Flow Software (Flow LLC Ashland, Oregon). We calculated absolute cell percentages based on flow cytometry data and complete blood counts. Six colors were used to analyze the surface phenotype and intracellular cytokine production of PBMCs. The antibodies used were CD3 PerCP Cy5/CD4 APC-Cy7/CD25 PE-Cy7/CD146 Alexa fluor 488 for cell surface staining and IL17A PE and FOXP3 APC for intracellular staining and cytokine staining.

Flow cytometry analysis was performed on the canto 2 flow cytometry (BD Bioscience, San Diego, CA, USA). Lymphocytes subsets were gated by first forward versus side light scatter to identify lymphocytes, then CD4+ T cells were gated using a dot plot showing CD3 versus CD4. First of all, TH17 cells were identified using a dot plot showing CD4 and IL17A and then TH17 cells expressing CD146 were gated using a dot plot showing IL17A and CD146. Frequencies of cells were determined using statistical option in FLOWJO V10.5.3.

In vitro sCD146 stimulation on PBMCs from patients with SSc. Fresh PBMCs from patients were used. 5 × 10 5 cells from each patient were incubated with 100 ng/ml of sCD146 [RPMI-1640 (Gibco, Carlsbad, CA, USA) with 5% AB + human serum, 10 mM HEPES, non-essential amino acids, sodium private, 2 mM L-glutamine (Sigma, St. Louis, MO, USA), 100 units/ml of penicillin and 100 μg/ml of streptomycin (Invitrogen, Carlsbad, CA, USA)] + 100 ng/ml sCD146, then cultured in a humidified incubator at 37 °C with 5% CO2 for 24 hours. For cytokine secretion assays, cells were stimulated overnight with PMA-Ionomycine-Golgi Stop culture medium. And the cells were stained with CD3 PerCP Cy5/CD4 APC-Cy7/CD146 Alexa fluor 488 antibodies for cell surface staining and IL17A PE antibody for cytokine staining.

Flow cytometry analysis was performed on the canto 2 flow cytometry (BD Bioscience, San Diego, CA, USA). Lymphocytes subsets were gated as described above in flow cytometry analysis section.

Finally, using these cells stimulated or not with sCD146, total RNA was extracted and cDNA was synthesized as described above (2.6) and Q-RT PCR was performed using CD146 primers (Table 2).

Statistical analysis.

Results presented as means ± standard deviation. Statistical analyses were performed using Prism (Graph Pad Software, San Diego, CA). A p value < 0.05 was considered statistically significant.

Correlation and linear regression are the most commonly used techniques for quantifying the association between two parameters. The analyses are based on linear relationship between the two variables. A scatter plot is essential before embarking on any correlation-regression analysis to show that this is indeed the case. In our study, correlation and linear regression let us study correlation between sCD146 concentrations, IL17A concentration and TH17 expressing CD146 cells percentages.

Results

Epidemiologic study. We included 50 patients with SSc from the internal medicine department of university hospital RABTA Tunis, having a score of 9 or higher according to the ACR/EULAR criteria. Our cohort was composed of 43 women and 7 men and a mean age of the onset of illness equal to 60 ± 16 years [28-68 years]. Rodnan's score ranged from one to 41 in our cohort and averaged 17. Then, clinical data highlighted several inaugural events such as Raynaud's syndrome, skin involvement, arthralgia, cutaneous sclerosis, gastro-esophageal reflux. Pulmonary Arterial Hypertension (PAH), is one of the most serious disorders of systemic sclerosis, and is diagnosed by cardiac Doppler ultrasound after exercise dyspnea. Another serious manifestation is pulmonary fibrosis demonstrated by Chest CT examination. Our cohort was constituted by 20 patients with PAH and 19 patients with pulmonary fibrosis (Table 1).

Soluble CD146 and IL17A concentrations are increased in sera of patients with SSc.

Serum concentrations of sCD146 and IL17A were measured in patients with SSc (n = 50) and compared to age -and sexmatched controls (n = 50). As shown in Fig. 1A,B, sCD146 and IL17A concentrations were significantly higher in patients with SSc as compared to healthy controls (respectively, 329.0 ± 24.90 ng/ml versus 168.4 ± 19.39 ng/ ml, p < 0, 0001 and 20.05 ± 1.97 ng/ml versus 3.32 ± 0.54 ng/ml, p < 0, 0001). Regarding clinical association, we found a significant association between low sCD146 concentrations and pulmonary fibrosis (p = 0.04) as shown in Fig. 1C, while there was no correlation with other clinical disorders described in Table 1. For IL17A, no correlation was observed with clinical disorders. Interestingly, we found a positive correlation between sCD146 and IL17A concentrations (p = <0.0001), as illustrated in Fig. 1D. The number of CD146+ TH17 cells is increased in patients with SSc. We analyzed the expression of IL17A and of the master transcription factor of TH17 cells, RORγT, in patients with SSc as compared to control group.

The mRNA expression of RORγT and IL17A was first quantified by qRT-PCR in peripheral blood mononuclear cells (PBMCs) of patients with SSc and healthy controls. RORγT and IL17A mRNA expression were significantly higher in patients with SSc as compared to healthy controls (p = 0, 0013 and p = <0.0001) (Fig. 2A,B). For RORγT and IL17A mRNA expression, no significant association was observed with clinical features.

In a second step, we studied the proportion of TH17 cells over the total CD4+ cells population using a flow cytometry analysis (Fig. 3A). We found a higher percentage of TH17 cells in patients with SSc (Mean 16.95 ± 0.8482), as compared to healthy controls (Mean 9.135 ± 0.2522, p < 0.05) as shown in Fig. 3B.

Then, we analyzed CD146 expression on TH17 cells, we found a higher percentage of TH17 cells expressing CD146 (CD146+ IL17A+ subset) in patients with SSc as compared to healthy controls (Fig. 4A,B). Interestingly the percentage of TH17 cells expressing CD146 was inversely correlated to the pulmonary fibrosis (4 C) but no association with other clinical features was detected.

To better understand the link between TH17 cells and sCD146, the ratio between CD146+ TH17 cells and total TH17 cells was calculated (percentage of TH17 cells expressing CD146/TH17 cells). We found a significant correlation between sCD146 concentration and this ratio (p = 0, 0135), as shown in Fig. 4D. sCD146 affects T lymphocytes in PBMCs from patients with SSc. We evaluated sCD146 effects on TH17 cells after sCD146 treatment (100 ng/ml for 24 hours) of PBMCs of patients with SSc. Interestingly, an up regulation of the percentage of TH17 cells expressing CD146 (CD146+ IL17A+ subset) was found after sCD146 treatment (Fig. 5A). The number of cells was determined at T0, and after 24 h of sCD146 treatment with 492 million +/-6088 at T0 and 491 million +/-6441 at T24, no significant difference was observed (p = 0.8992) attesting for the absence of cell proliferation. In addition expression of CD146 was up regulated after sCD146 treatment as demonstrated by fluorescence intensity analysis (Fig. 5B). Besides, after treatment with sCD146, mRNA expression of CD146 was increased in PBMCs treated with sCD146 as compared to untreated cells (Fig. 5C).

Discussion

In systemic sclerosis, we recently reported a major role of CD146, a molecule ubiquitously distributed on vascular cells, but also expressed on a subset of lymphocytes involved in the pathogenesis of the disease, TH17 cells. In this study we demonstrated for the time that TH17 lymphocytes expressing CD146 are increased in patients with SSc, suggesting their involvement in the pathology.

In these patients, we found a higher percentage of TH17 cells expressing CD146 and a correlation between sCD146 and IL17A serum concentrations. Interestingly the ratio of TH17 cells expressing CD146/Total TH17 cells in patients with SSc was inversely correlated to pulmonary fibrosis. Finally, in vitro treatment of TH17 cells with sCD146 led to an up regulation of CD146 expression. Afterward, PBMCs treatment with sCD146 induced an increase in the mRNA expression of CD146 as compared to untreated cells.

In a previous study, unfavorable evolution of SSc was associated with a decrease of sCD146 concentrations. We recently showed that sCD146 concentrations were increased in patients with SSc and that severe manifestations of the disease, as pulmonary fibrosis, were associated with low sCD146 concentrations [START_REF] Kaspi | Identification of CD146 as a novel molecular actor involved in systemic sclerosis[END_REF] . Patients producing high and sustained levels of sCD146 may be protected against the disease complications, accounting for the reduction in the concentration of the soluble form in the case of pulmonary fibrosis [START_REF] Kaspi | Identification of CD146 as a novel molecular actor involved in systemic sclerosis[END_REF] . In the present study, we confirm these data, highlighting sCD146 as a novel biomarker for the assessment of the disease. In addition, we evidence a significant correlation between sCD146 and IL17A concentrations. This positive correlation could explain the protective role of sCD146, interfering with the counterbalance of inflammation and fibrosis mediated by IL17A.

In patients with autoimmune disease, an increased proportion of TH17 cells were described in the peripheral circulation and at the sites of active inflammation [START_REF] Bedoya | TH17 Cells in Immunity and Autoimmunity[END_REF] . Several studies have shown that in SSc, TH17 cells could affect the inflammatory response of the disease, and potentially the fibrosis process [START_REF] Truchetet | Increased frequency of circulating Th22 in addition to TH17 and Th2 lymphocytes in systemic sclerosis: association with interstitial lung disease[END_REF] . Our results confirm a higher percentage of TH17 cells in patients with SSc and show for the first time that this increase is partially due to a higher percentage of TH17 cells expressing CD146. We propose that in patients, the up-regulation of this sub-population could be related to the sera sCD146 increase. In accordance with this hypothesis, we showed an in vitro increase of TH17 cells expressing CD146 after sCD146 treatment. This increase was attributed to an augmentation of CD146 expression since we showed an up-regulation of fluorescence intensity without TH17 proliferation. Such an increase of membrane CD146 expression has been already reported in endothelial cells [START_REF] Bardin | Soluble CD146, a novel endothelial marker, is increased in physiopathological settings linked to endothelial junctional alteration[END_REF] . We also showed that sCD146 concentrations correlate with the TH17 expressing CD146/TH17 total cells ratio in patients with SSc.

Altogether our results led us to hypothesize that sCD146 could be involved in TH17 cells differentiation and their homing to the site of the disease. Along this line, it has been described that CD146 positive TH17 cells was the sub-population of TH17 cells with an enhanced ability to bind to, and to cross through, vascular endothelium. In addition, CD146 was described as a marker of mouse NK cell maturation [START_REF] Despoix | Mouse CD146/MCAM is a marker of natural killer cell maturation[END_REF] . At last, TH17 expressing CD146 are able to migrate from the peripheral circulation to sites of active inflammation [START_REF] Dagur | MCAM-expressing CD4+ T cells in peripheral blood secrete IL-17A and are significantly elevated in inflammatory autoimmune diseases[END_REF] . Further studies will be conducted to confirm the role of this sub-population CD146+ TH17 in the fibrotic process, to get more insights into mechanistic pathways and to investigate the potential interest for therapeutic applications.

In conclusion, TH17 cells expressing CD146 could represent a new component of the adaptive immune response and could be potential targets for therapy, opening the way for the generation of new tools for the management of SSc.

Figure 1 .

 1 Figure 1. Serum level of soluble CD146 (sCD146) and circulating IL17A in patients with Systemic sclerosis (SSc) and healthy controls (HC). (A) Soluble CD146 (sCD146) concentration was significantly higher in patients with SSc (n = 50) than in HC (n = 50) [329.0 ± 24.90 ng/ml versus 168.4 ± 19.39 ng/ml, p < 0,0001]. (B) The cytokine IL17A concentration was significantly increased in patients with SSc (n = 50) than in (HC) (n = 50) [20.05 ± 1.97 ng/ml versus 3.32 ± 0.54 ng/ml, p < 0, 0001]. (C) A significant association was observed between pulmonary fibrosis (n = 19) and low levels of sCD146 (p = 0.0147). (Yes = have pulmonary fibrosis/ No = have other involvement than pulmonary fibrosis). (D) A correlation was observed between the IL17A and soluble CD146 (sCD146) concentrations in patients with SSc (p < 0.0001).

Figure 2 .

 2 Figure 2. Relative mRNA expression of IL17A and RORγT in PBMCs from patients with Systemic sclerosis as compared to healthy control. (A) The expression of the transcription factor RORγT mRNA was higher in patients with SSc than in HC (p = 0, 0013). (B) The expression of the cytokine IL17A mRNA was higher in patients with SSc than in HC (p < 0.0001).

Figure 3 .

 3 Figure 3. Flow cytometry analysis of TH17 cells in patients with systemic sclerosis as compared to healthy controls. (A) Gating strategy of TH17 cells in HC as compared to PBMC of Patients with SSc by FACS: cells were activated with PMA-Ionomycine-Golgi Stop overnight and F c blocking (Humain TrueStain FcX TM Ozyme FRANCE) was used as an isotype control. (B) The percentage of TH17 cells in patients with SSc was found to be increased as compared to HC (p = 0.0019).

Figure 4 .

 4 Figure 4. Flow cytometry analysis of TH17 expressing CD146 in Systemic sclerosis. (A) Gating strategy for analysis of CD146 expression on TH17 total cells in HC compared to patients with SSc. (B) The mean percentage of TH17 cells expressing CD146 (CD146+ TH17cells) and non expressing CD146 (CD146-TH17) among HC and patients with SSc: Percentage of TH17 cells expressing CD146 (CD146 + TH17) is higher in case of SSc than in healthy controls (p = 0.0019). (C) The percentage of TH17 cells expressing CD146 (CD146+ TH17) is inversely correlated to pulmonary fibrosis (p = 0.04) (Yes = have pulmonary fibrosis/No = have other involvement than pulmonary fibrosis). (D) A correlation is observed between the ratio (CD146+ TH17/TH17 Total) cells and sCD146 concentration in patients with SSc (p = 0.0004).

Figure 5 .

 5 Figure 5. Expression of CD146 on TH17 cells in vitro; Expression of CD146 on TH17 cells was analyzed in peripheral blood mononuclear cells (PBMCs) from healthy controls (HC) and patients with SSc. (A) Flow cytometry analysis of TH17 cells expressing CD146 in PBMCs from patients with SSc treated with sCD146: in each subpopulation, expression of CD146 on TH17 cells was compared between untreated PBMCs (squares) and treated ones (triangles) in patients with SSc. (B) Flow cytometry analysis of TH17 cells expressing CD146 in PBMCs from patients with SSc treated with sCD146: Fluorescence intensity of CD146 is higher on TH17 cells from PBMCs treated with sCD146 compared to untreated ones. (C) The expression of the transcription factor RORγT mRNA and IL17A mRNA in PBMCs of patients with SSc before and after treatment with sCD146: there is no difference in RORγT mRNA expression (p = 0.7994) while IL17A mRNA expression was lower after treatment (p = 0.0053). (D) CD146 mRNA expression in PBMCs of patients with SSc before and after treatment with sCD146: there is an increase in CD146 mRNA expression after treatment (p = 0, 0232).

SSc patients n = 50 Median

	Median age (years)	60 (28-68)
	Male/female	7/43
	Clinical Features	n (%)
	Limited cutaneous form	24(48)
	Diffuse cutaneous form*	26 (52)
	Ulcers, scars, gangrene	17 (65)
	Pulmonary fibrosis (PF)	19 (73)
	Pulmonary arterial Hypertension (PAH)	20 (76)
	Digestive involvement	13 (50)
	Kidney involvement	8 (30)
	Immunological Results Positive for anti-nuclear antibodies at -80 °C. Total RNA quality and quantity was assessed by absorbance at 260 nm using a nucleic acid and protein n (%) 48 (96) -centromeric staining (ACA) 24 (47) analyzer. This RNA was reverse-transcribed in a total volume of 25 µL with 200 units of Moloney murine leu-kemia virus reverse transcriptase (Invitrogen, US) in the presence of 1.5 µL of 5X First Strand Buffer (Invitrogen,
	-positive for anti-topoisomerase I (anti-topo I) US) (250-mM Tris-HCL; 375-mM KCL; 15-mM MgCL2) in addition to 10 units of RNAsine (Invitrogen, US), 26 (49)
	1.25 µL of 100 mM DTT and 1.25 µL of each desoxynucleotide triphosphates (Promega, US) at 10-Mm. After that, all these reagents were incubated at 37 °C for 60 min and then cooled at -20 °C. The integrity and the quantity of Table 1. [A] Demographic data and disease characteristics of SSc patients. n: number of subjects. *According to the classification of ACR 2013. the cDNA samples were tested by amplifying β-actin cDNA.
	Quantitative RT-PCR. Real-time PCR was performed on a The Step-One-Plus Real-Time PCR (Applied Biosystems, CA, USA) using the SYBR Green qPCR Kit (Power Up ™ SYBR ™ Green Master Mix A25742 Applied Biosystem Thermofischer FRANCE), and continuous fluorescence monitoring. A reaction volume of 25 µl (1.0 µl
	cDNA) was amplified for 40 cycles after initial denaturation (95 °C, 10 min) with the following parameters: 95 °C
	for 10 s, 60 °C for 15 s, and 72 °C for 20 s.

Table 2 .

 2 Primer sequences. 1 -βactin (promega). 2-RORδT (eurobio). 3-IL17A A (eurobio). 4-CD146 (eurobio).

	Primer

Scientific RepoRtS | (2019) 9:17721 | https://doi.org/10.1038/s41598-019-54132-y

Acknowledgements

We would like to thank: All the members of cytometry platform at the faculty of medicine of Tunis for flow cytometry analysis. All the members of Laboratory of toxins and venoms at the Institute Pasteur Tunis. All the members of internal medicine at the RABTA hospital Tunis, La Timone Hospital Marseille and La conception Hospital Marseille for their cooperation. This work was supported by the Ministry of Higher Education and Scientific Research of Tunisia.

Author contributions

Competing interests

The authors declare no competing interests.


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PhytoCOTE project : State of organic and inorganic contamination on vineyard soils

Manon Pierdet a,b , Juliette Gaillard b , L. Denaix a , H. Budzinski b Viticulture is one of the agricultural crops that use the most pesticides in France, in particular fungicides. These regular inputs may lead to a long-term contamination of ecosystems and thereby affect fauna and flora. Different processes in soils play a role in pesticides retention and transfer. In order to improve the knowledge about the evolution in time and scale of different chemicals contaminants within different soil types, a state of contamination level in soils surfaces and a characterisation of traces elements availability were assessed. 53 plots with important pedological diversity were sampled over the 0-15 cm horizon. The soils were characterised (organic matter, Fe and Al oxyhydroxides, CEC, granulometry, pH) and content measurments of total copper, cadmium, lead, zinc and 205 organic molecules were made. The characterisation of trace elements availability were made using passive samplers (DGT).

A copper contamination due to the past and current use of Bordeaux mixture (copper sulphate) has been shown on the experimental site (until 197 mg/kg of dry soil). About organic pesticides, a high molecules diversity at different level of concentration were found depending on crops. The results analysis will allow us to show if (1) the copper contamination level play a role on the molecules degradation and contamination level, (2) the soils physical and chemical parameters play a role on the molecules degradation and on the copper and molecules retention and (3) the past and current soils use impact the contamination levels.


Hélène Fargeon

Nicolas Martin-Stpaul

François Pimont

Miquel De Cáceres

Julien Ruffault

Thomas Opitz

Denis Allard

Jean-Luc Dupuy

Assessing the increase in wildfire occurrence with climate change and the uncertainties associated with this projection

Increasing temperature and drought duration/intensity in the Mediterranean area o Expected increase in fire activity, as highlighted by projections of fire danger indices

Introduction: an expected increase in fire danger with climate change

From [START_REF] Bedia | Forest fire danger projections in the Mediterranean using ENSEMBLES regional climate change scenarios[END_REF]

Uncertainties

• What are the differences between climate models?

• What are the differences between climate scenarios?

• How strong are these differences compared to current variations of climate?

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Hélène FARGEON / 8th ICFFR

  What will an increase in fire danger imply in terms of fire activity? • Are spatial patterns of FWI representative of current fire activity? • Is FWI a correct metric to predict fire activity

Hélène FARGEON / 8th ICFFR 12/11/2018

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Application

In the revised INRA feeding system for ruminants (INRA, 2018), it is now possible to take into account bodyweight variation for rationing beef cows.

Introduction Maintenance requirements of beef cows account for at least 70% of total energy requirements (Ferrell and Jenkins, 1984), compared to 40 to 50% for dairy cows. Energy for maintenance corresponds to the energy required to ensure the integrity of life functions, to maintain muscle tone, renal excretion, secretion of enzymes and hormones. Traditionally, maintenance requirements were defined as the minimum energy required for body weight gain or body energy balance equal to zero, but this is difficult to estimate directly. Estimates reported in literature are highly variable (91 to 180 kcal/kg 0.75 , Ortigues et al., 1993), due to the measurement methodologies used and factors linked to the animal (breed, level of production, body composition) and its environment (especially the feeding level). In the context of improvement of efficiency and robustness of beef production, a better knowledge of energy use, and in particular the partition of energy intake and its variation to physiological expenditure components, is needed.

Material and methods

Beef cows were submitted to a nutritional challenge in order to measure their ability to cope with nutritional changes through the partition of available energy between productive and non-productive (NP) functions. From a systemic approach, the cow is considered as a dynamic system that takes up energy from the environment to maintain its function over a productive cycle. Changes in energy partition are known to be modulated by the stage of lactation and other various metabolic pathways (e.g. protein accretion and mobilisation or/and lipogenesis/lipolysis). The energy balance can be estimated over the duration of the nutritional challenge and summarises the partition of energy fluxes. Energy changes expressed in net energy for lactation (UFL; INRA, 2018) is: UFLreq = UFLreq_MY + UFLreq_ΔBR + UFLreq_gest + UFLreq_NP, where UFLreq is the total net energy requirements, UFLreq_MY is the energy requirements for milk production, UFLreq_ΔBR is the net energy equivalent to body reserves changes, UFLreq_gest is the energy requirements for pregnancy and UFLreq_NP is the net energy required to NP functions; all the terms are expressed in UFL/day. By definition, when body weight (BW) change is null, ΔBR is null and UFLreq_NP corresponds to maintenance requirements. The relative importance of UFLreq_NP was determined by quantitative analysis of the INRA "Beef Cows" database. It included 33 INRA challenge trials (from 1975 to 2014) on late-maturing 'continental' beef cows with simultaneous measurements of diet intake, BW, BW changes corrected for variations in DMI, body reserves changes (ΔBR) assessed by adipose cell measurements, milk yield assessed by the weigh-suckle-weigh method. Within this database, the energy equivalents were expressed in UFL (INRA, 2018) using the metabolic efficiency coefficients (kpf, kg and kls for protein and fat deposition, gestation and lactation, respectively). Feed (ingredient) intake was also expressed in UFL calculated according to INRA (2018). The database included 101 treatments differentiated by the production stage and the parity of cows: 54 treatments related to lactating multiparous cows, 26 to lactating primiparous cows, and 21 to dry or pregnant multiparous cows. Special attention was paid to the energy of NP functions linked to the lipid and protein composition of BW change.

Results

From the quantitative analysis of the database, it was shown that: i) energy intake or mobilized from BR was mainly allocated to milk production whatever the level of feeding, ii) net energy equivalent of BW change was on average 2.4 UFL/kg BW (38 % lipids) in multiparous cows and 1.8 UFL/kg BW (21 % lipids) in primiparous cows, iii) NP functions can be expressed as the sum of maintenance requirements and energy to support BR changes. The net energy required for maintenance amounted to 0.043 and 0.049 UFL/kg BW 0.75 /d in dry pregnant and in lactating cows, respectively. These values must be increased by 10 and 20 % in free-stalls and pasture environments, respectively.

Conclusion

With this work, we have upgraded the beef cows' energy requirements for the revised feeding system, using a larger number of observed data. The improved knowledge about chemical composition of body reserves changes help to provide a more detailed and original model of energy partitioning. Although the model was derived using late-maturing 'continental' breeds, this approach can be used generically and adapted to other breeds.

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Patrice Peyre
email: patrice.peyre@ensam.eu

Laser Shock Processing on Metal

Introduction and Scope

Since its invention in the late 1960s, and the pioneering work on metal strengthening in USA during the late 1970s, laser shock processing (LSP) has become a reliable surface treatment for improving the mechanical or corrosion resistance of metallic materials. Moreover, laser-induced shock waves can also be envisaged for the investigation of dense matter's behavior-including phase transformations-under ultra-high strain rate loading (up to 107 s -1 ) using dedicated diagnostics (VISAR, etc.).

This Special Issue on LSP aims at providing a rather exhaustive and up-to-date state of the art on LSP based upon the most recent research works. The following fields are covered in the seven selected papers: materials' behavior and phase transformations under high strain rate (Amadou et al. [START_REF] Amadou | Laser-Driven Ramp Compression to Investigate and Model Dynamic Response of Iron at High Strain Rates[END_REF]), new loading conditions with ultra-short pulses (Petronic et al. [START_REF] Petronic | Picosecond laser shock peening of Nimonic 263 at 1064 nm and 532 nm wavelength[END_REF]) surface modifications induced by laser peening including recrystallization effects (Zhou et al. [START_REF] Zhou | Nanocrystallization effects in the Duplex Ti-6Al-4V Alloy processed by multiple laser shock peening[END_REF]) and warm laser peening (Huang et al. [START_REF] Huang | Residual Stress Distribution and Microstructure Evolution of AA 6061-T6 Treated by Warm Laser Peening[END_REF], Chen et al. [START_REF] Chen | Effects of Warm Laser Peening on Thermal Stability and High Temperature Mechanical Properties of A356 Alloy[END_REF]), and novel applications of LSP such as water droplet erosion resistance (Gujba et al. [START_REF] Gujba | Water droplet Erosion of laser-shock peened Ti-6Al-4V[END_REF]) or impact spot welding (Liu et al. [START_REF] Liu | Investigation of a novel laser impact spot welding[END_REF]). The wide variety of topics related to LSP highlights the extraordinary dynamism and enthusiasm of the growing international LSP community.

Contributions

Seven papers have been published in this special issue of Metals on Laser Shock Processing. They can be roughly separated in four distinct topics which are presented here below.

LSP-Induced Nano-Recrystallization Effects

The ability of laser-induced shock waves to generate nano-recrystallization effects was mostly underestimated in the past decades. An example is shown here by Zhou et al. [START_REF] Zhou | Nanocrystallization effects in the Duplex Ti-6Al-4V Alloy processed by multiple laser shock peening[END_REF] of microstructural modifications at the nanoscale range obtained on a Ti-6Al-4V (α + β) titanium alloy. Using the Scherrer-Wilson method for analyzing X-ray diffraction peak broadening, microstrains and average grain sizes of laser-peened diffracting zones were estimated and subjected to TEM microstructural observations. Above three LSP impacts, a minimum grain size of 100 nm was assumed (compared with 1-2 µm before LSP), which was kept rather constant with subsequent loading steps. An interesting input of TEM was to show that multiple laser impacts tend to promote more homogeneous nano-grain distributions, and that α and β phases exhibit specific plastic deformation modes (regular dislocation lines and mechanical twins for α and dense and tangled dislocations on β).

Warm Laser Peening Applied to Aluminum Alloys

In its usual form, laser peening is carried out at room temperature. However, recent developments have shown a possible benefit of performing LSP treatments at elevated temperature, mostly to combine the benefits of laser peening and dynamic strain aging. Two papers have been selected to put a focus on this advanced surface strengthening technique. They provide interesting insights into the beneficial effects of temperature on laser-shock-induced plastic deformations. Considering LSP temperature ranges up to 160 • C and a 6061 (Al0.9Mg0.6Si) alloy, warm laser peening (WLP) trials were carried out with 2 mm diameter impacts, a 50% overlap, and a glass confining medium [START_REF] Zhou | Nanocrystallization effects in the Duplex Ti-6Al-4V Alloy processed by multiple laser shock peening[END_REF]. Such WLP trials evidence a 20% decrease of surface compressive stresses, but deeper affected layers and large hardening effect (up to +55% HV for a WLP at 130 • C). Microstructural TEM and optical microscopy (OM) analysis reveal a higher dislocation density and more pronounced grain refinement when LSP is performed at elevated temperature. Many dislocations tangled to strengthening precipitates are also shown, which are assumed by Huang et al. [START_REF] Huang | Residual Stress Distribution and Microstructure Evolution of AA 6061-T6 Treated by Warm Laser Peening[END_REF] to be beneficial for improving residual stress stability. This last aspect was confirmed for a similar WLP procedure by Chen et al. [START_REF] Chen | Effects of Warm Laser Peening on Thermal Stability and High Temperature Mechanical Properties of A356 Alloy[END_REF], considering an A356 (Al7Si0.3Mg) cast aluminum alloy. After a 220 • C-100 min thermal treatment, WLP samples exhibited more stable residual stress fields than room temperature peening. Due to the enhanced dynamic strain aging and grain refinement effect, hardness levels were also found to be systematically higher and more stable than after conventional laser peening.

New Applications and Loading Conditions for Laser-Shock Processing

Novel potential applications have recently emerged either for laser peening [START_REF] Gujba | Water droplet Erosion of laser-shock peened Ti-6Al-4V[END_REF], or more broadly, for laser-induced shock waves [START_REF] Liu | Investigation of a novel laser impact spot welding[END_REF]. Besides, new LSP loading conditions like those using ultra-short pulses in the picosecond [START_REF] Petronic | Picosecond laser shock peening of Nimonic 263 at 1064 nm and 532 nm wavelength[END_REF] or femtosecond range have appeared.

The mitigation of water droplet erosion (WDE) with laser peening was addressed by Gujba et al. [START_REF] Gujba | Water droplet Erosion of laser-shock peened Ti-6Al-4V[END_REF] using the MIC-Lawrence Livermore laser facility, delivering squared laser impacts. The main application envisaged is the leading edge of Ti-6Al-4V compressor blades in gas turbines. Considering cumulative mass loss during water erosion for impact speeds of 150 to 350 m/s, the comparison between laser peened and as-machined samples (T-shaped flat or Airfoil-like) does not indicate a beneficial effect of LSP. This is mostly attributed to the limited work-hardening imparted by LSP, in spite of deep and intense residual stresses. Indirectly, such a research work provides interesting insight into the physical mechanisms involved in WDE, which are not directly controlled by fatigue behavior.

Apart from the well-known strengthening effect of laser peening, laser-induced shock waves can also be envisaged for generating spot-welding, especially when considering dissimilar alloys such as titanium and copper. The original work by Liu et al. [START_REF] Liu | Investigation of a novel laser impact spot welding[END_REF] demonstrates the feasibility of Ti-Cu joining by laser induced spot welding, through the dynamic collision between a thin laser-accelerated titanium flyer plate and a copper substrate, separated by an optimized stand-off distance. An interesting point to notice is the need of jetting formation behind the flyer plate to provide a cleaned impact surface by removing contaminants and ensuring optimum contact between Ti and Cu. With optimized LSP conditions, wavy interfaces can be obtained, similar to those shown in conventional explosive welding. Such interfaces are shown to favor anchoring effects and improve bond strengths.

Even though the use of very short pulses is not common in the field of LSP applications, Petronic et al. [START_REF] Petronic | Picosecond laser shock peening of Nimonic 263 at 1064 nm and 532 nm wavelength[END_REF] shows that 170 ps pulses carried out with a large number of local impacts (up to 200) can generate interesting microstructural modifications and resulting hardness levels on a Ni-based superalloy. Even if some questions remain unanswered about the precise thermal + mechanical loading submitted to surfaces, this work opens a new range of investigations on new ranges of materials' dynamic loading.

Phase Transformation under Severe Laser-Shock Loading

Following earlier works, the kinetics of phase transformation on iron was investigated by Amadou et al. [START_REF] Amadou | Laser-Driven Ramp Compression to Investigate and Model Dynamic Response of Iron at High Strain Rates[END_REF] under laser-induced ultra-high strain rate, using a dedicated beam smoothing system to deliver a uniform spatial loading. Using Doppler velocimetry diagnostics and severe loading conditions (several tenths of GPa, dε/dt ≈ 10 7 s -1 ), useful data have been measured such as elastic-plastic transitions or α-ε phase transformation in terms of shock pressure thresholds. One of the most interesting inputs of this paper is the integration of real physical deformation mechanisms such as twinning or dislocation slip into constitutive laws to provide a predictive modelling of shock propagation and phase transformation kinetics. Such an approach appears to be particularly important because there is still a lack a reliable and physically consistent constitutive equations to model real-time behavior of laser-impacted materials and resulting residual effects.

Conclusions

In the seven original research papers presented in this special issue on LSP, the reader will benefit from a representative overview on current LSP works, which demonstrate the versatility and capability of laser-induced shock waves to reinforce or probe the dense matter. Various pure metals (Fe) or alloys (C263 Ni-base, A356 and 6061 aluminum alloys, Ti-6Al-4V, Cu) have been considered, thus confirming the wide range of possible applications for LSP. Moreover, as shown by at least three papers presented in this review, many questions remain, especially concerning microstructural modifications induced by laser peening surface treatment, especially including dislocations' sub-structure and their relation to hardening levels or residual stress distribution. It is sure that these seven papers will contribute to the dissemination of novel ideas and concepts related to LSP.

Conflicts of Interest:

The author declares no conflict of interest.


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Effect of lipid supplements on milk fat yield and composition: a direct comparison between cows and goats responses

Hélène Fougere, Carole Delavaud, Laurence Bernard

To cite this version:

Hélène Fougere, Carole Delavaud, Laurence Bernard. Effect of lipid supplements on milk fat yield and composition: a direct comparison between cows and goats responses. Lipid fraction is a major determinant of the nutritional quality of milk and efficiency of production that could be modulated by nutritional factors such as lipid supplementation.

However, previous data mainly from indirect comparison among species showed that cows and goats differ in their susceptibility to diet-induced milk fat depression (MFD).

The objective of this study is to characterize, in cows and goats, the effects on milk fat plasticity of 3 types of diets chosen to induce a depression or an augmentation of milk fat content in cows and for which the effects in goats are absent or unknown. Twelve Holstein cows and 12 Alpine goats were fed diets containing forage and concentrate (45:55) (CTL) or the same diet supplemented with corn oil and wheat starch (COS), or marine algae powder (MAP) or hydrogenated palm oil (HPO) according to a 4X4 latin square design with 28-d experimental periods. Milk yield was recorded and milk samples were collected over 4 consecutive milking on d21 and d22 of each experimental period. Data were subjected to ANOVA for a 4X4 latin square design using the Mixed procedure of SAS, with statistical model including period, species, experimental diet and interaction between species and diets, as fixed effects.

Dietary treatments had no significant effects (P>0.05) on milk yield in cows and goats. In cows, milk fat content decreased with COS and MAP (-45% and -22%, respectively; P<0.001) and increased with HPO (+12%, P<0.001) compared with CTL. In goats only MAP decreased (-15%, P< 0.001) milk fat content. In cows, percentages variation in milk fat yield were of similar magnitude to that for milk fat content with a decrease for COS and MAP (-50% and -26%, respectively; P<0.001) and an increase for HPO (+9%; P<0.001). Conversely, in goats no significant effect (P>0.05) of dietary treatments was observed on milk fat yield.

These results provide evidence of species differences in the response to diets that induced MFD (high-starch diets containing plant oils or addition of marine algae) and milk fat augmentation (hydrogenated palm oil) in cows. This nutritional study which led to contrasted milk fat plasticity responses offers an original model that will be used to decipher the mechanisms underlying the specificities of the lipid metabolism among ruminant species.

02734466 Effect of Lipid Supplements on Milk Fat Yield and Composition: A Direct Comparison between Cows and Goats Responses

	H. Fougère, C. Delavaud, L. Bernard
	UMR1213 Herbivores, INRA, Vetagro Sup, F-63122, St-Genès-Champanelle, France.

15. Euro Fed Lipid Congress, 'Oil, fats and lipids: New Technologies and Applications for a Healthier Life', Aug 2017, Uppsala, Sweden, 27-30 august 2017, p. 87, Sweden. 1 p. hal-


Clémence Fraslin

Christos Palaiokostas

Nicolas Dechamp

Maria Bernard

Tatiana Rochat

Jean Francois Bernardet

Ross Houston

Edwige Quillet

Resistance to Flavobacterium psychrophilum in rainbow trout : QTL detection reveals effect of infection route and epistatic interactions

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

RESISTANCE TO

Bacterial cold-water disease, caused by Flavobacterium psychrophilum (Fp) is one of the most important diseases affecting rainbow trout (Oncorhynchus mykiss). Previous studies that used injection as route of infection have revealed that resistance to Fp is controlled by several QTL. However, skin and mucus are recognized as important components of fish defense against infections. Immersion challenge is expected to be closer to natural infection by Fp, and may reveal new defense mechanisms. The objective of this study was to identify and compare QTL associated with resistance to Fp using the two routes of infection.

Three experimental families, one F2 progeny and two gynogenetic doubled haploid, were produced in RE-SIST and FISHBOOST projects (FUI 15 French and FP7 European) from 6 doubled haploid grand-parents belonging to 6 isogenic lines with opposite resistance to Fp (3 resistant, 3 susceptible). Around 300 to 600 fish/family were individually tagged and challenged using either intramuscular injection or immersion (survival rate from 7% to 50% for injection; from 30% to 80% for immersion). Fish were genotyped with 1,200 to 9,600 markers using the double-digest-RAD or RAD-sequencing methodology. QTL for different resistance traits (i.e. survival, tolerance or endurance) were detected separately for each family and each infection route, using linkage analysis and interval mapping method.

Several chromosome-and genome-wide significant QTL (P<0.01 and P<0.05, respectively) associated with resistance to Fp were detected. Most of them were family specific but the most significant were detected in several families. Many QTL were detected after either immersion or injection. Only a few were found after both infectious challenges, suggesting that different defenses mechanisms may be triggered depending on the infection route. Interestingly, epistatic interactions among resistance QTL were evidenced, highlighting the complexity of the underlying immune pathway.

This study confirms the complex genetic architecture of trout resistance to Fp and demonstrates that different defense mechanisms can be modulated by the infection protocol. Some QTL with large effect were identified consistently across different genetic backgrounds and infectious protocols, paving the way for a better understanding of host defense mechanisms and further implantation of selective breeding for resistance to Fp. BCWD, QTL, Resistance, Route of infection, Epistasis § Corresponding author: Tel.: + 33 1 34 65 24 36; E-mail address: clemence.fraslin@inra.fr


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MIXED of SAS (SAS Institute Inc., Cary, NC), and effects of diet, species, and their interaction were determined. Whatever the species, and compared to Ctrl, FO increased plasma CE (+31%, P<0.001), Chol (+33%, P< 0.001) and PL (+22%, P<0.05), whereas SOS increased plasma NEFA (+55%, P<0.05) and Chol (+23%, P<0.001). Irrespective of dietary treatments, plasma lipid content was higher (+39%, P<0.001) in cows than in goats, with more Chol (+86%, P<0.001), CE (+43%, P<0.001) and PL (+44%, P<0.001), but less TG (-68%, P<0.001). In conclusions, we show for the first time differences in plasma lipid composition among goats and cows fed similar diets supplemented or not with lipids. These data will contribute to better understand the differences in mammary lipogenic response to MFD diets observed in cows and goats.

  Lipid classes content and composition in plasma of dairy goats and cows fed similar diets supplemented or not with lipids Hélène Fougere, Laurence Bernard, Yves Chilliard, Pablo Toral, Vincent Dietz, Carole Delavaud To cite this version: Hélène Fougere, Laurence Bernard, Yves Chilliard, Pablo Toral, Vincent Dietz, et al.. Lipid classes content and composition in plasma of dairy goats and cows fed similar diets supplemented or not with lipids. 15. Euro Fed Lipid Congress, 'Oil, Fats and Lipids: New Technologies and Applications for a Healthier Life', Aug 2017, Uppsala, Sweden. 1 p. hal-02734579 Lipid classes content in plasma of dairy goats and cows fed similar diets supplemented or not with lipids H. Fougère 1 , L. Bernard 1 , Y. Chilliard 1 , P. Toral 2 , V. Dietz 1 , C. Delavaud 1 1 UMR1213 Herbivores, INRA, Vetagro Sup, F-63122, St-Genès-Champanelle, France, 2 Instituto de Granaderia de Montaña (CSIC-ULE), 24346 Grulleros, Leõn, Spain The objective of this work was to determine plasma lipid content and composition on dairy cows and goats fed diets that induced milk fat depression (MFD) in cows, but not, or to a lesser extent, in goats. Twelve cows and 14 goats, at a similar lactation stage, were assigned to a 3x3 latin square design, and successively fed a basal diet without addition of lipids (Ctrl), or supplemented with sunflower oil plus wheat starch (SOS), or fish oil (FO). Plasma lipids were extracted by modified Folch procedure from individual blood collected before morning feeding at the end of each nutritional 26-d period. Lipid classes were separated by HP-TLC (High Performance-Thin Layer Chromatography, Camag, Switzerland). After derivatization and heating, the plates were analyzed by densitometry (VideoScan, Camag, Switzerland). In order to quantify the major lipid classes, 5 standard curves with increasing quantities of pure C17:0-phospholipid (PL), Cholesterol (Chol), C23:0-fatty acid (NEFA), cis9-C18:1-triglyceride (TG) and C15:0cholesteryl ester (CE), were deposited on each plate with individual samples, and concentrations were expressed in µg/µL of lipid extract. Data were analysed with proc


Even though animals are from the same genetic line, farmers have to cope with variability both in sows and piglets. In sows, variability is observed in traits such as parity, prolificacy, appetite, body weight (BW) and back fat thickness (BF). For instance, at the beginning of gestation, variability in body condition among sows can be high due to parity and age. In addition, at a given age, variability in litter size, milk potential, and appetite results in different nutrient requirements and consequently in variability of changes in maternal body reserves. Variability in BF can be a problem as several studies have indicated that too high or too low BF values are to be avoided at farrowing as well as at weaning. In both cases, the longevity of the sow is impaired, and farmers are advised to manage the sows toward a target BF depending on the physiological stage, associated with an age-dependant BW, increasing with age up to mature BW. In addition, variation in sow's body condition at farrowing and in prolificacy influences the new born and weaning piglet traits. Compared to less prolific sows, high-prolific sows farrow more piglets, which are both lighter on average and more heterogeneous. Compared to normal birthweight piglets, the survival rate of low birthweight piglets is lower. Providing additional care around birth helps these piglets to survive, but subsequent housing and feeding management have to be adapted to deal with the variability in their growth potential. Nutritional strategies (based on modelling approaches that take into account criteria that influence requirements) are suggested to optimise the expression of the animals' potential, but most often without an intention to reduce inter-individual variability in growth performance. In order to control or reduce variability, other solutions have been evaluated in experimental studies that focus on the level and the dynamic of the feeding plan and the quality of the diet. The challenge is now to validate these solutions in production units, which will be more or less easy depending on the existing housing and feeding systems, and the economic, welfare and environmental context.

Genetic bases of individual performance variation in pigs

L. Bodin 1 , N. Formoso-Rafferty 2 and H. Gilbert 1 1 INRA, GenPhySE, 31326 Castanet-Tolosan, France, 2 Universidad Complutense de Madrid, Fac. de Veterinaria, Dept. of Animal Production, Madrid, Spain; loys.bodin@inra.fr Part of the variability of performances in animal production is due to the genetic variation in the level of a trait between animals; that is the keystone of all genetic improvement programs and has to be maintained to guarantee the long-term sustainability of the selection progress. Another part of the performance variability comes from the individual variation of responses to various environmental conditions, called environmental sensitivity, which can have a highly negative impact on the final profitability of the production. Joint estimations of breeding values (EBV) and genetic parameters of the individual environmental sensitivity of a trait can be obtained along with those of the level of the trait. Moreover, use of these two kinds of EBV in genetic programs partly depends on their correlations as well as their genetic relationships with other traits. In pigs, homogeneity of litter size is desired and in many cases its mean level should not decrease. Similar relationships between objectives exist for the birth weight components (mean and variability); homogeneity is desired to ensure good perinatal viability and subsequent growth, but the mean birth weight should also not decrease. The genetic parameters and relationships between components (mean and variability) of birth weight and litter size in pigs will be presented along with similar parameters estimated in a mice experiment. Furthermore, similar estimations were obtained for production traits, such as residual feed intake and growth, which will be discussed in the perspective of producing more efficient and robust animals.


Folkert L Kuipers

Jan Freark De Boer

Bart Staels

Title Microbiome modulation of host's adaptive immunity through bile acid modification

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

Text

The intestinal tract is populated by a diverse, dynamic and metabolically-active microbial flora, the microbiome. It is well-established that an imbalanced microbiome ("dysbiosis") contributes to onset and progression of diseases of the host, including cardiovascular disorders and inflammatory bowel disease (IBD), i.e., disorders associated with inflammation. Indeed, the microbiome modulates the immune system of the host through complex interactions in which metabolites produced by bacteria signal to intestinal epithelial cells and immune cells. A broad spectrum of metabolites is formed from diet components, such as short-chain fatty acids (SCFA), synthesized directly by bacteria, or produced by the host and modified by bacteria [START_REF] Postler | Understanding the Holobiont: How Microbial Metabolites Affect Human Health and Shape the Immune System[END_REF] . Secondary bile acids (BA) belong to the last group. BA are increasingly recognized as important signaling molecules impacting on glucose, lipid and energy metabolism. BA exert these actions through activation of membrane-bound (G proteincoupled BA receptor 1 (GPBAR1/TGR5)) and nuclear receptors (Farnesoid-X-Receptor (FXR) and Vitamin D Receptor (VDR)) [START_REF] De Boer | New insights in the multiple roles of bile acids and their signaling pathways in metabolic control[END_REF] . BA activation of TGR5, FXR and VDR has been implicated in shaping innate immune responses [START_REF] Schubert | Interactions between bile salts, gut microbiota, and hepatic innate immunity[END_REF] . Song et al. [START_REF] Song | Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis[END_REF] now report a role of BA and VDR in the adaptive immune system, particularly in the modulation of a specific population of FOXP3 + regulatory T (T reg ) cells expressing the transcription factor RORγ (RORγ + T reg ) that are present in the colonic lamina propria.

The circulating BA pool consists of a mixture of primary and secondary BA with varying physicochemical characteristics and signaling functions 2 . Primary BA are synthesized from cholesterol in the liver and conjugated to either taurine of glycine prior to secretion into bile. After a meal, BA are expelled into the small intestine to facilitate fat absorption. BA are efficiently maintained within the enterohepatic circulation by ileal and hepatic transporter systems. A small fraction of BA escapes ileal absorption and enters the colon to interact with the microbiome. Bacteria alter BA in a sequential process that is initiated by deconjugation and followed by epimerization, dehydroxylation and/or oxidation of -OH groups, generating secondary BA. A part of these modified BA is reabsorbed from the colon, enabling them to exert signaling functions in the host. In general, more hydrophobic secondary species show greatest potency for FXR and VDR activation [START_REF] Makishima | Vitamin D receptor as an intestinal bile acid sensor[END_REF] . Important differences in BA metabolism exist between mice and humans, e.g., mouse-specific C6 hydroxylation reactions that generate hydrophilic muricholic acids 2 , which caution against direct translation of results obtained in mice to the human situation.

Song and colleagues [START_REF] Song | Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis[END_REF] searched for the mediators of diet-dependent induction of a distinct population of FOXP3 + T reg cells which express RORγ, RORγ + T reg cells, that are critical in maintaining immune homeostasis in the colon. Specified pathogen-free (SPF) mice fed a "rich", fiber and cholesterolcontaining diet from weaning onwards showed higher levels of these cells than mice fed a "minimal" diet without cholesterol or germ-free mice fed the rich diet, implying a role of the microbiome. After exclusion of SCFA as potential mediators, the authors showed that concentrations of both primary and secondary BA in the colon were higher in mice fed rich as compared to minimal diets, possibly related to induction of hepatic BA synthesis by dietary cholesterol [START_REF] Jolley | Induction of bile acid synthesis by cholesterol and cholestyramine feeding is unimpaired in mice deficient in apolipoprotein AI[END_REF] . Germ-free mice showed even higher colonic BA concentrations, but solely conjugated primary BA, indicating that bacterial BA transformations are essential for induction of RORγ +-T reg cells. Transfer of BA-metabolizing microbiota from minimal diet-fed mice to rich diet-fed germ-free mice restored the RORγ + T reg cell population. To directly assess the role of BA, individual and mixtures of unconjugated primary and secondary BA were added to drinking water of minimal diet-fed mice for 28 days. Only certain mixtures of primary and secondary BA were found to regulate RORγ + T reg cell abundancy. However, interpretation of these results is difficult since very high concentrations of BA were added to drinking water, above the reported maximal solubility of several species used [START_REF] Hofmann | Bile acid solubility and precipitation in vitro and in vivo: The role of conjugation, pH, and Ca2+ ions[END_REF] , corresponding to an estimated daily BA intake exceeding the size of the endogenous BA pool by a factor 3 [START_REF] Kok | Enterohepatic Circulation of Bile Salts in Farnesoid X Receptor-deficient Mice[END_REF] . Unfortunately, colonic concentrations of administered BA and their metabolites were not reported and comparison to levels found in rich diet-fed mice is not possible. Yet, bacterial species capable of inducing RORγ + T reg cells lost this ability after genetic deletion of BA hydrolase, the enzyme responsible for BA deconjugation, the first and essential step in secondary BA formation. Studies employing several BA receptor knock-out mouse models revealed that BA-activated VDR mediates RORγ + T reg cell induction. Indeed, VDR is expressed at relatively high levels in the RORγ + T reg population. Studies in T reg -specific VDR knock-out mice supported the role of VDR in programming of colonic RORγ + T reg cells. The pathophysiological relevance of the BA-VDR signaling axis in these cells was demonstrated by showing that mice fed the minimal diet were more vulnerable to dextran sodium sulfate (DDS)-induced colitis than mice fed the rich diet and that BA supplementation tended to normalize RORγ + T reg cell numbers and alleviated colitis. Lack of VDR in T regs worsened DDS-induced colitis. Importantly, BA supplementation did not cure established colitis but prevented its development.

This interesting work leaves us with a number of questions. The identity of the components that functionally differentiate the rich from the minimal diet as well as the BA metabolites responsible for activation of VDR in this system remain unknown. Makishima et al. [START_REF] Makishima | Vitamin D receptor as an intestinal bile acid sensor[END_REF] demonstrated that LCA and 3oxo-LCA are the most potent BA activators of VDR, while primary BA appear inactive. This delineates the likelihood that primary BA upon their supplementation require conversion into LCA. Indeed, conversion to LCA has been reported to be critical for the protective effects of ursodeoxycholic acid (UDCA) in DSS-induced colitis [START_REF] Ward | Ursodeoxycholic acid and lithocholic acid exert antiinflammatory actions in the colon[END_REF] . It also remains to be established by which mechanism(s) the colonic shift of the BA pool occurs and how activated VDR modulates the RORγ + T reg cell population.

This study, complemented by recent work 10 that identified two LCA metabolites as modulators of T h 17 and T reg differentiation, underscores the (patho)physiological importance of BA as signaling molecules, now also in regulation of adaptive immunity. It will be important to assess whether the existing large inter-individual variations in human BA pool size and composition translate into interindividual differences in adaptive immunity.

Acknowledgements

FK is supported by the Netherlands Heart Foundation (IN CONTROL, CVON2018-27) and the Noaber Foundation, Lunteren, The Netherlands, JFdB is supported by the Nutrition & Health initiative of the University of Groningen, BS is supported by grants from "European Genomic Institute for Diabetes" (E.G.I.D., ANR-10-LABX-46), European Commission and Agence Nationale pour la Recherche (ANR-FXREn) and holds a "European Research Council advanced Grant" (694717)