Nugue Denis Habauzit
email: denis.habauzit@univ-rennes1.fr

Guillaume Nugue

Frédéric Bourbon

Catherine Martin

Flavia Del Vecchio

Séverine Maunoir-Regimbal

Thomas Poyot

Marco Valente

Rachid Jaoui

David Crouzier

Yves Le Dréan

Jean Claude Debouzy

Dr Denis Habauzit Transcription

Evaluation of the effect of chronic 94 GHz exposure on gene expression in the skin of hairless rats in vivo

Millimeter waves (MMW) are broadband frequencies that have recently been used in several applications in wireless communications, medical devices, and nonlethal weapons (i.e., the nonlethal weapon called Active Denial Systems, ADS, operating at 94-95 GHz, CW). However, little information is available on their potential effects on humans. These radiofrequencies are absorbed and stopped by the first layer of the skin. Therefore, our study aimed to evaluate the effects of 94 GHz on the gene expression of skin cells. Two rat populations consisting of 17 young animals and 14 adult ones were subjected to chronic long-term 94 GHz MMW exposure. Each group of animals was divided into exposed and sham subgroups. The two independent exposure experiments were conducted for 5 months with rats exposed 3 h per day for 3 days per week to an incident power density of 10 mW/cm², which corresponded to twice the ICNIRP limit of occupational exposure for humans. At the end of the experiment, skin explants were taken, and RNA was extracted. Then, the modifications to the whole gene expression profile were analyzed with a gene expression microarray.

Without modification of the animal's temperature, long-term chronic 94 GHz-MMW exposure did not significantly modify the gene expression of the skin on either the young or adult rats.

Introduction

Radiofrequencies constitute a form of nonionizing radiation with a frequency range between 3 kHz and 300 GHz. These frequencies are now required for devices in common use, such as in cooking (induction table and microwave), telecommunications (mobile phone, Wi-Fi, radio, and radar), medicine (cancer radiofrequency ablation and MRI), and security (airport body scanner). Among these frequencies, millimeter waves (MMWs) are broadband frequencies between 30 and 300 GHz that are utilized in civilian or military applications, such as telecommunications, medical devices, security and weapon development. For example, nonlethal weapons such as the active denial system (ADS), which produces a strong heat sensation for people in the beam, operates at the 94 GHz band. The evolution of such military technologies involves the use of high output power, which increases the risk of inadvertent exposure of personnel. To date, very few studies have been published on the effects of such waves used in military applications on human health. At these frequencies, the energy deposition on biological tissues penetrates into the first millimeter of the body surface [START_REF] Zhadobov | Millimeter waves as a source of selective heating of skin[END_REF][START_REF] Zhadobov | Microscale temperature and SAR measurements in cell monolayer models exposed to millimeter waves[END_REF], which implies that the exposure mainly concerns the epidermal and cutaneous regions of the skin. Therefore, the skin is the primary target of interest for the study of the potential biological effects of MMWs. Experiments conducted on human volunteers showed that very short exposure (3 s) at high power 94 GHz (1.8 W/cm²) was sufficient to increase the temperature at the surface of the skin [START_REF] Walters | Heating and pain sensation produced in human skin by millimeter waves: comparison to a simple thermal model[END_REF]. These acute high-power exposures lead to a sensation of pain or, when prolonged, to burns. It was also found that prolonged exposure at 94 GHz with high power intensity (75 mW/cm²) induces such extreme peripheral heat on rats that it could induce cardiovascular failure [START_REF] Jauchem | Cardiovascular and thermal responses in rats during 94 GHz irradiation[END_REF][START_REF] Millenbaugh | Comparison of blood pressure and thermal responses in rats exposed to millimeter wave energy or environmental heat[END_REF]. It is important to note that these exposure conditions dramatically exceed the allowed limits for people. Reference levels for whole body exposure at millimeter frequencies are 5 mW/cm² for occupational exposure and 1 mW/cm² for the general public [START_REF]Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz). International Commission on Non-Ionizing Radiation Protection[END_REF].

To gain insight into the biological effects of MMWs, several studies examined changes in global gene expression caused by exposure. For instance, the Millenbaugh group observed the modification of the mRNA content in rat skin tissue after exposure to 35 GHz at 75 mW/cm² [START_REF] Millenbaugh | Gene expression changes in the skin of rats induced by prolonged 35 GHz millimeter-wave exposure[END_REF]. By using microarray analysis, these authors found 56 and 58 genes differentially expressed after 6 h and 24 h of exposure to MMWs, respectively. The majority of these genes were related to heat shock response, oxidative stress, inflammation, wound repair or tissue matrix recovery. A study carried out on human keratinocytes showed that MMW exposure at 20 mW/cm² led to an increase in temperature and to an important alteration of gene expression in vitro [START_REF] Habauzit | Transcriptome analysis reveals the contribution of thermal and the specific effects in cellular response to millimeter wave exposure[END_REF]. However, this modification of the whole gene expression profile was mainly associated with the thermal effect of MMWs. Therefore, when the temperature was artificially maintained constant by decreasing the incubator temperature, no additional change was observed in whole gene expression profile after A c c e p t e d M a n u s c r i p t MMW exposure compared to the sham (control) gene expression [START_REF] Habauzit | Transcriptome analysis reveals the contribution of thermal and the specific effects in cellular response to millimeter wave exposure[END_REF]. This study is in agreement with others that suggest that, if care is taken to avoid MMW-associated thermal effects, acute low-power exposures have no notable effect on gene expression [START_REF] Zhadobov | Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins[END_REF][START_REF] Quément | Impact of 60-GHz millimeter waves and corresponding heat effect on endoplasmic reticulum stress sensor gene expression[END_REF][START_REF] Quément | Wholegenome expression analysis in primary human keratinocyte cell cultures exposed to 60 GHz radiation[END_REF].

The purpose of the present study was to verify whether repeated and prolonged MMW exposure at low power can have an impact on gene expression in rat skin. Exposure was set to 94 GHz at an incident power density (IPD) of 10 mW/cm² to mimic the exposure of personnel handling ADS weapons.

Materials and methods

Animals

During this study, 2 exposure experiments were conducted on groups of 30 hairless male rats (CD Hairless rats, from Charles River Laboratories, Ecully, France). The rats were housed in pairs during the experiment and maintained in a temperature-controlled room (22°C) with an automatic 12 h light/dark cycle (06:00 18:00). Water and diet were given ad libitum. All animal procedures were approved by the Animal Care and Use Committee of the French Biomedical Research Institute (2019/13.0) and were in compliance with the guidelines of the European Union (directive 2010/63/EU and revisited Appendix A of European Treaty Series 123), implemented into French law (decree 2013-118) regulating animal experimentation. All efforts were made to reduce the number of animals used and animals were daily observed.

Exposure system, dosimetry and experimental setup

The 94 GHz generator (Figure 1A), which contains an EIK subsystem consisting of an EIK with 100 watt CW operation, a 94 GHz air-cooled EIK, an RF source with an integrated power supply, RF input and output components, and an integrated device cooling and protection system, was designed by the Communications & Power industries (CPI Europe Ltd., Walton-on-Thames, UK). This system was connected with a waveguide to a cornet antenna (Figure 1B) in an anechoic chamber in which the rats were exposed (Figure 1C). The temperature of the rats was monitored before and after exposure with a FLIR A320 IR camera (FLIR France, Issy-les-Moulineaux, France), and the temperature data were integrated with FLIR Quickreport software.

A c c e p t e d M a n u s c r i p t

The dosimetry (Figure 2) was realized using the 262 W cornet antenna connected with circular waveguides in the TE11 mode (Flange W387) at 94 GHz. The simulation was performed with CST MicroWaveStudio software (Figure 2) in the FDTD resolution mode. The optimal distance, considering a gain of 26 dBi, to obtain a homogeneous exposure field was calculated. The distance between the antenna and the rats was 71 cm, creating an area of 1 m with a homogeneous beam of 100 W/m² (equivalent to 10 mW/cm², which is twice the ICNIRP limit for occupational exposure).

Animal exposure and sample recovery

Two exposure experiments were conducted. In the first experiment (E1), adult CD hairless male rats (average weight 521 ± 44 g, 14 weeks old) were used, and in the second experiment (E2), young CD hairless male rats (6 weeks old rat) that weighed an average of 301 ± 11 g at the beginning of the experiment were used. This choice of two age groups results on the question of the potential differential action of electromagnetic field betwenn ages. Younger animal are usually considered more sensitive toward environmental insults. For each experiment, the animals were divided into two distinct groups. The first group corresponded to the sham (control) (15 rats), and the second group was composed of rats (n = 15) exposed to MMWs at an incident power density of 10 mW/cm² for 3 h per day and 3 days per week for 5 months. Three groups of 5 rats were exposed or sham exposed while in the anechoic chamber shown in Figure 1C. The temperature in the anechoic room was maintained with air conditioning at a constant 20 ± 1°C level, and the temperature of each exposed or sham-exposed rat was monitored by the IR camera at 2 time points. The rectal temperature of each rat was also monitored before and after each exposure. Every week, each rat was weighed. During E2, one animal in each group died during the experiment. At the end of the exposure series, the rats were sacrificed by decapitation, and then, skin explants were extracted and conserved in RNAlater (Thermofischer) for use in the analysis.

RNA extraction and microarray

Rat skin biopsy samples were stored at -80°C in RNAlater (Thermo Fisher Scientific, Saint Aubin, France). Between 15 and 20 mg of the biopsy sample was first cut into small fragments and then mixed with Qiazol solution (Invitrogen, Thermo Fisher Scientific, Saint Aubin, France), and then, it was crushed with a micro pestle and homogenized at 6300 rpm with ceramic beads in a Precellys apparatus (Bertin Technology, France) for 5 cycles of 23 s with 2 min on ice between each cycle. The total homogenate containing the skin extract and 1 ml of Qiazol was mixed with 300 µl of chloroform A c c e p t e d M a n u s c r i p t and then vortexed for 30 s and centrifuged for 15 min (14000 rpm, 4°C). Equal amounts of the aqueous phase were taken, mixed with 70% ethanol and transferred to a Qiagen column from an RNeasy lipid tissue kit (Qiagen, Hilden, Germany) for purification according to the manufacturer's instructions. Finally, the RNA was recovered and quantified on a Nanodrop 1000 spectrophotometer (Nanodrop Technology, Cambridge, UK), and the integrity of the RNA samples was assessed on a Bioanalyzer 2100 (Agilent Technologies, Les Ulis, France).

From the two sets of 30 animals exposed during E1 and E2, 15 and 17 animals were randomly selected, respectively, for the microarray experiments. The mRNA from the animals was analyzed using a one-color whole gene expression microarray with an Agilent SurePrint G3 Rat Gene Expression v2 8x60K microarray kit (Agilent Technologies, Les Ulis, France) according to the manufacturer's instructions. One sample in the E1 experiment was excluded due to low-quality feature extraction. Therefore, the data from the two experiments E1 (14 animals: 8 sham and 6 exposed) and E2 (17 animals: 8 sham and 9 exposed) were randomly analyzed with Genespring GX software (Agilent Technologies, Les Ulis, France). The fluorescence intensity of each microarray was log2-transformed, and the baseline was normalized by quantile. Finally, 30367 probes were detected on the microarrays. Each microarray was then filtered on the basis of the expression level (expression greater than 125). Finally, 15168 and 14853 unique probes were found for experiments 1 (E1) and 2 (E2), respectively. A total of 14721 probes were typically found for the two groups. The complete data set was deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo, GEO series accession numbers GSE132816, GSE132817 and GSE132818).

Statistics

The microarray analyses were performed with Genespring software (Agilent Technologies, Les Ulis, France). The probes were filtered by fold change (absolute fold change above 1.5) and then analyzed by two-tailed Mann-Whitney tests. The P-values were adjusted by controlling for the false discovery rate (FDR) with the Benjamini-Hochberg (BH) correction for multiple testing. Genes were considered differentially expressed if the adjusted P-value was below 0.05 with or without BH correction.

Results

A c c e p t e d M a n u s c r i p t

Effect of MMW exposure on animal weight

The MMW exposure did not affect the body weight of the adult or young rats (Figure 3). The weight of the adult animals (E1) remained constant throughout the experiment, regardless of whether or not the animals were exposed. The weight of the young animals (E2) increased steadily until reaching a plateau that corresponded to the normal adult weight. The differences between the sham and exposed rats were not significant.

Rat temperature before and after each exposure.

Since rats have been exposed to twice the level of the recommended limit for humans, thermal effects were possibilities. Therefore, during exposure, the temperature of each rat was monitored by an IR camera. Figure 4A clearly shows that exposure significantly increased the temperature of the skin surface. The IR camera measurements show an average increase of 1.0±0.5°C for the adult animals (E1) and 0.5±0.5°C for the young rats (E2). Nevertheless, this clear increase in body surface temperature was not found for internal temperature levels (Figure 4B). The rectal temperature was assessed for each rat before and after each exposure. According to these internal measurements, the difference in temperature after exposure was only +0.2±0.5°C for the first experiment (E1) and -0.2±0.3°C for the second experiment (E2) (Figure 4B). The internal temperature of the Sham control group is modified in the same magnitude as the Exposed one (data not shown). These measurements clearly indicate that physiological compensation phenomena maintained the constant internal temperature during exposure.

Effect of low-intensity long-term exposure on gene expression in the skin

After normalization and filtration of the expression levels, 15168 (E1) and 14853 (E2) probes were selected. More than 97% of the detected probes were common to both experiments (E1 and E2). The first quality control test was conducted to verify that the 1°C increase observed on the skin surface did not induce tissue damage or a heat shock response. Genes from the heat shock protein family (HSP) and genes known to be involved in skin inflammation and tissue repair [START_REF] Sass | Transcriptomic responses to wounding: meta-analysis of gene expression microarray data[END_REF] were extracted based on the microarray data, and their overall expression was assessed. As shown in Figures 5 and6, these genes were continuously expressed whether the animals had been exposed or not. Taken together, these observations suggest that the slight temperature increase due to exposure did not have an impact on the skin.

A c c e p t e d M a n u s c r i p t

Then, we analyzed the differentially expressed genes between the two groups of animals (Sham E1 vs Sham E2). We observed with a MW test and a BH correction on the 15 210 common gene entities that 336 genes were differentially expressed between this two groups (Data not shown). Considering that we have difference in gene expression in the two groups we therefore analyzed data in each subgroup independently by filtering the microarray data by fold changes from each experiment. Approximately 1% of the detected probes (156 probes for E1 and 120 probes for E2) had a fold change greater than 1.5 between the sham and exposed group (Table I). The Mann-Whitney test with Benjamini-Hochberg (BH) correction was performed on this list of probes, and in both cases, no genes were shown to be differentially expressed between the sham and exposed groups. By removing the BH correction, 14 and 4 genes from the E1 and E2, respectively, were found to be differentially expressed between the two groups. These genes are described in detail in Table II. However, in the absence of the BH correction, some false positives for genes might represent contaminants among the sensitive genes listed. Since there seems to be little likelihood that a false positive would be found twice in the two independent analyses, we cross-referenced the lists of genes obtained from the two exposure experiments. There was no common gene between the two lists. This finding suggests that we did not detect strict exposure-sensitive genes that should have been differentially expressed in the 2 experiments. Therefore, we can conclude that long-term lowpower 94 GHz exposure did not modify the gene expression in rat skin.

Discussion

Applications using high-power millimeter waves already exist, implying that people using these technologies may be inadvertently exposed. Several publications have studied some peculiar frequencies used in millimeter range such as at 30, 42, 60 GHz and now 94 GHz [START_REF] Quément | Wholegenome expression analysis in primary human keratinocyte cell cultures exposed to 60 GHz radiation[END_REF][START_REF] Alekseev | Millimeter wave effects on electrical responses of the sural nerve in vivo[END_REF][START_REF] Gapeyev | Features of anti-inflammatory effects of modulated extremely high-frequency electromagnetic radiation[END_REF][START_REF] Makar | Effect of millimeter waves on cyclophosphamide induced suppression of T cell functions[END_REF][START_REF] Makar | Effect of cyclophosphamide and 61.22 GHz millimeter waves on T-cell, B-cell, and macrophage functions[END_REF][START_REF] Samsonov | The effect of a 94 GHz electromagnetic field on neuronal microtubules[END_REF][START_REF] Mahamoud | Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome[END_REF][START_REF] Radzievsky | Electromagnetic millimeter wave induced hypoalgesia: frequency dependence and involvement of endogenous opioids[END_REF]. All of these frequencies may be studied as the main difference in the physical action on skin is the penetration depth. Indeed these frequencies did not permit to reach the same skin layer (dermis, epidermis). For instance, at 30 GHz the penetration depth is near 0.9 mm and at 94 GHz, it is near 0,37 mm (1). To gain inside knowledge about the safety of these applications, this research aimed to evaluate the long-term chronic exposure of 94 GHz millimeter waves. The treatment chosen was midrange exposure, i.e., for 3 h per day and 3 days a week at an IPD of 10 mW/cm². This condition A c c e p t e d M a n u s c r i p t corresponds to a worst case scenario of exposure for personnel handling ADS weapons because this IPD is twice the limit recommended by the ICNIRP for workers [START_REF]Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz). International Commission on Non-Ionizing Radiation Protection[END_REF]. Moreover, to characterize the potential effect of these frequencies better, two different rat populations (young and old) were chosen.

Considering that little information is available on the impact of these waves on animals and humans, data on general physiological effects were monitored throughout the 5-month exposure period. First, the exposure did not significantly modify the body weight of the rats. The IPD used was low enough that it did not induce tissue damage or heat shock response. During the exposure, the skin surface temperature increased by 1°C on average, but this increase had no or very little impact on the internal temperature. Moreover histologic approach did not evidence any difference in the skin morphology (data not shown).

Then, we analyzed the gene expression to verify whether chronic exposure could have an impact on gene expression. The gene expression signature reflects the cell identity in a particular environmental context. Since the MMWs do not penetrate deeply into the body, we used skin as a model. The whole gene expression profile as analyzed by microarray was not modified by the MMW treatment, and only a few genes were observed to be modified when the statistical correction was not included in the analysis. Two independent exposure experiments were conducted, and no common gene was found to be differentially expressed in either experiment. This finding emphasizes a lack of reproducibility, which is likely explained by the fact that, in the absence of statistical correction, false positives may have emerged. Despite this risk of false positive, we chose to present the genes identified with this uncorrected statistical test. This in order to help researcher who would like to develop a meta-analysis on experiments performed in microarray. Moreover, no correlation was observed between these uncorrected differentially expressed genes and genes identified in previously published microarray experiments either in animal models [START_REF] Millenbaugh | Gene expression changes in the skin of rats induced by prolonged 35 GHz millimeter-wave exposure[END_REF] or in cells [START_REF] Habauzit | Transcriptome analysis reveals the contribution of thermal and the specific effects in cellular response to millimeter wave exposure[END_REF][START_REF] Quément | Impact of 60-GHz millimeter waves and corresponding heat effect on endoplasmic reticulum stress sensor gene expression[END_REF][START_REF] Mahamoud | Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome[END_REF]. In parallel no modified genes were involved in membrane permeability, nor in protein involved in channels, although some recent MMW effects were found associated with cell membrane permeability in keratinocytes [START_REF] Pogam | Untargeted metabolomics unveil alterations of biomembranes permeability in human HaCaT keratinocytes upon 60 GHz millimeter-wave exposure[END_REF]. We can therefore assume that, under the conditions of our study, MMW exposure had no impact on gene expression in the skin. These results are in agreement with those studies listed in the bibliography that show that acute and low-power MMW exposure does not dramatically change the whole gene expression profile in the primary cultures of human keratinocytes [START_REF] Habauzit | Transcriptome analysis reveals the contribution of thermal and the specific effects in cellular response to millimeter wave exposure[END_REF][START_REF] Mahamoud | Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome[END_REF][START_REF] Quément | Wholegenome expression analysis in primary human keratinocyte cell cultures exposed to 60 GHz radiation[END_REF]. Nevertheless the gene expression modification is not the only possible effect in chronic action of the MMW. Indeed, no information of epigenetic modifications or of DNA adducts are available in the literature. Moreover if some transitory effect are induced during the treatment time we cannot see it as the manipulation was design for an end point recovery.

A c c e p t e d M a n u s c r i p t

In summary, our experiments did not reveal any deleterious effect on skin in vivo after chronic MMW exposure at power twice as high as that recommended by the ICNIRP for workers.

Since skin is the main target of millimeter waves, these results are important in the context of risk assessment. However, we must not lose sight of the fact that our study was focused on the skin and that we did not investigate the possibility of a systemic response that activates nerve endings or induces secretion of factors into the blood circulation [START_REF] Ziskin | Millimeter waves: acoustic and electromagnetic[END_REF][START_REF] Zhadobov | Evaluation of the Potential Biological Effects of the 60-GHz Millimeter Waves Upon Human Cells[END_REF].

Figure 1 :

 1 Figure 1: Exposure system: A) generator, B) anechoic chamber, C) horn antenna and exposed rats.

Figure 2 :Figure 3 :

 23 Figure 2: Dosimetry of the 94 GHz wave Figure 3: Animal weight during chronic exposure. The body weight of each animal was measured every week (n=31) in each group (sham and exposed). E1 = first exposure experiment on adult rats. E2 = second exposure experiment on young animals. The data are expressed as the mean of the values ± standard deviation.

Figure 4 :

 4 Figure 4: A) External temperature changes before exposure (initial T) and after exposure (final T) for the exposed groups of rats during the first (E1) and the second (E2) experiment. B) Internal temperature change before and after exposure in the exposed groups. The box plot presents a summary of the mean temperature of each animal throughout the chronic exposure period. The results are significant for P-value < 0.05 (*), p-value < 0.01 (**), or p-value < 0.001 (***) based on t-tests. NS = not significant.

Figure 5 :

 5 Figure 5: Expression of Heat Shock Protein (HSP). The data are expressed as the means ± standard deviation.

Figure 6 :

 6 Figure 6: Expression of selected genes involved in skin inflammation, wound repair and tissue remodeling. From Sass et al., 2017. (Data are expressed as the means ± standard deviation)

Figure 1 :

 1 Figure 1: Exposure system: A) generator, B) anechoic chamber, C) horn antenna and exposed rats.

  A c c e p t e d M a n u s c r i p t

Figure 2 :

 2 Figure 2: Dosimetry of the 94 GHz wave

Figure 3 :

 3 Figure 3: Animal weight during chronic exposure. The body weight of each animal was measured every week (n=31) in each group (sham and exposed). E1 = first exposure experiment on adult rats. E2 = second exposure experiment on young animals. The data are expressed as the mean of the values ± standard deviation.

Figure 4 :

 4 Figure 4: A) External temperature changes before exposure (initial T) and after exposure (final T) for the exposed groups of rats during the first (E1) and the second (E2) experiment. B) Internal temperature change before and after exposure in the exposed groups. The box plot presents a summary of the mean temperature of each animal throughout the chronic exposure period. The results are significant for P-value < 0.05 (*), p-value < 0.01 (**), or p-value < 0.001 (***) based on t-tests. NS = not significant.

A c c e

  p t e d M a n u s c r i p t

Figure 5 :

 5 Figure 5: Expression of Heat Shock Protein (HSP). The data are expressed as the means ± standard deviation.

  A c c e p t e d M a n u s c r i p t

Figure 6 :

 6 Figure 6: Expression of selected genes involved in skin inflammation, wound repair and tissue remodeling. From Sass et al., 2017. (Data are expressed as the means ± standard deviation) A c c e p t e d M a n u s c r i p t

Table Legends Table I : Microarray data on the two experiments of exposure. Table II: Differentially expressed genes obtained by Mann-Whitney test without BH correction.

 LegendsI

Table II : Differentially expressed genes obtained by Mann-Whitney test without BH correction.

 II

					E1
	Probe	P-	Regulation	FC	Gene	GenBank	Gene Name
	Name	value		(abs)	Symbol	Accession
	A_44_	0.010	Up	1.66	Fam86a	NM_	Family with sequence
	P142850					001106975	similarity 86, member A
	A_64_	0.039	Up	2.89	LOC102557336	XM_	Keratin-associated
	P105342					006223705	protein 5-4-like
	A_44_	0.039	Up	1.59	LOC291863	NM_	Carboxylesterase-like
	P555689					001013889
	A_64_	0.039	Up	1.52	Mest	NM_	Mesoderm specific
	P032683					001009617	transcript
	A_42_	0.039	Up	1.59	Myl4	NM_	Myosin, light chain 4
	P643349					001109495
	A_44_	0.028	Up	2.78	Olr1229	NM_	Olfactory receptor 1229
	P522461					001000444
	A_44_	0.039	Up	2.41	Vsig8	NM_	V-set and
	P522461					001105972	immunoglobulin domain
							containing 8
	A_64_	0.028	Up	2.47	CCTGGTAACTGTAGGGGCCTTTTTAGCTAGCAAGTGA
	P137704
	A_64_	0.039	Up	2.43	CTGCTGTGCCCTTGAGTGCTGCCAGTGCAAGATATGA
	P063913
	A_64_	0.028	Up	2.63	TGCTGCAAACCCTGCTGCTGTCAGTCCAGCTGCTGCA
	P095642
	A_44_	0.028	Up	2.66	TGTTGCAAGCCCTGCTGCTGTCAGTCCAGCTGTTGTA
	P636423
	A_64_	0.020	Down	1.51	Prpg1	NM_172064	Proline-rich
	P061524						proteoglycan 1
	A_64_	0.014	Down	1.56	RGD1564482	NR_036617	RGD1564482
	P096589
	A_64_	0.007	Down	1.51	Siglec10	NM_	Sialic acid binding Ig-like
	P117433					001106250	lectin 10
					E2

Probe Name P-value Regulation FC (abs) Gene Symbol GenBank Accession Gene Name

	M a n u s c r i p t
	A_64_ P108389 A_44_ P288015 A_64_ P086284 A_42_ P536702 A c c e p t e d 0.021 Up 1.73 0.003 Up 2.10 0.043 Up 2.30 0.016 Down 1.62	Plekhh1 Mrps10 Rptn Sftpc	NM_ 001108036 NM_ 001008859 XM_ 001054767 NM_017342	pleckstrin homology domain containing, family H (with MyTH4 domain) member 1 mitochondrial ribosomal protein S10 repetin surfactant protein C

Acknowledgments

We are most grateful to the Biosit Health Genomics core facility of Biogenouest Genomics for its technical support. The English was checked and verified by American Journal Expert.

Funding

This work was supported by the French National Program for Environmental and Occupational Health of Anses (EST-2016-2-RF-02) and by the Delegation Generale de l'Armement Grant (PEA 08CO601).


Ryad Bouzouidja

Maeva Sabre

David Ramier

Patrice Cannavo

Rémi Claverie

Rémi Claverie Green

Green roof set-up analysis: Monitoring and simulation of water and thermal transfer using HYDRUS-1D

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

• We conduct observations and simulations of temperature and hydraulic behaviour of green roof in Nantes city, France.

• Our work provide a better understanding of the coupled thermal and hydrological processes in green roof technology.

• Our simulations showed that the green roofs has an original behavior: (i) great variations in hot and cold thermal performance, (ii) a strong impact of water inside GR opposite the thermal behavior and (iii) the daily variability of the ET is well observed. The measurement with the evapotranspiration chamber also makes it possible to apprehend the seasonal variability. • Further simulation of actual evapotranspiration will be performed using Hydrus-1D, incorporating the impact of rock present in the soil using double porosity models. • Store and release water with a delay. Storage capacity = 40 to 80% of the total annual rainfall volume depending on the substrates properties (i.e. thickness, characteristics and proportion of its organic and mineral components) but also on type of plants and vegetation (Bengtsson et al., 2005).

Ryad

• Reduce urban air temperatures by 1.4°C on average 3.2°C at best, which may lead to a reduction in UHI amplitude (Jaffal et al., 2012).

But GR are also living systems that are submitted to a significant evolution with time capability to retain water and refresh the climate.

HYDRUS-1D

In this study the HYDRUS-1D model is used to simulate the thermal behavior and water movement in the GR. The model is capable of simulating the water movement in unsaturated, partly saturated or fully saturated porous media (Simunek et al., 2008).

ET EVALUATION

The ET calculated as residual of the water balance shows the total ET of 0.4 mm in 24 hours. Measured ET for the same period is comparable to WB.

INFLUENCE OF LAND SURFACE

• Regarding the variation of water content during time illustrates the variably temperature conditions inside the substrate. The model slightly overestimates (+6°C) the surface temperature and soil temperature at 6 cm depth (+0.5°C) during the warm period. During the cold period, the model underestimates the surface temperature during nighttime (-2°C) and perfectly simulates the temperature during daytime. • It suggest that the presence of water and modification of land surface affect the temperature and should be calculated more accurately.

EVOLUTION OF THERMAL BEHAVIOR OF GREEN ROOF

R 2 =0.89 R 2 =0.95 Simulated Observed VWC1 VWC2
The ET simulated by Hydrus is overestimated and higher than the measured ET (Evapotranspiration chamber). It suggest that the water content should be simulated more accurately.

RESOLUTION

The soil water content θ is modeled using the van Genuchten-Mualem (VGM) model (simple porosity model) (Mualem, 1976;van Genuchten, 1980) θ(h) = ൞ θ r + θ s -θ r 1 + αh n m , h < 0 θ s h ≥ 0 ET = R -Q out ± 𝑆

The water balance of GR can be written as below:

January 2012 July 2012

  surfaces poorly covered by vegetation → Increasing water runoff 55% in urban area vs 10% in natural area. Urban Heat Island (UHI) = Man's modification of the atmospheric environment → the temperature increase may reach up to 10°C. Green roofs (GR) = part of the solution for urban water and thermal management because they can:

•

  of GR water and thermal performances under different land covers: i) bare soil, ii) sedum album, iii) Festuca glauca and iv) Dianthus deltoides.SUMMARY*Contact: Ryad BOUZOUIDJA ryad.bouzouidja@gmail.comVARIATION OF HYDROLOGIC PERFORMANCES• The corresponding simulated data (using HYDRUS-1D simulation tool) and measured hydrographs for 401 l m -2 of cumulated rainfall (January to July 2012) show that the simulated soil water content value of the simulated water content is in quite good agreement with the experimental data • The figure shows that the model correctly calculates the internal state of the substrate. The water content increases over time during rainfall, and decreases during dry periods. Soil moisture,• Air and soil temperature,• Relative humidity,• Wind speed,• Global radiation,• Rainfall

  content [m 3 m -3 ]; h soil pressure head [m]; t time [hour]; z spatial coordinate [m]; positive downward; K(h) unsaturated hydraulic conductivity [m h -1 ]; S sink term, accounting for plant water; Cp and Cw volumetric heat capacity of the solid and liquid phases [J m -3 °C-1 ]; T temperature [°C]; λ(θ) heat conductivity [W m -1 °C-1 ]; q total water flux density [m s -1 ]; θ s and θ r saturated and residual water content [m 3 m -3 ]; K s saturated hydraulic conductivity [m h -1 ]; α [m-1], n[-] and [m] are adjustable parameters; ET evapotranspiration [mm]; R Rainfall [mm]; Q out drainage outflow [mm]. + Contact: Maeva SABRE Maeva.sabre@cstb.fr REFERENCE • Bengtsson, J., Ahnström, J., Weibull, A.C., 2005. The effects of organic agriculture on biodiversity and abundance: a meta-analysis. J. Appl. Ecol. 42, 261-269 • Jaffal, I., Ouldboukhitine, S., Belarbi, R., 2012. A comprehensive study of the impact of green roofs on building energy performance. Renewable Energy 43, 157-164. • Mualem Y. A new model for predicting the hydraulic conductivity of unsaturated porous media. Water • Resour. Res. (1976) 12:513-522. • Simunek J, Sejna M, Saito H, Sakai M, van Genuchten MT, 2008. The HYDRUS-1D software package for simulating the one-dimentional movement of water, heat, and multiple solutes in Variably-saturated Media: Department of environmental sciences university of California riverside, California. • van Genuchten MT. A Closed-form Equation for Predicting the Hydraulic Conductivity of Unsaturated • Soils. Soil Sci. Soc. Am. J. (1980) 44:892-898. evapotranspiration chamber developed at Cerema (Figure below) with an area of 1 m² and a height of 30 cm:

EPHor, AGROCAMPUS OUEST, IRSTV, 49042, Angers, France

Université de Nantes, UMR 6112 LPG-Nantes (Laboratoire de Planétologie et Géodynamique de Nantes)

CAPE, Centre Scientifique et Technique du Bâtiment, Nantes, France

Cerema Dter IdF, Trappes, France

CEREMA DTer Est, Tomblaine, France