Clément Delannoy

Chin Huang

Bernadette Coddeville

Jian-You Chen

Dounia Mouajjah

Sophie Groux-Degroote

Anne Harduin-Lepers

Kay-Hooi Khoo

Yann Guerardel

Elisabeth Elass-Rochard

Mycobacterium bovis BCG infection alters the macrophage N -glycome

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche

Introduction

The glycome of immune cells comprises a complex mixture of glycans carried on proteins and lipids that decorate the cell surface, or secreted [START_REF] Moremen | Vertebrate protein glycosylation: diversity, synthesis and function[END_REF][START_REF] Maccioni | Organization of the synthesis of glycolipid oligosaccharides in the Golgi complex[END_REF], and is fundamental to initiating proper host defense [START_REF] Pinho | Glycosylation in cancer: mechanisms and clinical implications[END_REF][START_REF] Rabinovich | Glycobiology of immune responses[END_REF][START_REF] Johnson | The regulatory power of glycans and their binding partners in immunity[END_REF]. It is now well-appreciated that the biological activities of various glycosylated immune molecules such as the immune receptors (Toll-like receptors, mannose-receptor), IgG antibodies, cytokines, integrins and ligands of selectins, are dependent on their glycosylation status [START_REF] Semel | Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors[END_REF][START_REF] Su | Glycosylation influences the lectin activities of the macrophage mannose receptor[END_REF][START_REF] Kataoka | Roles of N-linked glycans in the recognition of microbial lipopeptides and lipoproteins by TLR2[END_REF]. Significant glycomic changes are noted in a wide range of pathological states including cancer [START_REF] Pinho | Glycosylation in cancer: mechanisms and clinical implications[END_REF], inflammatory diseases [START_REF] Schulz | Glycosylation of sputum mucins is altered in cystic fibrosis patients[END_REF], and bacterial infection [START_REF] Barel | Host glycosylation pathways and the unfolded protein response contribute to the infection by Francisella: Glycosylation and UPR in Francisella infection[END_REF][START_REF] Hare | Mycobacterium tuberculosis Infection Manipulates the Glycosylation Machinery and the N-Glycoproteome of Human Macrophages and Their Microparticles[END_REF], and often positively correlated with regulated expression of glycosyltransferases (GTs) genes. In particular, alterations in both the terminal capping and elongation of glycans may affect interaction of cells with host immune lectins, such as selectins and galectins that transduce a multitude of functional effects including modulation of cell activation, survival and migration, or microbial lectins that mediate adhesion of pathogen [START_REF] Magalhães | Helicobacter pylori chronic infection and mucosal inflammation switches the human gastric glycosylation pathways[END_REF]. In turn, bacteria may manipulate host glycosylation to their own benefit. Several of these microorganisms invade macrophages in which they multiply and further control numerous cell-signaling pathways including production of cytokines and reactive oxygen species, fusion of phagosomes with lysosomes, cell apoptosis and autophagy [START_REF] Marakalala | Macrophage Heterogeneity in the Immunopathogenesis of Tuberculosis[END_REF][START_REF] Sampath | Monocyte Subsets: Phenotypes and Function in Tuberculosis Infection[END_REF][START_REF] Siqueira M Da | Autophagy and Its Interaction With Intracellular Bacterial Pathogens[END_REF]. Modulated gene expressions of galactosyltransferases, sialyltransferases, neuraminidases and fucosidases in macrophages exposed to Mycobacterium tuberculosis (Mtb) have been reported [START_REF] Silver | Human alveolar macrophage gene responses to Mycobacterium tuberculosis strains H37Ra and H37Rv[END_REF][START_REF] Kumar | Genome-wide analysis of the host intracellular network that regulates survival of Mycobacterium tuberculosis[END_REF]. Alteration of sialylation was also detected in murine macrophages activated with purified mycobacterial cell wall glycolipids, such as trehalose-dimycolate (TDM) [START_REF] Mercurio | Glycolipids of the mouse peritoneal macrophage. Alterations in amount and surface exposure of specific glycolipid species occur in response to inflammation and tumoricidal activation[END_REF][START_REF] Afroun | Modifications of glycosylation patterns in macrophages upon activation[END_REF]. To more precisely define the glycosylation changes based on these findings, we have investigated by a mass spectrometry approach how M. bovis BCG may modulate the glycome of infected human primary blood monocytes-derived macrophages (MDM) and the macrophage-like THP-1 cells, both commonly served as relevant cell models to study immunomodulatory functions of infected phagocytes [START_REF] Mendoza-Coronel | Comparative evaluation of in vitro human macrophage models for mycobacterial infection study[END_REF][START_REF] Chanput | THP-1 cell line: An in vitro cell model for immune modulation approach[END_REF]. Our in depth analysis revealed a significant increased expression of terminal fucosylated epitope, Le x , carried on complex type N-glycans, while the glycosphingolipids (GSL) profiles remained largely unaffected.

Experimental

Cell culture: Differentiated THP-1 cells: Human monocytic THP-1 leukemia cells (ECACC n° 88081201) were differentiated into macrophage-like cells with 20 nM phorbol-12-myristate-13-acetate (PMA) for 72 h, in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine and 20 µM b-mercaptoethanol, as previously described [START_REF] Delannoy | Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages[END_REF][START_REF] Tsuchiya | Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester[END_REF]. Non-adherent cells were removed by washing with Phosphate Buffered Saline (PBS).

Human primary blood monocytes-derived macrophages (MDMs):

Human blood samples from healthy donors were collected at the local blood transfusion center (Etablissement Français du Sang, Lille). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation (Lymphoprep, AbCys). Monocytes were purified from PBMC by immunomagnetic selection with magnetic beads coupled to CD14, according to the instructions of the manufacturer (Miltenyi Biotech). Monocyte preparations were more than 95% pure, as checked by flow cytometry. MDMs were obtained by incubating freshly isolated monocytes (106 cells/mL) in complete RPMI 1640, 10% fetal calf serum (FCS) supplemented with 10 ng/mL of GM-CSF for 6 days, as previously described in literature [START_REF] Ohradanova-Repic | Differentiation of human monocytes and derived subsets of macrophages and dendritic cells by the HLDA10 monoclonal antibody panel[END_REF]. Fresh medium with GM-CSF was added after 2 and 4 days.

Mycobacterial cell culture

M. bovis BCG (Pasteur strain) were obtained from Pasteur Institute (Lille, France). Mycobacterium were grown in Sauton medium on shaker at 37°C. Sauton medium was prepared as described by Larsen and collaborators [START_REF] Larsen | Laboratory maintenance of Mycobacterium tuberculosis[END_REF]. Infection and activation of macrophage After 6 days of monocytes differentiation into M0, macrophages adherent cells were infected with M.bovis BCG, at MOI 5:1 (5 mycobacteria per macrophage) or activated by 0,5 µg/ml of mycobacterial lipopeptide PAM3Lp19 and maintained in a humidified atmosphere with 5% CO2 at 37°C for 48h [START_REF] Schromm | Influence of serum on the immune recognition of a synthetic lipopeptide mimetic of the 19-kDa lipoprotein from Mycobacterium tuberculosis[END_REF][START_REF] Riendeau | THP-1 cell apoptosis in response to Mycobacterial infection[END_REF]. The activation of differentiated THP-1 cells was performed in similar conditions.

Flow cytometry analysis of cell surface markers during cell activation:

The activation of both PMA THP-1 cells and MDM, by either BCG-infection or lipopeptide stimulation, was controlled, by measuring the cell surface antigens expression (CD80, CD40, CD54) in unstimulated and activated cells, by flow cytometry. First, cells were pre-incubated 20 min at 4°C with Fc-receptor blocking reagent (Innovex Biosciences, Richmond, CA, USA) to decrease the nonspecific binding of immunoglobulin to cells. Cell surface immunostaining was directly stained, for 40 min at 4°C in PBS/ 0.1% BSA with fluorescein-conjugated mouse anti-human CD80, anti-CD40 Abs or PE-conjugated mouse anti-CD54 (BD Biosciences, Le Pont de Claix, France). Both FITC-or PE-conjugated mouse isotype control IgG (BD Biosciences) was used as negative control. Data were monitored on a flow cytofluorimeter (FACSCalibur, BD Biosciences) and analyzed with the CellQuest software (Mountain View, CA, USA).

Quantitation of pro-infllammatory cytokines secretion by ELISA:

After 8h or 24h activation, culture supernatants were collected and analyzed for the detection of TNF-α, and IL-1β, respectively, by sandwich ELISA, according to the manufacturers' instructions (Ozyme S.A.). Cytokine concentrations were determined using standard curves obtained with recombinant human TNF-α or IL-1β. Statistical significance was determined using the Student t test, with GraphPad Prism 5 (only values of p <0.05 were considered to be significant).

Glycolipids extraction and purification:

Infected or uninfected cells were detached from T75 flasks with cell dissociation non-enzymatic solution and washed twice with PBS. Cells (5x10 6 ) were lyophilized and extracted three times with CHCl3/CH3OH (2:1, v/v) and once by CHCl3/CH3OH (1:2, v/v) using intermediary centrifugations at 2500 g for 20 min. Combined supernatants were dried under a nitrogen stream, subjected to mild saponification in 0.1 M NaOH in CHCl3/CH3OH (1:1, v/v) at 37°C for 2 h and evaporated to dryness. Samples were reconstituted in CH3OH/0.1% TFA in water (1:1, v/v) and applied to a reverse phase C18 cartridge (Waters, Milford, MA, USA) equilibrated in the same solvent. After washing with CH3OH/0.1% TFA in water (1:1, v/v), GSL were eluted by CH3OH, CHCl3/CH3OH (1:1, v/v) and CHCl3/CH3OH (2:1, v/v). The elution fraction was dried under nitrogen stream prior to structural analysis.

Release and purification of N-glycans:

Cells were resuspended by Triton X-100 extraction buffer (1% Triton X-100 in PBS buffer). To lyse cells, suspension was sonicated 30 min. Debris and insoluble fraction were pelleted down by centrifugation at 13000 rpm for 10 min at 4°C, and supernatant was kept. 1/10 volume of 0,1M dithiothreitol were added to a final concentration of 10mM and incubated at 37°C for 1 hour, followed by addition of 1/10 volume of 0,5M iodoacetamide 1 hour in the dark at 37°C. The reduced/alkyled glycoproteins were precipitated with 1/9 volume of 100% trichloroacetic acid to final 10% at -20°C for 30 min. Pellet was precipitated by centrifuge at 13000 rpm for 10 min at 4°C, then the supernatant was discarded. Pellet was resuspended and washed with 1 mL of cold acetone, then centrifuged at 13000 rpm for 10 mins at 4°C, and repeat this step three times. Sample was incubated with trypsin (Sigma-Aldrich) in 50 mM NH4HCO3, pH 8.4, overnight at 37°C. The reaction was quenched by boiling at 100°C for 5 min. N-Glycans were released by N-glycosidase F (BioLabs) digestion at 37°C for 1 day and N-glycans and O-glycopeptides were separated by C18 Sep-Pak chromatography. C18 Sep-Pak was equilibrated in 5% aqueous acetic acid and washed in the same solvent. Sample was loaded on the C18 Sep-Pak and the released N-glycans were eluted with 5% aqueous acetic acid and the bound peptides with 20%, 40% and 60% (v/v) propanol in 5% aqueous acetic acid, pooled and lyophilized.

Mass spectrometry analysis of Glycans:

Glycans and glycolipids were permethylated according to the method of Ciucanu and Kerek [START_REF] Ciucanu | A simple and rapid method for the permethylation of carbohydrates[END_REF] prior to mass spectrometry analysis. Briefly, samples were incubated with DMSO/NaOH/ICH3 during 2h under agitation. The derivatization was stopped by addition of water and the permethylated glycans were extracted in CHCl3 and washed at least seven times with water. Then, permethylated N-glycans were purified on C18 Sep-Pak. Samples were washed with ACN/0.1% TFA in water (1:9, v/v) and eluted with ACN/0.1% TFA in water (8:2, v/v). Permethylated glycans were solubilized in ACN/H2O (1:1, v/v) and mixed with 2,5dihydroxybenzoic acid matrix solution (10 mg/mL dissolved in ACN/H2O (1:1, v/v) and spotted on MALDI plate. MALDI-TOF and MALDI-TOF/TOF spectra were acquired using a 4800 TOF/TOF spectrometer (Applied Biosystems, Framingham, MA, USA) in reflectron positive mode. The laser was set at 337nm and a frequency of 200Hz with 5000 shots per spot was used for MS and MS/MS data acquisition. Additional permethylated glycan samples were analyzed by nanoLC-MS/MS on an Orbitrap Fusion Tribrid system (ThermoFisher Scientific), using the same reverse phase C18 nanoLC conditions, instrument settings, MS 2 -product dependent MS 3 data acquisition, processing and analysis methods described previously [START_REF] Hsiao | Advancing a High Throughput Glycotope-centric Glycomics Workflow Based on nanoLC-MS 2 -product Dependent-MS 3 Analysis of Permethylated Glycans*[END_REF]. To identify potential changes in a2-3 vs 2-6-sialylation, the released glycan samples were subjected to dimethylamidation prior to permethylation [START_REF] Jiang | Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS[END_REF] and mapped against the nondimethylamidated samples first by MALDI-MS on a MALDI TOF/TOF 5800 system (AB Sciex) and then by nanoLC-MS/MS analyses. Dimethylamidation was performed by adding 25 μL of freshly prepared reagent containing 250 mM EDC, 500 mM HOBt, and 250 mM dimethylamine in DMSO to the glycan samples and incubated at 60°C for 1.5 h. After drying down, samples were cleaned up by passing through C18 Sep-Pak cartridges equilibrated in 5% acetic acid before subjected to permethylation.

Detection of carbohydrate epitopes by flow cytometry:

To detect the variation of fucosylated epitopes at the cell surface of activated macrophages, the binding of FITC-conjugated plants lectins, such as LTA (Lotus tetranologobus agglutinin) and UEA-I (Ulex europaeus I agglutinin), which have a specificity for α-Lfucose. was investigated by flow cytometry [START_REF] Sughii | Further immunochemical studies on the combining sites of Lotus tetragonolobus and Ulex europaeus I and II lectins[END_REF][START_REF] Yan | Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le(x) determinant[END_REF]. Untreated and infected cells (250 000/well) were incubated with each lectin at 15 µg/ml, for 40 min at 4 °C in PBS with calcium and magnesium, containing 0.05% BSA which has been previously pretreated by sodium metaperiodate. Furthermore, the non-specificity binding of LTA and UEA-I to cells was evaluated in presence of α Lfucose. After washing, fluorescence analysis was performed on a FACSCalibur, as previously described above. The log of fluorescence intensities in arbitrary units is plotted against cell number. For each lectin, the ratio of mean fluorescence intensity (MFI) of stimulated cells versus MFI of unactivated cells, was determined from three independent experiments performed in triplicate, with MDMs obtained from three blood donors. Statistical significance between untreated and stimulated cells was determined, using the Student t test. Values of p <0.05 were considered to be significant.

Results & Discussion

PMA-treated THP-1 cells and human primary monocytes-derived macrophages (MDMs) were used as models of study because they are known to display similar phagocytic capacity toward M. bovis BCG, leading to up-regulation of expression of cell surface markers and pro-inflammatory cytokines secretion [START_REF] Mendoza-Coronel | Comparative evaluation of in vitro human macrophage models for mycobacterial infection study[END_REF]. Prior to glycomic analysis, we have checked that macrophages (PMA THP-1 and MDMs) infected by M. bovis BCG or incubated with the lipopeptide exhibited a pro-inflammatory phenotype through the increased expression of cell surface antigens and increased production of TNF-α and IL1-b (SIFigure 1).

GSL profiles -Glycosphingolipids were then extracted and purified by solid phase extraction (SPE) on C18 cartridge and analysed by MALDI-MS in permethylated forms based on the calculated composition of glycan ceramide moieties and the prior knowledge of biosynthetic pathways of human GSLs (Fig. 1A). The MS/MS fragmentation analyses show that neutral GSLs are composed of (iso)globosides (i)Gb3 (Gala1-3/4Galb1-4Glcb1-Cer) and Gb4 (GalNAcb1-3Gala1-3/4Galb1-4Glcb1-Cer), as well as neutral ganglioside GA1 substituted by major ceramides [d18:1-C16:0 (Cer*)] and [d18:1-C24:0 (Cer**)] (SIFigure 2C). Among sialylated GSLs, ganglioside GM3 (Neu5Aca2-3Galb1-4Glcb1-Cer) is the major structure, but other mono-sialylated structures such GM1, GD3 and GD1a were also detected. Infection by M. bovis BCG and activation by the lipopeptide PAM3-LP19, did not modify the GSL patterns of THP1 cells (Fig. 1B, SIFigure 2A and 2B). Compared to PMA THP-1, MDM exhibited a much simpler GSL profile, dominated by GM3 associated with [d18:1/C16:0 (Cer*)] and [d18:1/C24:1 (Cer**)] (Fig. 1C), with traces of GM1 in some batches of MDM. As observed for PMA THP-1, M. bovis BCG infection did not induce any modification of the GSL profiles of MDM (SIFigure 3).

N-glycan profiles -

The N-glycans isolated from THP-1 and MDM cells were similarly profiled by MALDI-MS analysis of permethylated derivatives (Fig. 2A and Fig. 3A). In both cell types, the lower mass range signals (m/z 1500 to 2500) were dominated by high-mannose N-glycans that represent more than 50% of the total N-glycans. The remaining compounds were identified as complex-type N-glycans differently sialylated, branched and fucosylated (Fig. 2A and Fig. 3A; sTable 1). The relative amounts of individual N-glycans signals were calculated by integrating their intensities and dividing them by the cumulated intensities of all signals and integrated into different groups sharing similar features (Fig. 2B and Fig. 3B). After infection by M. bovis BCG, PMA THP-1 and MDM did not show significant variation on the ratio of high mannose type versus complex type N-glycans. Similarly, the prevalence of sialylation among complex type N-glycans in THP-1 and MDM was not significantly modified. To ascertain if there is any significant change in the a2,3/a2,6-sialyl linkages, the N-glycans from MDM cells, were subjected to linkage-specific dimethylamide modifications prior to permethylation and MS analysis. Initial MALDI-MS profiling indicated that after dimethylamidation, most of the peaks assigned as sialylated complex type N-glycans were visibly accompanied by additional peaks at 13 u higher (SIFigure 4), consistent with the presence of a2,6-sialyl linkage [START_REF] Jiang | Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS[END_REF]. For signals carrying multiple sialic acids, multiple degrees of +13 u were observed showing variable degree of a2,6-sialylation (bottom panels SIFigure 4). However, in all cases, there is a substantial amount of sialylated peaks remain unaffected, which indicates that sialylation on the N-glycans of MDM comprises both a2,3 and a2,6 linkages. Based on the relative intensities of the in source-generated oxonium ions at m/z 825 and 838 (SIFigure 5A), it could be further inferred that there is more a2,3 than a2,6 linkages but, most importantly, their relative amount was not appreciably affected upon M. bovis BCG treatment (SIFigure 5B).

Increased fucosylation -Contrary to sialylation pattern, infection by M. bovis BCG induced reproducible variations (over four replicates of THP-1 cell cultures and three MDM donors) of antennae and fucosylation levels (Fig. 2B and Fig. 3B) on complex Nglycans. In MDM the proportion of mono-and multi-fucosylated N-glycans increases from 15 to 20% and from 12 to 17%, respectively. MALDI-MS/MS analysis of selected compounds showed that fucose residues could be attached to both chitobiose core and LacNAc extensions (data not shown) of fucosylated N-glycans. However, these analyses could not clearly establish how the fucosylation pattern was modified following infection. In order to further delineate the nature and the extent of the glycosylation changes, we subjected the samples isolated from MDM cells to a semi-quantitative glycotope-centric glycomic mapping of total N-glycome [START_REF] Hsiao | Advancing a High Throughput Glycotope-centric Glycomics Workflow Based on nanoLC-MS 2 -product Dependent-MS 3 Analysis of Permethylated Glycans*[END_REF]. Zooming in on fucosylated complex type N-glycans, it is clear that upon BCG treatment, not only were several mono-fucosylated peaks detected at slightly higher intensity than the non-fucosylated counterparts, but also those carrying a second Fuc (m/z 2508, 2795, 2883, 3244, 3833, 3419, 3606) became more apparent (SI Figure5). The second Fuc was confirmed to be associated with terminal Hex-HexNAc by the oxonium ion at m/z 638, which was only produced by MALDI in source fragmentation from sample treated with BCG (Fig 4). The detection of the range of terminal glycotopes by means of MALDI in source prompt fragmentation and the notable increase in fucosylation were corroborated by LC-MS/MS analysis of the permethylated sample under acidic conditions. Focusing only on the biantennary N-glycans that were better resolved, it is clear that those structures assigned as carrying 2 to 3 fucose residues were expressed at a higher level upon BCG treatment, after being normalized to the Man9-high mannose peak (Fig. 4). Their structures were further verified by manual examining their respective HCD-MS 2 spectra (not shown), which produced the diagnostic MS 2 ion at m/z 638, (Fuc1Hex1HexNAc + ), 825 (NeuAc1Hex1HexNAc + ) but not m/z 999 (NeuAc1Fuc1Hex1HexNAc + ).

Increased Le x expression -

To resolve further the isomeric Fuc1Hex1HexNAc1 glycotopes, the data dependent mode of MS 2 acquisition during the LC-MS/MS run was programmed to be coupled with product-dependent MS 3 , targeting the MS 2 ion at m/z 638. In other words, whenever m/z 638 was detected among the MS 2 ions, it would be automatically taken through to an MS 3 event on the fly. Le x would be unambiguously identified if MS 3 ion at m/z 432 was detected. In addition to manually interpreting several of the MS 2 /MS 3 spectra acquired, the entire range of distinctive glycotopes expressed in MDM with and without BCG treatment, and their relative abundance, were mapped out based on the summed intensities of their respective diagnostic MS 2 ions (Fig. 4). The results indicated that Le x was indeed more represented among the terminal glycotopes after BCG treatment. So was the core fucose as represented by the diagnostic MS 2 ion at m/z 468. Moreover, there was no significant amount of LacdiNAc or Le y /Le b (m/z 812), but a low level of sialyl fucosylated Hex-HexNAc (m/z 999) and diLacNAc (m/z 913) was detected. The increased expression of cell-surface fucosylated epitope was further confirmed by flow cytometry using two fluorescent α1,3fucose specific LTA and α1,2-fucose specific UEA-I lectins [START_REF] Sughii | Further immunochemical studies on the combining sites of Lotus tetragonolobus and Ulex europaeus I and II lectins[END_REF][START_REF] Yan | Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le(x) determinant[END_REF]. In agreement with MS analyses that showed an increased Le x expression, LTA displays a higher binding on PMA-THP1 (x2.1) (Fig. 5A) and MDMs (x4.5) (Fig. 5B) after infection (p < 0,001, n=3). Inversely, UEA signal associated to cells did not show any modification following infection. This recognition is carbohydratedependent, since fucose inhibits the LTA binding to cells (Fig. 5 A & B, peaks 3).

Functional relevance -We have shown that infection by M. bovis BCG modifies the N-glycan patterns of human macrophage PMA-THP-1 cell line and primary MDM. Most notably, we observed a modification in the average number of antennae and an increase in the surface expression of Le x antigen. On the contrary, activation by Pam3Lp19, an agonist of TLR2, increases the level of biantennary N-glycans but does not alter their fucosylation. This subtle difference suggests that mycobacterium-driven changes are not restricted to a TLR-2 dependent pathway. Our results are in agreement with Li and collaborators who suggested that only terminal and subterminal fucosylation, but not core fucosylation (linked a1,6), may be considered as a hallmark of M1 inflammatory macrophages [START_REF] Li | Inhibition of Fucosylation Reshapes Inflammatory Macrophages and Suppresses Type II Collagen-Induced Arthritis: Inhibition of Fucosylation in Arthritis[END_REF]. Moreover, the biosynthesis of GDP-Fuc nucleotide or its intracellular transport in macrophage could be modified upon BCG infection, as previously reported in inflammation, tumorigenesis or apoptosis processes [START_REF] Niittymäki | Differential gene expression of GDP-L-fucose-synthesizing enzymes, GDP-fucose transporter and fucosyltransferase VII[END_REF][START_REF] Moriwaki | The effect of epigenetic regulation of fucosylation on TRAIL-induced apoptosis[END_REF]. Indeed, M. bovis BCG may induce a partial apoptosis of macrophages [START_REF] Wojtas | Mannosylated lipoarabinomannan balances apoptosis and inflammatory state in mycobacteria-infected and uninfected bystander macrophages[END_REF]. Fucosylated N-glycans also participate in tissue remodeling by reducing the activity of tissue inhibitor of metalloproteinases-1 (TIMP-1) [START_REF] Kim | The presence of outer arm fucose residues on the N-glycans of tissue inhibitor of metalloproteinases-1 reduces its activity[END_REF]. Le x is a stage specific embryonic antigen-1 (SSEA-1) and a marker for human myeloid cells that contributes to leukocytes recruitment to inflammatory sites by selectins and interacts with the immune receptor DC-SIGN. Several reports established that along differentiation and maturation of phagocytes, a dynamic regulation in expression of sLe x and Le x occurs, which may be due to transcriptional or post-translational changes of sialyltransferases and neuraminidases expression [START_REF] Silva | Sialyl Lewisx-dependent binding of human monocyte-derived dendritic cells to selectins[END_REF]. Indeed, both differentiation of monocytic THP-1 cells into macrophages and the maturation of DC reduce sLe x expression at the cell surface [START_REF] Delannoy | Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages[END_REF][START_REF] Julien | Sialyl-Lewis(x) on P-selectin glycoprotein ligand-1 is regulated during differentiation and maturation of dendritic cells: a mechanism involving the glycosyltransferases C2GnT1 and ST3Gal I[END_REF]. The sLe x epitope, expressed preferentially on molecules of monocytes such as PSGL1, CD43 and CD44, mediates the recruitment and adhesion of circulating leukocytes to endothelium by interactions with E-selectin [START_REF] Silva | Sialyl Lewisx-dependent binding of human monocyte-derived dendritic cells to selectins[END_REF].

MS-analysis also revealed an increase in the proportion of bi-antennary N-glycans upon M. bovis BCG infection. LacNAc epitopes are regulators of immunity since they are preferential ligands for galectins, a family of lectins including 15 members with various fines specificities [START_REF] Vasta | Functions of galectins as 'self/non-self'-recognition and effector factors[END_REF]. Galectins exert multifunctional immunomodulatory effects by regulating phagocytosis, apoptosis and by inducing or suppressing cell effector functions. Among those, binding affinity of galectin 3 expressed by macrophages might be affected by the apparent modulations of antennae and fucosylation. Furthermore, mycobacterial lectins may also recognize glycoconjugates on macrophages and thus be involved in the adhesion and internalization of pathogens [START_REF] Abhinav | Archeal lectins: An identification through a genomic search[END_REF]. Bacterial lectins could also potentially manipulate the immune response, by competing with the host lectins for the glycans present at the surface of immune cells. In particular, a ricin-type b-trefoil lectin, encoded by Rv1419 gene, has been detected in pleural effusions and granulomas of patients with active TB [START_REF] Nogueira | Mycobacterium tuberculosis Rv1419 encodes a secreted 13 kDa lectin with immunological reactivity during human tuberculosis[END_REF]. Interestingly, Rv1419 modulates adhesion to murine and human derived macrophages and influences the intracellular growth of Mtb [START_REF] Bafica | A mycobacterial lectin promotes bacilli adhesion to macrophages and influences pathogen growth during infection (INM3P.409)[END_REF]. Although its ligands remain unknown, it is expected to interact with Galβ1,4GlcNAcβ patterns present in the human N-glycans. We cannot exclude that the modification of N-glycans antenna in infected macrophages modulates the affinity of this lectin to cells.

Conclusion

In conclusion, we have directly identified and highlighted alterations in the N-glycome of human macrophages upon infection with M. BCG bovis, the most significant of which is the increased expression of Le x epitope. Further research is clearly needed to identify the spectrum of glycoproteins carrying the Le X epitope and to determine to what extent glycosylation changes are driven by mycobacteria to promote infection or/and by host-response for protection. Changes in fucose metabolism could be analyzed by click-chemistry in BCG-infected macrophages using GDP-FucAz. The effect of the 6-alkinyl-fucose inhibitor on macrophages infection by Mycobacterium should also be investigated. The corresponding dataset from THP-1 cells were also plotted for side-by-side comparison). The relative peak areas were normalized to Man9 peak serving as internal reference (taken as 1.0 in the bar chart, and 100% in the XIC plots). (B). The characteristic HCD MS 2 ions indicative of each of the annotated glycotope were extracted from all MS 2 spectra acquired within the elution time of the N-glycans identified. Their respective ion intensities were summed and then calculated as % total for comparative analysis purpose only and not to be taken as true quantification. The charts for m/z 638, 812 and 999 (boxed) were magnified to show clearly the increase in m/z 638 for the MDM dataset. The THP-1 dataset was similarly plotted for comparison. Despite the significant increase in m/z 638, the relative proportion of which that would yield m/z 432 by MS 3 remained roughly constant. MS 3 ion at m/z 606 is indicative of H type 2 glycotope but may also be partly contributed by Le x . A small amount of MS 3 ion at m/z 402 suggests that a very low level of Le a may also be present but the overall amount is negligible.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: MALDI-MS analyses of N-glycans isolated from THP1-cells. (A) MALDI-MS profile of permethylated N-glycans purified from uninfected PMA-differentiated THP-1 cells. The compositions of major glycan signals were deduced from the m/z values of [M+Na] + adducts. Position of Fuc/Sia residues and branching patterns were not established at this stage and are only indicative. Complete list of identified Nglycans is provided in SITable 1. (B) Differential profiling of complex N-glycans, extracted from PMA activated THP-1 cells, before (light blue) and after M. bovis BCG infection (deep blue). Top panel, proportions of multi-antennary complex-type N-glycans (bi, two LacNAc; tri, three LacNAc; tetra/penta, four and five LacNAc); middle panel, proportions of mono and multi-fucosylated complex type N-glycans; bottom panel, proportions of sialylated complex N-glycan. Statistical significance compared with untreated cells (**p<0.01, (*p<0.05) was determined by a two-tailed t-test using GraphPad Prism 5 software. Data represent median ± interquartile range from 4 independent experiments from different cell cultures.

Figure 3 :

 3 Figure 3: Mass spectrometry analysis of N-glycans isolated from human MDM cells. (A) MALDI-MS profile of permethylated N-glycans purified from MDM cells. The composition of major glycan signals were deduced from the m/z values of [M+Na] + adducts. Position of Fuc/Sia residues and branching patterns were not established at this stage and are indicative. Complete list of identified N-glycans is provided in SITable 1. (B) Differential profiling of complex N-glycans before (light green) and after M. bovis BCG infection (dark green). Top, proportions of multi-antennary complex-type N-glycans (bi, two LacNAc; tri, three LacNAc; tetra/penta, four and five LacNAc); middle, proportions of mono and multi-fucosylated complex type N-glycans; bottom, proportions of sialylated Nglycan. Statistical significance compared with untreated cells (**p<0.01, (*p<0.05) was determined by a two-tailed t-. Data represent median ± interquartile range from three independent experiments with different donors.

Figure 4 .

 4 Figure 4. LC-MS/MS analysis of macrophages N-glycans with and without BCG treatment. (A) The relative abundance of the five major sialylated and/or fucosylated biantennary N-glycans were inferred from extracted ion chromatogram plots (right panel, showing one set of data out of the three replicates) and relatively quantified based on their respective peak areas (left panel, averaged from 3 replicates, those of significance by statistical analysis were marked by *).The corresponding dataset from THP-1 cells were also plotted for side-by-side comparison). The relative peak areas were normalized to Man9 peak serving as internal reference (taken as 1.0 in the bar chart, and 100% in the XIC plots). (B). The characteristic HCD MS 2 ions indicative of each of the annotated glycotope were extracted from all MS 2 spectra acquired within the elution time of the N-glycans identified. Their respective ion intensities were summed and then calculated as % total for comparative analysis purpose only and not to be taken as true quantification. The charts for m/z 638, 812 and 999 (boxed) were magnified to show clearly the increase in m/z 638 for the MDM dataset. The THP-1 dataset was similarly plotted for comparison. Despite the significant increase in m/z 638, the relative proportion of which that would yield m/z 432 by MS 3 remained roughly constant. MS 3 ion at m/z 606 is indicative of H type 2 glycotope but may also be partly contributed by Le x . A small amount of MS 3 ion at m/z 402 suggests that a very low level of Le a may also be present but the overall amount is negligible.

Figure 5 .

 5 Figure 5. Binding of plant lectins to macrophages THP-1 and MDM, after infection by M. bovis BCG :(A) PMA-THP1 and (B) MDM were infected by M. bovis BCG for 48h and washed with PBS. FITC-conjugated LTA or UEA-I lectins that interact specifically with a L-fucose, were incubated with untreated (ctrl) or stimulated cells, as described in "Method section". For each lectin, the mean fluorescence intensity (MFI) was determined by flow cytometry and the ratio MFI of infected cells versus MFI of untreated cells was determined. Binding of LTA lectin to unstimulated (grey peak 1) or to activated cells (peak 2) was illustrated in fluorescence histograms. The addition of L-fucose to cells inhibits the LTA interaction with activated cells (peak 3). These data shown are representative of three independent experiments with similar results ( *** p<0,001).

Acknowledgements

The work was supported by the Ministère de l'Enseignement Supérieur et de la Recherche (to CD), by VisonnAIRR grant from Région Hauts-de-France (to DM), and an Academia Sinica Investigator Award grant AS-IA-105-L02 (to KHK). We are grateful to Marlène Mortuaire for technical advice in cell culture. We are indebted to the Research Federation FRABio (Univ. Lille, CNRS, FR 3688, FRABio, Biochimie Structurale et Fonctionnelle des Assemblages Biomoléculaires) and to PAGés platform (http://plateformepages.univ-lille1.fr) for providing the scientific and technical environment conducive to achieving this work. We further acknowledge LC-MS/MS data acquisition at the Academia Sinica Common Mass Spectrometry Facilities for Proteomics and Protein Modification Analysis located at the Institute of Biological Chemistry, Academia Sinica, supported by Academia Sinica Core Facility and Innovative Instrument Project (AS-CFII-108-107).

Conflicts of interest

There are no conflicts to declare.

Abbreviations

Dendritic Cell-Specific ICAM-Grabbing non-integrin (DC-SIGN), Glycosyltransferases (GTs), Glycosphingolipids (GSL), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), LTA (Lotus tetranologobus agglutinin) Mass spectrometry (MS), Monocytes-Derived Macrophages (MDM), Mycobacterium (M.), Mycobacterium tuberculosis (Mtb), N-glycans (NG), Phorbol-12-Myristate-13-Acetate (PMA), Toll-Like Receptors (TLR), Trehalose-DiMycolate (TDM), Sialic acid binding Ig-like Lectins (SIGLECS), Stage Specific Embryonic Antigen-1 (SSEA-1), UEA-I (Ulex europaeus I agglutinin)


N Savalle

J Blanc-Gonnet

E Vincens

S Hans

Keywords: Earthquake engineering, masonry, shaking table tests, dry stone, seismic behaviour, harmonic shaking, DSRW

In this paper, an experimental study aiming at understanding the seismic behaviour of dry stone retaining walls is presented. Harmonic shaking table tests have been carried out on scaled-down dry-joint retaining walls involving parallelepiped bricks. It is found that a thicker wall is more resistant and that a given retaining wall is less sensitive to higher frequencies. For those higher frequencies, the walls accept larger displacements before collapsing. The displacements start to occur from a given threshold, which depends on the wall geometry but not on the frequency of the base motion. The typical toppling failure is observed for slender wall and/or low frequency inputs. For less slender walls or higher frequency inputs, walls experience local sliding failures until the complete collapse of the system. The acceleration at failure reported during the dynamic tests have been compared to the corresponding pseudo-static resistance, enabling a conservative estimate of the seismic behaviour coefficient for pseudo-static analysis of this class of retaining walls. This novel experimental dataset is aimed to serve as a validating framework for future numerical or analytical tools in the field.

Introduction

Dry stone retaining walls (DSRWs) are present worldwide and constitute an important cultural heritage for numerous territories. Today, this old heritage is generally damaged due to weathering but also due to a lack of maintenance. In France, most of the DSRWs have been erected before World War I and no more walls were built after World War II. Since this damaged heritage can be considered as an economical asset for these territories (tourism, agriculture, transportation), the local authorities have some great concerns in relation to their conservation. The required interventions on this heritage are however difficult to make in a context where the behaviour of DSRWs is not completely known.

A lot of studies have been carried out on DSRWs since the first known experimental work performed by Lieutenant Sir John Burgoyne in 1834 [START_REF] Burgoyne | Revetments of retaining walls[END_REF] who studied the impact of the section geometry on CONTACT E. Vincens. Email: eric.vincens@ec-lyon.fr

The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10. 1080/19648189.2020.1855477 the resistance of DSRWs. These first experiments have been afterwards modelled using analytical methods [START_REF] Husband | Structural engineering[END_REF][START_REF] Capper | The mechanics of engineering soils[END_REF][START_REF] Clayton | Earth pressure and earth-retaining structures[END_REF] or numerical models using the Discrete Element Method (DEM) [START_REF] Harkness | Numerical modelling of full-scale tests on drystone masonry retaining walls[END_REF][START_REF] Powrie | Deformation and failure modes of drystone retaining walls[END_REF][START_REF] Claxton | Rigid block distinct-element modelling of dry-stone retaining walls in plane strain[END_REF]. At the time, DEM codes had already been used to successfully model the free standing walls of Great Zimbabwe site (UNESCO) [START_REF] Walker | Stability of medieval dry stone walls in Zimbabwe[END_REF][START_REF] Dickens | Use of distinct element model to simulate behaviour of dry-stone walls[END_REF]. Further experiments on slope DSRWs were carried out much more recently when [START_REF] Villemus | Experimental assessment of dry stone retaining wall stability on a rigid foundation[END_REF] and [START_REF] Colas | Full-scale field trials to assess drystone retaining wall stability[END_REF]Colas et al. ( , 2013a) ) aimed at finding the critical height of a backfill that triggers the collapse of a DSRW. They developed analytical methods [START_REF] Villemus | Experimental assessment of dry stone retaining wall stability on a rigid foundation[END_REF][START_REF] Colas | Yield design of dry-stone masonry retaining structures -Comparisons with analytical, numerical, and experimental data[END_REF][START_REF] Colas | Yield design modelling of dry joint retaining structures[END_REF] to create charts included in design guides for the French dry stone masons [START_REF] Capeb | Pierres sèches : guide de bonnes pratiques de construction de murs de soutènement[END_REF][START_REF] Entpe | Technique de construction des murs en pierre sèche : Règles professionnelles[END_REF]. These works have been complemented by analytical refinements [START_REF] Terrade | Upper bound limit analysis of masonry retaining walls using PIV velocity fields[END_REF] as well as more sophisticated numerical models [START_REF] Oetomo | Modeling the 2d behavior of dry-stone retaining walls by a fully discrete element method[END_REF]. In other European countries, the behaviour of slope DSRWs is also a new field of research [START_REF] Walker | Plane strain numerical model for drystone retaining walls[END_REF][START_REF] Alejano | Stability of granite drystone masonry retaining walls: II. Relevant parameters and analytical and numerical studies of real walls[END_REF][START_REF] Alejano | Stability of granite drystone masonry retaining walls: I. Analytical design[END_REF][START_REF] Pulatsu | Advanced analysis of masonry retaining walls using mixed discrete-continuum approach[END_REF]. In addition, other experimental [START_REF] Mundell | Limitequilibrium assessment of drystone retaining structures[END_REF][START_REF] Mundell | Behaviour of drystone retaining structures[END_REF][START_REF] Mccombie | Drystone retaining walls: Ductile engineering structures with tensile strength[END_REF][START_REF] Le | Assessing the three-dimensional behaviour of dry stone retaining walls by full-scale experiments[END_REF], analytical [START_REF] Le | 3d homogenised strength criterion for masonry: Application to drystone retaining walls[END_REF] and numerical [START_REF] Quezada | 3d failure of a scale-down dry stone retaining wall : a DEM modelling[END_REF] studies have been carried out on 3D failure of DSRWs induced by a concentrated load on backfill top.

All these studies focused on the static behaviour of DSRWs. However, the assessment of the seismic stability of these structures is a necessary step to guide the repair of old structures, as well as to design new ones, since they may be located in seismic zones. [START_REF] Fukumoto | The effects of block shape on the seismic behavior of dry-stone masonry retaining walls: A numerical investigation by discrete element modeling[END_REF] carried out an experimental study on the seismic behaviour of DSRWs but focused on the optimal shape of dressed stones for the wall seismic resistance through centrifuge scaled-down tests. A pseudo-static study on slope DSRWs was carried out by Savalle et al. (2018a) and [START_REF] Savalle | Seismic behaviour of dry stone retaining walls: experimental and numerical pseudo-static studies[END_REF] with the aim at developing a practical analytical tool for the design of DSRWs in seismic areas. Although seismic recommendations are given for the design of slope DSRWs in low seismic risk zones, their method failed to give realistic recommendations for higher seismic risk zones, as pseudo-static approaches are known to be over conservative in that case [START_REF] Richards | Seismic Behavior of Gravity Retaining Walls[END_REF][START_REF] Bellezza | A New Pseudo-dynamic Approach for Seismic Active Soil Thrust[END_REF]Savalle et al., 2018a), thus requiring a displacement design approach [START_REF] Newmark | Effects of earthquakes on dams and embankments[END_REF][START_REF] Huang | Seismic Displacement Criterion for Soil Retaining Walls Based on Soil Strength Mobilization[END_REF][START_REF] Gaudio | A performance-based approach to design reinforced-earth retaining walls[END_REF]. Therefore, dynamic approaches must be followed to get a better insight into the seismic behaviour of slope DSRWs.

Dynamic studies using shaking table tests are common in geotechnical engineering: numerous studies of slope stability [START_REF] Sugimoto | Dynamic characteristics of reinforced embankments with steep slope by shaking model tests[END_REF][START_REF] Bathurst | Geosynthetic-reinforced soil walls and slopes-seismic aspects[END_REF][START_REF] Lo Grasso | Shaking table tests and analysis of reinforced slopes[END_REF][START_REF] Huang | Dynamic behavior of reinforced slopes: horizontal acceleration response[END_REF][START_REF] Srilatha | Effect of frequency on seismic response of reinforced soil slopes in shaking table tests[END_REF] and of unreinforced (or reinforced) retaining walls [START_REF] Ishibashi | Dynamic earth pressures with different wall movement modes[END_REF][START_REF] Fairless | Seismic performance of reinforced earth walls[END_REF][START_REF] Iai | Similitude for shaking table tests on soil-structure-fluid model in 1g gravitational field[END_REF][START_REF] Koseki | Shaking and tilt table tests of geosynthetic-reinforced soil and conventional-type retaining walls[END_REF][START_REF] Bathurst | Geosynthetic-reinforced soil walls and slopes-seismic aspects[END_REF][START_REF] Bathurst | Shaking table model study on the dynamic response of reinforced soil walls[END_REF][START_REF] Huang | Seismic Displacement Criterion for Soil Retaining Walls Based on Soil Strength Mobilization[END_REF][START_REF] Wilson | Shake table lateral earth pressure testing with dense c-φ backfill[END_REF] have been carried out in the past thirty years. However, none of them dealt with the specificity of DSRWs, where blocks can move during the base motion and hence can dissipate energy before collapsing.

In order to pave the way to numerical and analytical dynamic studies, a vast and novel experimental campaign of shaking-table tests involving dry stone retaining walls is envisioned in this work. First, shaking table tests on scaled-down dry-joint brick retaining walls are presented. Then, the influence of the slenderness of the wall as well as the frequency of the ground motion on the behaviour of the retaining walls are investigated. Finally, a comparison with pseudo-static calculations is conducted and a conservative value for the seismic behaviour coefficient r is proposed.

Experimental set-up

Shaking-table tests

Principle of the tests

Herein, more than forty tests on a shaking table have been carried out using scaled-down models of dry-joint brick walls retaining a sandy backfill. The walls were erected using small paralelepiped shaped clay bricks. The mock-up was loaded by a horizontal harmonic ground motion whose amplitude was increased from 0 until the collapse of the wall.

One could note that the parallelepiped bricks utilised in this work are quite different in shape from the undressed blocks used in the field. This latter point has already been studied by other authors. First, [START_REF] Fukumoto | The effects of block shape on the seismic behavior of dry-stone masonry retaining walls: A numerical investigation by discrete element modeling[END_REF] found that, using a trapezoidal shape for stones, the results of centrifuge tests were actually improved compared to more simple shapes like parallelepipeds. However, they attributed this improvement to the change in the backfill-wall interface friction angle and not directly to the shapes of stones. In addition, all numerical and analytical static studies modelling the actual irregular blocks by parallelepiped blocks have been proved relevant to found the key parameters driving the behaviour of actual dry stone walls as well as their ultimate capacity [START_REF] Dickens | Use of distinct element model to simulate behaviour of dry-stone walls[END_REF][START_REF] Harkness | Numerical modelling of full-scale tests on drystone masonry retaining walls[END_REF][START_REF] Powrie | Deformation and failure modes of drystone retaining walls[END_REF][START_REF] Claxton | Rigid block distinct-element modelling of dry-stone retaining walls in plane strain[END_REF] Figure 1. Mock-up of the dry joint retaining wall before a shaking-table test. [START_REF] Villemus | Experimental assessment of dry stone retaining wall stability on a rigid foundation[END_REF][START_REF] Colas | Yield design of dry-stone masonry retaining structures -Comparisons with analytical, numerical, and experimental data[END_REF][START_REF] Mundell | Limitequilibrium assessment of drystone retaining structures[END_REF][START_REF] Colas | Yield design modelling of dry joint retaining structures[END_REF][START_REF] Oetomo | Modeling the 2d behavior of dry-stone retaining walls by a fully discrete element method[END_REF][START_REF] Savalle | Comportement sismique des murs de soutènement de talus en pierre sèche[END_REF][START_REF] Pulatsu | Advanced analysis of masonry retaining walls using mixed discrete-continuum approach[END_REF]. It means that, as long as the dry stone walls are constructed following the state of the art of dry stone construction, the shape of the blocks does not seem to play a significant role on the wall stability.

Experimental devices

The erected retaining walls were B = 34mm wide, L = 400mm long and H = 50 to 140mm high and were made of clay bricks of dimensions: height×width×length = 11mm×17mm×34mm. They were placed in the aperture of a container of dimensions height×width×length = 250mm×400mm×400mm (Figure 1). To reduce the significance of the two walls ends (potentially the most vulnerable parts), the wall length L has been chosen sufficiently large. Indeed, according to [START_REF] Dewoolkar | Experimental developments for studying static and seismic behavior of retaining walls with liquefiable backfills[END_REF], above a wall ratio L/H = 2.0, end effects do not alter the plane strain conditions. The backfill was made of a dry Hostun sand S281 , filling the container and having an equivalent volume of height×width×length = 50-140mm×400mm×400mm (Figures 1 and2). Using an analytical pseudo-static approach, the backfill width W (Figure 2) was designed so that the failure surface in the backfill did not interfere with the vertical back wall of the container (Savalle et al., 2018a). This aspect was validated throughout the shaking-table tests.

The whole mock-up (30-60kg) was placed on an electrodynamic shaking table of dimensions 60cm×60cm that can handle accelerations of 8g for loads of 600N, (Serial number: Sonic S201LS3-340/SPA203). In order to absorb harmonic waves reaching or originated from the vertical back wall of the container, this latter was covered by an absorbing foam which was intended to reproduce free-field conditions (Figure 2). Moreover, a gap was introduced between the brick wall ends and the lateral container walls to avoid any disturbing friction at this location. Two plastic sheets were placed in this gap to prevent the seepage of the backfill sand (Figure 1). The first layer of the wall was fixed in order to model a rigid foundation with an infinitely resistant abutment (Figures 1 and2). In the following, the wall height (H) refers to the height of the free wall and does not take into account this first fixed layer. Therefore, the base friction of the wall was the one of the brick-brick contact itself. In addition, a Linear Variable Displacement Transducer (LVDT6 in Figure 2) was used to monitor the settlement of the backfill. Then, a laser sensor (serial number: FT 50 RLA-40-F-L4S of SensoPart company) was used to measure the displacement of the second to last row of bricks. It will be used later as an indicator of failure for the wall.

Finally, eight accelerometers (ACC) with a minimum precision of 10mV/g have been placed at different locations on the container to quantify its 3-dimensional dynamic response (Figure 2). The accelerometer labeled ACC2, located on the floor of the container, was a special low-frequencies sensor that has been used as an input for the shaking-table tests (special precision of 1000mV/g). The accelerometer labeled ACC10 was a triaxial sensor and all the other sensors (ACC3, ACC4, ACC5, ACC7, ACC8, and ACC9) were mono-directional and oriented in the direction of shaking ( X), perpendicular to the wall face (Figure 2).

Experimental protocol

Preliminary results

It has been observed that the container had not a simple response with neither fully rigid nor fully deformable pattern which would not make future numerical simulations easy. Then, an analysis of the response of the container is presented in this part.

Using a white noise input signal given to the base of the model (ACC2), the Transfer Functions2 of the responses of all other accelerometers have been analysed (Figure 3). Though only the response of the empty container is shown, the response of the container filled with different backfill heights (5cm, 8cm, 10cm, 15cm and 20cm) has been found similar. It is observed that in the range of frequencies between 10Hz and 30Hz, the container can be likened to a rigid container. Indeed, all accelerometers in the X-direction (ACC3, ACC4, ACC5, ACC7, ACC8, ACC9 and ACC10X) had a frequency response equal to the one of the input signal, while there were no junk vibrations in the other directions (ACC10Y and ACC10Z). Analysing the data from sensors ACC4 and ACC9 (not placed at the top of the container), and considering that the top surface of the backfill was never above the sensor ACC9, the mock-up can be considered as rigid up to the investigated frequency of 70Hz.

Then, the response of the backfill itself has been studied. The first resonant frequency of the backfill layer has been evaluated using the classical free-field shear layer model of soils which gives the frequency of the first mode:

f 0 = Cs 4H (1)
The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10. 1080/19648189.2020.1855477 with H the height of the backfill and Cs the speed of shearing waves in the backfill, given by

Cs = G ρ = E 2(1 + ν) ρ (2)
with G the shear modulus of the sand, E its Young's modulus, ν, its Poisson's ratio and ρ its unit mass. The elastic modulus E = 18MPa was evaluated using the formula from [START_REF] Biarez | Elementary mechanics of soil behaviour: saturated remoulded soils[END_REF], e = 0.72 being the sand's void ratio and p = 0.7kPa the confining pressure:

E (in MPa) = 450 e × p (in MPa) (3)
Using a classical Poisson's ratio of ν = 0.3, a backfill height H ranging between 5cm and 15cm and the experimental unit weight of ρ = 1540kg.m -3 , the first eigen-frequency of the backfill is expected to range between 100Hz and 300Hz. In addition, one should note that the validity of Equation 3 for low confined soil is not established. Indeed, close to the soil surface, this equation predicts an excessively deformable material which does not hold any physical meaning. Therefore the actual value might be larger than the predicted one.

Therefore, even at 70Hz, the resonance of the sand layer is not likely to be triggered throughout the experiments. However, this aspect could not have been checked, since the precision of the laser sensor (≈ 0.1mm) was too poor compared to the amplitude of the signal (≈ 0.01mm at 70Hz).

Description of the protocol

First, the wall was erected in the aperture of the container. Then, the sand was carefully deposited from a zero drop-height in order to obtain an homogeneous loose backfill. As the backfill was expected to settle due to the subsequent shakings, a preliminary step for all shaking-table tests was applied to stabilise it to a final state, turning the loose backfill into a dense backfill 3 . It consisted in applying a five-minutes duration white noise to the wall-backfill system (constant spectral density of 0.001g 2 /Hz in the range of leading to a mean acceleration of 0.6g). This white noise input has been found sufficient to achieve a dense and stable state for the backfill, that would not evolve afterwards. The wall was maintained in place by a wood panel fixed to the container, so that no failure of the wall could happen throughout this preliminary stage.

An actual seismic signal is a combination of multiple harmonic waves, whose amplitude is varying in time but also in frequency. In a research context, this complex motion needs to be first simplified in order to understand the basic dynamic features, instead of using actual earthquake recordings where the influence of all parameters is difficult to extract. Therefore, in a first step, the authors chose harmonic waves as input motions in the shaking-table tests, before more complex signals that will be addressed in a future work. Four frequencies compatible with the response of the container have been used (Figure 3): 10Hz, 30Hz, 50Hz and 70Hz, which is in accordance with the classical frequency scaling law given by [START_REF] Ishibashi | Dynamic earth pressures with different wall movement modes[END_REF][START_REF] Krawinkler | Scale effects in static and dynamic model testing of structures[END_REF][START_REF] Fairless | Seismic performance of reinforced earth walls[END_REF][START_REF] Huang | Dynamic behavior of reinforced slopes: horizontal acceleration response[END_REF]:

f scaled-down = f full-scale * 1 K (4)
where f scaled-down is the frequency of shaking in the scaled model, f full-scale is the frequency of shaking at full scale and K is the scale ratio (approximately equal to 1/20 = 0.05 in these experiments). Given that seismic central frequencies mainly range between 1Hz and 20Hz, one ends up with scaled frequencies between 4.5Hz and 89.4Hz, which is close to the chosen frequency range .

The equation of the harmonic ground motion S(t) is given by:

S(t) = A × t × sin(ωt) (5)
with A a constant (see Appendix A for details about its values), t the time throughout the experiment and ω the angular frequency defined as ω = 2πf , f being the frequency of the motion (Figure 4). The duration of the tests was 30 seconds, which is a typical, though long, duration for an actual quake.

Material properties of the mock-up

Retaining brick walls had a width of B = 34mm and a variable height H (50mm to 140mm), enabling the slenderness of the studied walls to range between 1.3 and 4. Only one wall has been erected using a bigger width: B = 51mm and a height of H = 104mm. A typical assemblage is presented in Figure 5. assemblages used to construct the walls can be found in [START_REF] Savalle | Experimental and numerical studies on scaled-down dry-joint retaining walls: Pseudo-static approach to quantify the resistance of a dry-joint brick retaining wall[END_REF]. They were based on dry stone state-of-art rules and enabled stable masonries: headers were placed frequently and all the bricks were installed with staggered joints.

In this study, the mechanical parameters were derived from previous experimental works that used the same materials [START_REF] Amirouche | Dispositifs absorbants à base de matériaux à double porosité dans des champs acoustiques complexes[END_REF][START_REF] Quezada | 3d failure of a scale-down dry stone retaining wall : a DEM modelling[END_REF]Savalle et al., 2018a). Table 1 gives all the data in relation to the materials. The mean unit weight of the wall as well as the unit weight of the backfill were measured during the experiments and were found to be equal to 14.4kN/m -3 and 15.4 ± 0.1kN/m -3 respectively.

The internal friction angle ϕ sand of the backfill has been evaluated using several indirect approaches since typical geotechnical tests can hardly be used due to the very low pressures involved in these experiments (approximately p = 0.7kPa at mid-height). Moreover, in the literature there are only few studies focused on low confined sand, and no specific ones on the S28 Hostun sand at dense state. Yet, using literature's analytical expressions and the experimental confining pressure p = 0.7kPa, a value The first one consisted in adding a raised floor (Figure 6) and a vertical wood panel instead of the brick wall. After filling the container with the loose sand, the container was shaken by the same white noise as herebefore. Then, while removing the wood panel, a failure surface developed in the backfill and dense sand flowed out of the container. Along this failure surface, it has been observed that the sand experienced strong shearing strains leading to the generation of dilative volumetric strains. Therefore, the obtained sandy slope corresponded to the angle at the critical state which is close to the angle of repose for loose S28 sand (Figure 6). Then, a layer (approximately 4cm) of loose sand laying on the dense sand slope (Figure 7) was carefully removed with a brush. Out of three different tests, nine measurements of the slope angle were performed leading to an average value of

ϕ sand = 45 • ± 2 • .
The second experimental approach consisted in using an inverse method with the help of results coming from a previous study (Savalle et al., 2018a). In that study, an analytical tool was developed to predict the tilting angle at failure of an identical mock-up made of a brick wall retaining a sandy backfill. The only difference arises from the density of the backfill which was in a loose state. To evaluate ϕ sand , the container was first filled with the brick wall (H = 8cm and B = 3.4cm) and with the loose Hostun S28 sand. Then, the preliminary process that lead to a dense backfill was applied. At the end of the shakings, the mock-up was carefully placed on a tilting plateform. After the removal of the vertical wood panel that maintained the brick wall stable, the mock-up was progressively inclined and the tilting angle at failure for the wall was measured. Two tests were carried out for repeatability. Using a trial and error method for the analytical method, the unknown being the internal friction angle of the backfill, values equal to ϕ sand = 41.5 • and ϕ sand = 43 • were found. These values are close to the internal friction angle evaluated with the previous experimental approach (45 • ± 2 • ) and are both very different from the one estimated from the literature analysis. Therefore, in the following, an internal friction angle of ϕ sand = 45 • was considered.

The interface (wall-backfill) friction angle δ sand has not been experimentally evaluated but was deduced from past results. Indeed, in a previous study, Savalle et al. (2018a) showed that the interface friction angle can be related to the backfill internal friction angle ϕ sand through the relationship:

δ sand ϕ sand = 0.71 (6)
which is close to the common value of two third typically considered for this kind of interfaces. Thus, the interface friction angle δ sand was such that:

δ sand = 0.71 * ϕ sand = 32 • (7)

Analysis of the experimental data

To get back the acceleration corresponding to the triggering of the wall failure, both the data of the laser sensor (Figure 2) and the data of the harmonic waves are required. The laser sensor registered the displacement of the second row of bricks from top wall (an example is shown in Figure 8). The time t fail associated to the wall failure has been defined as the time required to reach a given displacement threshold, herein defined as 5% of the wall height H (dashed line in Figure 8). This definition is consistent with the definition of [START_REF] Wu | On seismic displacements of rigid retaining walls[END_REF] who considered that an acceptable displacement for a rigid retaining wall is generally below 2% of its height, while a displacement characterising its failure is about 10% of it. In this study, the wall being discrete in nature, a smaller value of 5% was chosen as a limit value corresponding to failure.

Then, the amplitude A of the sinusoidal acceleration at time t fail was found as a root mean square value of the imposed time-acceleration S(t), recorded by ACC2, in a time window of duration T = 1/f centred on t fail (f being the frequency of the input signal used for the particular test: 10Hz, 30Hz, 50Hz or 70Hz)

Experimental results

Repeatability of the tests

To have more insight into the repeatability of the experiments, two different walls (base width of 3.4cm and height equal to 7.9cm or 11.3cm) have been studied. Three identical tests were carried out for each of the signal frequencies of 10Hz, 30Hz, 50Hz and 70Hz for the wall of 7.9cm height following the protocol described in Section 2.2.2. This repeatability test was restricted to the frequency 10Hz for the wall with 11.3cm height.

Table 2 gives the acceleration at failure for all the repeatability tests. One can note that the tests are quite repeatable for all frequencies with a coefficient of variation of about 10%. The higher value of 11.2% was found for the imposed frequency signal of 70Hz. In the following, the study focuses on frequency 10Hz, as seismic frequencies are usually low: 10Hz in the model corresponds to actual seismic frequency of 2.2Hz (Equation 4).

Influence of H/B

The influence of the wall slenderness (i.e. its aspect ratio) on its dynamic resistance was first studied. Ten wall geometries with slenderness ranging from H/B = 1.3 to H/B = 4.0 have been tested. Nine of them had a base width of B = 34mm and various heights, ranging from H = 44mm to H = 135mm and one of them had a width of B = 51mm and a height of H = 104mm. Figure 9 gives the results in terms of acceleration at failure time t fail . A significant decrease of the wall dynamic resistance with respect to its slenderness can be observed, which was expected. The test with a thicker wall enabled to have arrangements of bricks with no full headers, which is more representative of actual arrangements in DSRWs. Its height was chosen in order to obtain a typical slenderness of H/B = 2.0. This first test (though needed to be confirmed by other tests) leads to the conclusion that slenderness is the main geometrical factor responsible for the dynamic resistance of dry-stack retaining walls, which has also been noticed for pseudo-static tests (Savalle et al., 2018a[START_REF] Savalle | Seismic behaviour of dry stone retaining walls: experimental and numerical pseudo-static studies[END_REF]. Moreover, the two typical failure modes of DSRWs were experimentally retrieved: sliding (crosses in Figure 9) occurred for the less slender walls whereas toppling (circles in Figure 9) occurred for the more slender ones. A mixed mode of failure was obtained for walls with slenderness ranging between 2 and 2.5 which is the typical range of actual DSRWs. Figures 10 and 11 give snapshots of two walls through the development of toppling and sliding modes of failure. Generally, toppling was easier to identify compared to sliding since for the former type of failure, the wall kept a monolithic behavior until collapse. It was not the case for the sliding mode where local relative displacements occurred in different locations. Walls were considered to experience a sliding mode of failure when the top bricks slid and fell one by one until the total collapse of the retaining wall.

Influence of the motion frequency

In a second study, the influence of the frequencies of shakings has been investigated. 50Hz and 70Hz. The results are shown in Figure 12, while full details can be found in the Appendix A. One can note that the walls resistance increases dramatically with the frequency of the base motion. Moreover, for the slenderness of 3.3, the mode of failure was found independent of the signal frequency. It is not the case for the two other studied slendernesses which were smaller and where sliding was generally triggered for higher frequencies. As previously mentioned, sliding generally occurs with local failures of bricks. However for slenderness H/B = 2.7 and frequency 70Hz, a monolithic slide started to occur at the bottom right corner wall and then a toppling mode of failure propagated through the wall (Figure 13). One could note that this failure did not develop following plane strain assumptions, on the contrary to the vast majority of toppling failed walls. This can easily be explained by local material and geometrical heterogeneities, and especially at the two ends, leading to more vulnerable parts.

The increase of resistance with the frequency may be linked to the following points:

• The duration of each cycle of loading is shorter (though more frequent),

• The velocity amplitude is smaller; indeed the Peak Ground Velocity (PGV) is known to be a relevant parameter to caracterised the destructive power of a seismic loading [START_REF] Richards | Seismic Behavior of Gravity Retaining Walls[END_REF]),

• The maximum dynamic earth pressure is lower due to the decrease of the wave length of the input signal with the increase of the frequency [START_REF] Baziar | Sliding stability analysis of gravity retaining walls using the pseudo-dynamic method[END_REF][START_REF] Bellezza | A New Pseudo-dynamic Approach for Seismic Active Soil Thrust[END_REF][START_REF] Pain | Seismic stability of retaining wall-soil sliding interaction using modified pseudo-dynamic method[END_REF]. Indeed, though at f = 10Hz, the wave length is approximately equal to λ = Cs f ≈ 6m which is much larger than the wall height (H ≈ 10cm), at f = 70Hz, the wave length decreases to λ ≈ 0.9m]. For a wall of height H ≈ 10cm, the local dynamic earth pressure may not be any more constant along its height.

• The smaller displacements (at each cycle, as well as total displacements) are more endurable by the structure.

To illustrate this latter point, the author propose hereafter an analysis of a single brick based on Newmark's theory [START_REF] Newmark | Effects of earthquakes on dams and embankments[END_REF]). Newmark's analysis is here intended as a explaining tool for the increase of failure acceleration with the increase of the frequency of the base motion. Basically, Newmark's theory consists in integrating twice the acceleration record. Therefore, it leads to a displacement amplitude divided by ω 2 (ω being the angular frequency in rad/s), compared to the acceleration amplitude. In order to obtain the total displacement, we sum the displacements occurring for each cycle, thus multiplying one cycle's displacement by the number of cycles, which is proportional to the angular frequency ω. In the end, the total displacement is inversely proportional to the frequency f of the base motion. The following developments give complementary details.

For the sake of simplicity and in a context where just qualitative results are expected, a very basic modelling is chosen, neglecting for instance the earth pressure. The analysis is focused on the stability of one brick of the last layer, since it has been observed that in case of sliding failures, bricks of the topmost layer fell the first. The pseudo-static critical acceleration amax is taken as the tangent of the brick-brick friction angle. Then, a Newmark's scheme is adopted, where at every moment the input acceleration S(t) is bigger than the threshold amax = g * tan(ϕ brick ), the total displacement of the studied brick d is computed with a Matlab home code integrating twice the unbalanced acceleration S(t) -amax. Since the brick can only move if the acceleration is directed outward, the double integration is carried out only for S(t) > amax and not for S(t) < -amax.

Using the parameters of the experiments (Equation 5with amax = 0.62g and A = 1.0g s -1 ), one can obtain the following fitted relationship:

d = 0.18 × (t -33) 2.75 f (8)
with f the frequency of the signal and t the reference time of the test. The numeric values (0.18, 33 and 2.75) enable to fit the original curve, which basically contains steps associated to stable phases. These numerical values depend on the parameters amax and A, though no simple relationships have been found. One could notice however that using constant amplitude harmonic signal, the value 33 is replaced by 0 and the 2.75 one is replaced by 1.

During the experiments, the failure has been detected through a displacement criteria d crit = 5% * H. Then, inverting Equation 8 for a given wall height H, one can find that the failure time t fail is a root function of the frequency f of the input. Therefore, it partially explains why sliding failures were triggered later for higher frequency inputs.

In Figure 14, the displacements measured throughout the tests for the three walls subjected to the four frequency input signals have been plotted. Firstly, it is noted that irrespective of the frequency, noticeable wall displacement (above 1mm) occur at the same moment, i.e. at the same acceleration level. Therefore, until a certain acceleration level, the input frequency does not play a significant role: the dynamic behaviour of the wall is not yet mobilised. This acceleration threshold only depends on the wall's static resistance and is slightly higher than the pseudo-static resistance of the wall. Secondly, the rate of increase of the displacement through time is slower for higher frequencies. In addition, one can note that though the wall shaken by 10Hz harmonic waves can indeed be considered as near collapsing when reaching the 5% displacement threshold, this is not the case when it is shaken with higher frequency signals. In fact, the behaviour of the wall shaken by 10Hz waves is a rigid one with no displacement in a first phase until a threshold after which huge displacements and brittle failure occur. Conversely, the larger the frequency is, the more ductile its response seems to be: walls can handle huge displacements without collapsing and these displacements are noticeable well before the wall collapse. This capacity to experience stable irreversible displacements is clearly linked to the capacity of the wall to dissipate seismic energy, and thus to resist to an earthquake. This is particularly interesting for actual DSRWs, where their actual resistance could be much higher than the one evaluated through classical pseudo-static approaches.

To conclude, the retaining wall seems to be less vulnerable to higher frequencies. This extra resistance is permitted by small and stable relative movements of the bricks and/or the whole wall. These ones are irreversible, enabling to dissipate the energy of the input signal. Analysing again the experimental results of [START_REF] Huang | Dynamic behavior of reinforced slopes: horizontal acceleration response[END_REF], sand slopes seem also to be more resistant to higher frequency motions, though this had not been highlighted by the authors. [START_REF] Huang | Dynamic behavior of reinforced slopes: horizontal acceleration response[END_REF] carried out shaking-table tests on three identical sand slopes using tapered input waves of growing amplitude. The three sand slopes were subjected to three different frequency waves: 3Hz, 6Hz and 15Hz. From Figure 6 of their article, it appears that higher frequency tapered waves were required to trigger the slope failure.

The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www. tandfonline.com/10.1080/19648189.2020.1855477 Table 3. Comparison between resistance to 10Hz harmonic motion and pseudo-static resistance of dry-joint walls depending on the slenderness of the wall. The failure mode, sliding (S) or toppling (T), is also depicted.

Slenderness of

Comparison with a pseudo-static study

The results obtained throughout the experimental dynamic tests have been compared to results obtained throughout a pseudo-static approach of the problem in order to evaluate how much this latter is conservative. The pseudo-static method considered herein is based on the limit equilibrium of a Coulomb's wedge of soil, that includes the pseudo-static force modelling the seismic action. Then, the stability of the wall is checked according to two possible modes of failure, sliding and toppling. This method has been validated on pseudo-static experiments (Savalle et al., 2018a).

Table 3 gives the relative difference, denoted gap, between the acceleration at failure for the dynamic approach (for 10Hz input signals) and the one derived analytically from the pseudo-static approach according to the wall slenderness.

First, the results confirm that the pseudo-static approach (which is equivalent to a 0Hz frequency input motion) is generally conservative since almost only positive values were obtained. The special case where slenderness is equal to 1.3 should require more tests before confirmation. When the walls tended to fail by toppling (high slendernesses), the relative difference lies around 45%, which is very significant. On the other hand, when the walls tended to fail by sliding (low slendernesses), the relative difference falls down to 15%. Typical slendernesses for DSRWs ranges between H/B = 2 and H/B = 2.7 where the relative difference decreased from 43% to 12%. Then, depending on the properties of the studied DSRW, a pseudo-static approach could be just conservative or very conservative.

In fact, these experimental results have to be analysed in the light of the conclusions made previously:

• Increasing the frequency of the motion increases the resistance of a dry-joint retaining wall, and since natural seismic motions usually have a frequency content that extends beyond the frequency of 2.2Hz (equivalent to the 10Hz frequency input in the shaking-table tests), the relative difference gap may be most of the time higher than those noted in Table 3 (Table 4).

• From some other tests not shown here, it has been noted that the larger the duration of the base motion was, the lower the wall's resistance was. Earthquakes often last less than 30 seconds, which is the average duration of the harmonic input used herein. Therefore, the actual seismic resistance of slope DSRWs may probably be higher than that identified in this work.

Finally, a seismic behaviour coefficient r = 1.2 can be proposed from the present experimental campaign. This can already be used in conjunction with a pseudo-static approach. As explained above, this value can still be considered as too conservative in many cases. Therefore, further numerical simulations involving true seismic motions would be helpful to support a higher value for this coefficient. On the other hand, one could conduct Newmark's analysis on the displacement of the retaining wall, as it is commonly performed for geosynthetic reinforced earth retaining walls [START_REF] Masini | An interpretation of the seismic behaviour of reinforced-earth retaining structures[END_REF][START_REF] Gaudio | A performance-based approach to design reinforced-earth retaining walls[END_REF]. For these particular systems, since it has been found that the failure mode predicted by pseudo-static analysis was similar to the one developed throughout dynamic simulations [START_REF] Masini | An interpretation of the seismic behaviour of reinforced-earth retaining structures[END_REF], pseudo-static methods can be very efficient for an infield application, since less time-consuming than dynamic simulations. However, back to DSRWs, the validity of Newmark's analysis for this class of retaining walls still needs to be demonstrated, and above all regarding the ability of Newmark's method to handle toppling failure modes.

Conclusion

The seismic behaviour of slope dry stone retaining walls has been studied through a simplified case of harmonic shaking-table tests where the input acceleration is a sinus associated to a single frequency. Experiments have been carried out on a model of actual dry stone retaining walls composed of parallelepiped bricks. Their resistance to shaking have been investigated for input signals of increasing amplitude, with different harmonics.

As expected, their resistance was found decreasing with slenderness. Classical toppling failures have been observed for slender walls, while less slender ones experienced local failures of bricks that could be akin to a sliding failure. As long as the frequency of the harmonic input wave increases, the wall's resistance increases as well and the toppling mode of failure is progressively replaced by sliding ones. It has been noted that a dry-stack retaining wall can experience small local relative displacements throughout the shaking, particularly when subjected to higher frequency motions. It may be an advantage since relative displacements contribute to dissipate energy. Based on these experimental results and on a pseudo-static approach, the seismic behaviour coefficient has been derived for the experimental walls. A reasonable value of r = 1.2 can be selected for a pseudo-static analysis of DSRWs. Finally, this experimental campaign paves the way for future studies in the field, either for numerical computations furnishing a complete, detailed and wide experimental data set. It also sets the basis for similar experiments with a consolidated and accurate protocol.

To conclude, the contribution of the paper to the field can be summarised as follows:

(1) It provides the first experimental data set of shaking-table tests involving dry-stack masonry retaining walls, which will be helpful for numerical validations. It should lead to a better understanding of the seismic behaviour of Dry Stone Retaining Walls (DSRWs).

(2) It gives details about the best way to conduct shaking-table tests on retaining structures with high reproducibility, with some insights about the way to handle a densifying backfill or to identify the internal friction angle of a low confined sand.

(3) It brings to light the capacity of dry-stack retaining walls to experience small stable relative movements, and especially when the frequency of the input signal is high. It leads to a greater seismic capacity of walls for higher frequencies.

(4) It evidences that for a given wall, irrespective of the frequency of the base motion, noticeable displacements starts to occur for the same acceleration threshold.

(5) It identifies a first and conservative seismic behaviour coefficient for pseudo-static studies of DSRWs, which is equal to 1.2. This behaviour coefficient is generally sensitive to the wall slenderness, the duration of the motion or the frequency content.

TheFigure 2 .Figure 3 .

 23 Figure 2. Scheme of the mock-up (top and cross-section views). Accelerometers (ACC), laser sensor and Linear Variable Displacement Transducer (LVDT) are depicted.

Figure 4 .

 4 Figure 4. Input acceleration signal S at the base of the model: two frequencies are partially shown (total duration is ≈ 30s). The amplitude of the signal increases linearly.

Figure 5 .

 5 Figure 5. Picture of the assemblage used for each layer: the wall was constructed layer by layer.

  Figure 6. (a) A raised floor is added to the container to ensure that (b) the S28 sand that flows falls away from the stable slope.

Figure 7 .Figure 8 .

 78 Figure 7. 2D scheme of the stable slope after removing the vertical wood panel. A layer of loose S28 sand stands in front of the dense sand.

Figure 9 .Figure 10 .Figure 11 .

 91011 Figure 9. Acceleration at failure for the dry-joint brick retaining wall depending on its slenderness throughout experiments.

TheFigure 12 .Figure 14 .

 1214 Figure 12. Influence of the frequency on the acceleration at failure for three different walls.

  gap =acc dynamic (10Hz) -acc pseudo-static acc pseudo-static

  The detailedThe Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477

	0.2
	0.15
	0.1
	0.05
	0
	-0.05
	-0.1
	-0.15
	-0.2
	0	0.02 0.04 0.06 0.08	0.1	0.12 0.14 0.16 0.18	0.2

Table 1 .

 1 The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477 Mechanical parameters of the experiments. Symbol refers to[START_REF] Amirouche | Dispositifs absorbants à base de matériaux à double porosité dans des champs acoustiques complexes[END_REF]; symbol to[START_REF] Quezada | 3d failure of a scale-down dry stone retaining wall : a DEM modelling[END_REF]; symbol † toSavalle et al. (2018a) and symbol to the present study.

		Brick wall Dense backfill Interface Absorbing foam
	Mean unit weight (kN.m -3 ) 14.4 †	15.4	-	0.52
	Friction angle ( • )	32 •	45 •	32 • †	-
	Cohesion (kPa)	0	0	0 †	-
	Damping	-	-	-	0.14

  The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477

	Removed wood panel	Loose sand	ϕ sand	Dense sand	Raised bottom
			ϕ loose

Table 2 .

 2 4 .The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477 Repeatability tests: comparison of the accelerations at wall failure for identical tests. g = 9.81m.s -2 refers to the natural gravity on earth.

		H/B = 3.3 H/B=2.3 H/B=2.3	H/B=2.3	H/B=2.3
		H=11.3cm H=7.9cm H=7.9cm H = 7.9cm H=7.9cm
		at 10Hz	at 10Hz	at 30Hz	at 50Hz	at 70Hz
	Test 1 (% g)	0.22	0.34	0.48	0.56	0.81
	Test 2 (% g)	0.20	0.30	0.44	0.65	0.86
	Test 3 (% g)	0.20	0.34	0.46	0.60	0.69
	Mean acceleration (% g)	0.21	0.33	0.46	0.60	0.79
	Standard deviation (% g)	0.02	0.02	0.02	0.04	0.09
	Standard deviation (%)	8.1%	7.1%	4.1%	7.0%	11.2%

  The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477

			Range of slenderness
			for actual DSRWs
	0.5
	0.45
	0.4
	0.35
	0.3
	0.25
	0.2
	0.15
	0.1
	1	1.5	2	2.5	3	3.5	4	4.5

For three different walls with three different slendernesses which are typical range of slendernesses of actual DSRWs (H/B = 2.3, H/B = 2.7 and H/B = 3.3), shaking table tests have been carried out for four signal frequencies 10Hz, 30Hz,

Table 4 .

 4 The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477 Comparison between resistance to harmonic motion of varying frequency and pseudo-static resistance of dry-joint walls of slenderness H/B = 2.3.

	Frequency of the test (Hz) Relative difference e (%)
	10	18%
	30	60%
	50	110%
	70	176%

Table A1 .

 A1 The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477 The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477 Ending accelerations of the shaking-table tests.

	Slenderness of the wall Ending acceleration (% g)
	1.3	15
	1.7	10
	2.0	10
	2.3	10
	2.7	10
	3.0	5
	3.3	5
	3.7	3
	4.0	2

Table A2 .

 A2 Complementary details of the experimental tests carried out using different frequencies: duration and failure acceleration.

	Slenderness of Frequency (Hz) Duration (s) the wall (H/B)	Failure acceleration (% g)
			25	0.34
		10	25	0.34
			22	0.30
			33	0.47
		30	30	0.44
	2.3		32 38	0.46 0.56
		50	43	0.65
			40	0.60
			53	0.81
		70	46	0.69
			56	0.86
		10	24	0.33
	2.7	30 50	30 41	0.39 0.57
		70	54	0.78
			31	0.22
		10	28	0.20
	3.3	30	27 29	0.20 0.22
		50	44	0.34
		70	59	0.46

The S28 sand had minimum and maximum void ratios equal to e min = 0.689 and emax = 1.031[START_REF] Combe | Comportement du sable d'Hostun S28 au triaxial axisymétrique[END_REF].

The Transfer Function corresponds to the Fourier Transform of the response of an accelerometer divided by the Fourier Transform of the input signal (ACC2), which is herein characterised by a plateau between

3Hz and 70Hz.

Mean void ratios and relative densities of e loose = 0.97, R D, loose = 19%, e dense = 0.72 and R D, dense = 91% have been found throughout the tests.

A = 2× < S 2 (t) >

Acknowledgements

The authors want to thank the French Ministry of Higher Education and Research for their financial support through the PhD scholarship. The authors want also to acknowledge the support of the technical team of the ENTPE, and particularly the help of Stéphane COINTET. The authors valued a lot the conversations had with Dr. Thiep DOANH and Dr. Denis BRANQUE (ENTPE) in relation to the behaviour of sands at low confining pressures.

AUTHOR ACCEPTED MANUSCRIPT

Dynamic behaviour of drystone retaining walls: shaking table scaled-down tests

The Version of Record of this manuscript has been published and is available in European Journal of Environmental and Civil Engineering. 24th November 2020. DOI: http://www.tandfonline.com/10.1080/19648189.2020.1855477

Appendices Appendix A. Ending acceleration of shaking-table tests

In this appendix the test protocol is more detailed. The nominal test, whose a full record is given in Figure A1, lasted 1 min (from 0.01% g to 1.02% g), with the failure occurring approximately around 30 seconds. However, the ending acceleration of the shaking was varying depending on the wall's expected resistance. Table A1 sums up the ending accelerations of each test depending on the aspect ratio (H/B) of the wall. The relatively short duration of each test enables further numerical simulations of the experiments. Similarly, full details of the tests carried out using different frequencies are given in Table A2. In this table, the duration refers to the duration between the beginning of the test and the failure of the wall, including the 5 seconds of automatic calibration of the shaking-table at low-level shaking (Figure A1). Then, the acceleration is linearly growing during the second phase. At around t = 25s, the wall collapses, generating huge noise in the acceleration record. Finally, the test is manually stopped at t = 29s.

Appendix B. Evaluation of the friction angle of the sand

In this appendix, the authors sum up a literature analysis on friction angle of low confined sand, with the aim of having some reference values. Sands are known for increasing their friction angle as confining pressure decreases [START_REF] Lancelot | Comportement du sable d'Hostun sous faibles contraintes[END_REF]. Some analytical formulas [START_REF] Bolton | The strength and dilatancy of sands[END_REF]Maksimovic, 1989) have been calibrated on low-pressure experiments [START_REF] Lancelot | Comportement du sable d'Hostun sous faibles contraintes[END_REF] involving Hostun RF sand, which is very close to Hostun S28 sand used in the present work [START_REF] Combe | Comportement du sable d'Hostun S28 au triaxial axisymétrique[END_REF]. Some analytical equations have also been calibrated on experimental results for Toyoura sand [START_REF] Chakraborty | For analytical expression, these values were derived using p = 0.7kPa, as it was the mean pressure along the experiments[END_REF]. Finally, very low-pressure tests have been conducted out of gravity on Ottawa sand [START_REF] Sture | Mechanics of granular materials at low effective stresses[END_REF][START_REF] Alshibli | Strain localization in sand: plane strain versus triaxial compression[END_REF]. All the results from these studies are gathered in Table B1, leading to values well above ϕ sand = 50 • . From these results, the value found by the authors of ϕ sand = 45 • is plausible.

Authors

Type Sand ϕ ( • ) Comments [START_REF] Lancelot | Comportement du sable d'Hostun sous faibles contraintes[END_REF] and Anal. Hostun RF 55 • validated up to a minimum (Maksimovic, 1989) pressure of 20kPa [START_REF] Lancelot | Comportement du sable d'Hostun sous faibles contraintes[END_REF] Anal. Hostun RF 55 • validated up to a minimum and [START_REF] Bolton | The strength and dilatancy of sands[END_REF] pressure of 20kPa [START_REF] Chakraborty | For analytical expression, these values were derived using p = 0.7kPa, as it was the mean pressure along the experiments[END_REF] Mean from two tests at [START_REF] Sture | Mechanics of granular materials at low effective stresses[END_REF] 0.52kPa. Friction rapidly [START_REF] Alshibli | Strain localization in sand: plane strain versus triaxial compression[END_REF] dropped from 1% strain reaching 38 • at 5% strain