Françoise Illien Malik Zibouche

Françoise Illien

Jesus Ayala-Sanmartin
email: jesus.ayala-sanmartin@sorbonne-universite.fr

Annexin A2 expression and partners

Keywords: Actin, Annexin A2, Caveolin, Epithelial cell differentiation, E-cadherin

come

Introduction

The members of the annexin family of calcium-and phospholipid-binding proteins participate in different cellular processes [START_REF] Gerke | Annexins: from structure to function[END_REF]Rescher and Gerke 2004;[START_REF] Monastyrskaya | The annexins: spatial and temporal coordination of signaling events during cellular stress[END_REF]. They bind to negative phospholipids of membranes with different Ca 2+ sensitivity [START_REF] Monastyrskaya | Annexins as intracellular calcium sensors[END_REF]. Annexin A2 (AnxA2) has been involved in many membrane related processes: exocytosis, endocytosis, cell junction formation and actin cytoskeleton stabilisation and dynamics [START_REF] Grieve | Annexin A2 at the Interface of Actin and Membrane Dynamics: A Focus on Its Roles in Endocytosis and Cell Polarization[END_REF]. Two molecules of AnxA2 are able to bind to a S100A10 dimer forming a heterotetramer with high calcium sensitivity for membrane binding. It is accepted that the tetrameric AnxA2 form is functional in cells but functional roles for the monomeric AnxA2 have also been suggested [START_REF] López-Rodríguez | Molecular dissection of the membrane aggregation mechanisms induced by monomeric annexin A2[END_REF][START_REF] Taylor | Heterotetrameric annexin A2/S100A10 (A2t) is essential for oncogenic human papillomavirus trafficking and capsid disassembly, and protects virions from lysosomal degradation[END_REF].

Actin was described as the first partner of AnxA2. The binding of annexin A2 to actin filaments is calcium dependent [START_REF] Gerke | Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to nonerythroid spectrin and F-actin[END_REF] and seems to be mediated by the Cterminal end of the protein [START_REF] Filipenko | The C Terminus of Annexin II Mediates Binding to Factin[END_REF]. AnxA2 not only binds actin filaments but also regulates actin filaments bundling in vitro [START_REF] Jones | A nonapeptide to the putative F-actin binding site of annexin-II tetramer inhibits its calcium-dependent activation of actin filament bundling[END_REF][START_REF] Ma | In vitro modulation of filament bundling in F-actin and keratins by annexin II and calcium[END_REF]) and in vivo [START_REF] Falsey | Actin microfilament aggregation induced by withaferin A is mediated by annexin II[END_REF]. Its role in actin dynamics has been demonstrated during actin-driven macro-pinocytosis rocketing [START_REF] Merrifield | Annexin 2 has an essential role in actin-based macropinocytic rocketing[END_REF], its accumulation in actin enriched pedestals during bacterial-plasma membrane interaction [START_REF] Zobiack | Cellsurface attachment of pedestal-forming enteropathogenic E. coli induces a clustering of raft components and a recruitment of annexin 2[END_REF][START_REF] Miyahara | Enterohemorrhagic Escherichia coli effector EspL2 induces actin microfilament aggregation through annexin 2 activation[END_REF], and cortical actin remodelling and stress fibres re-arrangements [START_REF] Hansen | Molecular Mechanism for Orienting Membrane and Actin Dynamics to Nascent Cell-Cell Contacts in Epithelial Cells[END_REF][START_REF] Hayes | Regulation of actin dynamics by annexin 2[END_REF][START_REF] Hayes | Annexin A2 at the interface between F-actin and membranes enriched in phosphatidylinositol 4,5,-bisphosphate[END_REF][START_REF] Rescher | Tyrosine phosphorylation of annexin A2 regulates Rho-mediated actin rearrangement and cell adhesion[END_REF]. The AnxA2 phospholipid ligand phosphatidylinositol-4,5-bisphosphate (PI2P) has been suggested to be a participant of this AnxA2-actin-membrane interactions during membrane ruffles and bacterial pedestals formation (Rescher et al. 2004), and macro-pinocytosis [START_REF] Hayes | Annexin A2 at the interface between F-actin and membranes enriched in phosphatidylinositol 4,5,-bisphosphate[END_REF].

Caveolin 1 is a protein found in caveolae which are cholesterol-enriched invaginated membrane domains [START_REF] Murata | VIP/caveolin is a cholesterol-binding protein[END_REF]. Caveolin 1 has been suggested to bind AnxA2 by co-localization in detergent resistant membrane domains [START_REF] Corvera | Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterol[END_REF] and coimmunoprecipitation [START_REF] Uittenbogaard | Cholesteryl Ester Is Transported from Caveolae to Internal Membranes as Part of a Caveolin-Annexin II Lipid-Protein Complex[END_REF][START_REF] Smart | Annexin 2-caveolin 1 complex is a target of ezetimibe and regulates intestinal cholesterol transport[END_REF]). Liao and collaborators suggested recently that caveolae are signalling platforms for AnxA2-induced cell proliferation [START_REF] Liao | Annexin A2-modulated proliferation of pulmonary arterial smooth muscle cells depends on caveolae and caveolin-1 in hepatopulmonary syndrome[END_REF]).

Yamada and collaborators showed that annexin A2 moves from the cytosol to the lateral plasma membrane of epithelial cells and co-localizes with the adherens junction protein Ecadherin (Yamada et al. 2005a). They showed that the inhibition of AnxA2 expression blocks the formation of E-cadherin dependent junctions. In endothelial cells a similar behaviour was observed [START_REF] Heyraud | Contribution of Annexin 2 to the Architecture of Mature Endothelial Adherens Junctions[END_REF][START_REF] Su | Annexin 2 Regulates Endothelial Morphogenesis by Controlling AKT Activation and Junctional Integrity[END_REF]. Moreover, AnxA2 has been involved in intestinal cell differentiation and polarity [START_REF] Hein | The dual role of annexin II in targeting of brush border proteins and in intestinal cell polarity[END_REF]. Interestingly, depletion of cell membrane cholesterol induced AnxA2 membrane dissociation and disruption of adherens junctions (Yamada et al. 2005a;[START_REF] Heyraud | Contribution of Annexin 2 to the Architecture of Mature Endothelial Adherens Junctions[END_REF]. These results suggest that annexin A2 modulates the association of junctional proteins with cholesterol-enriched domains. The involvement of annexin A2 in anchoring structures to the cholesterol-enriched membrane domains was supported by experiments showing that clusters of AnxA2 and CD44 in membrane "rafts" favours the directionality of actin bundles in mammary epithelial cells [START_REF] Oliferenko | Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton[END_REF].

Annexin A2 can be phosphorylated in residue Tyr23, and it is also phosphorylated by PKC on serine residues in a calcium and phospholipid dependent manner [START_REF] Johnsson | Functionally distinct serine phosphorylation sites of p36, the cellular substrate of retroviral protein kinase; differential inhibition of reassociation with p11[END_REF][START_REF] Khanna | Phosphorylation of p36 in vitro by protein kinase C[END_REF]) (for review [START_REF] Grindheim | Protein phosphorylation and its role in the regulation of Annexin A2 function[END_REF]). Tyrosine phosphorylation of AnxA2 has been involved in the regulation of cofilin-dependent actin cytoskeletal dynamics [START_REF] Graauw | Annexin A2 Phosphorylation Mediates Cell Scattering and Branching Morphogenesis via Cofilin Activation[END_REF]). Jost and Gerke [START_REF] Jost | Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexin II[END_REF] showed that the principal sites of serine phosphorylation are Ser11 and Ser25 on the AnxA2 N-terminal domain. Moreover, PKC induced phosphorylation has been involved in chromaffin granules membrane fusion [START_REF] Regnouf | In vitro" phosphorylation of annexin 2 heterotetramer by protein kinase C. Comparative properties of the unphosphorylated and phosphorylated annexin 2 on the aggregation and fusion of chromaffin granule membranes[END_REF] and in mRNA binding [START_REF] Grindheim | Effect of Serine Phosphorylation and Ser25 Phospho-Mimicking Mutations on Nuclear Localisation and Ligand Interactions of Annexin A2[END_REF]. However, the role of AnxA2 phosphorylation during cell junction formation has not been determined.

In this work, we studied AnxA2 movements and looked for its partners during epithelial cell differentiation. The results indicate that actin, caveolin 1 and E-cadherin are the principal partners of annexin A2-S100A10 in epithelial cells and that serine phosphorylation of the Nterminal domain does not strongly affect epithelial differentiation.

Materials and Methods

Materials

DMEM with and without Ca 2+ and foetal calf serum were from Life Technologies Inc. Rabbit anti human AnxA2 was produced in the laboratory. Mouse monoclonals anti-human S100A10 and anti-flotillin 2, rabbit anti-caveolin 1 and rat monoclonal anti-E-cadherin were from BD biosciences. Goat anti-actin was from Santa Cruz and rat monoclonal anti-ZO1 was a kind gift of M. Maurice (INSERM, CHU Saint Antoine, Paris, France). Secondary antibodies for immuno-fluorescence and WB (anti-mouse, -rabbit and -rat coupled to FITC, CY3 and CY5) were from Jackson ImmunoResearch and Sigma. Restriction enzymes SmaI and T4 polymerase were from Boheringer Mannheim and PpuMI, NheI and T4 DNA ligase from New England Biolabs.

Cell culture

Madin Darby Canine kidney (MDCK) cells type II provided by Dr. M. Maurice were cultured as described in [START_REF] Maniti | Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin[END_REF]. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated foetal bovine serum, penicillin (0.1 i.u./ml) and streptomycin (100 mg/ml). MDCK cells were grown at 37°C in a 5 % CO2/air atmosphere. The medium was changed every two days. Caco2 cells were cultured as described in [START_REF] Ayala-Sanmartin | Homodimerization of Annexin 2. Role of the N-terminal tail and modulation of membrane aggregation properties[END_REF]). Briefly the cells were maintained in DMEM supplemented with 20% heat-inactivated foetal bovine serum, 1% non-essential amino acids, 0.1 i.u./ml penicillin and 100 mg/ml streptomycin. Caco2 cells were grown at 37°C in a 10% CO2/air atmosphere. The medium was changed every day.

For calcium-induced differentiation and de-differentiation strategy we followed the protocol described in [START_REF] Illien | Lipid organization regulates annexin A2 Ca(2+)-sensitivity for membrane bridging and its modulator effects on membrane fluidity[END_REF]. To obtain differentiated epithelial MDCK cells, the cultures were collected 2 days after confluence. Undifferentiated cells were obtained using the Ca 2+ depletion strategy [START_REF] Hansen | Molecular Mechanism for Orienting Membrane and Actin Dynamics to Nascent Cell-Cell Contacts in Epithelial Cells[END_REF]. Briefly confluent MDCK cultures were incubated in a Ca 2+ -free medium for two days. This medium was prepared with Ca 2+ -free DMEM and serum dialyzed against PBS.

Construction and cell transfection of AnxA2-GFP wild type and phosphorylation mutants.

AnxA2-GFP expression vector pEGFP-C1-AnxA2 was a kind gift of Drs Stephen Moss and Matthew Hayes (University college London, UK) [START_REF] Merrifield | Annexin 2 has an essential role in actin-based macropinocytic rocketing[END_REF]. The N-terminal phosphorylation site mutants of AnxA2-GFP were obtained from the previously constructed pCDNA3 vectors containing the S11A-S25A and S11E-S25E replacements [START_REF] Ayala-Sanmartin | N-Terminal Domain of Annexin 2 Regulates Ca(2+)-Dependent Membrane Aggregation by the Core Domain: A Site Directed Mutagenesis Study[END_REF]. The restriction fragments containing the mutations were used to replace the wt residues of the pEGFP-C1-AnxA2 plasmid. Briefly, the pCDNA vectors containing the annexin A2 phosphorylation mutants were cut with the restriction enzymes SmaI (position 907) and PupMI (position 1778). The resulting 870 bp fragment was purified by agarose gel electrophoresis. The pEGFP-C1-AnxA2 was cut with the restriction enzyme NheI (position 591) and after purification, the sticky ends were filled with the T4 polymerase to be compatible for blunt end ligation with a SmaI fragment. The vector was cut with PpuMI (position 1433) and the fragment containing the vector part of the plasmid was purified by agarose gel electrophoresis. The vector and the mutant containing fragments were ligated (DNA ligase) and the products were transfected in E. coli DH5α strain by the standard calcium transfection method. The absence of undesired mutations was verified by DNA sequencing. MDCK and Caco2 cells were transfected by the standard lipofectamine (Thermo Fisher) protocol.

S100A10 affinity column.

S100A10 protein was purified as previously described [START_REF] Ayala-Sanmartin | Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca(2+)-induced membrane bridging[END_REF]. Four mg of S100A10 protein were attached to a HiTrap NHS activated HP (Sigma-Aldrich) column (1 ml) as recommended by the furnisher. For control experiments a "void" column was used. The control column was treated and blocked as the S110A10 containing column but in the absence of the protein. Before the cell extract loading, and to avoid unspecific protein binding, the columns were saturated in PBS pH 7 with PVP 1%. The samples for control and S100A10 affinity columns were prepared as follows. Cells from two Petri dishes (10 cm diameter) were lysed in PBS added with protease inhibitors (Sigma Aldrich) cocktail with or without calcium by passing them through needles (18G, 23G and 26G successively). Then the samples were centrifuged (800g) for 10 minutes and the supernatant was sonicated and passed through a 45 µm filter. Then, the samples were passed through the column in closed circuit overnight in PBS with or without calcium. Columns were washed with PBS buffer and the bound proteins were recovered by elution with Glycine 0.1 M pH 2.5. Elution fractions (0.5 ml) were neutralized with Tris 1 M pH 9. The column was washed with PBS containing sodium azide and conserved at 4°C.

Immunofluorescence and confocal microscopy.

Cells were cultured on glass cover-slips in 24 well plates as described in [START_REF] Illien | Lipid organization regulates annexin A2 Ca(2+)-sensitivity for membrane bridging and its modulator effects on membrane fluidity[END_REF].

The cells were washed with PBS, and fixed with paraformaldehyde (3.7% ) in PBS for 15 minutes at room temperature [START_REF] Ayala | Developmental and regional regulation of rab3: a new brain specific "ras-like" gene[END_REF]). Then they were washed three times with PBS, NH4Cl 50 mM and post-fixed and permeabilized with cold methanol (-20°C) for 1 minute and washed as before. Fixed cells were saturated with PBS, NH4Cl 50 mM, BSA 1%

for 20 minutes at room temperature. The first antibodies were added at the right dilution during 1.5 hours at room temperature followed by three washes in saturation solution. The secondary antibodies were incubated and washed as the first antibodies. Finally, the coverslips were dried and mounted in Mowiol (Sigma-Aldrich). The fluorescence confocal images were acquired with a TCS SP2 laser-scanning spectral system (Leica, Wetzlar, Germany) attached to a Leica DMR inverted microscope at the specific excitation wavelengths and appropriate emission channels for each fluorophore. Optical sections were recorded with a 63/1.4 or 100/1.4 immersion objectives.

Protein partners identification by mass spectrometry.

The pull down proteins from S100A10 affinity columns were identified by mass spectrometry. After polyacrylamide gel electrophoresis of column fractions, the coomassie blue-stained bands of interest were cut from gels and processed by trypsin digestion for identification by MS as described [START_REF] Munera | Recruitment and membrane interactions of host cell proteins during attachment of enteropathogenic and enterohaemorrhagic Escherichia coli[END_REF]. The proteins were identified by peptide mass fingerprinting using MALDI-TOF-MS. Peptide sequences were obtained by MS/MS. The data was analysed by using the MASCOT search algorithm. Molecular weight search (MOWSE) scores attained for individual protein identifications were inspected manually and considered significant only if at least two peptides were matched for each protein. The MS identification was performed for two independent extracts and for each cell type and differentiation state (differentiated and undifferentiated).

Sucrose density gradients.

Sucrose solutions at 20% and 50% (W/V) were prepared in buffer Hepes 40 mM pH 7, KCl 30 mM EGTA 1 mM, and free Ca 2+ 0.2 mM. The gradients were prepared in 12 ml polypropylene Beckman tubes with a mixer (Amersham) and the help of a peristaltic pump.

The cell extracts were laid on the top of the gradients and centrifuged 35 000 g during 16 hours at 4°C in a Beckman rotor SW41.Ti. 24 fractions (0.5 ml) were collected. The refraction index of all fractions was measured with a refractometer and their density was calculated with a reference table [START_REF] Evans | Preparation and Characterisation of Mammalian Plasma membranes[END_REF].

Miscellaneous procedures.

Standard protein quantification, gel electrophoresis and western blot were performed as described in [START_REF] Borot | Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells[END_REF]. DNA purification for AnxA2-GFP mutants construction and transfections was performed as described previously [START_REF] Ayala-Sanmartin | Stability of ColE1-like and pBR322-like plasmids in Escherichia coli[END_REF]. The statistical analysis; unpaired, two-tailed t-test was performed with GraphPad Prism software.

Results

Annexin A2 expression before and after epithelial cell differentiation.

To characterise AnxA2 expression in epithelial cells, we performed cell cultures of MDCK cells (kidney) and Caco2 cells (intestinal epithelium). We plated 50 000 dissociated MDCK cells or 100 000 Caco2 cells in 60 mm Petri dishes and recovered the cultures after 2, 4, 6, 8 and 10 days for MDCK and after 3, 7, 11, 16 and 21 days for Caco2 cells. The cells were lysed and the protein content was quantified by BCA.

Annexin A2 co-localizes with E-cadherin in adherent junctions

Yamada and collaborators have shown that AnxA2 was involved in adherens junctions formation (Yamada et al. 2005a). Therefore we followed its localization during MDCK cellcell contacts formation. We plated dissociated MDCK cells at different cell densities (2 000 to 30 000 cells per well) and fixed them four days later. Fig 2A shows clear co-localization of AnxA2 with E-cadherin at the level of early cell contacts as well as in cells highly confluent and differentiated (Fig S2A). Lee and collaborators reported that AnxA2 co-localized with tight junctions in a MDCK II cell line [START_REF] Lee | Annexin A2 heterotetramer: role in tight junction assembly[END_REF]) . However, we saw no localization with the marker of tight junctions ZO1 during in vitro cell differentiation (Fig 2B andS3).

The ZO1 signal was very thin and close to the membrane zones enriched in AnxA2 but not in contact with them.

To study the distribution of annexin A2, S100A10 and E-Cadherin, we performed triple antibody labelling. As shown in Fig 2C andS2B, the previously observed co-localization of AnxA2 with E-cadherin was confirmed and the co-localization of S100A10 with both proteins were evidenced suggesting that AnxA2 is in the heterotetrameric form.

Expression of AnxA2-GFP during epithelial cell differentiation.

An MDCK cell line expressing the chimeric protein AnxA2-GFP was obtained. The expression level of the AnxA2-GFP chimeric protein was evaluated. Cell extracts were subjected to an affinity column containing the S100A10. Western blot revealed that the expression of AnxA2-GFP chimeric protein was 35% and the endogenous protein of 65% (Fig S4A). This fact rise the possibility that the total annexin A2 content in the transfected cells would be higher compared to the wild type cells. However it is also possible that the cell regulates the expression of the endogenous protein in order to maintain normal total annexin A2 levels. To characterize the AnxA2-GFP calcium dependent movement to the membranes we treated the cells with the calcium ionophore ionomycin. After 10 minutes of ionophore addition, the association of the protein with the plasma membrane due to the calcium entry into the cells was clearly observed (Fig S4B), as previously reported with other pore forming peptides [START_REF] Lamaziere | Nonmetabolic membrane tubulation and permeability induced by bioactive peptides[END_REF]. We also obtained a Caco2 cell line expressing AnxA2-GFP.

The chimeric protein presented a similar behaviour during epithelial cell differentiation compared to MDCK cells and showed strong co-localization with S100A10 (Fig S5 ).

We followed the expression of AnxA2-GFP, S100A10 and E-cadherin by confocal microscopy and observed co-localization of AnxA2-GFP with S100A10 and E-cadherin during junction formation and in differentiated cells (Fig 3).

Annexin A2 localization during differentiation and de-differentiation of MDCK cells.

In order to obtain a high quantity of cells in the undifferentiated state, we performed cell cultures at a high density in the absence of calcium by using the calcium switch protocol. We characterized the cellular movements of AnxA2, S100A10 and E-cadherin during differentiation and de-differentiation. As shown in Fig S6, the undifferentiated cells express the 3 proteins mainly in the cytosol. After addition of calcium the cellular junctions established and the three proteins move from the cytosol to the plasma membrane at the level of cell-cell contacts. Co-localization of AnxA2 with S100A10 and E-cadherin was observed.

Annexin A2-S100A10 partners in differentiated and undifferentiated Cells.

To find out AnxA2 partners in MDCK and Caco2 cell lines, we used a S100A10 affinity column. The elution fractions were collected and subjected to gel electrophoresis. To confirm the S100A10 binding specificity of the pull down proteins, we compared the bands with those of a control (void) column. Figure S7 show several examples of gels indicating that the S100A10 column specifically pull down S100A10 and AnxA2 binding proteins. After gel electrophoresis we identify the bands of interest by mass spectrometry. Two independent experiments were performed for each cell line. Several annexin A2-S100A10 partner candidates were identified. The proteins identified with the best scores were β-actin, heat shock 70 cognate protein (HSC70) and α-Enolase 1 from MDCK cells, and β-actin, β-tubulin and α-Enolase 1 from Caco 2 cells.

The partner candidates from the affinity column were verified by western blot. Three independent experiments were performed. As shown in Fig 4, actin was detected as a S100A10-AnxA2 partner in differentiated and undifferentiated cells. We were unable to detect HSC70, Enolase 1 and tubulin by western blot. On the contrary, caveolin 1 and flotillin 2, two proteins which are, similarly to AnxA2, associated to cholesterol enriched membrane domains, were pulled down with the S100A10 affinity column. Caveolin 1 was detected in differentiated and undifferentiated cells and flotillin 2 only in undifferentiated cells.

We also performed a study of protein co-migration in sucrose density gradients. As shown in Fig 5, in undifferentiated cells, AnxA2 and S100A10 were found in high quantity in fractions of density 1.09 and 1.17 and in minor quantity in fraction of density 1.15. The actin and AnxA2 profiles were very similar. E-cadherin was found in fraction 1.17, 1.15 and 1.09. On the contrary, flotillin 2 was present mainly in the 1.17 fraction and caveolin 1 was present in fractions of density from1.11 to 1.16. After cell differentiation, we observed that AnxA2 in the 1.09 and 1.17 fractions diminished but its amount increased in fraction 1.13 giving a plateau 1.13 to 1.19. S100A10 was present in fractions 1.08, 1.15. 1.17 but was absent in the 1.13 fraction. The actin profile was similar to that of AnxA2 and E-cadherin was also enriched in the zone 1.11 to 1.19. Flotillin 2 (1.17) and caveolin 1 (1.13 to 1.17) show only small changes in density profile after cell differentiation. Overall, the data show that actin and E-cadherin co-migrate with AnxA2 before and after cell differentiation. On the contrary after cell differentiation, the degree of co-migration of AnxA2 and caveolin 1 strongly increases. In order to study the role of serine phosphorylation on the N-terminal domain of AnxA2, we constructed chimeric AnxA2-GFP double mutants on serine residues 11 and 25. We replaced serine by alanine residues to avoid phosphorylation (S11A-S25A) and by glutamic acid residues to mimic "permanent phosphorylation" (S11E-S25E). The physicochemical characterisation of the mutants showed that they are able to bound S100A10 in vitro [START_REF] Ayala-Sanmartin | N-Terminal Domain of Annexin 2 Regulates Ca(2+)-Dependent Membrane Aggregation by the Core Domain: A Site Directed Mutagenesis Study[END_REF]. Fig 8 shows that the mutants did not perturbed the formation of junctions neither the co-localization of AnxA2-GFP with E-cadherin, actin and caveolin 1.

Discussion

In the present paper we studied the localisation of AnxA2, S100A10 and its putative partners during epithelial differentiation of kidney and intestinal cells lines in vitro. MDCK and Caco2 cell lines changed their morphology and underwent full polarization showing formation of adherens and tight junctions and reorientation of cytoskeletal elements [START_REF] Bacallao | The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium[END_REF].

We searched for annexin A2-S100A10 partner proteins during epithelial cell differentiation by analysing the AnxA2 binding proteins in cells before and after cell differentiation. By using immunofluorescence, mass spectrometry and western blot analyses after S100A10 affinity column separation, we had identified several AnxA2 partner candidates. We also performed co-immunoprecipitation experiments but unfortunately without success. Probably because the interaction of annexin A2 with membranes and partners diminish the availability of annexin A2 surface for antibody recognition. We also performed protein crosslinking of cell extracts before column affinity pull down but it was also difficult to obtain accurate MS data. The putative partner candidates detected after S100A10 affinity column can be considered as partners of the tetrameric form of the protein (Annexin A2-S100A10) because in the column, the most important protein associated to S100A10 is AnxA2 (Fig S7). The association of some putative candidates obtained by MS after column affinity such as the αenolase-1, the chaperon protein HSC70 and tubulin were not validated by other techniques such as immunofluorescence and sucrose gradient separation. However, it have been reported that enolase-1 is present in caveolae and co-precipitates with Cav-1 and AnxA2 [START_REF] Zakrzewicz | The interaction of enolase-1 with caveolae-associated proteins regulates its subcellular localization[END_REF]. These data support the interaction observed herein by affinity pull-down. Three proteins were clearly associated to AnxA2: E-cadherin, actin and caveolin 1.

Cell junctions are very complex structures [START_REF] Vogelmann | Fractionation of the Epithelial Apical Junctional Complex: Reassessment of Protein Distributions in Different Substructures[END_REF]. The adherens junctions are calcium-dependent molecular complexes necessary for epithelial cell differentiation. The role of AnxA2 during cadherin junctions formation has been demonstrated in epithelial and endothelial cells (Yamada et al. 2005a;[START_REF] Heyraud | Contribution of Annexin 2 to the Architecture of Mature Endothelial Adherens Junctions[END_REF].

The E-cadherin protein associates to catenins that bind the actin cytoskeleton. It has been suggested that the adherens complex is modulated by the sphingomyelin cholesterol-enriched domains in the membranes [START_REF] Bryant | The ins and outs of E-cadherin trafficking[END_REF]. Herein, we observed by immunolocalisation and membrane separation by sucrose density gradients that AnxA2 and Ecadherin are associated during the adherens junction formation in kidney (MDCK) and intestinal (Caco2) cells. However, we were unable to detect E-cadherin in affinity columns suggesting that the association of AnxA2 with cadherin could be mediated by other proteins or lipids in the junctional zone.

The actin-AnxA2 binding has been well documented (for reviews see [START_REF] Hayes | Annexin-Actin Interactions[END_REF][START_REF] Grieve | Annexin A2 at the Interface of Actin and Membrane Dynamics: A Focus on Its Roles in Endocytosis and Cell Polarization[END_REF]). In the present work we found an association of annexin A2 and actin by affinity column and corroborate this association by immunofluorescence and sucrose density gradients. The association of annexin A2 with E-cadherin could be mediated by actin.

However the direct association of actin with E-cadherin has been questioned by experiments which demonstrated that catenins, are not associated to actin and cadherin at the same time [START_REF] Drees | α-Catenin Is a Molecular Switch that Binds E-Cadherin-β-Catenin and Regulates Actin-Filament Assembly[END_REF]Yamada et al. 2005b). In such a case, another molecule could be responsible for the association with the junctional complex. This partner could be a protein but we did not exclude that the lipids in the form of particular membrane domains enriched in cholesterol, phosphatidylserine or PI2P play an important role in this process.

Caveolin 1 was the third identified partner. This protein is considered to be present in cholesterol enriched membrane domains [START_REF] Murata | VIP/caveolin is a cholesterol-binding protein[END_REF][START_REF] Harder | Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains[END_REF] and has been shown to co-localize with AnxA2 [START_REF] Corvera | Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterol[END_REF][START_REF] Uittenbogaard | Cholesteryl Ester Is Transported from Caveolae to Internal Membranes as Part of a Caveolin-Annexin II Lipid-Protein Complex[END_REF][START_REF] Liao | Annexin A2-modulated proliferation of pulmonary arterial smooth muscle cells depends on caveolae and caveolin-1 in hepatopulmonary syndrome[END_REF]. In spite of the fact that a study concerning the interaction of cav-1 and annexin A2 has been retracted [START_REF] Smart | Annexin 2-caveolin 1 complex is a target of ezetimibe and regulates intestinal cholesterol transport[END_REF], three other reports and our own study demonstrate a relationship between both proteins. In endothelial cells caveolin 1 and AnxA2 bound to low density cholesterol-enriched membranes after formation of cellular junctions [START_REF] Corvera | Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterol[END_REF]. Moreover, it is known that the presence of cholesterol in membranes increases the calcium sensitivity for AnxA2 membrane binding [START_REF] Ayala-Sanmartin | Cholesterol enhances phospholipid binding and aggregation of annexins by their core domain[END_REF][START_REF] Ayala-Sanmartin | Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca(2+) concentration[END_REF]. Therefore, the Caveolin-AnxA2 association seems to depend on membrane lipid composition.

Overall the data shows that annexin A2-S100A10 is able to associate to molecular complexes containing actin, caveolin 1 and flotillin 2 before epithelial differentiation and to complexes containing E-Cadherin, actin and caveolin 1, but not flotillin 2 after cell differentiation. It is of interest to note that after cell differentiation, AnxA2 and other proteins move to compartments of light density and that the increase of cholesterol at the level of the plasma membrane could be responsible for this change in density. At present we know that annexin A2 can bind actin but it is less clear whether the association with the other proteins is directly or indirectly mediated by other proteins or membrane domains. Considering that AnxA2 is essentially a membrane phospholipid binding protein sensitive to cholesterol content, we must consider the membrane domains as mediators of these complex interactions. In fact, it has been shown that membrane clusters of AnxA2 and CD44 depend on cholesterol and favour the direction of actin bundles to these membrane domains [START_REF] Oliferenko | Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton[END_REF]. It is of interest to note that there is a correlation between the actin filament membrane attachment and plasma membrane ordered domain formation [START_REF] Dinic | Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains[END_REF]. In smooth muscle cells, a membrane domain separation of stiff actin binding and flexible caveolar domains that bind annexin proteins at different calcium concentrations have been observed [START_REF] Babiychuk | Annexins in cell membrane dynamics. Ca(2+)-regulated association of lipid microdomains[END_REF]. A similar behaviour could be present in epithelial cells in which AnxA2 bind to different membrane domains (caveolar, actin rich cell junctions) at different calcium concentrations and/or with different membrane phospholipids. For example, PI2P which is a ligand of AnxA2, is involved also in actin filaments reorganisation and changes in cell shape [START_REF] Takenawa | Phosphoinositides, key molecules for regulation of actin cytoskeletal organization and membrane traffic from the plasma membrane[END_REF]. Martin-Belmonte and collaborators showed that annexin A2 expression in the apical surface of MDCK cells overlapped with actin and suggest that this is an important event during the establishment of apical membrane identity [START_REF] Martin-Belmonte | PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42[END_REF]. Moreover, AnxA2 is targeted to PI2P sites of the membrane enriched in cholesterol where actin accumulates (Rescher et al. 2004;[START_REF] Gokhale | Phosphoinositide specificity of and mechanism of lipid domain formation by annexin A2-p11 heterotetramer[END_REF] and the localisation of AnxA2 to cell-cell contacts and actin re-organisation seemed to require PI-3 kinase activity suggesting the participation of lipids and not only proteins in these complex cellular processes [START_REF] Hansen | Molecular Mechanism for Orienting Membrane and Actin Dynamics to Nascent Cell-Cell Contacts in Epithelial Cells[END_REF]. Secondly, membrane cholesterol seems to play an important role because its decrease abolished both, the AnxA2 movement to the membrane and the concentration of E-cadherin to the nectin cell-cell contact sites (Yamada et al. 2005a;[START_REF] Heyraud | Contribution of Annexin 2 to the Architecture of Mature Endothelial Adherens Junctions[END_REF]).

In the present study we were unable to observe differences in behaviour and localisation of annexin A2 serine phosphorylation mutants. However, other studies showed that Tyrosine phosphorylation is an important event in actin mediated changes in cell morphology associated with the control of cell adhesion in BHK cells [START_REF] Rescher | Tyrosine phosphorylation of annexin A2 regulates Rho-mediated actin rearrangement and cell adhesion[END_REF]) and in nicotine induced chromaffin cell secretion [START_REF] Gabel | Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells[END_REF]. New experiments designed to study the dynamics of serine phosphorylation-dephosphorylation must be performed to deeply investigate the role of annexin A2 serine phosphorylation during epithelial cell differentiation.

In conclusion, the association of AnxA2 with other proteins and lipids is a very complex field to be developed accurately. The participation of AnxA2 in different cellular processes seems to be mediated by different proteins but, the association with the membranes lipids organized in different domains play certainly a very important role. Therefore, the development of new experimental approaches such as lipidomics of Annexin A2 membrane binding sites must be considered in the future research. Confocal images of AnxA2-GFP transfected MDCK cells after four days in culture plated at different densities. AnxA2, S100A10 and caveolin 1 clearly co-localize in lateral membranes involved in cell contacts (top). Actin and AnxA2 co-localizes in cell membranes of isolated cells and in contacts (middle). They co-localize with E-cadherin (bottom). Bars, 20 µm.

  Fig 1A shows that MDCK cell line arrive at confluence on day 6 and Caco2 cells at day 11. Microscopy observation showed that at day 2 (and 3) the cultures showed small cell aggregates, at day 4 (and 7) the cultures were close to confluence and at day 6 (and 11) they were fully confluent (data not shown). Specific protein expression was analysed by western blot. Fig 1 shows that AnxA2 and S100A10 expression increases slightly during in vitro epithelial cell differentiation. However, S100A10 expression increase was not statistically significant in MDCK cells. Cellular localization of AnxA2 and its principal partner S100A10 were followed by immunofluorescence and confocal microscopy. Fig S1 shows a strong co-localization of both proteins in MDCK cells during all steps of epithelial differentiation. However, in fully differentiated cells, we observed an enrichment in AnxA2 in the apical pole of the cells and that both proteins co-localize approaching the apical region. In the basolateral region, annexin A2 was probably present also in monomeric form because S100A10 showed low expression in this zone (Fig S1).

Flotillin 2

 2 seemed not to be sensitive to cell differentiation and AnxA2 movements. Cellular localisation of Annexin A2-S100A10 partners candidates. Immunofluorescence experiments were performed to characterize the localization of AnxA2 partner candidates during epithelial cell differentiation. In MDCK cells, no clear colocalization with tubulin was observed. HSC70 does not co-localizes with AnxA2 in membranes involved in cell-cell contacts and only small co-localization was observed in peripheral membranes of undifferentiated cells (Fig S8A). On the contrary we observed clear co-localization of caveolin 1 with AnxA2 and S100A10 (Fig 6) in the cell-cell contact zones. We also performed cellular localization experiments with the AnxA2-GFP cell line. As for wild type MDCK cells, we did not observe localization of AnxA2 with tubulin (Fig S8B). On the contrary, we observed co-localization with S100A10, actin, caveolin 1 and E-cadherin (Fig 7).

  INSERM, France) for helping with AnxA2-GFP cloning in MDCK and Caco2 cells. Anne Marie Juniaux (INSERM, France) for technical assistance.

Figure 1 .

 1 Figure 1. Annexin A2 and S100A10 expression during epithelial cell differentiation. A; Kinetics of cell culture growth of MDCK and Caco2 cell lines followed by total protein expression as a function of time. B; Expression of annexin A2 and S100A10 by western blot. Each well contains contained 7.5 µg of MDCK extracts and 15 µg of Caco2 extracts. Quantitative analysis of protein expression for MDCK cells (C) and Caco2 cells (D). Gel quantification was performed by normalizing the bands intensities in percent; the strongest band for each gel being the 100% of expression. This normalization allows the statistical comparison of different experiments. Notice the slight increase in annexin A2 and S100A10 expression during cell contacts formation. Means ± SEM from 3 to 4 experiments. * = P<0.05, ** =P<0.01, continuous and dotted lines for AnxA2 and S100A10 respectively.

Figure 2 .

 2 Figure 2. Annexin A2, E-cadherin, ZO1 and S100A10 expression in MDCK cells.Confocal images of MDCK cells after four days in culture and plated at a low density (5 000 cells per well). Annexin A2 and E-cadherin are co-localized in the lateral membrane (A). Annexin A2 and the

Figure 3 .

 3 Figure 3. AnxA2-GFP, S100A10 and E-cadherin expression in MDCK cells.Confocal images of MDCK cells after 3 days in culture plated at 5 000 (A) and 20 000 cells per well (B). AnxA2-GFP, S100A10 and E-cadherin are co-localized in the lateral membranes. Bars, 20 µm.

Figure 4 .

 4 Figure 4. S100A10-AnxA2 binding proteins in undifferentiated and differentiated epithelial cells.Western blots of cellular fractions from undifferentiated and differentiated epithelial MDCK cells eluted from S100A10 affinity (S100A10) and control (ctrl) columns.

Figure 5 .

 5 Figure 5. Protein profiles on sucrose density gradients in undifferentiated and differentiated MDCK cells. (A), non-differentiated cells. (B), differentiated cells. Annexin A2 (black continuous line •), S100A10 (black dotted line ○), actin (red ▲), E-cadherin (blue ■), caveolin 1 (green continuous line ♦) and flotillin 2 (green dotted line ◊). The profiles are means of 2 to 6 independent experiments.

Figure 6 .

 6 Figure 6. Annexin A2, S100A10 and caveolin expression in MDCK cells.Confocal images of MDCK cells after four days in culture and plated at a low density (5 000 cells per well). AnxA2, S100A10 and caveolin 1 are clearly co-localized in the lateral membranes involved in cell contacts. Bars, 20 µm.

Figure 7 .

 7 Figure 7. Annexin A2, S100A10, actin, E-cadherin and caveolin 1 expression in AnxA2-GFP MDCK cells.

Figure 8 .

 8 Figure 8. Localization of E-cadherin, caveolin 1 and actin in AnxA2-GFP phosphorylation mutants. The cellular localization of the AnxA2 phosphorylation mutants S11A-S25A (AA) and S11E-S25E (EE) is similar to the wild type protein. The cellular localization of actin, E-cadherin and caveolin was not modified by the presence of mutant proteins. Bars, 20 µm.

Acknowledgments

Dr Stephen Moss and Dr Matt Hayes (University college London, UK) for AnxA2-GFP expression vector. Dr Michelle Maurice (INSERM, CHU Saint Antoine, Paris, France) for ZO1 antibody and MDCK cells. Drs Christelle Lenoir and JL Delaunay (CHU Saint Antoine,

Figure legends


Nahum Maso

Cecile Marcelot

• Laëtit Ia Fabre

Jean-Baptiste Fruhauf

Pascal Dufour

Christophe Tenailleau

Bénédicte Warot-Fonrose

Etienne Snoeck

Sophie Guillemet-Fritsch

Microstructure and electrical properties of (Ba 0 . 6 Sr 0 . 4 ) 0 .ssBio.1 TiO 3 ceramics prepared by single-step, liquid-phase, solid-state reactive sintering

Keywords: Liquid-phase, Solid-state reactive sintering, Electrical properties, CBao.6Sr0.4)o_85Bio., TiO 3 ceramics

est destinée au dépôt

Introduction

Barium titanate, BaTiO 3 , and strontium titanate, SrTiO 3 , are the parent materials for a range of commercial electroceramic devices. They form a continuous solid solution [START_REF] Mcquarrie | [END_REF]2] with general formula Ba 1 _ x Sr x TiO 3 : 0 :5 x :51. On increasing x, the crystal structure at room temperature changes from tetrag onal to cubic at x::::: 0.30; the temperatures of rhombohedral orthorhombic, orthorhombic-tetragonal and tetragonal-cubic phase transitions of BaTiO 3 decrease linearly and coalesce at x::::: 0.80 to give a single transition with T max at ~ 115 K; a relaxor behaviour is observed on further increasing x; [Refs 3 -7] and, finally, SrTiO 3 is regarded as a quantum paraelectric. ln addition, Ba 1 _ x Sr x TiO 3 ceramics may also contain a wide variety of dopants in or der to further control the temper ature of the maximum of the permittivity vs. temperature pro file as well as the temperature coefficient of capacitance. ln par ticular, in Bi-doped Ba 1 _ x Sr x TiO 3 ceramics, a relaxor be haviour near room temperature is observed for low Sr and Bi content, i.e. (Ba 1x SrJ 1-uyBi y TiO 3 with 020 :Sx :5 0.40 and y:50.10 [Refs 8 -13].

Commonly, Ba 1 _ x Sr x TiO 3 ceramics are obtained in a two step process which involves, first, decarbonation and initial reaction of the raw powder reactants, i.e. BaCO 3 , SrCO 3 and TiO 2 , at low temperature, e.g. ~1000 °C, and, next, sintering of the ceramic body at temperature above e.g. ~1300 °C. ln addition, sintering agents, e.g. Si 0 2 , or an excess of one of the reactant, e.g. TiO 2 , are also added into the formulation since they promote the formation ofa liquid phase which enhances the reaction and grain growth during sintering. The use of a liquid phase during sintering is regarded in the literature as Liquid-Phase Sintering [14] (LPS) or Solid-State Reactive Sintering [15] (SSRS).

The company SCT (Société des Céramiques Techniques) is interested on manufacturing (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 ceramics capacitors in a single-step, reproducible approach. In order to do so, we have used the LPS/SSRS approach taking advantage of the low melting temperature of Bi 4 Ti 3 O 12 . The influence of the sintering temperature on the chemical composition, microstructure and electrical properties are reported here.

Experimental

The starting raw material was a granulated (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 powder provided by Ferro. The powder was first dispersed in Darvan C. Then, 1 wt.% PEG 4000 and 2 wt.% Poval 205C were added and the resultant solution was atomised at 315 °C. After powder conditioning, pellets were fabricated by uniaxial compaction at 600 bar of pressure, repressed isostatically at 1000 bar, placed on a bed of sacrificial powder (MgO doped ZrO 2 ) and sintered in air at several temperatures as follows: 1150 °C for 6 h; 1250 °C, 1300 °C and 1350 °C for 2 h. After sintering, the edge and the faces of the pellets were polished ~0.05 mm and ~0.30 mm, respectively, in order to remove any contamination from the sacrificial powder and/or furnace. The final dimensions of the pellets were 4.75-5.15 mm thick and 20.00-20.20 mm diameter. For statistical purposes, three pellets were characterised for each sintering temperature.

The density of the pellets was determined by the Archimedes method, using water as the immersion liquid. Pellets were first dried at 180 °C overnight, then cooled down to room temperature into a desiccator under vacuum before the density measurement.

Samples were analyzed by X-ray powder diffraction (XRD) at room temperat ure using a Bruker D4 Diffractometer CuKα radiation. Lattice parameters were determined by Le Bail method for reflections in the range 20 < 2θ < 100°using the software GSAS. X-Ray Fluorescence (XRF) used a Bruker S2 Ranger.

For dilatometry, a pellet, ~10 mm thick and ~6 mm diameter, was heated at 1400 °C in air, ~20 cm 3 min 1 , at 2 °C min 1 in a Setaram Setsys Evolution 16/18. For thermogravimetry (TG), ~100 mg of powder were heated at 1400 °C in air, ~100 cm 3 min 1 , at 2 °C min 1 in a Netzsch STA 409 C thermobalance.

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed with a JEOL JSM6510LV and FEI CM20, respectively, both equipped with Oxford EDS detectors.

Dielectric and impedance data were collected with an Agilent 4294A impedance analyser over the frequency range 40 Hz to 1 MHz, with an ac measuring voltage of 100-500 mV and over the temperature range -60 °C to 650 °C on heating and cooling cycles. For dielectric measurements pellets ~5 mm thick and ~20 mm diameter were analysed whereas impedance measurements used slabs of ~1 to 2.5 mm thick and ~100 mm 2 of area which were obtained by cutting the pellets perpendicularly to both faces. Electrodes were fabricated with Ag paint that was annealed at 760 °C. Data were corrected for the overall pellet geometry and, consequently, resistivity and permittivity data are reported in units of Ω cm and F cm 1 , respectively.

Results and discussion

Figure 1(a) shows the mass loss of the green powder and the linear contraction of a green pellet as a function of temperature. Between room temperature and ~400 °C, a weight loss of ~4 % occurred which was associated with evaporation of water and combustion of organic matter, i.e. binders. Between ~700 °C and ~1050 °C, no noticeable weight loss was observed whereas, above ~1050 °C, weight loss occurred gradually and became pronounced above ~1250 °C. No shrinkage was observed up to ~900 °C whereas, above ~900 °C, it became pronounced over the temperature range ~1075-1250 °C. An expansion was finally observed above ~1300 °C.

XRD performed on the raw powder of (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 used to prepare the ceramics contained a mixture of BaTiO 3 , SrTiO 3 and Bi 4 Ti 3 O 12 , Fig. 1(b). After sintering in the temperature range from 1250 °C to 1350 °C, ceramics were phase pure (Ba,Sr,Bi)TiO 3 whereas those sintered at 1150 °C contained, in addition to the main phase (Ba,Sr,Bi)TiO 3 , a small amount of unreacted SrTiO 3 , Fig. 1(c) and(d). Hereafter, we focus on the phase-pure samples determined by XRD.

Lattice parameters were a ≈ 3.961(1) Å and V ≈ 62.15(1) Å 3 in agreement with the literature data [12].The calculated theoretical density was ~5.79 g cm 3 . The relative density of the pellets (Table 1) decreased from ~98.1(2) % to ~96.9(3) % on increasing the sintering temperature from 1250 to 1350 °C, in agreement with pellet expansion observed in the dilatometry data, Fig. 1(a).

The chemical composition analyses by XRF on the surface of the pellets, Table 1, showed a good agreement between the theoretical and experimental concentration for ceramics sintered at 1250 °C. However, on further increasing the sintering temperature, the concentration of TiO 2 and BaO increased, the concentration of SrO remained constant whereas that of Bi 2 O 3 decreased.

Backscattered SEM images of the cross-section of the pellets were carried out on samples sintered from 1250 °C to 1350 °C. All samples showed the same microstructure; for clarity, we only show data for a pellet sintered at 1250 °C. Two microstructurally-and chemically-distinct regions were observed, Fig. 2. The main phase exhibited small, round grains (not shown) and appeared in whitish tones in the backscatter micrographs whereas the secondary phase exhibited large particles and appeared in blackish tones in the backscatter micrographs, Fig. 3. Mapping images revealed that the main phase contained Ba, Sr, Ti and Bi whereas the secondary phase contained essentially Ba and Ti, Fig. 3. According to the chemical analyses by EDS, the secondary phase appeared to be Ba 4 Ti 13 O 30 , a well-known phase present in the BaO-TiO 2 system [16].Since SEM images showed a noticeable presence of secondary phase which was not observed by XRD, these results indicated that the secondary phase appeared to be amorphous.

The estimated grain size of (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 by TEM images was in the range ~0.5 to ~2 μm in the sintering temperature 1250-1350 °C. EDS mapping by TEM on the (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 main phase showed that, independently of the sintering temperature, Sr, Ba and Ti were homogeneously distributed through the grains whereas Bi appears to be homogeneously distributed at 1250 °C but, above 1300 °C, evidence of Bi segregation at the grain boundaries was observed, Fig. 4 (regions 3 and 5) and Table 2. In addition, some Al was also present due to the milling media. We note that no Al was detected by XRF since the weak Al peak in the XRF spectrum could not be resolved from that of the Pd XRF source.

Electrical measurements showed that, independently of the sintering temperature, all samples exhibited a similar behaviour. As a representative set, Figs. 5 and6(a) show the relative permittivity, ε r ', and dissipation factor, tan δ, as a function of the temperature as well as the impedance complex plane plot, Z^vs Z', for a pellet sintered at 1350 °C for 2 h.

Thus, above room temperature, the permittivity decreased on increasing temperature and was frequency-independent whereas, below room temperature, it passed through a The values and errors represent the average and estimated standard deviation, esd, of three pellets † Theoretical mass concentration for (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 : BaO = 36.10%, TiO 2 = 36.87%, SrO = 16.27% and Bi 2 O 3 = 10.76% maximum, was frequency-dependent and showed a relaxor effect, Fig. 5(a). The dissipation factor was frequencydependent and, at 50 Hz, passed through a minimum over the temperature range -20 °C to 40 °C, Fig. 5(b). Table 1 summarises ε r ' at 20 °C and tan δ in the temperature range 10 to 30 °C as well as the TCC in the temperature range -25 °C to 100 °C as a function of the sintering temperature. Both ε r ' and tan δ were approximately independent of the sintering temperature with values around ~1780 and ~0.06 %, respectively. By contrast, TTC increased linearly from ~10 ppm o C 1 to ~21 ppm o C 1 on increasing the sintering temperature from 1250 °C to 1350 °C, probably due to an increase in grain size.

In Z^vs Z', Fig. 6(a), each electrically active region of the sample gave rise to a semicircle (ideally) from which the resistance values, R, were obtained from the intercepts on the Z' axis whereas the permittivity values were obtained by applying 2πfRC = 1 at the maximum of each semicircle. Thus, independently of the sintering temperature, all samples showed two semicircles from which the resistivity values R 1 and R 2 were obtained. From the magnitudes of the associated permittivity values, C 1 represented the bulk permittivity of the sample, e.g. ~45 pF cm 1 at ~350 °C, whereas C 2 was of the order of nanofarads and was attributed to a grain boundary effect. Bulk and grain boundary conductivity data (σ = R 1 ), Fig. 6(b) gave linear Arrhenius plots with similar conductivity and activation energy values independently of the sintering temperature/time. The dc conductivity at room temperature, extrapolated from the Arrhenius plots, was ~10 20 S cm 1 .

The results presented here show that (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 dense ceramics can be obtained in a single-step using stoichiometric amounts of BaTiO 3 , SrTiO [17][18][19][20] and, thus, creates a liquid phase which enhances the reaction. At lower temperatures, where no liquid phase forms, unreacted ceramics are obtained even with sintering times of 6 h, Fig. 1(c,d).

However, the presence of the liquid phase appears to contribute critically to (1) enhance the Bi loss from the ceramic as observed in the TG and XRF data, Fig. 1(a) and Table 1, in particular at temperatures ≥1300 °C, and (2) promote the formation of a secondary phase, Figs. 2 and3, although this is often observed in ceramics prepared by liquid-phase/solid-state reactive sintering whose microstructure consists of solid grains and a solidified liquid network [14] or a secondary phase [21].

Nevertheless, neither the secondary phase nor bismuth loss affect the electrical properties. Thus, independently of the sintering temperature, (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 ceramics exhibit similar values of relative permittivity, dissipation factor and bulk/grain boundary conductivity, Table 1 and Fig. 6(b), which are comparable to those reported in the literature for the same composition prepared in a two-step process [12]. However, among the sintering temperatures studied here, 1250 °C is the best option from the point of view of energy efficiency during manufacturing. These results should, therefore, be of interest to the ceramics industry.

  , 29 rue Jeanne Marvig, BP 94347, 31055 Toulouse Cedex 4, France 3 SCT Soci été des Céramiques Techniques, Zl Ouest Route d'Our sbelille, BP 9, 65460 Bazet, France

Fig. 1

 1 Fig. 1 (a) Mass loss and linear contraction as function of the temperature. XRD patterns of: (b) powder used to prepare the pellets; pellets sintered at (c) 1150 °C for 6 h and (d) 1300 °C for 2 h

Fig. 4 Fig. 2

 42 Fig. 4 Bright field TEM images of ceramics sintered at several temperatures. Areas analysed by EDS are shown

4 Conclusions(

 4 Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 ceramics can been obtained from a mixture of BaTiO 3 , SrTiO 3 and Bi 4 Ti 3 O 12 at 1250 °C with a relative density of ~98 %, ε r ' ~1780, tan δ ~0.07 % and TTC ~10 ppm o C 1 . However, above that temperature, Bi loss from the ceramic is enhanced. Independently of the sintering temperature, (Ba 0.6 Sr 0.4 ) 0.85 Bi 0.1 TiO 3 ceramics show similar bulk and grain boundary conductivities with an estimated dc conductivity at room temperature of ~10 20 S cm 1 .

Fig. 6

 6 Fig. 6 (a) Impedance complex plane plot at 525 °C of a ceramic sintered at 1350 °C and (b) Arrhenius plot of bulk, σ 1 , and grain boundary, σ 2 , conductivity for ceramics sintered at several temperatures. Activation energies, in eV, are noted beside each data set

Table 1

 1 Density, chemical composition, ε r ', tan δ and TTC as function of the sintering temperature

	Temp (°C)	Time (h)	Density (%)	Concentration (mass %) †			ε r '	tan δ (%)	TCC (ppm/°C)
				BaO	TiO 2	SrO	Bi 2 O 3
	1250	2	98.1(2)	35.7(3)	37.6(4)	16.06(5)	10.6(1)	1781(16)	0.07(3)	9.7(5)
	1300	2	97.5(2)	36.2(1)	38.1(7)	16.1(1)	9.7(7)	1790(20)	0.05(3)	14.6(6)
	1350	2	96.9(3)	36.2(2)	39.3(1)	16.1(3)	9.4(1)	1783(22)	0.05(3)	21(1)

Table 2

 2 EDS analyses on areas marked in Fig.4

	Area	Element (at.%)
		Ba	Sr	Bi	Ti	Al
	1	33.8	20.4	3.7	40.7	1.4
	2	32.9	19.4	4.7	42.1	0.9
	3	35.0	17.8	6.3	40.3	0.6
	4	25.6	22.0	4.2	46.8	1.4
	5	23.1	22.1	5.5	47.7	1.6

Acknowledgements This work was supported by Fonds Innovation Recherche du Conseil Départemental des Hautes Pyrénées [grant num ber 14051044].


Bassam Moujalled
email: bassam.moujalled@cerem.fr

Sylvain Berthault

Andrés Litvak

Valérie Leprince

Gilles Frances

Assessment of long-term and mid-term building airtightness durability: field study of 61 French low energy single-family dwellings

Keywords: Airtightness durability, field measurements, building envelope, low-energy house

The French ongoing research project "Durabilit 'air" (2016-2019) aims at improving our knowledge on the variation of buildings airtightness through onsite measurement and accelerated ageing in laboratory controlled conditions. This paper presents the final results of the second task of the project. This task deals with the quantification and qualification of the durability of building airtightness of single detached houses. It is done through field measurement at mid-term (MT) and long-term (LT) scales. We first present the field measurement protocol. For the MT campaign, a sample of 30 new single-detached houses has been selected nationwide. During the study, the airtightness of each building was measured once per year over a 3-year period. A part of this sample (5 houses) was also measured twice per year during two different seasons in order to investigate the impact of seasonal variation. In addition, the air permeability of a window was measured once per year over the 3-year period in 5 houses. The LT campaign was carried out with a second sample of 31 existing single-detached houses constructed during the last 10 years. The airtightness of each house was measured once. A specific measurement protocol was defined after a detailed literature review. The main challenge is to understand the variations of the airtightness and to identify whether it is related to the products/assembly ageing, the maintenance conditions or other factors such as the occupants' behaviour. The protocol is based on the standard ISO 9972 for the measurement method of building air permeability with additional requirements for the measurement conditions. It also includes a detailed qualitative leakage detection and questionnaire for occupants. Secondly, this paper presents the construction characteristics of both samples. All houses were tested upon completion. The air changes per hour at 50 Pa pressure difference (n50) of both samples show the same mean value of 1.4 h -1 , with larger variations among the LT sample. Finally, we discuss measurement results. Regarding MT sample, the air permeability slightly increases during the first year (mean increase by 18%), and then stabilizes during the second and third year. However, for some houses with exposed timber framing, n50 has increased by more than 100%. Regarding LT campaign, the air permeability (n50) show a similar increase after 3-10 years with a mean value of 20%. Measurements performed during two different seasons did not show a significant impact of seasonal variation. The results show globally an increase in the number of detected leakages for all houses, but this increase is not always correlated with the change in air permeability. For 10 houses of both samples, the building airtightness has improved. For 6 houses, this improvement is maybe due to the building material (wood), the maintenance of windows, or the sealing of leaks by occupants but for 4 houses, we have not been able to explain this improvement.

INTRODUCTION

The increasing weight of building leakages energy impact on the overall performance of lowenergy buildings led to a better understanding and characterization of the actual airtightness performance of buildings. Several European countries have already included in their Energy Performance regulation (EP-regulation) mandatory requirements regarding the building airtightness. This is the case in France, where the EP-regulation requires a limit airtightness level for residential buildings that must be justified by measurement. However, low expertise is available today on the durability of building airtightness and its evolution in mid-and longterm scales. The French ongoing research project "Durabilit'air" is conducted since 2016 for a 42-month period, in order to improve our knowledge on the variation of buildings airtightness through onsite measurement campaigns and accelerated ageing in laboratory controlled conditions. As part of this project, a comprehensive literature review about building airtightness durability was realized by [START_REF] Leprince | Durability of building airtightness, review and analysis of existing studies[END_REF]. This review showed an important evolution over time of the air permeability in real buildings, with an increase of more than twice in some cases. The air permeability seems to increase in the 3 first years and then stabilise. This paper is issued from the second task of the "Durabilit'air" project. This task deals with the quantification and qualification of the durability of building airtightness of single detached houses. It is done through field measurement at mid-term and long-term scales. This paper presents the results of both MT and LT measurements.

METHODOLOGY

Onsite measurements

In order to evaluate the durability of the building airtightness in real conditions at mid-and long-term scales, two field measurement campaigns were conducted: a mid-term (MT) campaign and a long-term (LT) campaign. The MT campaign aims at characterising the yearly evolution of building airtightness of new dwellings over a 3-year period. Therefore, a sample of 30 new single-detached low-energy houses, measured upon completion, has been selected nationwide. The following measurements were performed:

 The airtightness of each building was measured once per year over the 3-year period.

 Five buildings of this sample were measured twice per year in order to investigate the impact of seasonal variations.  For five buildings (four buildings from this sample plus one additional building), the airtightness of an installed window were measured once per year over a 3-year period. The LT campaign aims at characterising the evolution of building airtightness of existing dwellings over a longer period from 3 to 10 years. A second sample of 31 existing singledetached dwellings, measured upon completion, has been therefore selected. The dwellings have been constructed during the last 10 years. The airtightness of each dwelling was measured once. All dwellings were selected according to well-defined criteria to reduce uncertainties about main factors impacting building airtightness. In particular, all dwellings should be tested upon completion, and the test reports should be available and in accordance with the standard ISO 9972 [START_REF] Nf | Thermal performance of buildings -Determination of air permeability of buildings -Fan presurization method[END_REF] and its French implementation guide (FD P50-784, 2016). Information about the treatment of the building airtightness must also be available.

The main challenge of this project is to understand the variation of the airtightness and to identify whether it is related to the products/assembly ageing, the maintenance conditions or other factors such as the occupants' behaviour. Therefore, a specific measurement protocol was defined after a detailed literature review [START_REF] Leprince | Durability of building airtightness, review and analysis of existing studies[END_REF]. The protocol is mainly based on the standard ISO 9972 and its French implementation guide for the measurement method with additional requirements for the measurement conditions in order to reduce uncertainty due to measurement procedure:

 Each dwelling is to be measured under the same conditions as the first measurement upon completion as far as possible (same tester, same calibrated measurement device, same building preparation, same pressure difference sequences, and same season). Measurements are to be performed in both pressurization and depressurization. Deviations from the conditions of the first test are to be reported.  Detailed qualitative leakage detection is to be performed at each measurement according to the leaks categories of the French implementation guide of ISO 9972 (FD P50-784, 2016). In particular, an annual follow-up of leaks is to be performed for the dwellings of MT sample during the 3 years.  Questionnaires for occupants are to be filled at each measurement in order to identify the modifications of the building envelope due to the action of the occupants (i.e. drillings made in the air barrier after the first test, replacement of products…). At the total, 84 and 31 measurements of building airtightness were performed for MT and LT samples respectively, plus an extra of 10 measurements for the seasonal impact, and an another extra of 15 measurements for the airtightness of windows.

Results analysis

The results presented here are expressed according to the airflow at 50 Pa (q50) for which the measurement is more reproducible than at 4 Pa [START_REF] Delmotte | Interlaboratory tests for the determination of repeatability and reproducibility of buildings airtightness measurements[END_REF]. For the MT sample, four measurements are carried out on each building: just after completion of the building (reference measurement n0), then at 1 year (measurement n1), 2 years (measurement n2) and 3 years (measurement n3) after completion. For the LT sample, two measurements are carried out on each building: just after completion of the building (reference measurement n0), then at 3 to 10 years after completion (measurement nx). Boxplots are used to graphically summarise the main descriptive statistics of measured air leakage rates q50 for each measurement of both samples. It shows means alongside medians and quartiles. One-sided paired t-test (95% confidence level) is performed to analyse the statistical significance of the increase in the mean q50 between the reference measurement n0 and the other measurements of each sample. Shapiro test is also performed to check the normality of the samples of measurements. Multiple linear regression is performed to analyse the correlation between the evolution in the measured air leakage rates q50 and the evolution in the numbers of detected leakages. Regarding the measurements of seasonal variations and windows, no statistical analysis is performed as the sample size is small for both (5 buildings in each case). We will only look at the evolution of the measured air leakage rates per each building.

RESULTS

Main characteristics of buildings

Figure 1 shows the distribution of buildings of MT and LT samples according to the year of construction and buildings main material and type of air barrier. The measurements upon completion (measurement n0) of both samples showed the same mean value of the air changes per hour at 50 Pa n50 of 1.4 h -1 , with larger variations among the LT sample (standard-deviation of 0.65 h -1 for LT sample against 0.33 h -1 for MT sample).

For MT sample, 1 st -year measurements (measurements n1) started in November 2016 and finished in October 2018, thus from 1 to 3 years after the measurements n0 upon completion.

The measurements were delayed due to the difficulty in finding occupants willing to be involved in a 3 year-long measurement campaigns. Thus, the average timespan between measurements n0 and n1 is 1.7 years, and the average timespans between the following measurements are less than one year to compensate for the delay (0.7 year between n1 and n2, and 0.9 year between n2 and n3). For three houses, measurements n2 and n3 were not carried out because of the withdrawal of occupants during the project. Besides, a house was excluded from the MT sample because of problems during measurement n1.

For the LT sample, all measurements (measurements nx) were conducted in 2017, from 3 to 8 years after n0 (average timespan of 4.6 years between n0 and nx). Only 9 houses older than 5 years were measured. As for MT, it has been difficult to find houses, fulfilling selection criteria with volunteer occupants. A house was excluded from the LT sample because of problems during measurement nx.

Evolution of envelope air permeability

Figure 2 and Figure 3 show the boxplots of the measured air leakage rates at 50 Pa q50 for the different measurements of MT and LT sample respectively. For the measurements n0, tests were performed by either pressurisation or depressurisation unlike the other measurements where tests were performed by both pressurisation and depressurisation. Boxplots show the results of the measurements n1, n2 and n3 for MT sample, and nx for LT sample, for the same houses tested by pressurisation or depressurisation at n0, in order to be comparable to the reference measurements n0. For the MT sample, when considering both pressurisation and depressurisation measurements at n0, we can observe a significant increase in the mean q50 between the measurements n0 and n1 by 58.9 m 3 .h -1 , i.e. +18% (p-value = 0.037 < 0.05), than a stabilization of q50 at n2 and n3.

For the LT sample, we observe similar results as MT sample with a significant increase in the mean q50 between n0 and nx by 67.7 m 3 /h, i.e. +20% (p-value = 0.002 < 0.05). Figure 4 shows a lack of correlation between the evolution in q50 and the age of the houses for both MT and LT samples. Therefore, the air permeability does not seem to change with the age of the building; it varies mainly during the first two years of the building, and then stabilizes, as observed in previous studies [START_REF] Leprince | Durability of building airtightness, review and analysis of existing studies[END_REF]. Variations during the first two years may have several origins, including actions by the occupants when they move in the building (e.g. installing furniture, picture frames, downlight…), the first heating of the building or the first seasonal cycles.

Figure 4: Measured evolution in q50 for MT and LT samples according to the age of the houses (timespan between the measurement at completion n0 and the other measurements nx)

Analysis of explanatory factors

As there is almost no evolution between the results of n1, n2 and n3 for the MT sample, we will focus in the analysis on the evolution between n0 and n1. Tables 1 and2 show the evolution in q50 in relation to the main characteristics of the houses (constructor, number of levels, type of air-barrier, type of material, type of floor, type of roof, type of heating, specific HVAC equipment) and modifications by the occupants (modification on windows, modification on walls). The houses in the table are sorted in ascending order of the evolution in q50. They are classified into 4 categories depending on the evolution in q50:  significant decrease of q50 (< -50 m 3 .h -1 ): 5 houses for the MT and LT samples each;  no or little variations of q50 (-50 to +50 m 3 .h -1 ): 13 houses for MT sample and 8 houses for LT sample;  moderate increase of q50 (+50 to +150 m 3 .h -1 ): 6 houses for MT sample and 10 houses for LT sample;  strong increase of q50 (> +150 m 3 .h -1 ): 5 houses for MT sample and 7 houses for LT sample. It is difficult to make statistical analysis to identify the impact of different factors on the evolution in q50 due to the small size of the samples regarding the factors. For the MT sample, we are generally observing an upward trend of q50 for 2-storey detached houses with exposed wood frame. For the two houses with exposed wood frame of this sample (MICT06 & MICT19), MICT06 has become much leakier (q50 at n1 almost 4 times higher than n0), mainly because of leakages appearing at the junction between the wood and the plasterboard (shrinkage of mastic). While the airtightness level of MICT19 has remained almost stable between n0 and n1. Knowing that both houses are tighten with the same method, the conditions of implementation of the air-barrier seem to have an impact on the durability of the airtightness. Unfortunately, it was not possible for us in this study to collect information on the conditions of implementation; our knowledge was limited to the type of treatment of the airtightness from the technical plans, without having information about the products and their implementations. Therefore, it would be interesting to investigate this factor in future studies. For the LT sample, we observe that the airtightness of wooden houses (6 houses) has generally remained stable and even improved for 2 houses. It is interesting to notice that laboratory testing has come to same conclusion on wood structure [START_REF] Litvak | Assessment of the durability of airtightness products in laboratory controlled conditions: developement and presentation of the experimental protocol[END_REF] and it may be due to the expansion of wood with the humidity that would expand the wood and therefore reduce leakages. Regarding the modifications on the envelope made by occupants, the information was collected through questionnaires. We have identified mainly two categories of modifications:

-modification on windows (Modif_window): maintenance (M) or replacement (R) of some elements; -modification on the walls: replacement (R) of some elements or drilling the walls (D). As showed in Table 1, one window was modified in MICT28 (adjustment of the hinge) and another one in MICT21 (adjustment of the service door overlooking the garage), which probably explain the improvement of the airtightness. While the replacement of windows in MICT22 and MICT23 seem probably to deteriorate the airtightness. This is also the case of MILT16 of the LT sample in Table 2. Regarding the modifications of walls, all houses were generally modified by the occupants (drilling the walls for installing furniture, decoration, hood, downlight led...) whatever the evolution of the airtightness. In some cases (MILT 25 & MILT27), the degradation of the airtightness is may be due to the installation of a heat pump which required the piercing of the walls in order to be able to install the cables and the ducts. In other cases (MILT8 & MILT21), the improvement of the airtightness is may be due to the fact that the occupants have sealed the leaks detected during the test at completion at the junction between the ducts and the ceiling. However, it is difficult to draw general conclusions from these observations about the impact of the modifications by occupants on the evolution of the airtightness.

Evolution of leakages

Figure 5 and Figure 6 show the evolution in the number of leakages for MT sample and LT sample respectively for each category of leakages as defined in (FD P50-784, 2016). The houses are sorted in ascending order of the evolution of q50. The categories of leakages are the following:

 A: leakages through main envelope area;  B: leakages through wall, roof and floor junctions;  C: leakages through doors and windows;  D: leakages around penetration through envelope;  E: leakages through trapdoor;  F: leakages through electrical components;  G: leakages through junctions between wall and door/window;  H: other leakages. The figures show an increase in the number of leakages for doors and windows (C), electrical components (F), penetrations through envelope (D) and junctions between walls and doors/windows (G). However, multiple linear regression has been performed and has shown that the evolution in q50 is not correlated with the evolution in the number of leakages. As we can see on both figures, for some houses with a decrease in q50, there is an increase in leakages equivalent to houses with a strong increase in q50. Therefore, a thorough leakage location detection is not useful as long as it does not quantify leakages for the analysis of the onsite durability. Thus, new methods are needed to detect and to quantify leakages. Gathered information on the modification of the envelope has explained a part of the evolution in q50. However, neither leakages detection nor building characteristics are correlated with the observed evolution of q50. Therefore, there are probably other parameters not considered in this study, which have an impact on the durability of the airtightness. One guess is that the environmental conditions (temperature, dustiness) when the air barrier is implemented may have an impact on the durability of the airtightness. It was not possible for us to collect information on these parameters, but it would be interesting to investigate this parameter in future studies.

Impact of seasonal variations

Figure 7 shows the evolution of the measured air leakage rates at 50 Pa q50 depending on the moment of measurement for the five houses of the MT sample. Globally, we do not observe a seasonal variation in q50 except for MICT 22. In case of MICT22, q50 has strongly increased after the measurement at completion (SUM-14). But since the second measurement in winter 2017 (WIN-17), the house seems to be slightly more airtight in summer than in winter. Unfortunately, measurement n2 is missing between SUM-17 and SUM-18 to confirm this observation. It is interesting to notice that this is the only 2-storey house while the four others are 1-storey houses.

Evolution of windows air permeability

The window air permeability was measured with the PAM device as described in [START_REF] Fournier | In situ measurement of window air tightness : stakes, feasibility, and first results[END_REF]. Figure 8 shows the results of the measured air leakage rates at 100 Pa and the results of the leakages detection for the five windows. We note that the air permeability of the windows was not measured at completion. Thus, the reference measurements n0 are missing. We can only analyse the evolution of the airtightness between n1 and n3. Globally, we observe small variations between measurements n1, n2 and n3. As for the envelope, we do not observe any correlation between the evolution in detected leakages and the evolution in air leakage rates of the windows. In addition, the evolution in the air leakage rates of windows is not correlated with the evolution in the air permeability of the envelope.

CONCLUSIONS

The durability of building airtightness of low energy single-detached houses was assessed through two field measurement campaigns at mid-term (MT) and long-term (LT) scales.

The results showed that the airtightness of houses can deteriorate mainly during the first two years and then it seems to stabilise as: -For MT sample, the mean and median values of the air leakage rates q50 in years n1, n2 and n3 are equivalent; -MT and LT samples show the same mean evolution of the air leakage rate q50

(respectively +18% and +20%). However, as for other studies [START_REF] Leprince | Durability of building airtightness, review and analysis of existing studies[END_REF], we have observed that the building airtightness deteriorated significantly in some houses while in others it stabilised or even improved. With this study, it has not been possible to determine "where and why" new leakages are appearing however; it has led us to the following useful conclusions:

-One of the two houses with exposed wood-frame has become much leakier, mainly because of leakages appearing at the junction between the wood and the plasterboard (shrinkage of mastic). While the airtightness of the other house has remained almost stable. Therefore, the conditions of implementation of the air-barrier seem to have an impact on the durability of the airtightness. -It has not been possible to determine the location of the new leakages causing the deterioration of the airtightness. New methods are needed not only to locate but also to quantify more precisely leakages. A thorough leakage detection is not useful as long as it does not quantify leakages for the analysis of the onsite durability. -Observed variations of the air permeability are not due to seasonal variations and given the strict protocol applied in this study, they are probably not due to measurement uncertainty.

-The evolution of the airtightness does not appear to be correlated in this study with the following parameters: constructor, type of air-barrier, type of floor, type of roof, type of heating, specific HVAC equipment. The two parameters that seem to be correlated with the evolution of the airtightness are:

-The material: it seems that the airtightness of wood houses tend to stabilise or even improve over years, maybe due to the expansion of wood with humidity. -The number of levels: 2-storey houses seems to more deteriorate than 1-storey ones, which is maybe due to more important foundation settlement. Regarding the houses where the airtightness has improved (10 houses for both samples), this improvement is maybe due to the building material (2 wooden houses), the maintenance of windows (2 houses), or the sealing of leaks by occupants (2 houses). However, for the other four houses, we have not been able to explain it. Therefore, the results of this study do not stress the need to perform long-term study on the durability of airtightness, but on the contrary to better understand where and why leakages appear during the first year, which causes the deterioration of the building airtightness (very short-term ageing). Other parameters need to be considered, such as the environmental conditions (hygrothermal, dustiness) during the implementation of the air barrier or the evolution of the temperature and humidity inside the building during the first year. In addition, modifications made by occupants need to be known more closely. More frequent airtightness measurements (e.g. monthly measurements) could be performed on a small sample of houses over the period from the implementation of the air barrier till one year upon building completion, by recording at each measurement the aforementioned parameters.

Figure 1 :

 1 Figure 1: Distribution of buildings depending on the year of construction (left) and buildings main material and type of air barrier (right); Air barrier-A when the air barrier is ensured by vapour barrier, Air barrier-B by coating on the masonry, and Air barrier C by plasterboards and mastics at the inside facing of the walls The MT sample is composed of 30 new single-detached low-energy houses constructed mainly in 2014 and 2015, with 20 one-story houses and 10 two-story houses. The average floor area is 124.1 m² with a minimum of 87 m² and a maximum of 172.2 m², and the average volume is 217.1 m 3 with a minimum of 156.1 m 3 and a maximum of 363.9 m 3 . All houses are built of masonry with interior insulation (16 houses with concrete blocks and 14 with hollow bricks). The majority of roofs are made of light-frame wood truss (20 houses), against 8 houses with traditional wood frame and 2 houses with an exposed traditional wood frame. Single exhaust ventilation system with humidity control is installed in all houses. The airtightness is done through plasterboards and mastics at the inside facing of the walls in all houses (air barrier C). The LT sample is composed of 31 single-detached low-energy houses constructed between 2009 and 2015, with 7 one-story houses and 25 two-story houses. The average floor area is 147.9 m² with a minimum of 83.1 m² and a maximum of 269 m², and the average volume is 256.6 m 3 with a minimum of 138.9 m 3 and a maximum of 478.2 m 3 . The majority of houses are built of masonry with interior insulation (25 houses with hollow bricks and 3 with concrete blocks), against 6 wooden houses. The majority of roofs are made of light-frame wood truss (27 houses), against 5 houses with flat roof. A balance ventilation system is installed in 1 house while all other have a single exhaust ventilation system with humidity control. The airtightness of masonry houses of the LT sample is mainly done by coating on the masonry (air barrier B), while the airtightness of wooden houses is done by the vapour barrier (air barrier A).The measurements upon completion (measurement n0) of both samples showed the same mean value of the air changes per hour at 50 Pa n50 of 1.4 h -1 , with larger variations among the LT sample (standard-deviation of 0.65 h -1 for LT sample against 0.33 h -1 for MT sample).For MT sample, 1 st -year measurements (measurements n1) started in November 2016 and finished in October 2018, thus from 1 to 3 years after the measurements n0 upon completion.The measurements were delayed due to the difficulty in finding occupants willing to be involved in a 3 year-long measurement campaigns. Thus, the average timespan between measurements n0 and n1 is 1.7 years, and the average timespans between the following measurements are less than one year to compensate for the delay (0.7 year between n1 and n2, and 0.9 year between n2 and n3). For three houses, measurements n2 and n3 were not carried out because of the withdrawal of occupants during the project. Besides, a house was excluded from the MT sample because of problems during measurement n1.For the LT sample, all measurements (measurements nx) were conducted in 2017, from 3 to 8 years after n0 (average timespan of 4.6 years between n0 and nx). Only 9 houses older than 5 years were measured. As for MT, it has been difficult to find houses, fulfilling selection criteria with volunteer occupants. A house was excluded from the LT sample because of problems during measurement nx.

Figure 2 :

 2 Figure 2: Boxplot of the measured air leakage rates at 50 Pa q50 for the measurements n0, n1, n2 and n3 of the MT sample for the pressurization test, the depressurization test and the average between both tests.

Figure 3 :

 3 Figure 3: Boxplot of the measured air leakage rates at 50 Pa q50 for the measurements n0 and nx of the LT sample for the pressurization test, the depressurization test and the average between both tests

Table 1 :

 1 Evolution in q50 for MT according to buildings mains characteristics and modifications by occupants (M1: concrete block -M2: bricks -M3: wood construction -F1: floor on crawl space -F2: Slab on grade -F3: Cellar -R1: light-frame wood truss -R2: Traditional wood frame -R3: Exposed wood frame -R4: Flat roof -H1: Heat pump -H2: Gas boiler -H3: Wood stove -H4: Electric heating -E1: Thermodynamic water heater -E2: Heat pump water heater -E3: Electric heat water -E4: Solar thermal -E5: Solar PV -E6: DHW by gas boiler -M: Maintenance -R: Replacement -D: Drilling)

Figure 5 :Figure 6 :

 56 Figure 5: Evolution in detected leaks numbers for MT sample

Figure 7 :

 7 Figure 7: Evolution in q50 for the measurements with seasonal variations of the MT sample (WIN: Winter; SUM: Summer; AUT: Autumn; SPR: Spring)

Figure 8 :

 8 Figure 8: Evolution in the measured air leakage rates at 100 Pa (a) and the number of leakages (b) for the measured windows of the MT sample (C1: other leakage through window -C2: leakage through the junction between the window leaf and frame -G4: leakage through the junction between the window and the wall)

Table 2 :

 2

Evolution in q50 for LT according to buildings mains characteristics and modifications by occupants

ACKNOWLEDGEMENTS

This work was supported by ADEME and French ministry for construction. The views and opinions of the authors do not necessarily reflect those of ADEME and French ministry. The published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall authors, ADEME or French ministry be liable for damages arising from its use. Any responsibility arising from the use of this report lies with the user.