Dandan Yao

Limiao Shi

Zhipeng Sun

Mireille Blanchard-Desce

Olivier Mongin

Frédéric Paul

Christine O Paul-Roth
email: christine.paul@univ-rennes1.fr

New Fluorescent Tetraphenylporphyrin-Based Dendrimers with Alkene -Linked Fluorenyl Antennae designed for Oxygen Sensitization

Keywords: Porphyrin, fluorenyl, fluorescence, energy transfer, double bond, dendrimer, photodynamic therapy 1. Introduction

A new family of conjugated porphyrin-based dendrimers with fluorenyl arms has been synthesized. Their detailed luminescence properties and selected photophysical properties are discussed in the context of two-photon-induced theranostics.

There has been great interest in porphyrin systems because the peripheral substituents on the macrocyclic core allows to significantly modulate the photophysical properties at will. Thus, light-harvesting compounds can be easily obtained by attaching four strongly absorbing energydonor dendrons at the meso positions of the central porphyrin core which will play the role of peripheral "antenna" for the porphyrin. 1 In this respect, Fréchet and coworkers 2 have reported porphyrin systems with fluorene-containing oligoether-type dendrons as efficient one-and twophoton light-harvesting units and demonstrated that such an "antenna effect" was facilitated in dendritic architectures versus linear architectures. 2b-c More recently, related star-shaped porphyrins with fully conjugated oligofluorene arms have also been reported by Bo and coworkers and shown to behave as a remarkable light-harvesting systems. 3 In this context, we have previously reported the synthesis of porphyrin possessing four fluorenyl arms directly connected at the meso-positions. This compound (TFP; Figure 1a). 4 exhibited a high luminescence quantum yields (24%), demonstrating the good capacity of the fluorenyl units to enhance emission by increasing the radiative process. 5 Subsequently, we synthesized two series of substituted meso-porphyrin dendrimers with terminal fluorenyl arms, taking TPP as the central unit: (i) a non-conjugated family with flexible ether linkage 6 and, more recently, (ii) a conjugated family with rigid alkynyl linkage (Figure 1b). 7 We then could show that these molecular architectures were promising models for the design of new bophotonic photosensitizers for theranostics i.e. allowing to perform photodynamic therapy [START_REF]HRMS MALDI: m/z calcd for C228[END_REF] and fluorescence imaging after two-photon excitation. 7 Due to the practical advantages of twophoton excitation, this field has attracted attention and is rapidly expanding, several porphyrinbased such photosensitizers having been reported to date. 9,10,11 In order to gain additional insight about the potential of TPP-cored dendrimers such as TPP-T1 or TPP-T2 in this field, we have started systematically investigating the impact of various structural changes taking place in the peripheral light-harvesting antenna on the photophysical properties of these systems.

Given that 1,2-alkene spacers are known to promote electronic communication better than 1,2-alkyne ones, 12 we recently started exploring the optical properties of related dendrimers featuring alkene linkages. However this was exclusively done for the first generation dendrimer TPP-D1 (Figure 1c; M = 2H) resulting in a significant improvement in the linear and nonlinear optical properties vs. TPP-T1. 13 This statement prompted us to test higher generation dendrimers of this kind. Accordingly, we now report our efforts to isolate the second generation dendrimer TPP-D2 and the metalated species ZnTPP-D1 and ZnTPP-D2. Subsequently, their linear and nonlinear optical properties of interest will be disclosed as well as evidence for the very efficient energy-transfer process taking place from the conjugated dendrons to the porphyrin core in these new species. These data will then be compared to those of their known alkyne-containing analogues (or T series: Figure 1b).

Results and Discussion

Synthesis and characterization

This new family of dendrimers was prepared the generation of the required dendrons (D1-PhCHO and D2-PhCHO), followed by their condensation with pyrrole to give the desired freebase porphyrins as intermediates which were then metalated by Zn(II) to give the final dendrimers ZnTPP-D1 and ZnTPP-D2.

Dendron synthesis. The synthesis of the two generations of vinyl-bridged aldehyde-terminated dendrons D1-PhCHO and D2-PhCH is described in Scheme 1. First, commercial 1-bromo-3,5-bismethylbenzene was halogenated with benzyl by N-bromosuccinimide (NBS) using azobis-isobutyronitrile (AIBN) as radical initiator. Given that this bromination takes usually place non selectively, [14][15] the reaction conditions were optimized (temperature, time and solvent). The resulting conditions gave mainly the desired dibrominated product along with mono-and tri-brominated byproducts. The former byproduct could be isolated by chromatography (heptane), while the latter could not be fully separated from targeted dibrominated product (ratio tri/di of 1/4 by 1 H NMR). In the next step, this mixture containing 49% of desired product was reacted directly with excess of P(OEt)3 under reflux following a Michaelis-Arbuzov 14 protocol. The desired product 3 and its bromo-substituted byproduct were both formed and were subsequently separated by chromatography (Scheme 1). The unwanted byproduct could be easily eluted using CH2Cl2, while the target product 3 was collected using ethyl acetate as a colorless oil in 83% yield. After a subsequent Horner-Wadsworth-Emmons (HWE) reaction, 14 the compound 3 reacts with the previously prepared aldehyde 1 7a in presence of t-BuOK/THF to give the double-bonded precursor D1-PhBr in 89% yield. The aldehyde D1-PhCHO can then be obtained in two steps from this product in 86% yield. Repetition of the HWE reaction 14 between this new aldehyde and 3 gave access to the second generation intermediate D2-PhBr in 93% yield, and subsequently to the corresponding D2-PhCHO dendron in 65% yield (Scheme 1). Scheme 1. Synthetic routes for alkene-bridged dendrons D1-PhCHO 13 and D2-PhCHO.

Porphyrin synthesis. Two synthetic methods are most often used to synthesize porphyrins: the Adler-Longo 16 or the Lindsey reaction. 17 Both are efficient for synthesizing porphyrins substituted at their meso positions. Given that the first generation dendrimer TPP-D1 was previously isolated using the Adler-Longo approach, 13 these reaction conditions were used again to synthesize TPP-D2. This compound could however not be isolated pure after several purification attempts by chromatography and subsequent recrystallizations (CHCl3 and MeOH), the yield of crude TPP-D2 in the isolated solid being below 4%. Fortunately all these porphyrin dendrimers have good solubilities in common organic solvents, allowing for their easy metalation and this approach provided a way to access the pure zinc complex from the mixture in the case of ZnTPP-D2 (Scheme 2). Thus, the corresponding zinc complex ZnTPP-D1 was formed at 40 o C overnight from TPP-D2 using Zn(OAc)2 in CH2Cl2/MeOH and isolated pure in 60% yield (Scheme 2). Using similar conditions for metalation ZnTPP-D1 was isolated in 80% yield from TPP-D1. From a purely synthetic standpoint, the isolated yields in alkene-bridged dendrimers (D-type series; Figure 1c) were always lower than those of their alkyne-bridged analogues (T-type series; Figure 1b), although rigorously similar reaction conditions have been used (the isolated yields of TPP-T1 and TPP-T2 were 18% and 13%, respectively. 7 ), making the D-type series dendrimers more challenging to obtain via the Adler-Longo approach. 7,18

1 H NMR analysis. The aldehyde dendrons D1-PhCHO and D2-PhCHO, and the corresponding dendrimers, i.e. the free-base and metalated phorphyrins MTPP-D1 and MTPP-D2, were characterized by 1 H NMR analysis (Figures 234). The Figure 2 shows the full 1 H NMR spectra of the dendrons compared to those of their analogues with triple bondes (T1-PhCHO and T2-PhCHO). They all show three diagnostic signatures: (i) the aldehyde proton as a singlet, around 10 ppm; (ii) the aromatic protons located as multiplets, in region 7-8 ppm, belonging to protons of phenyl and fluorenyl, partially identified; (iii) four groups of alkyl protons Ha,b,c,d located at 0.5-2.1 ppm are assigned to n-butyl of fluorenyl. We can particularly notice that for double bonded dendrons D1-PhCHO and D2-PhCHO, we observe four additional alkene protons as a broad peak around 7.18-7.40 ppm. ACCEPTED MANUSCRIPT -CLEAN COPY 12

Photophysical properties

UV-visible absorption and emission spectra, after excitation in the Soret-band, 19 were recorded for the isolated dendrimers at room temperature (Table 1 andFigures 5 and6). The free-base tetraphenylporphyrin (TPP; Figure 1a) and the corresponding zinc complex (ZnTPP)

were chosen as references compounds. Their two-photon oxygen photosensitizing yields were subsequently evaluated and compared to those of their alkyne analogues (TPP-T1 and TPP-T2) to analyze the impact of this structural modification (Tables 23).

For the free-base porphyrins series, all absorption spectra are typical of porphyrin derivatives with an intense Soret-band around 430 nm and four Q-bands in the 520-650 nm range. 19a Compared to TPP, the dendrimers TPP-D1, TPP-T1 and TPP-T2 present an additional absorption around 300-400 nm, corresponding to the conjugated fluorenyl dendrons.

This band is of almost similar intensity for TPP-D1 and TPP-T1, most likely in reason of their identical terminal fluorenyl number, however, a red-shift is observed for TPP-D1 compared to TPP-T1, likely due to the improved conjugation beween core and arms in the former compound.

Concerning the Soret band, the vinyl-bridged dendrimer TPP-D1 present also a larger red-shift (15 nm) than its alkyne analogue TPP-T1 (9 nm) versus the Soret band of the reference TPP, which reflect the extension of the porphyrin  manifold 20 through conjugation with the peripheral arms at the meso positions.

The corresponding zinc complexes ZnTPP-D1 and ZnTPP-D2 exhibit characteristic changes in their electronic spectra compared to those of the corresponding the free-base porphyrins TPP-D1 and TPP-D2. 19a,21 Only two Q-band absorption are now observed around 552 nm and 635 nm due to the symmetry change from D2h to D4h symmetry upon metalation and an intense Soret band around 435 nm (Table 1 and Figure 6). An additional broad band is also observed in UV range (346 nm) which corresponds to π-π* absorption of the fluorenyl chromophores. This UV absorption is weaker for the first generation zinc(II) complex (ZnTPP-D1) than for the higher generation dendrimer (ZnTPP-D2) due to the smaller number (eight vs.

sixteen) of fluorenyl groups present in the peripheral arms. Upon excitation in their Soret-band, all these compounds exhibit the characteristic porphyrin emission peaks Q(0,0) and Q(0,1). 5,[START_REF] Gouterman | The Porphyrins[END_REF] After normalizing their emission spectra on their Q(0,0) peaks, all these compounds exhibit two emission peaks at similar wavelengths, but with different intensities (Figure 5). Compared to TPP, the Q(0,1) band of all dendrimers does not change in intensity for TPP-D1 but increases for TPP-T1 and TPP-T2. The emission spectra of zinc(II) porphyrin complexes usually consist of three sub-bands assigned to a vibronic progression from a Q state: Q(0,0), Q(0,1) and weak Q(0,2), the last one, near 720 nm

(a) (b)
being usually too weak to be observed. [START_REF] Paul-Roth | [END_REF] Presently, with ZnTPP-D1 and ZnTPP-D2 exhibits the two expected Q-bands around 603 nm and 653 nm (Figure 6), the blue shift of these bands compared to the corresponding free-bases being ascribable to the metal coordination. The quantum yields (F) were also measured for all these compounds (Table 1). While the free-base porphyrin-based dendrimers have similar quantum yields (F = 12-13%) than the TPP (F = 11%), these values drop drastically (3-6%) when a metal like zinc(II) is introduced in the porphyrin cavity, as also observed for the reference TPP. The existence of an energy transfer (ET) process, from the peripheral 2-fluorenyl donors toward the central porphyrin acceptor core was also studied. The emission spectra were measured from 450 to 800 nm, using two excitation wavelengths; the dendron absorption (325-351 nm) and the Soret-band absorption (419-434 nm). As expected, all dendrimers show exclusively Q-band emission around 650-720 nm in both cases with is no residual dendron emission observed around 400 nm. This suggests that the peripheral fluorenyl groups transfer very efficiently their energy to porphyrin core, given that any dendron emission is totally quenched (SI Figure S14). This very efficient energy transfer most likely corresponds to a so called "through-bond" energy transfer process (TBET). 23 Thanks to this very efficient "antenna effect", the dendron absorption band, when intense (as in ZnTPP-D2), might be efficiently used for exciting these compounds. b. Unless precised, experiments for fluorescence quantum yields were achieved in toluene (HPLC level) using TPP (=11%) as standard, by Soret band excitation. 5 c. Data obtained in CH2Cl2 (HPLC level).

Two-photon absorption

As these dendrimers exhibit good fluorescence properties, their intrinsic two-photon absorption cross-sections were determined by two-photon excited fluorescence (TPEF) in CH2Cl2. Measurements were performed with 10 -4 M solutions, using a mode-locked Ti:sapphire laser delivering femtosecond pulses, following the experimental protocol described by Xu and Webb. 24 A fully quadratic dependence of the fluorescence intensity on the excitation power was observed for each sample at all the wavelengths of the spectra (790-920 nm), indicating that the cross-sections determined are only due to TPA. A significant increase of their TPA cross-sections compared to that of TPP (12 GM at 790 nm) was observed for all porphyrins possessing fluorenyl dendrons (Table 2). Comparison between the free-base dendrimers TPP-D1 and TPP-T1 reveals that replacing triple bonds with double bonds in the peripheral dendrons leads to a significant increase of the TPA cross-sections. The zinc complexes of the D-type series (ZnTPP-D1 and ZnTPP-D2) also exhibit high TPA cross-section (2 = 260 and 450 GM, respectively) at 790 nm, ZnTPP-D2 being the best two-photon absorber of the series of compounds presently investigated. From the comparison between TPP-D1 and ZnTPP-D1, metalation by Zn(II) does not result in a significant change in cross-section at this wavelength and induces a slight decrease of 2 at higher wavelengths compared to the corresponding free-base porphyrin. This statement suggest that the crosssection of the missing TPP-D2 free-base porphyrin would be similar or slightly above that of TPP-D2 in the 790 -920 nm range. In this respect, the clear enhancement of 2 observed for ZnTPP-D2 relative to ZnTPP-D1 is particularly remarkable. First it reveals a more pronounced structural difference in two-photon cross-sections for the D-type series than for the T-type series, in favor of the largest dendrimers. Then it suggest that the free-base dendrimer TPP-D2 would be a better two-photon absorber than its TPP-T2 analogue in the investigated wavelength range.

The two-photon brightness (2.F) is a figure of merit allowing evaluating the potential of two-photon absorbers for fluorescence imaging. For the zinc complexes, any change in 2 is combined with a strong decrease in F leading to a decrease of this figure of merit for ZnTPP-D1 relative to that of its free-base analogue TPP-D1, which is the highest of the compound presently discussed. The ZnTPP-D2 dendrimer still exhibits an interesting two-photon brightness which is enhanced more than 20 times compared to TPP used as reference. Among free-base porphyrins, the two-photon brightness of TPP-D1 is also significantly higher than that of its analog TPP-T1, revealing the positive impact of replacing triple bond by double bonds in the peripheral dendrons for imaging purposes. b Maximum two-photon brightness in dichloromethane. c Enhancement factor: F2 max of the compound normalized to that of TPP.

d Data from lit. 25 The oxygen-photosensitizing properties of these dendrimers were also studied. Their quantum yields of singlet oxygen generation (∆) were determined and compared to those of analogous T dendrimers and TPP used as reference (Table 3). All these dendrimers exhibit values comprised between 0.55 and 0.64, comparable to that of reference TPP (0.60).

Interestingly, the free-base dendrimer TPP-D1 shows the highest value ∆ = 64%, whereas the two zinc complexes ZnTPP-D1 and ZnTPP-D2 show the lowest ones (59% and 55%, respectively). As previously noticed for T-type dendrimers 7,18 the increase in fluorescence quantum yield of the new free-base dendrimer TPP-D1 relative to TPP, is not obtained at the expense of the singlet oxygen production. c Enhancement factor: 2 max of the compound normalized to that of TPP.

Compound

In combination with the notable increase of the TPA cross-sections of the dendrimer TPP-D1 compared to its TPP-T1 analogue, significant enhancements of the figure of merit for the two-photon excited oxygen sensitization (2 max = 177 GM) can be achieved. For the zinc complexes, this value goes up to 153 GM for ZnTPP-D1 and even to 248 GM for larger ZnTPP-D2. The free-base TPP-D1 dendrimer exhibits a clearly higher enhancement factor than its T-type analogue, in relation with its higher 2 and its slightly increased This compound, easy to synthesis, appears therefore particularly promising for two-photon photodynamic therapy, and, considering its TPEF properties, also for theranostic applications provided it can be made water-soluble by proper functionalization. It should be emphasized that other porphyrin-based systems with more efficient conjugation between the sub-chromophoric units have often been shown to exhibit higher TPA cross-sections, but these are generally accompanied by strong modifications of their other photophysical properties such as the red shift of their linear absorption range, 10b,26 which somewhat limits their interest for theranostics.

Indeed, most often these highly efficient two-photon absorbers exhibit a modest to negligible fluorescence or some interfering residual one-photon absorption above 800 nm, which leads to the loss of the 3D resolution. In contrast, the dendrimers presently reported, with a more restricted -conjugation between the dendrons and the porphyrin core, [START_REF] Silvers | There is a large dihedral angle between the meso-aryl substituent and the macrocycle, which is more than 60° in the case of TPP[END_REF] exhibit an improved trade-off 7,18,28 between intrinsic TPA, fluorescence and photosensitizing properties.

Experimental section

General

Unless otherwise stated, all solvents used in reactions were distilled using common purification protocols, [START_REF] Perrin | Purification of Laboratory Chemicals[END_REF] except DMF and i Pr2NH which were dried on molecular sieves (3 Å). All chromatographic separations were effected on silica gel (40-60 μ, 60 Å). 1 H and 13 C NMR spectra were recorded on BRUKER Ascend 400 and 500 at 298K. chemical shifts are referenced to internal tetramethylsilane. High-resolution mass spectra were recorded on Bruker MicrOTOF-Q II in ESI positive mode in dried solvent at CRMPO in Rennes. Reagents were purchased from commercial suppliers and used as received. Element analyses were collected on a Microanalyser Flash EA1112. UV-visible absorption and photoluminescence spectroscopy measurements for all porphyrin dendrimers in toluene were performed on Edinburg FS920

Fluorimeter (Xe900) and BIO-TEK instrument UVIKON XL spectrometer at room temperature, respectively, toluene for spectral analysis was HPLC level.

Dendron synthesis and characterization

The two dendrons D1-PhCHO and D2-PhCHO, where obtained after a multistep synthesis from the brominated 1-bromo-3,5-xylene precursors and the corresponding 2-fluorenylaldehyde via Horner-Wadsworth-Emmons reactions followed by carbonylation using butyllithium and DMF.

1-bromo-3,5-bis(bromomethyl)benzene. Commercial 1-bromo-3,5-xylene (5.0 g, 3.67 mL, 27.02 mmol, 1 eq) was added into CH2Cl2 (100 mL, distilled), together with NBS (9.6 g, 54.04 mmol, 2 eq) and AIBN (220 mg, 1.35 mmol, 0.05 eq). The mixture was stirred for 30 min at room temperature, and then refluxed for 30 h. Then cooled in ice-water bath and filtered, washing residue with heptane. The solvents were evaporated and the residue was further purified by chromatography (heptane), collecting the target product (4.56 g, 49% yield)

admixed with 1-bromo-3-bromomethyl-5-methylbenzene (20%) as white powder, as well as Tetraethyl (5-bromo-1,3-phenylene)bis(methylene)diphosphonate (3). In a flask, the previously isolated [4:1] mixture of 1-bromo-3,5-bromomethyl-benzene and 1-bromo-3bromomethyl-5-methylbenzene (2.4 g, 7 mmol, 1 eq) and P(OEt)3 (2.4 mL, 14 mmol, 2 eq) were added respectively. The mixture was refluxed for 4 h at 140 o C. The excess of P(OEt)3 was removed under reduced pressure. Then the title product was purified by chromatography using CH2Cl2 to remove other byproducts and then collected by ethyl acetate, giving a colorless oil (2.66 g, 83% yield

Porphyrins synthesis and characterization

2.01 (t, J = 7.2 Hz, 64H), 1.12-1.01 (m, 64H), 0.65-0.51 (m, 160H).

Spectroscopic Measurements

All measurements have been performed with freshly-prepared air-equilibrated solutions at room temperature (298 K). UV/Vis absorption spectra were recorded on a BIO-TEK instrument UVIKON XL spectrometer or on a Jasco V-570 spectrophotometer. Fluorescence measurements were performed on dilute solutions (ca. 10 6 M, optical density < 0.1) contained in standard 1 cm quartz cuvettes using an Edinburgh Instruments (FLS920) spectrometer in photon-counting mode. Fully corrected emission spectra were obtained, for each compound, under excitation at the wavelength of the absorption maximum, with Aex < 0.1 to minimize internal absorption.

Measurements of singlet oxygen quantum yield (). Measurements were performed on a

Fluorolog-3 (Horiba Jobin Yvon), using a 450 W Xenon lamp. The emission at 1272 nm was detected using a liquid nitrogen-cooled Ge-detector model (EO-817L, North Coast Scientific Co). Singlet oxygen quantum yields  were determined in dichloromethane solutions, using tetraphenylporphyrin (TPP) in dichloromethane as reference solution ([TPP] = 0.60) and were estimated from 1 O2 luminescence at 1272 nm. NaOH using the well-established method described by Xu and Webb 25 and the appropriate solvent-related refractive index corrections. [START_REF] Werts | [END_REF] The quadratic dependence of the fluorescence intensity on the excitation power was checked for each sample and all wavelengths.

Two-Photon Absorption

Conclusions

We report here the synthesis, characterization and a photochemical study of two new zinc(II) complexes of TPP-based dendritic chromophores possessing 8 to 12 fluorenyl groups at their periphery (ZnTPPD1 and ZnTPP-D2). The corresponding free-base porphyrins are analogues of related dendrimers in which we have now replaced the alkyne linkages (T series) by E-alkene ones (D series) at their periphery (Scheme 1). While TPP-D2 could not be isolated pure, metalation of this free-base by Zn(II) provided a convenient mean to selectively access a representative of the higher generation dendrimer (ZnTPP-D2). Comparison with previously gathered data indicate that the optical properties of these dendrimers exhibit an obvious dependence on the dendrimer structure, E-alkene linkers being clearly better than 1,2-alkyne ones for enhancing the photophysical properties of interest (luminescence, 2PA cross-section and sensitization yields) for performing 2P-PDT and fluorescence imaging. Comparison between TPP-D1 and ZnTPP-D1 also reveals that metalation does not drastically affect the two-photon absorption cross-section nor improve the oxygen-sensitizing efficiency of these dendrimers.

Figure 1 a)

 1 Figure 1 a) Reference compounds; b) Previously reported alkynyl-bridged free-base porphyrin dendrimers (T series) based on TPP (TPP-T1 and TPP-T2); c) New porphyrin dendrimers (D series) based on TPP (MTPP-D1 and MTPP-D2; M = 2H, Zn).

Scheme 2 .

 2 Scheme 2. Synthesis of vinyl-bridged porphyrin dendrimers (D series) based on TPP-cored porphyrin (TPP-D1 and TPP-D2) and corresponding zinc(II) complexes (ZnTPP-D1 and ZnTPP-D2).

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Aromatic (a) and Alkyl (b) moieties 1 H NMR spectra (400 MHz) of D-style dendrons D1-PhCHO and D2-PhCHO compared to reported T-style dendrons T1-PhCHO and T2-PhCHO in CDCl3. 7

Figure 5 .

 5 Figure 5. Normalized absorption and emission spectra of selected TPP-cored dendrimers (TPP-D1, TPP-T1 and TPP-T2) compared to reference TPP in toluene (a). Detail of the Q bands of TPP-D1, TPP-T1 and TPP-T2 (b).

Figure 6 .

 6 Figure 6. Absorption and emission spectra of ZnTPP-D2 compared to ZnTPP-D1 in CH2Cl2 at 20 °C.

Figure 7 .

 7 Figure 7. Two-photon excitation spectra of D dendrimers TPP-D1, ZnTPP-D1 and ZnTPP-D2 and comparison with related T compounds TPP-T1, TPP-T2 and reference TPP in dichloromethane.

a

  Singlet oxygen production quantum yield determined relative to TPP in dichloromethane ([TPP] = 0.60). b 2 max : figure of merit of the two-photon excited singlet oxygen production in dichloromethane.

  pure 1-bromo-3-bromomethyl-5-methylbenzene (3.55 g) and 1-bromo-3-(bromomethyl)-5-(dibromomethyl)benzene (1.4 g). 1 H NMR (400 MHz, CDCl3, ppm):  7.47 (s, 2H), 7.34 (s, 1H), 4.41 (s, 4H).

  Experiments. To span the 790-920 nm range, a Nd:YLF-pumped Ti:sapphire oscillator (Chameleon Ultra, Coherent) was used generating 140 fs pulses at a 80 MHz rate. The excitation power is controlled using neutral density filters of varying optical density mounted in a computer-controlled filter wheel. After five-fold expansion through two achromatic doublets, the laser beam is focused by a microscope objective (10x, NA 0.25, Olympus, Japan) into a standard 1 cm absorption cuvette containing the sample. The applied average laser power arriving at the sample is typically between 0.5 and 40 mW, leading to a time-averaged light flux in the focal volume on the order of 0.1-10 mW/mm 2 . The fluorescence from the sample is collected in epifluorescence mode, through the microscope objective, and reflected by a dichroic mirror (Chroma Technology Corporation, USA; ''blue'' filter set: 675dcxru; ''red'' filter set: 780dxcrr). This makes it possible to avoid the inner filter effects related to the high dye concentrations used (10 -4 M) by focusing the laser near the cuvette window. Residual excitation light is removed using a barrier filter (Chroma Technology; ''blue'': e650-2p, ''red'': e750sp-2p). The fluorescence is coupled into a 600 µm multimode fiber by an achromatic doublet. The fiber is connected to a compact CCD-based spectrometer (BTC112-E, B&WTek, USA), which measures the two-photon excited emission spectrum. The emission spectra are corrected for the wavelength-dependence of the detection efficiency using correction factors established through the measurement of reference compounds having known fluorescence emission spectra. Briefly, the set-up allows for the recording of corrected fluorescence emission spectra under multiphoton excitation at variable excitation power and wavelength. TPA cross sections (σ2) were determined from the two-photon excited fluorescence (TPEF) cross sections (σ2.ΦF) and the fluorescence emission quantum yield (ΦF). TPEF crosssections of 10 -4 M CH2Cl2 solutions were measured relative to fluorescein in 0.01 M aqueous

Table 1 .

 1 Photophysical properties of the new dendrimers TPP-D1, ZnTPP-D1, ZnTPP-D2 compared to their alkynyl-bridged parents TPP-T1 and TPP-T2 and to TPP reference at 20 °C.7,18

			Absorption a (nm)	Emission a	Quantum yield b 
					Ex = Soret-band	F (%)
		Dendron	Soret-	Q-bands	Q(0,0) Q(0,1)
			band
	TPP	-	419	514, 548, 590, 649	652	719	11
	ZnTPP	-	421	556, 603	603	650	3
	TPP-D1	349	434	518, 552, 593, 649	652	719	13
	ZnTPP-D1 c	346	436	554, 594	604	654	3
	ZnTPP-D2 c	348	435	555, 595	602	652	6
	TPP-T1	325	428	518, 552, 593, 649	652	719	12
	TPP-T2	327, 349	428	518, 552, 593, 649	652	719	13

a. Unless precised, experiments were achieved in toluene (HPLC level) with the UV-visible absorption region from 287 to 800 nm and emission region from 450 to 800 nm.

Table 2 .

 2 Two-photon absorption and brightness properties of D dendrimers and related and related T compounds in dichloromethane.

	Compound	Fluorenes/	TPA max	2 max	F2 max	Two-photon brightness
		porphyrin	(nm)	(GM) a	(GM) b	enhancement factor c
	TPP	0	790	12 d	1.3	1
	TPP-D1	8	790	280	36	28
	ZnTPP D1	8	790	260	8	6
	ZnTPP-D2	16	810	450	27	20
	TPP-T1	8	790	200	24	18
	TPP-T2	16	790	290	38	29

a Intrinsic TPA cross-sections measured in 10 -4 M dichloromethane solutions by TPEF in the femtosecond regime; a fully quadratic dependence of the fluorescence intensity on the excitation power is observed and TPA responses are fully non-resonant.

Table 3 .

 3 Oxygen sensitization properties of double-bonded porphyrin dendrimers and related triple-bonded compounds.

Acknowledgements

The authors acknowledge China Scholarship Council (CSC) for PhD funding (DY, LS and XZ), This project was supported by the departmental committees CD35, CD28 and CD29 of the "Ligue contre le Cancer du Grand-Ouest".


M Cohen

J Plaza

Xavier

Annabelle Perrault

Laurence Chaby

Fabienne Bigouret

Dr Alice Oppetit
email: alice.oppetit@aphp.fr

David Cohen

Monique Plaza

Jean Xavier

Comprehension of conventional gestures in typical children, children with autism spectrum disorders and children with language disorders Compréhension des gestes conventionnels chez les enfants typiques, les enfants avec troubles du spectre autistique et les enfants ayant des troubles du langage oral

Keywords: Trouble du spectre autistique, troubles de la communication, troubles du langage, gestes conventionnels, gestes iconiques, gestes arbitraires Autism spectrum disorder, communication impairments, language disorders, conventional gestures development, iconic gestures, arbitrary gestures

  L'archive ouverte pluridisciplinaire

INTRODUCTION

To efficiently communicate and establish social interaction with others from an early age, individuals must be able to interpret both verbal and non-verbal messages [START_REF] Bernicot | L'usage Gestes Mots Chez L'enfant[END_REF][START_REF] Capirci | Gestures and words during the transition to two-word speech[END_REF]. Gestures constitute a universal and primary feature of human communication and a precursor to verbal language development [START_REF] Goldin-Meadow | Gesture's role in speaking, learning, and creating language[END_REF]. Co-verbal gestures are defined as the arm and hand movements that synchronise with speech [START_REF] Neill | Hand and mind: what gestures reveal about thought[END_REF]. They serve the purpose of supplementing language content, regulating speech flow, maintaining the attention between a speaker and listener during a conversation, shifting a conversational topic, facilitating the continuation of a topic, and emphasizing a particular topic or content [START_REF] Kendon | Gesture: Visible Action as Utterance[END_REF]. No categorisation of co-verbal gestures achieve unanimity between researchers. In this paper, we focus on conventional gestures (CG) [START_REF] Guidetti | Yes or no? How young French children combine gestures and speech to agree and refuse[END_REF], one of the 5 classes of co-verbal gestures according to the taxonomy of Ekman and Friesen [START_REF] Ekman | The repertoire of Non Verbal Behaviour, Categories, Origins, Usage and Coding[END_REF]. CG gestures are intentional, communicative acts which have a direct verbal translation usually consisting of a word or a phrase and which can easily be understood without speech by a certain culture or social group.

Conventional gestures during development

Previous research has shown that gesture both precedes and is tightly related to changes in early language development [START_REF] Özçalıkan | Gesture is at the Cutting Edge of Early Language Development[END_REF][START_REF] Iverson | Gesture paves the way for language development[END_REF]. Gesture comprehension emerges early (Morford & Goldin-Meadow, 1992) and its developmental trajectory is tightly linked with that of gesture production (Dimitrova, Özçalışkan, Adamson, 2017). However, research on gesture comprehension remains relatively scarce compared to the large literature documenting developmental changes in gesture production. Young children typically begin to gesture between 8 and 12 months [START_REF] Bates | The Emergence of Symbols: Cognition and Communication in Infancy[END_REF]. They first use deictic gestures (i.e. pointing gestures), whose meaning is given entirely by context and not by their form. Shortly after, between ages two to three, children produce and comprehend CG (Hodges, Özçalışkan & Williamson, 2017).

CG may be coded in an arbitrary (e.g., moving the hand to say 'goodbye') or iconic (e.g.a hand holding a glass to express drinking) way [START_REF] Ekman | Movements with Precise Meanings[END_REF]. Arbitrary means that the actual form of the symbol does not reflect the form of the thing or activity it symbolizes. On the other hand, iconic gestures show a close relation between the form or embodiment of the gesture and the meaning of its verbalisation. This type of gesture is produced when people move their arms and hands to produce a dynamic visual representation of the objects or events (action, size, motion, or shape) they wish to communicate [START_REF] Bernicot | L'usage Gestes Mots Chez L'enfant[END_REF][START_REF] Neill | Hand and mind: what gestures reveal about thought[END_REF]. Iconic gestures play an important role in precipitating early word learning [START_REF] Goodwyn | Impact of Symbolic Gesturing on Early Language Development[END_REF]. Communication skills and the expression of conventional gestures significantly improve between the ages of three and four years [START_REF] Kumin | Gestural Communication in Preschool Children[END_REF], with children acquiring a growing number of gestures that can be used while speaking. From the age of 5, they possess a plateauing repertoire of approximately one hundred gestures knowing that several gestures will disappear and will be replaced by lexical productions [START_REF] Guidetti | Aspects pragmatiques de la communication gestuelle et verbale chez le jeune enfant : une comparaison France/Côte-d'Ivoire, Pragmatic aspects of gestural and verbal communication in young children: A comparison France/Ivory Coast[END_REF]. When such lexicon is acquired, the remaining conventional gestures still perform several functions: they can replace, clarify or nuance language [START_REF] Guidetti | The emergence of pragmatics: forms and functions of conventional gestures in young French children[END_REF]).

According to Piaget's theory of symbolic development [START_REF] Piaget | Play, dreams and imitation in childhood[END_REF], one could suppose that iconic gestures are acquired earlier than arbitrary gestures because of their resemblance between the symbol and its referent Namy, Campbell and Tomasello [START_REF] Namy | The Changing Role of Iconicity in Non-Verbal Symbol Learning : A U-Shaped Trajectory in the Acquisition of Arbitrary Gestures[END_REF] conducted a study that examined mapping of iconic vs. arbitrary gestures in children aged 18 months, 26 months, and 4 years. They concluded that all ages were successfully able to map iconic gestures. However, only 18-month-olds and 4-year-olds, but not 26-month-olds, were able to map arbitrary gestures, revealing a U-shaped developmental function. The re-emergence of arbitrary gestures at 4 years is driven by a wider range of symbolic experiences and enhanced sensitivity to others' communicative intent.

Because CG contribute to communication and is a precursor of verbal language, their study and evaluation in children presenting neuro-developmental disorders including communication difficulties is crucial. According to DSM-5, communication disorders include several diagnosis among which language disorder (LD) and autism spectrum disorder. The former can affect both expressive and receptive language and is defined as a relatively 'pure' language impairment. ASD is characterised by severe deficits and pervasive impairment in several areas of development such as reciprocal social interactions, communication skills and stereotyped behaviors, interests and activities.

Pragmatics are defined as the use of language, prosody, and gesture for social communication. Deficits in pragmatics are considered a hallmark symptom of ASD and were once thought to distinguish ASD from LD. However, children with LD are a heterogeneous group in terms of linguistic abilities. Some children with LD may also have pragmatic difficulties [START_REF] Bishop | Conversational responsiveness in specific language impairment: evidence of disproportionate pragmatic difficulties in a subset of children[END_REF][START_REF] Rapin | Developmental language: nosological consideration[END_REF]. What about the acquisition of conventional gestures in these two groups ASD and LD ?

Conventional gestures in Children with Language Disorders

Children with LD should theoretically be able to use gesture as a compensatory mechanism providing an alternative mean to convey words or extend utterances [START_REF] Iverson | Gesture and motor skill in relation to language in children with language impairment[END_REF]. Within the literature, there is some debate as to how and when children with LD use gesture during spontaneous communication. Gesture is a complex skill that requires the combination of social, cognitive and motor skills. Impairments concerning these skills are often comorbid with LD [START_REF] Stothard | Language-impaired preschoolers: a follow-up into adolescence[END_REF]. Studies suggest that, compared to typically developing (TD) children, children with LD exhibit poorer performance on gross and fine motor tasks and have difficulty producing hand sequences [START_REF] Powell | Clumsiness and perceptual problems in children with specific language impairment[END_REF]. Some studies have found that children with LD produce significantly more gestures than their TD peers [START_REF] Mainela-Arnold | Gesture-speech integration in children with specific language impairment[END_REF]. On the other hand, others argue that children with LD do not gesture more frequently than TD children [START_REF] Blake | Gestures accompanying speech in specifically languageimpaired children and their timing with speech[END_REF], do not use gesture as a means of nonverbal communication, and, consequently, exhibit social difficulties [START_REF] Hart | The relationship between social behavior and severity of language impairment[END_REF].

Furthermore, some researchers have suggested that gesture and speech form an integrated communication system [START_REF] Neill | Hand and mind: what gestures reveal about thought[END_REF]. This theory would suggest that, similar to adults with aphasia for whom speech and gesture are impaired in parallel [START_REF] Neill | Hand and mind: what gestures reveal about thought[END_REF], children with language impairments may also display difficulties with gestural communication. Botting (Botting, Riches, Gaynor & Morgan, 2010) explored gesture production and comprehension abilities in relation to LD in school-aged children (4.3-7.4 years old). They found that the gesture production and motor control scores of the LD group did not differ significantly from a group of agematched TD controls. However, differences were found on a comprehension task combining speech and gesture.

The multimodal task consisted of a spoken sentence whose last word had been replaced with a gesture. Children were asked to identify the missing word from a choice of four pictures (including a semantic distractor, gesture distractor, and unrelated distractor). TD children achieved higher accuracy scores than peers with LD. In addition, when children failed to integrate gestural and spoken information in an utterance, the TD group was more likely to rely on the spoken cues and therefore was more likely to select the semantic distractor. In contrast, the children with LD were more likely to select the gesture distractor, suggesting that these children had failed to extract the intended gesture meaning. Finally, there were no gender differences in the gesture scores in either LD or TD group. Replicating this last study, Wray et al. [START_REF] Wray | Gestural abilities of children with specific language impairment[END_REF] found support for the hypothesis of gesture and speech forming an integrated system: children with LD scored significantly lower than TD children on gesture-production and on gesture-comprehension tasks [START_REF] Botting | Gesture production and comprehension in children with specific language impairment[END_REF]. They concluded that LD children failed either to extract the movement meaning or to integrate the gesture meaning with semantic context.

Conventional Gestures in Children with Autism Spectrum Disorders

Children with ASD produce less proto-declarative pointing and less communicative gestures than TD children, as described in studies based on familial home movies focusing on early signs of autism [START_REF] Saint-Georges | Signes précoces d'autisme: D'où vient-on? Où va-t-on?[END_REF]. However, they are able to formulate demands in protest by using proto-imperative gestures and unfamiliar gestures such as pushing an adult's hand towards a desired object or using the other as a tool [START_REF] Paul | Language Disorders from Infancy Through Adolescence: Assessment & Intervention[END_REF].

Clinicians have long reported abnormalities in both verbal and nonverbal forms of communication in ASD, including the 'clumsy' and 'inappropriate' nature of gestures described by Asperger in 1944 [START_REF] Wing | Asperger's syndrome: a clinical account[END_REF]. The presence of communicative gesture deficits in ASD is widely asserted in the clinical literature [START_REF] Özçalışkan | Early deictic but not other gestures predict later vocabulary in both typical development and autism[END_REF]. The impairments in ASD's gestures include (i) atypical gesture and/or developmental delay of the gesture [START_REF] Charman | Measuring early language development in preschool children with autism spectrum disorder using the MacArthur Communicative Development Inventory (Infant Form)[END_REF][START_REF] Luyster | Characterizing communicative development in children referred for autism spectrum disorders using the MacArthur-Bates Communicative Development Inventory (CDI)[END_REF] (ii) poor integration with speech, (iii) reduced frequency [START_REF] Bartak | A comparative study of infantile autism and specific development receptive language disorder. I. The children[END_REF] and (iv) poor variety [START_REF] Colgan | Analysis of social interaction gestures in infants with autism[END_REF]. Impairments in gesture are codified on gold-standard ASD diagnostic measures such as the ADOS [START_REF] Lord | The autism diagnostic observation schedule-generic: a standard measure of social and communication deficits associated with the spectrum of autism[END_REF], the Autism Diagnostic Interview [START_REF] Lord | Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders[END_REF], and the M-CHAT [START_REF] Robins | The Modified Checklist for Autism in Toddlers: an initial study investigating the early detection of autism and pervasive developmental disorders[END_REF]. In these questionnaires, the absence or infrequency of gesture is rated as symptomatic. Scoring criteria for these diagnostic measures suggest that individuals with ASD use less gestures than their typically developing (TD) peers. Many studies using only parent report [START_REF] Luyster | Characterizing communicative development in children referred for autism spectrum disorders using the MacArthur-Bates Communicative Development Inventory (CDI)[END_REF][START_REF] Mitchell | Early language and communication development of infants later diagnosed with autism spectrum disorder[END_REF] emphasized on pointing and showing gestures [START_REF] Camaioni | The communicative use of pointing in autism: developmental profile and factors related to change[END_REF][START_REF] Lebarton | Gesture development in toddlers with an older sibling with autism[END_REF]. Moreover, compared to gesture production, gesture comprehension in children with ASD remains an understudied research domain (Dimitrova, Özçalışkan, Adamson, 2017). Given the literature regarding conventional gestures of both LD and ASD, we believe that conventional gesture impairments could constitute a significant clinical dimension to better differentiate LD and ASD communication impairments. Early screening could also be useful in distinguishing the developmental trajectory of these two neurodevelopmental disorders as well as exploring their possible overlap that could aid clinicians in early diagnosis and tailored remediation.

Aims of the Study

To date, little is known about the development of CG comprehension especially in children with LD and ASD, and there is no established means of assessing it. Thus, the first aim of the present study was to analyse the developmental course of CG comprehension (from age 3 to 10 years) using an experimental protocol specifically designed for this purpose. The second aim was to analyse, from a clinical perspective, how children with developmental communication disorders can understand conventional gestures, insomuch as this ability can compensate for language weakness or absence. We formulated the following hypotheses: (i) among TD children, the comprehension of conventional gestures would quantitatively and qualitatively increase during development (ii) iconic conventional gestures would be acquired before arbitrary gestures in TD, ASD, and LD children (iii) among children with ASD, comprehension of iconic and arbitrary conventional gestures would be lower to that of TD children (iv) children with LD would correspond to an intermediate profile between ASD and typical children, showing better performance than children with ASD but worse or comparable performance compared to typical children of similar developmental age. The typical children were enrolled in the ordinary educational system and their educational levels corresponded to their age. We also recruited 55 children, from 4 to 8 years old, with communication impairments: 36 with ASD (mean= 6 ± 1.5 years; 11 girls and 25 boys) and 19 with LD (mean= 6 ± 1 years; 7 girls and 12 boys). In order to avoid interpretation bias, we included only homogenous LD children with phonological-syntactic dysphasia and good oral language comprehension skills, based on Rapin and Allen classification of LD [START_REF] Rapin | Developmental language: nosological consideration[END_REF].

METHODS

They were enrolled in medico-educational institutes, at-home intervention clinical services or in speech therapy private practices. Diagnoses were based on the DSM-5 criteria and were confirmed by clinicians in the Department of Child and Adolescent Psychopathology of the Pitié -Salpêtrière Hospital that have expertise in both ASD and LD [START_REF] Demouy | Differential language markers of pathology in Autism, Pervasive Developmental Disorder Not Otherwise Specified and Specific Language Impairment[END_REF].

Material

The computerised protocol was created by FB, a speech therapist, and implemented by a computer expert. The protocol consisted of thirty items divided into two categories: fifteen arbitrary gestures and fifteen iconic gestures (Table 1) that were defined according to the state of the art concerning typical children. The thirty gestures were realized by FB, without situational context, recorded on a video and introduced in the computerised protocol. These gestures were presented only visually i.e. without sound and word on a laptop computer in random order. The responses were presented visually (i.e. written) on the computer screen, read by the examiner and associated to colored tokens. The child could choose between three responses: the 'correct response', a 'literal response' and an 'incorrect response' corresponding to the same semantic field as the 'correct response' (Cf. Table 2). The presentation order of response modalities was randomised.

(PLEASE INSERT TABLE 1) (PLEASE INSERT TABLE 2)

Procedure

The testing of language gesture comprehension was performed on an individual basis. The child was alone in a quiet room with the examiner. He was placed in front of a laptop computer, and the examiner was sitting next to him. Videos with iconic or arbitrary gestures were presented to him. Each video sequence could be visualised several times, at the request of the child. Then, he was asked a question. For the iconic gestures, the child was asked, 'What is the person doing?' and, for the arbitrary gestures, 'What is the person saying?'. The child had to select the answer considered correct either by (i) clicking directly on the computer (ii) responding orally (iii) using the colored tokens. If the child answered spontaneously before the end of the response presentation, the examiner continued reading before selecting a final response to ensure consistency. When the child did not produce a response, the black box 'no response' was selected. For a wrong answer, the gesture was presented again and the software recorded the reaction time and responses.

Statistical Analysis

Statistical analyses were performed using R Software, Version 2.12.2. For every test, the level of significance, alpha, was fixed at 5%. To assess the variables associated with the comprehension of conventional gestures in typical children, a binomial general linear mixed model (GLMM) was applied to the mean performance (two scores per subject). The three following variables were entered in the model: age (continuous variable); type of gesture (arbitrary vs. iconic); and gender (girl vs. boys). For each variable, the normal distribution was checked.

To assess the performance of children with communication impairment, we used binomial general linear model (GLM) applied to the mean performance for arbitrary gesture then iconic gesture. The three following variables were entered in the models: age (continuous variable); type of group (TD vs. ASD vs. LD); and gender (girl vs. boys). Finally, we repeated the first analysis including individuals with communication impairments. To do so, we performed a binomial GLMM applied to the mean performance (two scores per subject). The four following variables were entered in the model: age (continuous variable); type of gesture (arbitrary vs. iconic); type of group (TD vs. ASD vs. LD) and gender (girl vs. boys).

separately, the GLM showed several significant effects: comprehension significantly improved with age (β=0.043, p<.001); comprehension was lower in children with ASD compared to TD children (β=-0.98, p<.001); comprehension was higher in children with LD compared to TD children (β=0.36, p=.023). However, there was no effect of gender (β=0.023, p=.61) and comprehension was higher for LD compared to ASD children (β= 1.34, p<.001). Finally, when we performed the binomial GLMM with both arbitrary and iconic gesture we found the same effects as for the model with typical children, only combined to the results found for children with communication impairments: (i) comprehension significantly improved with age (β=0.87, p<.001); comprehension was lower in children with ASD compared to TD children (β=-1.21, p<.001); comprehension tended to be higher in children with LD compared to TD children (β=0.51, p=.058) but was significantly higher when compared to ASD (β=1.72, p<.001); comprehension was easier for iconic gestures compared to arbitrary ones (β=0.42, p<.001). However, there was no effect of gender (β=-0.17, p=.15).

(PLEASE INSERT FIGURE 3)

DISCUSSION

The first aim of the present study was to analyse the developmental course of conventional gestures (CG) comprehension in typical children and in children with communication impairments (ASD and LD). As expected, conventional gesture comprehension increased during the early development of typical children, and the improvement of iconic gestures comprehension occurred earlier than that of arbitrary ones. The second aim of the study was, in a clinical perspective, to document how children with language impairments and developmental communication disorders can understand CG, to the extent that this ability can compensate for language weakness or absence. Children with ASD received lower CG scores than typical children and LD children. But, contrary to our fourth hypothesis, for each gesture taken separately, children with LD performed better than, not only ASD children, but typical children as well. Furthermore, when we performed the analysis with both arbitrary and iconic gesture, children with LD outperformed both children with ASD and TD children.

Developmental Course of Conventional Gesture Comprehension

In TD children from age 3 to 10 years, for both arbitrary and iconic gestures, gesture comprehension significantly increased during the children's development. This age effect is explained by the fact that the development of conjoint activity throughout daily interactions allows children to socialise with communicative intentions and, thus, progressively adapt the acquired codes to various social situations. These interaction patterns represent a fundamental resource to transfer from non-verbal communication to oral language. With the emergence and use of oral language, the child's gesture repertoire is transformed and enriched similarly to the lexical, semantic and pragmatic development of language [START_REF] Colletta | Age-related changes in co-speech gesture and narrative: Evidence from French children and adults[END_REF]. Due to acquired experiences during childhood, several CG subsist in older children and adults because they represent a common code [START_REF] Guidetti | Aspects pragmatiques de la communication gestuelle et verbale chez le jeune enfant : une comparaison France/Côte-d'Ivoire, Pragmatic aspects of gestural and verbal communication in young children: A comparison France/Ivory Coast[END_REF]. In addition, we found that comprehension of conventional gestures was influenced by gesture type and gender.

Concerning gesture type, in line with Dimitrova et al. (2017), comprehension was better for iconic than for arbitrary gesture. This result is consistent with the cognitive development of children in view of the concrete nature of iconic gestures [START_REF] Piaget | Play, dreams and imitation in childhood[END_REF]. The iconic gestures were easier to understand because they represent and directly suggest children's daily activities. Because children are confronted daily with iconic gestures at an early age, they quickly memorise the constant relationship between significant and signified, which facilitates comprehension. This situation is also the case for several arbitrary gestures ('congratulations', 'be quiet', and 'goodbye') that are often practiced during daily interactions and, thus, can be quickly memorised. In contrast, most arbitrary gestures are less frequently used in daily interactions and are less concrete, depending on the progress of memory capacity and the quality and intensity of child-adult interactions. Finally, our results are consistent with those of Namy [START_REF] Namy | The Changing Role of Iconicity in Non-Verbal Symbol Learning : A U-Shaped Trajectory in the Acquisition of Arbitrary Gestures[END_REF] showing that, contrary to iconic gestures, arbitrary gestures are not as well understood between 3 and 4 years old.

Concerning gender, our data showed a gender effect limited to iconic gestures. This result contrasts with Botting's study [START_REF] Botting | Gesture production and comprehension in children with specific language impairment[END_REF] which found no gender differences in gesture scores, but gender effect has been very seldom studied in a developmental way in the literature. We found that boys displayed better comprehension than girls who showed a faster improvement of comprehension with age (Figure 2). This could be due to the fact that girls tend to develop language earlier and better than boys [START_REF] Bouchard | Gender differences in language development in French Canadian children between 8 and 30 months of age[END_REF] and, thus, tend to use fewer gestures to communicate.

Boys compensate a slower maturation of oral language with a higher level of motor and gesture activity. In addition, parents play a key role in motor skills development by placing more importance on the physical development of boys [START_REF] Cerveau Rose | cerveau bleu: les neurones ont-ils uns sexe[END_REF]. Moreover, the sex of the parent and the playful context (e.g., use of certain toys) seem to have an impact on the language development in favour of girls [START_REF] Bailly | Les filles sont plus douées pour les langues? »: enquête autour d'une idée reçue[END_REF]. Thus, the variables concerning biology and social and developmental contexts could contribute to this gender effect [START_REF] Leaper | A meta-analytic review of gender variations in children's language use: talkativeness, affiliative speech, and assertive speech[END_REF].

Conventional Gesture Comprehension in Children with LD

All statistical analyses (whether they explored both arbitrary and iconic gestures or each gesture separately), reported that comprehension was higher for LD compared to both TD and ASD. The comparison with ASD is an expected result knowing the severe deficits in communication shown by children with ASD. The comparison with TD contradicts the theory of an integrated communication system [START_REF] Neill | Hand and mind: what gestures reveal about thought[END_REF] between gesture and speech that argue difficulties with gestural communication in children with LD. In this study, children with LD had rather good comprehension skills and mainly phonological-syntactic deficits. They appeared to rely on conventional gestures to understand and interpret the communicative intentions of others. All of these gestures that replace words and allow children to express information constitute a mechanism that compensates for language deficits [START_REF] Blake | Gestures accompanying speech in specifically languageimpaired children and their timing with speech[END_REF][START_REF] Botting | Gesture production and comprehension in children with specific language impairment[END_REF].

When children with LD must process simultaneously speech and gestures, they may have difficulties because of cross-modal processing limitations [START_REF] Wray | Gestural abilities of children with specific language impairment[END_REF][START_REF] Botting | Gesture production and comprehension in children with specific language impairment[END_REF] and, thus, they often prefer to interpret gestures. Moreover, children with LD more easily produce gestures than words when the context allows them to do so [START_REF] Blake | Gestures accompanying speech in specifically languageimpaired children and their timing with speech[END_REF]. At an early age, children with LD use symbolical gestures similar to the typical children; however, when growing up, they continue to depend on information produced by gestures more than by words because of their language delay. Similarly, children with LD produce more iconic gestures than the typical children [START_REF] Iverson | Gesture and motor skill in relation to language in children with language impairment[END_REF][START_REF] Mainela-Arnold | Gesture-speech integration in children with specific language impairment[END_REF]. The interpretation of conventional gestures allows them to facilitate social interactions that are disturbed but not hindered by their language impairment. To conclude, discrepancies in earlier studies regarding CG in LD (see introduction) may be related to language domain deficit (e.g. lexicon) or language comprehension deficit of children with LD included in these reports. Therefore, generalisation of our results needs the study to be replicated in a larger sample.

Conventional Gesture Comprehension in Children with ASD

Children with ASD performed worse in conventional gesture comprehension than typical children of similar developmental age. The association between action and its representation with iconic gestures did not facilitate comprehension in this group. It appeared that those children were unable to integrate the symbolic sense of conventional gestures and their meaning because they experienced difficulty using them outside social routines and generalising their use out of a particular context [START_REF] Bernabei | Developmental profile and regression in a child with autism: a single case study[END_REF]. In addition to communication abilities, the comprehension and use of CG require imitation skills. Imitation skills play a fundamental role in the development of communication and may constitute an initial social signature during interaction [START_REF] Boucenna | the Michelangelo Study Group. Extraction of social signatures through imitation learning between a robot and a human partner[END_REF]. Indeed, as long as children have no access to oral language, they use imitation to communicate [START_REF] Nadel | How imitation boosts development in infancy and Autism Spectrum Disorder[END_REF] and to learn using conventional gestures. Tomasello and Camaioni hypothesised that children with ASD have imitation difficulties, which impairs conventional gesture acquisition [START_REF] Tomasello | A Comparison of the Gestural Communication of Apes and Human Infants[END_REF]. This imitation deficit could be explained by a weak interest in social interaction and the presence of severe executive impairments, more than by a deficit per se of imitation [START_REF] Nadel | How imitation boosts development in infancy and Autism Spectrum Disorder[END_REF]. Children with ASD also show heterogeneous performance in facial emotion processing [START_REF] Vannetzel | Neutral versus emotional human stimuli processing in children with pervasive developmental disorders not otherwise specified[END_REF][START_REF] Xavier | A Multidimensional Approach to the Study of Emotion Recognition in Autism Spectrum Disorders[END_REF] that play a role in the comprehension of verbal and non-verbal messages. All of the communicative and emotional difficulties observed in children with ASD could be underlined by two factors. First, according to the 'weak central coherence theory' which was conceptualized by Happé and Frith [START_REF] Happé | The weak coherence account: detail-focused cognitive style in autism spectrum disorders[END_REF] and corresponds to the difficulties that children with ASD have when synthesizing stimuli into a coherent whole, children with ASD have impairments in multisensory processing [START_REF] Collignon | Reduced multisensory facilitation in persons with autism[END_REF]. Indeed, patients with ASD would not globally perceive the stimuli and, consequently, they would not establish the coherence of the object they observe. Information processing would be local and unorganised. Second, a deficit in TOM (theory of mind) could explain some aspects of communication disorders [START_REF] Baron-Cohen | Does the autistic child have a "theory of mind[END_REF]. Children with ASD have difficulties deducing the mental states of others, including their thoughts, intentions and emotions. They are occasionally capable of performing this function, however they do so later and less efficiently compare with typical children.

Study Limitations and Strengths

The results of the current study should be interpreted according to certain limitations. First, in contrast with the large sample of typical children, the group of atypical children was restricted. Specifically, the sub-analyses according to age had a restricted number of pathological cases, which limits the interpretation of clinical findings. Second, because the inclusion criteria precluded the existence of pointing gestures and a level of comprehension superior to children aged 3 years to ensure participation in the protocol, many young or severely pathological children were excluded from the study. In our procedure the gestures integrated different levels of complexity (e.g., sleeping gesture much easier than playing flute) that we did not take into account in our results.

According to the idea that gestures are a complex skill,in ecological situation as well as in our experimental setup, the majority of gestures accompany facial expressions, which give cues for interpreting and comprehending conventional gesture. For instance, in the current protocol, the experimenter's face expressed anger (e.g. 'things are going badly') or happiness (e.g. 'congratulations'). Facial expressions that were used to understand the psychological state of the interlocutor optimised the social interaction regulation. They sustained behaviour adjustment and facilitated communication between the child and others [START_REF] Ekman | Conceptual ambiguities. In : Emotion in Human Face[END_REF]. It can be hypothesised that children's comprehension of gestures benefits from these facial cues. Besides, although the children had similar school level, there were no systematic measures of the IQ, precluding controlling for IQ. In addition, although the team is specialised in the field of autism, the fact that the diagnoses of autism spectrum disorder were not established by standardised evaluation but only through clinical evolution can constitute a limitation.

On the other hand, this study presents several interesting preliminary findings. It shows the overall validity of the computer-based protocol from a clinical perspective. This study is now being finalised using a new protocol that combines conventional gesture comprehension and visual processing (eye tracking). The aim is to study (i) how children visually explore the videos to access conventional gesture comprehension (ii) whether the synchrony between correct responses and gaze direction in typical children and children with communication disorders (ASD or LD) may allow us to passively assess CG comprehension in young children with no language and no pointing gestures.

Conclusion

Among typical children, CG comprehension improves from 3 to 10 years old. The improvement of the CG comprehension occurs earlier for iconic gestures compared to arbitrary gestures. Among children with developmental communication impairments, two developmental profiles can be distinguished. First, children with ASD have lower performance compared to typical children. In contrast, children with LD perform better than both typical children and children with ASD. These results suggest a dissociation between oral language comprehension and language gesture comprehension in children with LD and a deficit in both oral language and language gesture comprehension in children with ASD.

FIGURE 1. Mean number of correct answers for arbitrary and iconic gestures in typical children

FIGURE 2. Probability of correct answers for iconic gestures in typical girls and boys

FIGURE 3. Mean number of correct answers for arbitrary and iconic gestures in typical children and children with ASD and LD

(

  Hodges, Özçalışkan & Williamson, 2017). In their study, Dimitrova et al. (Dimitrova, Özçalışkan, Adamson, 2017) found that, for 2-to 4-year-old children, comprehension of iconic gesture was better than that of arbitrary gestures. However, several studies indicate that children are good at acquiring both iconic and arbitrary gestures early in development, displaying no clear advantage for iconic gestures (Thompson, Vinson, Woll & Vigliocco, 2012).

Participants

  To perform a developmental analysis of the experimental procedure, we recruited 262 typical children (119 girls and 143 boys) from 3 to 10 years (3 y.o, n = 11; 4 y.o. n = 62; 5 y.o. n = 55; 6 y.o. n = 39; 7 y.o. n = 52; 8 -10 y.o. n = 37).

TABLE 1 . List of thirty conventional gestures (15 iconic and 15 arbitrary gestures) Iconic gestures Arbitrary gestures

 1

	She drinks	Goodbye
	She eats	I don't know
	She sleeps	You're crazy
	She cuts	Go away
	She calls	I have enough
	She drives	I love you
	She ties her shoe laces	Ouch
	She writes	Yes
	She combs her hair	No
	She plays piano	Come
	She brushes her teeth	Great
	She washes her hands	Quiet
	She reads	Stop
	She paints	Congratulations
	She plays flute	Things are going badly

TABLE 2 . Item and response examples for iconic and arbitrary gestures Iconic gestures Correct response She calls She sleeps She plays flute She drives

 2

	Literal response	She holds her	She tilts her head	She blows	She moves her
		fingers apart			hands
	Incorrect	She hangs up	She thinks	She plays guitar	She plays music
	response

Arbitrary gestures Correct response She says quiet She says you are crazy She doesn't know She says i love you

	Literal response She touches her	She puts her	She moves her arms	She touches her
		lips	finger on her head		fingers
	Incorrect	She says louder	She says it is	She says i know	She says you are
	response		good		mean

Acknowledgments

The study was supported by the Agence Nationale de la Recherche (ANR-12-SAMA-006), the Centre d'Activités en Psychiatrie Infanto-Juvenile and the Groupement de Recherche en Psychiatrie (GDR-3557).

Sponsors had no involvement in the study design, data analysis, or interpretation of the results.

RESULTS

Comprehension of Arbitrary and Iconic Gestures in Typical Children

Figure 1 summarises the comprehension of CG for both arbitrary and iconic gestures in typical children according to age. The GLMM showed several significant effects: (i) comprehension significantly improved with age (β=0.86, p<.001) ; the secondary analyses on iconic gestures and arbitrary gestures separately showed that this was the case for both types of CG (β=0.042, p<.001; β=0.043, p<.001, respectively) (ii) comprehension was significantly associated with the type of gesture: specifically, the comprehension of arbitrary gestures was significantly lower than that of iconic gestures (β=0.44, p<.001); the children were 1.6 (=e 0.44 ) times more likely to give a correct answer for iconic gestures vs. arbitrary ones; also, as shown in figure 1, the improvement of the comprehension of iconic gestures appeared earlier (nearly 6-month) than that of arbitrary ones (iii) comprehension tended to be significantly associated with gender, with boys showing better comprehension than girls: the secondary analyses on iconic gestures and arbitrary gestures separately showed that this was the case for iconic gestures only (β=2.22, p<.001), and not for arbitrary gestures (β= 0.07, p=.28). The interaction between age and gender was significant (β=0.04, p<.001), indicating that, in the case of iconic gestures, improvement of comprehension with age was faster for girls (Figure 2).

(PLEASE INSERT FIGURE 1) (PLEASE INSERT FIGURE2)

Comprehension of Arbitrary and Iconic Gestures in Children with Communication Impairments compared to TD children

Figure 3 summarises the comprehension of conventional gestures for both arbitrary and iconic gestures in typical children, children with ASD and children with LD For the iconic gestures separately, the GLM showed several significant effects: comprehension significantly improved with age (β=0.042, p<.001); comprehension was easier for girls (β=0.33, p<.001); comprehension was lower in children with ASD compared to TD Children (β=-1.15, p<.001); comprehension was higher in children with LD compared to TD children (β=0.97, p<.001); and comprehension was higher for LD compared to ASD children (β= 2.11, p<.001). For the arbitrary gesture

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.