Wen-Bin Zou

Arnaud Boulling

Atsushi Masamune

Prachand Issarapu

Emmanuelle Masson

Hao Wu

Xiao-Tian Sun

Liang-Hao Hu

Dai-Zhan Zhou

Lin He

Yann Fichou

Eriko Nakano

Shin Hamada

Yoichi Kakuta

Kiyoshi Kume

Hiroyuki Isayama

Sumit Paliwal

Radha Mani

Seema Bhaskar

David N Cooper

Claude Férec

Tooru Shimosegawa

Giriraj R Chandak

MD Jian-Min Chen

MD Zhao-Shen Li
email: zhaoshenli@hotmail.com

MD Zhuan Liao
email: liaozhuan@smmu.edu.cn

No Association Between CEL-HYB Hybrid Allele and Chronic Pancreatitis in Asian Populations

Keywords: Disease Susceptibility, Human Genetics, Nonsense-Mediated mRNA Decay, Pancreatic Acinar Cells

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

The etiology of chronic pancreatitis (CP) is complex and involves a subtle interplay between genetic and environmental factors. Most of the hitherto reported genes/loci affecting disease susceptibility encode proteins of the protease-antiprotease system of the pancreatic acinar cells. [1][2][3][4][5][6][7] A hybrid allele (CEL-HYB), involving the CEL gene (encoding pancreatic carboxyl ester lipase 8 ) and its tandemly linked pseudogene (CELP), was recently reported to be significantly overrepresented in CP cases as compared with controls, firstly in a discovery cohort of patients with familial CP and then in three replication cohorts of patients with idiopathic CP. 9 The CEL-HYB allele resulted from non-allelic homologous recombination (NAHR) occurring between CEL intron 10 and CELP intron 10′; replacement of the eleventh and last exon of CEL by CELP exon 11′ would yield a premature stop codon within the third "pseudo" 33-bp variable number tandem repeat (VNTR) 9 (Figure 1A). The mutant enzyme is more stable than its wild-type (WT) counterpart and induced autophagy in cellular models, 9 suggesting a novel pathogenic mechanism. However, since the patients analyzed in the original study 9 were solely of European ancestry, replication in independent populations of different ethnicity was warranted. 10 The detection of the CEL-HYB allele is dependent upon a long-range duplex PCR assay. 9 Using French CEL-HYB positive samples, we found that the originally described PCR assay 9 yielded better results under slightly modified conditions (see Supplementary Material and Supplementary Figure 1). Consequently, these modified conditions were employed to screen for the CEL-HYB allele in three Asian populations. We first analyzed a cohort of Han Chinese patients with idiopathic CP. The previously described CEL-HYBspecific 3.2-kb band was detected in 2.4% (19/799) of patients and 1.9% (20/1028) of controls (P = 0.64; Table 1). Sequencing of the 3.2-kb band-containing PCR products however revealed that none of these Chinese CEL-HYB alleles corresponded to that expected; instead, they invariably resulted from an NAHR event occurring within a 239-bp sequence tract affecting the intron 9/exon 10 boundary of CEL and the intron 9′/exon 10′ boundary of CELP (Supplementary Figure 2). Hereafter, we term the previously reported disease-associated CEL-HYB allele 9 as CEL-HYB1 so as to distinguish it from this newly described allele, CEL-HYB2.

Closer inspection of the 39 Chinese CEL-HYB2 alleles revealed that they could be divided into two subtypes by reference to three single nucleotide polymorphisms (SNPs) present within the substituting CELP sequence (Supplementary Figure 2); 38 harbored the G allele of rs10901232A/G, the T allele of rs10901233C/T and the G allele of rs671412A/G (termed CEL-HYB2a), the sole exception harboring the alternative alleles of these three SNPs (termed CEL-HYB2b). We further genotyped two idiopathic CP cohorts from Japan and India. Results from the Japanese cohort were remarkably similar to those from the Chinese cohort whereas in the Indian cohort, only one control carried a CEL-HYB allele, and this corresponded to CEL-HYB2b (Table 1). Note that a variant similar to CEL-HYB2 was mentioned in passing by Fjeld et al. 9 to be present in one European case and four controls, although no sequence details were presented. Sequencing of two corresponding French CEL-HYB positive samples revealed their identity to be CEL-HYB2b. In short, the PCR assay employed efficiently detects all three CEL hybrid alleles, the differentiation of which relies upon the sequencing of the respective PCR products.

Both CEL-HYB2a and CEL-HYB2b variants harbor premature stop codons within their chimeric exons 10 (Figure 1A); their corresponding mRNAs could therefore be subject to significant degradation by nonsense-mediated mRNA decay (NMD). However, this might not be the case for CEL-HYB1 because mRNAs that harbor a stop codon in the final exon usually escape degradation by NMD. 11 To explore this possibility, we first sought to compare the mRNA expression levels of CEL-HYB2a versus CEL-HYB1 in vitro (Supplementary Material and Supplementary Tables 1 and2). Briefly, we PCR amplified the full-length genomic CEL-HYB2a and CEL-HYB1 sequences from their corresponding carriers and cloned the resulting PCR products into the pcDNA3.1/V5-His-TOPO vector. Reverse transcription-PCR (RT-PCR) analyses of mRNAs from subsequently transfected HEK293T cells indicated lower CEL-HYB2a mRNA expression as compared to CEL-HYB1 (Figure 1B). Further quantitative RT-PCR analyses demonstrated that the mRNA expression of CEL-HYB2a accounted for only 60% of that of CEL-HYB1 (Figure 1C). Then, we tested whether the mRNA expression level of CEL-HYB2a could be increased by treatment of the transfected cells with cycloheximide, a known NMD inhibitor 12 and found this to be the case (Figure 1D). Hence, we conclude that mRNA expression from the CEL-HYB2a allele is significantly reduced by NMD. This conclusion probably also applies to CEL-HYB2b owing to its high sequence similarity with CEL-HYB2a (Figure 1A).

In summary, contrary to our expectation, we failed to identify the CEL-HYB1 allele in any of the three Asian cohorts. Based on the allele frequency of CEL-HYB1 in healthy German and French populations (0.4%), 9 the power to detect at least one CEL-HYB1 carrier among the cases is estimated to be >86%, even for the smallest cohort. Given that most rare variants (defined as a minor allele frequency of <0.5%) are population-specific, 13 CEL-HYB1 may be an ethnicity-specific disease risk factor, although this remains to be confirmed by replication in an independent cohort of European ancestry (e.g., the North American Pancreatitis Study 2 14 ). The other unexpected finding was the identification of an alternative CEL-HYB2 allele in all three Asian populations. The significant degradation of CEL-HYB2a mRNA by NMD and the observation that pancreatic exocrine function has been found to be normal in Cel-knockout mice 15 are consistent with the apparent lack of any association between CEL-HYB2a and CP. cycles of denaturation at 98°C for 10 s, annealing and extension at 68°C for 12 min, and a final extension step at 72°C for 10 min. PCR products of the expected size were purified using the NucleoSpin® Gel kit (Macherey Nagel) after gel electrophoresis. 3'-A overhangs were added to the purified products before being cloned into the pcDNA3.1/V5-His-TOPO vector (Invitrogen) in accordance with the manufacturer's instructions. Transformation was performed using XL10-Gold Ultracompetent Cells (Stratagene). Transformed cells were spread onto LB agar plates with 50 μg/ml ampicillin and incubated at 37°C overnight.

Plasmid constructs containing inserts in the right orientation were selected for simultaneously by two PCRs using the HotStarTaq Master Mix Kit (Qiagen). The first PCR amplifies a 235-bp fragment extending from the T7 promoter/priming site (located within the vector) to the exon 1/intron 1 boundary of CEL (i.e. primer pair A2 in Supplementary Table 1). The second PCR amplifies an approximately 1.3-kb fragment going from the beginning of CEL exon 10 to the BGH reverse priming site (located within the vector) (i.e. primer pair A3 in Supplementary Table 1). Selected plasmids were then sequenced using primer S10F (see Supplementary Figure 2) to pick up those carrying the desired CEL-HYB1 or CEL-HYB2a variant.

Cell Culture, Transfection and Cycloheximide Treatment

Human embryonic kidney (HEK) 293T cells were cultured in DMEM nutrient mixture with 10% fetal calf serum. 3.5 x 10 5 cells were seeded per well of 6-well plates 24 hours prior to transfection. For conventional RT-PCR analyses (see below), 1 μg pcDNA3.1-CEL-HYB1 or pcDNA3.1-CEL-HYB2a plasmid, which were mixed with 3 μL Lipofectamine 2000 Reagent (Invitrogen), were used for transfection per well. For real-time quantitative RT-PCR analyses (see below), 500 ng pcDNA3.1-CEL-HYB1 or pcDNA3.1-CEL-HYB2a plasmid were mixed with 500 ng pGL3-GP2 minigene for transfection. Forty-eight hours after transfection, total RNA was extracted using the RNeasy Mini Kit (Qiagen). For the NMD inhibition experiment, 50 μg/mL (final concentration) cycloheximide (Sigma) were added to the cells 4 hours before RNA extraction.

Reverse Transcription

Reverse transcription (RT) was performed using the SuperScript II Reverse Transcriptase (Life Technologies) with 1 µg total RNA, 500 µM dNTPs, 5 µM 20mer-oligo(dT) and 10 mM DTT (Eurogentec). The resulting cDNAs were treated with 2U RNAseH (Life Technologies) to degrade the remaining RNA.

Conventional RT-PCR and Sequencing of the Resulting Products

Conventional RT-PCR was performed in a 25-μl reaction mixture containing 0.5 U KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems), 1 μl cDNA and 0.3 µM each primer of primer pair A4 (Supplementary Table 1). Note that this primer pair was designed to amplify a full-length CEL-HYB1 or CEL-HYB2a transcript. The PCR program comprised an initial denaturation step at 95°C for 5 min, followed by 30 cycles of denaturation at 98°C for 20 s, annealing at 72°C for 15 s, and extension at 72°C for 3 min, and a final extension step at 72°C for 5 min.

RT-PCR products were cleaned using ExoSAP-IT® before being sequenced with five primers (Supplementary Table 2) by means of the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies).

Real-Time Quantitative RT-PCR

Real-time quantitative RT-PCR analyses were performed essentially as described elsewhere. 16 In brief, pcDNA3.1-CEL-HYB1 or pcDNA3.1-CEL-HYB2a was co-transfected with the previously constructed pGL3-GP2 minigene. 16 The target and reference genes were PCR amplified separately; target gene expression was determined using pGL3-GP2 minigene expression as a reference by means of the ∆∆Ct method in accordance with the Pfaffl efficiency-corrected calculation model. 18 Real-time quantitative RT-PCR was performed in a 25μL mixture containing 12.5 μL HotStarTaq Master Mix Kit (Qiagen), 1 μL 1:25 diluted cDNA, 0.5 µM SYTO9 (Life Technologies) and 0.3 μM each primer (primer pair A5 was employed for both CEL-HYB1 and CEL-HYB2a whilst primer pair A6 was used for the reference gene; Supplementary Table 1). The PCR program comprised an initial denaturation step at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 30 s and extension at 72°C for 30 s. Quantitative analysis of each RT-PCR amplification was performed in triplicate on a Lightcycler 480II (Roche, Paris, France) using "relative quantification" and "2nd derivative maxima" options.

The difference between the expression levels of CEL-HYB1 and CEL-HYB2a (results from six independent transfection experiments) and that between expression levels of CEL-HYB2a with and without cycloheximide treatment (results from three independent transfection experiments) were assessed for significance by the Student's t-test. The difference was regarded as being statistically significant when the P value was ≤ 0.05. nucleotide positions 12883-13599 and 28652-29371, respectively, from GenBank accession number AF072711.1.

Figure 1 .

 1 Figure 1. (A) Key differences between CEL-HYB1 (originally termed CEL-HYB 9 ), CEL-

Table 1 . Carrier frequencies of the CEL-HYB2 variant in Chinese, Japanese and Indian subjects with idiopathic chronic pancreatitis*

 1

	Populations	Case		Control		P value a
		+/n	%	+/n	%
	Chinese	19/799	2.4	20 (1)/1028	1.9	0.64
	Japanese	4 b /248	1.6	7 (2)/403	1.7	0.85
	Indian	0/280	0	1 (1)/225	0.4	0.91
	Combined	23/1327	1.7	28 (4)/1656	1.7	0.96
	* Number of subjects carrying the less frequent CEL-HYB2 subtype, CEL-HYB2b, is
	indicated in parentheses wherever applicable.
	a Two-tailed Fisher's exact test.

b

One patient is a homozygote.

Grant

Support WBZ is a joint PhD student between Changhai Hospital and INSERM U1078 and received a one-year scholarship (the year 2015) from the China Scholarship Council (No. 201403170271). Support for this study came from the National Natural Science Foundation of China (Grant Nos. 81470884, 81422010 [Z.L.]), the Shanghai Rising-Star Program (Grant No.13QA1404600 [Z.L.]), the HIROMI Medical Research Foundation (A.M.), the Mother and Child Health Foundation (A.M.), the Smoking Research Foundation (A.M.), the Pancreas Research Foundation of Japan (E.N.), the Ministry of Health, Labour and Welfare of Japan (Principal investigators: Yoichi Matsubara and Yoshifumi Takeyama), Council of Scientific and Industrial Research, Ministry of Science and Technology, Government of India (Grant No. BSC0121 [G.R.C.]), the Conseil Régional de Bretagne, the Association des Pancréatites Chroniques Héréditaires, the Association de Transfusion Sanguine et de Biogénétique Gaetan Saleun, and the Institut National de la Santé et de la Recherche Médicale (INSERM), contributions from EM, HW, XTS, LHH, DZZ, LH, EN, SH, YK, KK, HI

AM, GRC, ZSL and ZL recruited study subjects. AM, CF, GRC and ZL obtained the funding.

All authors approved the final manuscript.

Supplementary Material

Subjects

Chinese, Japanese and Indian patients with idiopathic chronic pancreatitis (i.e. absence of a positive family history and any known precipitating factor such as alcohol abuse) and healthy controls from their corresponding general populations were recruited by the three leading Asian pancreatitis groups. A clinical diagnosis of chronic pancreatitis was based on two or more of the following criteria: (i) presence of a typical history of recurrent pancreatitis; (ii) radiological findings such as pancreatic calcification and/or pancreatic irregularities revealed by endoscopic retrograde pancreatography or by magnetic resonance imaging of the pancreas; and (iii) pathological sonographic findings. Informed consent was obtained from each patient and the study was approved by the respective ethics committees of the three leading Asian pancreatitis groups.

Screening for the CEL-HYB Allele

Screening for the disease-associated CEL-HYB allele (termed CEL-HYB1 in the present study) was performed in accordance with the previously reported method 9 but with some modified reaction conditions. Specifically, we performed the PCR reactions in total volumes of 10 μl containing 2 × GC buffer, 0.4 μM primer L11F (5'-GTCCCTCACTCATTCTTCTATGGCAAC-3'), 0.2 μM of the primers IAR (5'-TCCAAAGCCCTAGCAGTAACGA-3') and CELP VNTR-rev (5'-CTGTGGAGGGCATGGAACT-3'), 0.4 mM each dNTP, 0.5 U LA Taq DNA polymerase and 10-50 ng genomic DNA. These conditions work better than those originally described for genotyping the French CEL-HYB1 positive samples (Supplementary Figure 1).

Sequencing the Positive Samples

Samples showing the CEL-HYB-specific 3.2-kb band 9 were subjected to Sanger sequencing using the previously published primers, S10F and S10R. 9 Crossover regions of the CEL-HYB alleles were assigned by evaluating the nucleotide positions that serve to discriminate the CEL and CELP sequences within the region of interest. To this end, we aligned CEL exon 10 and its partial flanking sequences against the corresponding CELP sequences, the discriminant nucleotides being clearly indicated (Supplementary Figure 2).

The assessment of significance of the differences between the carrier frequencies of the CEL-HYB alleles in patients and controls was performed by means of two-tailed Fisher's exact test using StatCalc (2 × 2 Tables) from Epi Info™ 7 (http://wwwn.cdc.gov/epiinfo/index.htm). The difference was regarded as being statistically significant when the P value was ≤ 0.05.

Construction of Expression Vectors for mRNA Expression Analysis in the Context of the Full-Length CEL-HYB1 and CEL-HYB2a Genomic Sequences

We PCR amplified the full-length (approximately 10-kb) genomic CEL-HYB1 and CEL-HYB2a sequences (going from the 5'-untranslated region of CEL to the 3'-untranslated region of the replacement CELP) from their corresponding carriers and cloned the resulting PCR products into the pcDNA3.1/V5-His-TOPO vector using previously described methods. 16,17 Specifically, the long-range PCR was performed using 50 ng DNA in a 50 μL reaction mixture with 2.5 U TaKaRa LA Taq DNA polymerase, 8 μL dNTP Mixture (400 µM final), 5 µL 10 × PCR buffer system, and 1 μM each primer of primer pair A1 (Supplementary Table 1). Thermal cycling conditions were: initial denaturation at 94°C for 1 min, followed by 30

Supplementary References

Supplementary Figure Legends

Supplementary Figure 1. Comparison of the originally reported PCR assay (A) and the currently used PCR assay (B) for detecting the CEL-HYB1 (as well as the CEL-HYB2b) allele (3.2 kb) in French heterozygotes. WT, wild-type (4.2 kb). NC, negative control. Supplementary Figure 2. Alignment of reference CEL and CELP sequences in the context of exon 10 (in bold) and partial flanking sequences. The crossover region for the CEL-HYB deletion allele 9 (termed here CEL-HYB1) is doubly underlined whereas that for CEL-HYB2 is singly underlined. The discriminant positions between the two paralogous sequences are indicated by numbered upwardly pointing arrows. The CEL-HYB2a variant harbors the G allele of rs10901232A/G, the T allele of rs10901233C/T and the G allele of rs671412A/G whereas the CEL-HYB2b variant harbors the alternative alleles of these three SNPs (the three affected sites are indicated by circles). The premature translation termination codon in the chimeric exon 10 of the CEL-HYB2b variant is indicated by a box. The two primers used for sequencing the breakpoint junctions of the CEL-HYB alleles, S10F and S10R, 9


Olivier Sentieys

Johanna Sepúlveda

Sébastien Le Beux

Jiating Luo

Cedric Killian

Daniel Chillet

Ian O'connor

Hui Li

Design Space Exploration of Optical Interfaces for Silicon Photonic Interconnects

Simula-on parameters q Models based on Gem5 q Quasi-cycle accurate simulator q Integration of C++ models of optical components q Based on Garnet NoC q Simulation characteristics q Ring-based optical interconnection q 8-tiles (ARM, 4-KB L1 cache) 32 bits channel width q 2 architectures : q 4 waveguides, 16 wavelengths for each waveguide q 16 waveguides, 16 wavelengths for each waveguide q Modulation speed: 10Gb/s q Real application traffic (SPLASH-2) q Communication between 2 pairs of Ips q One wavelength for each communication q Temporal and spatial conflicts in the waveguide q Laser power at nominal value q SNR î BER ì

IP i IP j IP m IP n 1 0 1 0 1 0 1 0 SNR 1 0 1 0 1 0 1 0 SNR

Conclusion and future works

q Optical NoC Interface design q critical issue in silicon photonics interconnects à need DSE at different levels q low level DSE/management q laser power and ECC q high level DSE/management q channel allocation / allocation protocol q Future works q tradeoff between Electric vs Optic energy performances q on-line strategy for channel allocation q thermal aware design method

•

  Photodetectors

inria.fr 18-mar-16 8 Channel size design tradeoff

	Channel size:
	number of
	waveguides and
	wavelengths per
	request, i.e.
	bandwidth
	Router	REQ
		0	t 1	t 2	t 3	t 4	t 5	t 6	t 7	t 8	t

data transfer time (optical domain) versus request latency (electrical domain) Best design option? http://inl.cnrs.fr http://www.inria.fr 18-mar-16 9

www.inria.fr 18-mar-16 10 Network size

	Controller Synthesis Results
		4 waveguides	16 waveguides
	Channel size	Area	Power	Area	Power
	(wg,wl)	(um 2 )	(mW)	(um 2 )	(mW)
	(16,16)	NA	NA	6292	2.09
	(8,16)	NA	NA	6629	2.21
	(4,16)	6292	2.09	7304	2.45
	(2,16)	6629	2.21	8781	2.95
	(1,16)	7304	2.45	13538	4.45
	(1,8)	8781	2.95	17022	5.06
	(1,4)	13538	4.45	21385	5.33
	(1,2)	17022	5.06	30110	5.86
	(1,1)	21385	5.33	47560	6.92

http://

http://www.inria.fr 18-mar-16

http://www.inria.fr

Op-cal layer

Impact of the waveguide sharing q How can we manage SNR and BER ?

q Laser power can be ì q SNR ì BER î q Or ECC can be included in the communication channel q SNR = BER î but BW î


Katarina Bartkova
email: katarina.bartkova@atilf.fr

Denis Jouvet
email: denis.jouvet@inria.fr

Elisabeth Delais-Roussarie
email: elisabeth.roussarie@wanadoo.fr

Prosodic Parameters and Prosodic Structures of French Emotional Data

Keywords: emotional speech, emotions, neutral style, prosodic parameters

The detection and modelling of emotions in speech remains a challenging issue in speech processing. The aim of the study presented here is to analyze and compare the use of several prosodic parameters in emotional speech in French. The data set used for the study contains utterances recorded in six emotional types: anger, fear, sadness, disgust, surprise and joy. The sentences of the emotional data are also recorded by the same speaker in a neutral reading style allowing a comparison between emotional and neutral speech. The prosodic analysis focuses to the main prosodic parameters such as vowel duration, energy and F0 level, and pause occurrences. The values of prosodic parameters are compared among the various emotional styles, as well as between emotional style and neutral style utterances. Moreover, the structuration of the sentences, in the various emotional styles, is particularly studied here through a detailed analysis of pause occurrences and their length, and of the length of prosodic groups.

Introduction

Over the last 30 years, many works showed that prosody conveys information on the linguistic content of the message, but also on speaker's attitudes [START_REF] Couper-Kühlen | An Introduction to English Prosody[END_REF] and emotional states (see, among others, [START_REF] Fonagy | La vive voix[END_REF], [START_REF] Fonagy | Emotional patterns in intonation and music[END_REF] and [START_REF] Scherer | Vocal correlates of emotion[END_REF]), leading to a distinction between linguistic prosody and emotional prosody. Note however that apprehending and distinguishing both prosodic domains is not an easy task, emotional and linguistic prosody being expressed by the same phonetic features (loudness, pitch, and speech tempo) and sometime interacting in a complex way [START_REF] Scherer | Vocal cues to speaker affect: Testing two models[END_REF]. As for perception, the robustness of the various prosodic parameters in the interpretation of speaker attitudes and emotions is still at issues in a given language as well as cross-linguistically (see, among others, [START_REF] Abelin | Cross linguistic interpretation of emotional prosody[END_REF]). Knowing how the different prosodic parameters are used to convey a particular emotion and how they interact with linguistic functions is crucial to provide a model for emotional prosody that can be used in speech technology. It requests however the development of methodological approaches.

Speech data bases that exhibit emotion styles are collected in various ways including in natural spontaneous speech, elicited emotional speech and simulated or acted speech [START_REF] Ververidis | A review of emotional speech databases[END_REF], [START_REF] Ververidis | Emotional speech recognition: Resources, features, and methods[END_REF]. Besides the traditional classification of emotions (anger, fear, disgust …) multi-dimensional representations are also used, as for example arousal (relaxed vs. aroused), valence (pleasant vs unpleasant) … [START_REF] Gunes | Emotion representation, analysis and synthesis in continuous space: A survey[END_REF]. In [START_REF] Erickson | Expressive speech: Production, perception and application to speech synthesis[END_REF] findings of previous research works relating prosodic and spectral features to the emotions are summarized.

Various sets of prosodic and spectral features as well as various classifiers have been used for emotion recognition [START_REF] Ayadi | Survey on speech emotion recognition: Features, classification schemes, and databases[END_REF], [START_REF] Lanjewar | Speech Emotion Recognition: A Review[END_REF]. Most of the approaches rely on large set of features that are represented by their statistics (min, max, mean, standard deviation, skewness, …,) [START_REF] Chen | Speech emotion recognition: Features and classification models[END_REF]; and best performance is achieved using statistics computed on the entire utterance segment rather than on word or syllable segments [START_REF] Koolagudi | Speech emotion recognition using segmental level prosodic analysis[END_REF].

In [START_REF] Schröder | Emotional speech synthesis: a review[END_REF] and [START_REF] Schröder | Expressive speech synthesis: Past, present, and possible futures[END_REF] various approaches that have been studied for expressive speech synthesis are described. Both prosodic and spectral aspects play a significant role for a correct perception of the emotions [START_REF] Bulut | Expressive speech synthesis using a concatenative synthesizer[END_REF], although the most critical one depends on the type of emotion. Concatenative speech synthesis has been used for emotional speech synthesis thanks to the recording of an emotion speech corpus for each of the desired emotions [START_REF] Iida | A corpus-based speech synthesis system with emotion[END_REF], [START_REF] Pitrelli | The IBM expressive text-tospeech synthesis system for American English[END_REF]; prosodic-phonology approaches have also been investigated for predicting fundamental frequency and duration through statistical models associated to linguistic units of prosody [START_REF] Pitrelli | The IBM expressive text-tospeech synthesis system for American English[END_REF]. Statistical speech synthesis (HMM-based) is also used for expressive speech synthesis. Performance (perception of emotion by listeners) is similar whether separate HMM-based models are developed for each emotion or whether the emotion information is included in the detailed context label (along with usual phonetic and linguistic information) [START_REF] Yamagishi | HMMbased expressive speech synthesis-Towards TTS with arbitrary speaking styles and emotions[END_REF]. When developing the speech synthesis models, using the emotion information corresponding to the intended emotion acted by the speaker or the emotion type as perceived by listeners leads to similar performance [START_REF] Tsuzuki | Constructing emotional speech synthesizers with limited speech database[END_REF]. Also, differences between neutral speech and emotion speech has been studied in order to produce emotion speech through "difference prosody models" [START_REF] Jiang | Prosody analysis and modeling for emotional speech synthesis[END_REF], or through conversion procedures to transform neutral speech into a target emotion speech [START_REF] Tao | Prosody conversion from neutral speech to emotional speech[END_REF], [START_REF] Inanoglu | Data-driven emotion conversion in spoken English[END_REF].

This study analyses the behavior of prosodic parameters on French speech data in six different emotion styles: anger, fear, sadness, disgust, surprise and joy. After a presentation of the speech data and associated features in Section 2, Section 3 focuses on the analysis of the main prosodic parameters (vowel duration, vowel energy and fundamental frequency), with comparisons between each emotion style and the neutral style. Section 4 focuses on the analysis of the pauses and of the prosodic structures in the various emotion styles, a topic which is not investigated in the literature. A short discussion in Section 5 summarizes the results.

Corpus and features

The speech corpus used contains French sentences in various emotion styles, as well as some neutral style speech material.

All the data are recorded from the same male speaker. About 50 utterances are recorded for each of the 6 emotion styles: anger, fear, sadness, disgust, surprise and joy. The same sentences are also recorded in a neutral reading style. Hence comparison between each emotion style and the neutral style can be performed on the same set of sentences.

For each sentence, the speech material is aligned with its corresponding text using an automatic speech-text forced alignment procedure (available at http://astali.loria.fr).

The approach relies on a statistical modeling of the speech sounds (Markov models). It provides the segmentation of each utterance into phone and silence (pause) units.

Prosodic features are then computed for each utterance. Segment duration is derived from the automatic phone level segmentation; whereas energy and fundamental frequency are extracted with the ETSI/AURORA front end [START_REF]Speech Processing, Transmission and Quality Aspects (STQ); Distributed speech recognition; extended advanced front-end feature extraction algorithm; compression Algorithms[END_REF].

Prosodic characterization

This section analyses for each vocalic segment duration, energy, and fundamental frequency in the six emotion styles. The behavior of these parameters is compared between emotion styles and also with respect to the neutral style.

Vowel duration

Vowel durations are measured in word non-final syllables as well as in word final syllables. Syllables in word non-final positions are in a non-stressed position, whereas those in word final position can be stressed and therefore lengthened. For every emotion style as well as for the neutral style, there is a significant difference for the mean duration of the vowels between word non-final and word final positions (Fig. 1). With respect to word non-final positions, the lowest vowel mean duration is observed in the fear emotion, and the longest one is found in the joy emotion. For word final positions, the longest vowel mean duration is observed in the anger emotion. For each emotion style, and each word position (i.e., nonfinal vs. final syllables), the duration of the vowels are compared to a reference vowel duration which corresponds to the vowel mean duration calculated on neutral style in same positions (i.e., respectively non-final vs. final syllables). Figure 2 reports the percentage of vowel occurrences that have longer duration than the corresponding reference vowel duration. Anger is the emotion which has the higher percentage of lengthened vowel durations (in both word nonfinal and final positions). The smallest percentage of lengthened vowels is observed in the fear and disgust emotion styles. The lengthening of the vowels in word non-final syllables has been quantified on a four degree scale, as defined in the PROSOTRAN prosodic annotator [START_REF] Bartkova | Prosotran: a tool to annotate prosodically non-standard data[END_REF]. The first degree (1) corresponds to vowels that are longer than the mean duration plus one time the standard deviation. The second degree (2) corresponds to vowels that are longer than the mean duration plus two times the standard deviation. Degree 3 is defined in a similar way; and degree 4 corresponds to vowels that are longer than the mean duration plus four times the standard deviation. Figure 3 illustrates, for each emotion style, the amount of vowels in word non-final position that are lengthened (according to the above scale). It thus displays only the percentages of vowels that are longer than the mean duration plus at least one time the standard deviation. The sadness emotion style contains mostly moderate vowel lengthening while the anger emotion style contains the higher percentage of heavily lengthened vowels (degree 4).

Figure 3: Percentage of vowel occurrences with respect to the four degree lengthening scale (from 1moderate lengthening to 4 -heavy lengthening).

Vowel energy

An analysis similar to the one done for vowel lengthening is carried out for vowel energy, considering vowels in word nonfinal position. A four degree scale is also defined for quantifying the level of increase of vowel energy. The first degree (1) corresponds to vowels whose energy is higher than the mean vowel energy plus one time the standard deviation and similarly for degrees 2, 3, and 4. Figure 4 illustrates, for each emotion style, the percentage of vowel occurrences in word non-final position that have an increase in energy (according to the defined scale). It thus considers only the vowels for which the energy is higher than the vowel mean value plus N times the standard deviation. For sadness, all vowels, with higher energy than the mean reference value, are

Percentage of occurrences (%)

Vowel lengthening degree sadness disgust suprise fear joy anger only slightly higher than the reference value. Also, but to a lesser extent, a similar behavior is observed for the disgust emotion. On the other hand anger has the highest percentage of vowels with a large increase in energy (degree 4: energy strongly higher than the reference value).

Figure 4: Percentage of vowel with respect to the four degree scale of energy increase.

F0 values

F0 values vary according to the general emotional state of the speakers. Values are higher when the speaker is angry and lower when the speaker is sad. Figure 5 displays the F0 values for a given sentence uttered in angry and in neutral styles: the angry melody (red curve) is much higher all over the sentence than the neutral melody (black curve). Mean F0 values are calculated for each emotion style. Results are displayed in Figure 6 along with mean F0 value calculated on the neutral style (red line). Mean F0 value on the sadness emotion is the closest to mean F0 value of the neutral style; although surprise emotion (slightly higher mean F0) and disgust emotion (slightly lower mean F0) are also very close to the mean F0 value of the neutral style. The three remaining emotions (fear, joy and anger) have higher mean F0 values than the neutral style.

To get a more detailed analysis of F0 values, their distribution is reported in Figure 7 for each emotion style (blues curves). On each graph, the orange curve reports the distribution of F0 values on the same sentences uttered in a neutral style. These statistics rely on the quantification of the F0 values on a 14 degrees scale defined according to the F0 range of the speaker. For the disgust emotion, although its mean F0 value is slightly lower than the mean F0 value of the neutral style (see Figure 6), the F0 distribution curve is slightly enlarged to the right (i.e., towards higher F0 values) compared to the neutral curve. The distribution curves of fear, joy and surprise emotions are also moved to the right side having at least half of their F0 values higher than those measured in neutral style. As far as anger is concerned, there is almost no intersection between the distribution curves representing the neutral style (orange curve) and the anger emotion (blue curve). As for the sadness emotion, it consists of one exception for which the F0 distribution is slightly enlarged to the left (i.e., towards lower F0 values) compared to the neutral style.

Pauses and prosodic structures 4.1. Pauses

The number of pauses is compared between emotion data and the data uttered in a neutral style. For analysis purpose, the pauses are classified into three categories: short pauses (shorter than 250 ms), mid pauses (shorter than 400 ms) and long pauses (longer than 400 ms). Table 1 reports for each emotion the percentage of short, mid and long pauses in the columns "E". The columns "N" reports the percentages calculated on the same sentences uttered in a neutral style. It shows that the greatest difference is observed between anger and neutral styles, and between joy and neutral styles. Indeed, the anger emotion uses a higher amount of short pauses and fewer long pauses. As for the joy emotion, a higher amount of short and mid pauses is used at the expense of long pauses. As for the total number of pauses in the various emotion styles compared to the neutral style, their number is much higher in anger emotion while sadness and surprise are very close to the neutral style, and joy and fear emotions contain significantly less pauses than the neutral style.

Prosodic groups

A segmentation of utterances into prosodic groups is carried out using an automatic approach [START_REF] Bartkova | Automatic detection of the prosodic structures of speech utterances[END_REF] based on [START_REF] Martin | Prosodic and rhythmic structures in French[END_REF], in which the decision on prosodic group frontiers relies on F0 slope, F0 level and vowel duration. Figure 8 displays, for each emotion style, the percentage of prosodic groups with respect to their length expressed in syllables. The results show that the anger emotion gives a preference to shorter prosodic groups compared to the other emotion styles. The disgust and the sadness emotion styles are the ones that use the longest prosodic groups. Table 2 summarizes the results of the comparison of the length of the prosodic groups between each emotion style and the same sentences uttered in a neutral style. Prosodic groups in the anger style are much shorter than prosodic groups in the corresponding sentences uttered in a neutral style. Sentences in surprise style contain slightly shorter prosodic groups. The sentences in the joy, fear and sadness emotions have longer prosodic groups than the corresponding sentences uttered in a neutral style. As for the disgust style, prosodic groups have the same length as in neutral speech.

Discussion

Table 3 summarizes the behavior of the parameters analyzed in this study. It displays for each emotion style the degree of lengthening (L) or shortening (S) for vowel and pause durations as well as the lengths of the prosodic groups, compared to the neutral style. It also displays the degree of higher (H) or lower (L) energy and F0 values measured on the vowels in comparison with the neutral style. For the anger emotion, all the parameters studied have a very different value in comparison to neutral data: heavy lengthening of vowel duration, short pauses, much higher energy and F0 values, and much shorter prosodic groups. Joy and fear emotion styles are also prosodically marked and are quite different from the neutral style. The disgust and sadness emotions have shorter vowel durations than neutral style, and the vowel energy is either lower than in the neutral style (as for sadness), or very similar to neutral style (as for disgust).

Conclusions

This paper provides an analysis of the behavior of the prosodic parameters on French speech data for various emotion styles: anger, fear, sadness, disgust, surprise, and joy. Beside the analysis of vowel duration, vowel energy and fundamental frequency in comparison to neutral reading style, the behavior obtained for pausing and prosodic phrasing is also investigated on the six emotional styles. This latter prosodic dimension is a phenomenon that deserves further investigation in order to correctly predict certain emotional prosodic features in speech synthesis systems. Indeed, statistical speech synthesis systems take benefit from adaptation techniques for modeling the impact and taking into account prosodic parameters; however such modeling relies on the sequence of units (sequence of phones and pauses, with their associated linguistic labels) as input both for training and for synthesis. Hence, pause distribution and lengths of the prosodic groups need to be predicted beforehand.

Figure 1 :

 1 Figure 1: Vowel mean durations in word non-final and word final syllables.

Figure 2 :

 2 Figure 2: Percentage of vowel occurrences, for the six emotion styles, that are longer than the corresponding mean duration (estimated on the neutral style).

Figure 5 :

 5 Figure 5: F0 values measured on neutral (black) and on angry emotion (red) styles for a French sentence (text at bottom of figure, meaning "Are you a prick or what? Slow down. You've almost run over the kid.").

Figure 6 :

 6 Figure 6: Mean F0 value for the six emotion styles (bars), and for the neutral style (red line).

Figure 7 :

 7 Figure 7: Distribution of F0 values for each emotion style, and comparison to F0 values for the corresponding sentences in neutral style. The points on the curves indicate the frequency (vertical axis) of quantified F0 values that fall in each degree of the 14 degree scale (horizontal axis).

Figure 8 :

 8 Figure 8: Percentage of prosodic groups (vertical axis) with respect to the length in syllables (horizontal axis) for each emotion style.

Table 1 .

 1 Percentage of pause occurrences in emotion (columns "E") and neutral styles (columns "N").

	anger disgust joy fear surprise sadness	Short pauses E N 63 17 12 8 39 9 35 32 12 12 36 30	Mid pauses E N 8 6 1 1 16 6 9 3 4 3 1 4	Long pauses E N 27 76 85 90 44 84 55 63 83 84 61 65

Table 2 .

 2 For each emotion style, comparison of the length of the prosodic groups with respect to the same sentences uttered in a neutral style.

	anger joy fear surprise disgust sadness	shorter (++) longer (+) longer (+) shorter same longer (+)

Table 3 .

 3 Summary of the parameter behavior in the different emotion styles, compared to the neutral style (see text for notations).

	duration pauses energy F0 L+++ S++ H++ H+++ S+++ Prosodic groups L++ S+ H+ H++ L+ S++ S++ H+ H++ L+ surprise L+ anger joy fear -H+ H+ S+ disgust S+ ----sadness S++ -L+ -L+

Acknowledgements

This study consists of a pilot study carried out within the ANR-15-CE23-0015 project SynPaFlex (PI: Damien LOLIVE, 2015 edition).


Viatcheslav Jouikov

Jacques Simonet

Electrochemical conversion of glassy carbon into a poly-nucleophilic reactive material. Applications for carbon chemical functionalization. A mini-review

Keywords: Glassy carbon, Poly-nucleophile, Surface modification via S N 2, Alkylation, Carboxylation

During the last decade, various carbon modifications were achieved via radical addition of substituted aryl radicals produced at the surface by chemical or/and electrochemical reduction of corresponding aryldiazonium salts. Very recently, surface reactivity of carbons towards electrophilic halo-derivatives with long alkyl chains had been developed. Surface nucleophilicity of carbons was induced by their specific cathodic charge (at E < -1.7 V in non-aqueous electrolytes) affecting the nano-crystallites (mainly graphite) contained in glassy carbons due to the mode of its manufacturing (high temperature carbonization of phenolic resins). This new paradigm of surface decoration was successfully applied using alkyl halides, carbon dioxide, dioxygen, sulfonyl chlorides etc. and proved efficient with allotropic varieties of carbon (in particular graphites and graphene).

Introduction

For the electrochemical community, glassy carbon (GC) is primarily a convenient electrode material for carrying out analyses and electrolyses within the cathodic range [1][START_REF] Schäfer | Comprehensive Organic Synthesis[END_REF]. Very often, this material was considered as non-reactive and was used as an advantageous substitute of mercury and platinum [START_REF] Peters | Organic Electrochemistry[END_REF]. However, most of the materials named glassy carbons, produced by carbonization of phenolic resins [START_REF] Jenkins | [END_REF] at temperatures up to 2500 °C, were reported to contain large amounts of graphite-like inclusions. They should therefore be regarded as complex materials containing reticulated C sp3 zones in contact with the areas where sp 2 carbon atoms predominate. Recent studies, focalized on large nano-and micro-inclusions of graphitized, graphenized, and fullerenized incorporates in the body of tetrahedral carbons, emphasize this point [5][START_REF] Bernier | Le carbone dans tous ses états[END_REF]. Structure and conductivity of commercial GCs are thus strongly dependent on the mode of their manufacturing and especially on the carbonization temperatures. There follows that common glassy carbons correspond to complex redox conglomerates gathering aromatic structures that provide the conductivity by electron hopping between them.

As a matter of fact, in polar aprotic solvents most of GCs can show own cathodic response very similar to that of highly oriented pyrolytic graphite (HOPG) and more generally to that of natural graphites. Thus, under cathodic polarization, graphites are well known to be charged with Li + [START_REF] Bernier | Le carbone dans tous ses états[END_REF]. More recently, HOPG was reversibly charged (or "reduced") in the presence of tetraalkylammonium salts (TAAX) to form intercalation compounds leading to well-defined structures depending on applied potentials (E < -1.7 V vs. Ag/AgCl) and on the bulkiness of TAA + cations used [START_REF] Besenhard | [END_REF]. These carbon insertion stages [C p -,TAA + ] formed by electron uptake and concomitant insertion of tetraalkylammonium salts were considered as "graphite salts", rather similar to TAA amalgams, and used (under inert atmosphere) as reducing species and poly-nucleophilic materials. Large exfoliation of graphite [8] and subsequently the exposure of the formed carbon salts, acting as nucleophilic species, to CO 2 permitted, for the first time, carboxylating HOPG to a large extent [9].

Electrochemical properties of HOPG can easily be transposed to GC, almost counter-intuitively bearing out that the latter can manifest a great deal of interfacial reactivity of graphite. In other words, GC surface could be perceived as a zone with strong nucleophilic reactivity when quite negative potentials (-1.7 V) are reached. In fact, this is not so surprising when considering aromatic inclusions in GC as more loaded congeners of benzenoid aromatics (benzene, anthracene etc) whose reduction to give nucleophilic species is well documented [START_REF] Heinze | Organic Electrochemistry[END_REF]. Therefore, the reactivity of GC interface (ET versus S N 2-like reactions) toward electrophilic organic derivatives is worth being re-considered. Given the above said, new paradigm of nucleophilic modification of carbons can be developed using this chemistry.

Evidence of cathodic charge of carbons at E < -1.7 V

The presence of graphite nano-crystallites in many kinds of glassy carbons is well-recognized [1], but the intrinsic cathodic behaviour of GC (i.e. its cathodic charge, principally in the interfacial area) is rarely considered or taken into account. A specific charge/discharge process beyond -1.7 V at a Carbone-Lorraine GC macro-electrode helps to interpret the way of trapping primary alkyl iodides [START_REF] Jouikov | [END_REF]. Figure 1A exhibits the charge of such CG electrodes as compared to curves B where a much poorer in (but not void of) C sp2 inclusions GC surface is covered by a thin layer of natural graphite (curve 2). The almost perfect correspondence of voltammetric responses in A and B2 is striking. Let us remark that a large presence of graphite in GC would provide an advantage for obtaining very dense coverages of grafting on carbons. Charging phenomenon, analogous to that of graphite, was also observed for graphenes deposited on carbons (and also on Au, Ag, Cu or Pt; in the latter case, very dry organic solvents are needed). However, fullerenes and carbon nanotubes did not exhibit any cathodic charging before -2.3 V [12] because surface-related ion-pairing in these systems, akin to the stabilization of the anion radicals of aromatic hydrocarbons by cations of the supporting salts [13], is less efficient for negative charge stabilization than charging with in-depth TAA + insertion.

The influence of a large incorporation of graphite (or graphene) in the GC may quite certainly influence reduction potentials of acceptors (here, electrophiles like 1-iodoalkanes [14]) in non-aqueous solvents as shown in Figure 1, C1-C2. This undermines a widespread view of GC as an almost perfect material without any catalytic activity compared to other electrode materials such as silver, palladium or copper. Thus, curve C1 (Tokai carbon having lower graphite content) has E 1/2 = -1.92 V while graphite-reach GC (Carbone-Lorraine) provokes an anodic shift, E 1/2 = -1.78 V. In the latter case (C2), the threshold of graphite charging and the reduction potential of 1-iodoalkane are identical (-1.6 V) meaning that graphite charge may induce a kind of redox-catalytic interface. This assumption was entirely verified with gold electrodes with deposited thin layers of natural graphite or graphene.

Surface modifications based on GC cathodic charge

Nucleophilic surface modification of GC takes on different aspects: i) an irreversible prodding of the initial surface by charge, ii) TAA + cation insertion to burst the graphite inclusions in the vicinity of the surface along with iii) chemical reactivity of nano-nucleophilic centres disposed at (or very close to) the interface. This phenomenon has been clearly demonstrated [9] during the charge of HOPG in the presence of CO 2 in N,N-dimethylformamide (DMF) using several TAA + cations. Consequently, one may expect the formation of a quite rough surface until a thick semi-porous interface is created leading to apparent superficial concentrations of the grafted groups up to hundred times the values expected for perfectly smooth surfaces. These main features are illustrated in Figure 2, part 1. The inherent to this process "over-grafting" was often interpreted by certain reviewers as indicating "multilayer" configurations of the immobilized layers.

The creation of poly-nucleophilic micro volumes remains for us the key to the surface reactivity excluding, therefore, the implication of radical chemistry at the potentials of GC charging; this is a principal difference opposing nucleophilic surface modification to the grafting of aryl radicals [15] generated at E > -1V. Several two-steps processes demonstrate this: charging GC (10 -3 C mm -2 ) followed by its transfer (under argon) to another vessel containing the solution of an electrophile to react for 10-20 s. Figure 2, part 2 exemplifies these S N 2-type reactions with ω-iodoalkylferrocenes, sulfonyl chlorides and carbon dioxide where the immobilization of specific redox groups could be shown.

Mechanism and examples of surface functionalization

Once the GC superficial zone is cathodically charged (Scheme 1, reaction I), its [C - p TBA + ] sites can act both as nucleophilic and as reducing species towards electrophilic substrates RX present at this interface. Two pathways are then possible: electron transfer (path II) or an S N 2 reaction (path III). Let us underline that beyond the potential of the threshold of carbon charging, the stability of free radicals is low and their specific immobilization is improbable.

Scheme 1 summarizes several type III surface nucleophilic displacement reactions [16]. The immobilization of CO 2 [17], which can be realized on most types of carbons (including graphite and graphene), enables a facile synthesis of poly-carboxylated GC eventually covered with graphene [22]; strongly hydrophilic, such interfaces can be readily transformed into redox materials by simple reactions of the carboxylate group. Also, the use of non-Kolbe oxidation of the polycarboxylate (at + 1.3 V) easily allows [12] the efficient conversion of GC into a poly-electrophilic material with a wide range of interesting reactions that could be regarded as a new chapter in the field of surface electrophilic displacement reactions. Deposition of graphene onto solid surfaces (GC, Au, Ag) followed by carboxylation led to three-dimentional electrodes with functional properties easily tuneable by ensuing chemical modification [23].

Besides the surface modifications displayed in Scheme 1, part B, several similar functionalizations of GC were shown [12] to occur. For example, let us quote a series of functional bromoalkanes Br-(CH 2 ) n -F (n > 2) with F being CN, NH 2 , OH, and aryl groups. The sole condition for the choice of the function F is that it should not be reducible prior to GC charge.

Lastly, α,ω-diiodoalkanes (X-(CH 2 ) n -X with X = I) can be used for immobilization of graphene or nano-graphite flakes onto GC at potentials where both GC and graphene are charged. Such an interface could be considered as a kind of redox polymer [24] quite obviously suitable for further modifications using multiple reactive possibilities of graphene (see Scheme 1, C).

Conclusion

The present compilation accentuates the possibility of taking advantage of the electrochemical charge of glassy carbons at E < -1.7 V using polar organic solvents containing non-electrophilic TAAX salts and aiming specific surface modification for unsaturated fractals are unavoidably present in such materials. The series of nucleophilic surface-related reactions (recalled or reported for the first time in this study) was possible due to the inclusions of graphite, graphene, and fullerene-like structures in carbon. Special care was taken to connect the cathodic reactivity of carbon to that of graphite and graphene deposited in very thin layers not only on GC but also on gold or silver. We stress the usefulness of this process for creating poly-nucleophilic materials (in mass or in thin (or ultra thin) supported layers) that offer -under quite simple conditions and using commercially available electrophilic reagents-very wide possibilities. Expanding the preliminary observations made with GC (published since the end of 2011), a very large panel of possibilities obviously emerges from using graphite(s) and graphene(s), especially underlaid with metals. Work in progress could underline the interest of this approach in electrochemistry and materials science.

Experimental section

6.1. Electrode materials: Carbon samples were purchased from Tokai Carbon Co (GC Rod) and from Carbone-Lorraine (large plates, VD 1500 and 2500, 3 mm thick). Polishing of GC was achieved with Struers waterproof silicon carbide papers (P#500 and then #1200) and Emery papers 02 and 03. After polishing, the electrodes were sonicated for eliminating grease and carbon dust.

Solid materials (GC, gold) could as well be used (for comparison) for deposition of graphite and graphene.

6.2. Solvents and electrolytes: Voltammetry discussed in the present review mostly concerns the solutions of tetra-n-alkylammonium salts (0.1 M) in DMF. Solvents and salts (Aldrich) were used as received. All the experiments described here did not need special treatments of the electrolytic solutions.

Electrochemical experiments: All potentials are referred to aqueous Ag/AgCl (sat) placed in a bridge containing the corresponding {solvent/electrolyte} solution. The electrochemical instrumentation has been previously reported [22].

Most of electrodes used had an apparent surface area of 0.8 mm 2 . Coulometric measurements and electrolyses were carried out using separated with fritted glass three-electrode cells with a total catholyte volume of about 5 to 7 mL. Argon bubbling was completed in all experiments. Modified electrodes were sonicated during 2 min in water and then rinsed with acetone. (2) Charge of GC and its reactivity with electrophilic reagents (10 -2 mol L -1 in DMF) after transferring (under argon) to another flask. The voltammograms illustrate specific responses of the grafted redox groups after sonication of the modified electrodes. DMF / 0.1M Et 4 NBF 4 .

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Scheme 1

Modification of GC surfaces by means of miscellaneous electrophilic reagents: A) General mechanism; B) Some examples of grafting through S N 2 surface processes (see text); C) Immobilization of graphene particles at GC surface via α,ω-diiodoalkanes.

Figure 1

 1 Figure 1 Voltammetry of glassy carbons under cathodic charge. DMF / 0.1 M Et 4 NBF 4 . Scan rate 50 mV s -1 . (A) Carbone Lorraine GC electrode (7 mm 2 ). (B) Poorer in graphite Tokai GC: bare (curve 1) and covered by an ultra thin layer of natural Ceylon graphite (curve 2). (C) Reduction of 1-iodo-octane (11 mmol L -1 ) at two different GCs. (C1) Tokai GC (0.8 mm 2 ); (C2) Carbone Lorraine GC (7 mm 2 ).

Figure 2 ( 1 )

 21 Figure 2(1) Cathodic charging of GC with progressive swelling and exfoliation caused by insertion of TAA + into surface-located graphitic inclusions.(2) Charge of GC and its reactivity with electrophilic reagents (10 -2 mol L -1 in DMF) after transferring (under argon) to another flask. The voltammograms illustrate specific responses of the grafted redox groups after sonication of the modified electrodes. DMF / 0.1M Et 4 NBF 4 .

Figure 1 A 2 Figure 2

 122 Figure 1

Graphical Abstract

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

A new process based on the cathodic charge of carbons at the potential E < -1.7 V vs. Ag/AgCl > In aprotic organic solvents containing non-electrophilic salts (for example: tetraalkyammonium salts), the charge of carbons permits creating poly-nucleophilic materials with high reactivity of the interface > In the contact with electrophilic organic reagents (such as CO 2 , organic acid halides, functionalized alkyl halides, etc…), nucleophilic reactivity of charged carbon leads to specific modified interfaces.