Maud Soty

Julien Chilloux

Sylvie Casteras

Alexeï Grichine

Gilles Mithieux

Dr Amandine Gautier-Stein
email: amandine.gautier-stein@univ-lyon1.fr

New insights into the organisation and intracellular localisation of the two subunits of Glucose-6-phosphatase

Keywords: CFP, cyan fluorescent protein, ER, endoplasmic reticulum, FRET, fluorescence resonance energy transfer, FLIM, fluorescence lifetime imaging, G6P, glucose-6 phosphate, G6Pase, glucose-6 phosphatase, G6PC, G6Pase catalytic subunit, G6PT, G6P translocase, GSD-1, glycogen storage disease, Pi, inorganic phosphate, YFP, yellow fluorescent protein

New insights into the organisation and intracellular localisation of the two subunits of glucose-6-phosphatase.

Introduction

Glucose-6 phosphatase (G6Pase) catalyses the last enzymatic step before the release of glucose into the blood: the hydrolysis of glucose-6 phosphate (G6P) into glucose and inorganic phosphate (Pi). G6Pase activity is restricted to the liver [START_REF] Ashmore | Studies on carbohydrate metabolism in rat liver slices. VI. Hormonal factors influencing glucose-6phosphatase[END_REF], the kidney cortex [START_REF] Wirthensohn | Renal substrate metabolism[END_REF] and the small intestine [START_REF] Rajas | The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats[END_REF] and confers on these three organs the capacity to release glucose into the systemic circulation. This enzyme plays a key role in glucose homeostasis since a deficiency in G6Pase activity leads to a severe metabolic disorder mainly characterised by hypoglycaemia in the post-absorptive state: glycogen storage disease type 1 (GSD-1) [START_REF] Chen | Glycogen storage diseases[END_REF]. From human genetic studies, GSD-1 patients have been classified in two types based on mutations of the G6Pase catalytic subunit (g6pc) or the G6P translocase (g6pt). GSD-1a results from mutations in g6pc and GSD-1b from mutations in g6pt. Both proteins are thought to be anchored in the endoplasmic reticulum (ER) and also expressed in nuclear membranes [START_REF] Arion | Evidence for the participation of independent translocation for phosphate and glucose 6-phosphate in the microsomal glucose-6-phosphatase system. Interactions of the system with orthophosphate, inorganic pyrophosphate, and carbamyl phosphate[END_REF]. This localisation was suggested from biochemical and histochemical studies based on phosphatase enzymatic activity [START_REF] Arion | The specificity of glucose 6-phosphatase of intact liver microsomes[END_REF][START_REF] Chiquoine | The distribution of glucose-6-phosphatase in the liver and kidney of the mouse[END_REF][START_REF] Shin | Electron microscopy on activity and localization of glucose-6-phosphatase in liver cells[END_REF]. However, no recent data based on the detection of G6Pase proteins have confirmed this intracellular localisation.

Since G6PC and G6PT are sufficient to completely account for G6Pase activity [START_REF] Van Schaftingen | The glucose-6-phosphatase system[END_REF][START_REF] Veiga-Da-Cunha | How many forms of glycogen storage disease type I ?[END_REF], the current G6Pase substrate transport model proposes that the active site of G6PC is exposed to the ER lumen [START_REF] Arion | On the involvement of a glucose 6-phosphate transport system in the function of microsomal glucose 6phosphatase[END_REF] and G6PT shuttles intracellular G6P across the ER membrane into the lumen, where it is hydrolysed. This model, based on two proteins, takes into account the kinetic characteristics of the G6Pase system. Indeed, in intact microsomal membranes (which correspond to an intact G6Pase system), G6P is specifically hydrolysed at a moderate rate and with high affinity. In solubilised ER membranes, in which catalytic activity is not dependent on substrate transport, G6Pase hydrolyses G6P at a high rate and affinity but, it decreases its substrate specificity and is therefore able to hydrolyse other sugar-6 phosphate moieties [START_REF] Mithieux | New knowledge regarding glucose-6 phosphatase gene and protein and their roles in the regulation of glucose metabolism[END_REF]. Moreover, disruption of microsomal membranes of livers from GSD-1b patients permits the recovery of normal G6Pase activity, whereas intact GSD-1b liver microsomes exhibit very low G6Pase activity [START_REF] Burchell | Molecular pathology of glucose-6-phosphatase[END_REF][START_REF] Narisawa | A new variant of glycogen storage disease type I probably due to a defect in the glucose-6-phosphate transport system[END_REF]. This set of data has suggested that G6PT is necessary for G6P transport in intact ER membranes and confers substrate specificity to the G6Pase system.

The importance of both proteins for optimal enzymatic activity has been confirmed in animal models. Using mice knocked out for g6pc (g6pc -/-), Lei et al. have suggested that a loss of g6pc gene expression annuls both G6P transport and hydrolysis [START_REF] Lei | Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse[END_REF]. In g6pc -/-mice, a single administration of an adenovirus containing the cDNA encoding g6pc (AdCMV-G6PC) improved the majority of GSD-1a metabolic abnormalities. The AdCMV-G6PC infusion restored 19% of normal G6Pase activity in the liver and, interestingly, corrected G6P transport deficiency in hepatic microsomes of g6pc -/-mice [START_REF] Chou | Adenovirus-mediated gene therapy in a mouse model of glycogen storage disease type 1a[END_REF]. The same authors examined microsomal G6P transport in transient expression studies in COS-1 cells and have shown that G6P transport is increased in cells transfected with either the g6pc or g6pt cDNA compared with mock-transfected cells. Moreover, G6P transport was more efficient in cells which co-expressed both genes [START_REF] Hiraiwa | Inactivation of the glucose 6-phosphate transporter causes glycogen storage disease type 1b[END_REF]. These studies provide direct evidence that G6PT and G6PC are tightly interdependent for global G6Pase activity.

Concerning their protein characteristics, neither proteins of the G6Pase system have been purified to homogeneity, but their individual structures within the membrane have been predicted using proteolytic digestion of tagged proteins [START_REF] Pan | Transmembrane topology of glucose-6-phosphatase[END_REF][START_REF] Pan | Transmembrane topology of human glucose 6phosphate transporter[END_REF]. We have reported that [ 32 P]G6PC migrates at an apparent molecular mass of 37 kDa in SDS-PAGE under denaturing conditions. This is consistent with its molecular mass determined by immunoblotting [START_REF] Daniele | Phosphatidylinositol 3-kinase translocates onto liver endoplasmic reticulum and may account for the inhibition of glucose-6-phosphatase during refeeding[END_REF]. Using radiation inactivation analysis, Ness et al.

suggested that the molecular weight of functional G6Pase ranged around 75-100 kDa, in both intact and solubilised ER membrane [START_REF] Ness | Radiation inactivation analysis of rat liver microsomal glucose-6phosphatase[END_REF]. These observations have suggested that the catalytic subunit (37 kDa) could be associated with another polypeptide through disulphide bonds, and that this association could be maintained under some denaturing conditions. This has raised the hypothesis that G6PT might be able to interact with G6PC, resulting in the formation of a complex.

The first aim of the present study was to document the intracellular localisation of G6Pase subunits. The proteins of the G6Pase complex were thus singly labelled on the C or N terminus with cyan fluorescent protein (CFP) for G6PC or yellow fluorescent protein (YFP) for G6PT. We also took advantage of these constructs to test the hypothesis of a direct interaction between both subunits of the G6Pase enzymatic system. To this purpose, we used fluorescence resonance energy transfer (FRET) with CFP as the donor and YFP as the acceptor. The second aim of this work was to test a putative direct interaction between the G6PT cytoplasmic termini. We thus developed a G6PT protein doubly labelled on the C and N termini with CFP and YFP, respectively. Using this chimera protein, we obtained further information on the structural conformation of G6PT within the membrane. Finally, we studied whether the presence of GSD-1 mutations affected the localisation of both G6Pase subunits and/or the interaction between the C and N termini of the G6PT protein.

Materials and methods

Generation of constructs

We used pSVK3-G6PC and pSVK3-G6PT constructs which contained the coding sequence for human g6pc (Genbank number NM 000151) and g6pt (Genbank In the case of the CFP-G6PT-YFP chimera protein, the DNA fragment encoding G6PT-YFP fusion protein was obtained after digestion of pG6PT-YFP plasmid at XhoI / HpaI restriction sites. For an easy cloning strategy, this cDNA was inserted to XhoI / HpaI in pECFP-Nuc vector. This cloning strategy induced the removal of the nuclear localisation signal of the simian virus large T-antigen contained in the pECFP-Nuc vector and produced a chimera protein containing a linker of 13 amino acids between the CFP and G6PT sequences (See supplemental figure 1).

The sequences of all plasmid constructions were controlled by sequencing (GenomExpress).

Cell culture and cDNA transfection

HeLa cells (ECACC 85060701), HepG2 cells (ECACC 8511430) and NRK cells (ECACC 86032002) were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) with 1g/L or 5g/L D-glucose, respectively, and supplemented with 10% foetal bovine serum (Invitrogen), 5 mM glutamine, streptomycin (1µg/µL) and penicillin (1 U/mL) at 37°C in a humidified 5% CO 2 /95% air atmosphere. For transient transfection, 1 day before transfection, 100,000 cells were plated out in two-well cell culture plates (Labteck, NUNC). The complete medium was refreshed 1 h prior to transfection. Cells were transfected using the Exgen 500 reagent (Euromedex) with 200 ng of each construct. After 48 hrs of incubation, the transfection efficiency was about 60% (data not shown).

Immunoblotting and immunohistochemistry

Whole proteins extracts were prepared from cells homogenised in 10 mM HEPES, pH 7.3 by ultrasonication coupled with freeze-thaw cycles to disrupt cell membranes. Proteins (40 µg) were separated by SDS-10% polyacrylamide electrophoresis gel and then transferred to Immobilon-P membrane (Millipore).

Analysis of G6PC expression was performed as previously described using an antibody raised against the C-terminal part of the protein [START_REF] Rajas | Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation[END_REF].

HepG2 hepatoma cells were fixed with 2% paraformaldehyde in PBS and permeabilised with 0.1% saponin in PBS. The cells were incubated for one hour at room temperature with a G6PC antibody (used at 1:1000) as previously described [START_REF] Rajas | Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation[END_REF]. The cells were rinsed and then incubated with goat anti-rabbit coupled to Alexa-546 (used at 1:1000, Invitrogen) for 1 h at room temperature.

Cell microscopy and FRET measurement

Cells were observed using an inverted two-photon laser scanning microscope Axiovert 200M (LSM510 NLO META, Carl Zeiss) equipped with a tunable IR femtosecond Ti:Sa laser (Tsunami, Spectra-Physics). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 using an on-stage incubator (PeCon, Germany). Confocal images of cyan and yellow fluorescence were detected with the emission band-pass filters 480/40 and 535/30 nm, respectively, using an excitation wavelength of 420 nm. Images were acquired using a 63×/1.4 oil immersion objective.

Pearson's coefficient was calculated using the JACoP plugin for ImageJ [START_REF] Bolte | A guided tour into subcellular colocalization analysis in light microscopy[END_REF].

FRET efficiency in living cells was assessed in confocal mode using the acceptor photobleaching method and using fluorescence lifetime imaging (FLIM) after two-photon excitation. Confocal detection of CFP/YFP was performed using 458/514 nm excitation wavelengths and 480-520/545-610 nm spectral filters, respectively. Repeated scans with unattenuated 514 nm illumination were used to photobleach YFP.

After complete YFP bleaching, fluorescence intensities of CFP and YFP were measured using the same parameters as before photobleaching. FRET efficiency was calculated using the formula E = 1-(I DA /I D ). I is the emission intensity of the donor (CFP) in the presence (I DA ) or in the absence (I D ) of the acceptor (YFP). E is the percentage of the increase of the donor fluorescence after bleaching the acceptor. For FLIM, the twophoton laser was tuned to 840 nm with a pulse duration of 150 fs. This wavelength was settled to obtain the best fluorescence excitation of CFP without a noticeable contribution of cellular autofluorescence. The irradiation intensity on the sample was 500 μW at 2% transmission of the acousto-optical tunable filter (AOTF). The fluorescence lifetime of CFP was measured by the time-correlated single photon counting technique (TCSPC) using the counter SPC 830 (Becker & Hickl GmbH, Germany).

G6Pase activity assay

Cells were homogenised in 10 mM HEPES, pH 7.3 by ultrasonication coupled with freeze-thaw cycles to disrupt cell membranes. Cell extracts were then suspended (1:20 dilution) in 10 mM HEPES and 0.25 M sucrose, pH 7.3. As previously described, we used a specific assay for G6Pase determination, a method involving glycerophosphatase activity determinations to determine the contribution of nonspecific phosphatase activities from G6Pase activity [START_REF] Rajas | The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats[END_REF].

Results and discussion

Intracellular localisation of G6Pase subunits.

The intracellular localisation of G6PC and G6PT, obtained from biochemical and histochemical studies, suggested that both proteins localised in the ER membrane [START_REF] Arion | The specificity of glucose 6-phosphatase of intact liver microsomes[END_REF][START_REF] Chiquoine | The distribution of glucose-6-phosphatase in the liver and kidney of the mouse[END_REF][START_REF] Shin | Electron microscopy on activity and localization of glucose-6-phosphatase in liver cells[END_REF]. However, no recent data based on the detection of G6Pase proteins have confirmed this intracellular localisation. To address this question, we chose to label the cytoplasmic termini of both G6Pase subunits with fluorescent proteins. The predicted model for the G6PC structure suggests that its N terminal part is located on the luminal side of the ER and the C terminal faces the cytoplasm, whereas both termini of G6PT are predicted to be in the cytoplasm (Figure 1 and2).

The enzymatic activity of G6Pase depends on the integrity of the amino acid sequences of G6PC. Therefore, we first checked that the addition of a fluorophore to the endogenous G6PC protein had no effect on its activity and cellular localisation. Cells transfected with G6PC-CFP (CFP fused the C-terminus of G6PC) conserved notable G6Pase activity (Figure 1a). This activity was approximately 50% lower than that of cells transfected with the wild-type protein (Figure 1a). This was in agreement with a 50% lower expression of G6PC-CFP compared to G6PC in HeLa cells (Figure 1b).

Altogether, this suggested that the fusion of CFP to the C-terminus of G6PC did not substantially alter the intrinsic G6Pase activity of the protein. As previously suggested for the wild-type protein [START_REF] Shin | Electron microscopy on activity and localization of glucose-6-phosphatase in liver cells[END_REF], we showed that G6PC-CFP was located in the nuclear membrane and in vesicular structures surrounding the nucleus, which are characteristics of ER membranes (Figure 1c). Surprisingly, G6PC-CFP was also located in punctuate vesicles within the cytoplasm and also close to the surface of the cell (Figure 1c). To confirm that the cellular localisation of the G6PC-CFP construct was the same as the endogenous one, we used an antibody specific for G6PC allowing its detection by immunohistochemistry in G6PC-expressing cells [START_REF] Rajas | Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation[END_REF]. We showed that endogenous G6PC expressed by the human hepatoma cell line HepG2 exhibited the same intracellular localisation as G6PC-CFP overexpressed in HeLa cells (Figure 1d). Finally, we used a time lapse process, which allowed us to follow up the movement of a point during a determinate time, to determine whether G6PC-CFP fluorescent points were mobile vesicles. We showed that G6PC-CFP expression points moved rapidly (0.5 µm/s) with no orientated trajectory (Movie 1).

Concerning G6PT, the addition of the YFP fluorophore to the C-terminal part of G6PT led to a punctuate pattern in the vicinity of the nucleus similar to that of G6PC-CFP (Figure 2a). Some vesicles were present in the cytoplasm but were localized nearer the nucleus compared to the G6PC-CFP vesicles (Figure 2a). In addition, it must be noted that localisation near the plasma membrane was not observed with G6PT-YFP.

Experiment with insertion of the YFP to the N-terminal part of G6PT was also carried out. However, the addition of the YFP fluorophore to the N-terminal part of the protein led to a broader and more diffuse localisation (Figure 2b). YFP is highly hydrophilic.

Thus, the presence of the fluorescent protein on the N terminus of G6PT likely perturbed its co-translational insertion process in the ER, thereby explaining the misaddressing of the chimeric protein YFP-G6PT. In agreement with the latter rationale, mutations inducing a destabilisation of G6PT proteins are mainly localised in the N-terminal part and in the first loop of the protein [START_REF] Pan | Transmembrane topology of human glucose 6phosphate transporter[END_REF]. An exogenous signal peptide could have been added to the N-terminus end of the YFP-G6PT construct in order to force N-terminal insertion in the membrane. However, such a chimera protein might probably not behave as the endogenous G6PT, and results obtained with such a protein would have been difficult to interpret. For further experiments, we thus used G6PT-YFP chimeras. Finally, using time lapse process, we analysed the behaviour of G6PT-YFP fluorescent points. On the contrary to G6PC-CFP, G6PT-YFP expression points did not move throughout cytoplasm (data not shown).

Both proteins were overexpressed in HeLa cells to analyse their putative colocalisation. The superposition of G6PC-CFP and G6PT-YFP fluorescence patterns suggested that both proteins were colocalised in the perinuclear area (Figure 3c). We calculated the Pearson's coefficient (PC) of correlation to quantify the degree of colocalisation between G6PC-CFP and G6PT-YFP. The PC measures the intensity of fluorescence of each pixel in both channels and compares the channels to determine the amount of correlation. A PC of 1.0 indicates complete colocalisation; a PC of 0 means no correlation and a PC of -1.0 means they are negatively correlated [START_REF] Bolte | A guided tour into subcellular colocalization analysis in light microscopy[END_REF]. G6PC-CFP showed a high degree of colocalisation with G6PT-YFP based on PC values of 0.86 ± 0.08 (Figure 3). To resume, the G6Pase complex was not affected by the labelling of either G6PC or G6PT proteins on their C-termini. Using G6PC-CFP and G6PT-YFP chimera proteins, we demonstrated for the first time that both G6Pase subunits strongly colocalised within the cell. In addition, the presence of G6PC, but nor G6PT within punctuate structures close to the plasma membrane was also detected. We then took advantage of these fluorescent chimeras for investigating a direct interaction between G6Pase subunits.

Analysis of the interaction between G6PC and G6PT using FRET.

To assess the putative FRET between G6PC-CFP and G6PT-YFP proteins, we determined the energy transfer efficiency by acceptor photobleaching approaches.

Acceptor photobleaching approaches with CFP-and YFP-tagged proteins are simple and prevent problems associated with any putative difference in expression levels: when FRET occurs, the fluorescence of the CFP donor increases after bleaching the YFP acceptor chromophore, and this is recognised as a relevant signature for FRET [START_REF] Sekar | Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations[END_REF].

HeLa cells were transfected with G6PC-CFP and G6PT-YFP proteins and subjected to intense illumination at 514 nm to bleach YFP (100 scans). In the bleached region, we observed a complete loss of yellow fluorescence (compare figure panels 4d with 4b) but no increase in cyan fluorescence (compare figure panels 4a with 4c), indicating the absence of FRET from CFP to YFP before bleaching (Figure 4e). Thus, these experimental conditions did not allow us to demonstrate the existence of an intermolecular FRET between labelled G6PC and G6PT.

The absence of detected FRET could be due to the cell model used. HeLa cells do not express endogenous G6Pase and could not offer the conditions necessary for an interaction between G6PC and G6PT specific to gluconeogenic cells. To test this hypothesis, we performed the same experiment in a renal cell line (NRK cells, which are able to produce glucose; Soty and Mithieux, unpublished data). However, as in HeLa cells, these experimental conditions did not allow us to demonstrate FRET between G6PC and G6PT (data not shown).

Another explanation for the absence of detectable FRET could be that the rapid movement of G6PC-CFP-labelled vesicles perturbs image acquisition and FRET measurements. Indeed, we showed that G6PC-CFP expression points moved rapidly (0.5 µm/s) (3.1, Movie 1). To detect FRET using acceptor photobleaching approaches, CFP images must be taken before and after YFP photobleaching. Between these two acquisitions, CFP labelled vesicles might disappear or change their localisation due to their putative movements in the x, y or z spatial plans. To test this hypothesis, we performed the detection of FRET by the single photon counting (FLIM), a method insensitive to the variation of fluorescence intensity. FLIM measures the effect of the acceptor on the excited state lifetime of the donor. When FRET is occuring between the donor and the acceptor, the donor lifetime is reduced [START_REF] Hayek | Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation[END_REF]. The mean lifetime of the double exponential decay of G6PC-CFP was measured to be 1.9 ns in the perinuclear endoplasmic reticulum either in the absence or in the presence of the G6PT-YFP, indicating no FRET between these labels (Supplemental figure 2, white circles). One should notice, however, the strong variation in the G6PC-CFP lifetime values in vesicular structures close to the cell periphery, precluding quantitative FRET analysis using FLIM (Supplemental figure 2).

Analysis of the interaction between G6PT termini.

The predicted model for the G6PT structure suggests that G6PT is anchored in the ER membrane by 10 transmembrane helices and has both termini facing the cytoplasm [START_REF] Pan | Transmembrane topology of human glucose 6phosphate transporter[END_REF]. The interaction between these termini has not been addressed yet. To study the conformational behaviour of G6PT ends, we generated a G6PT protein tagged on the C and N termini with CFP and YFP, respectively (CFP-G6PT-YFP).

The intracellular distribution of the doubly labelled G6PT subunit (CFP-G6PT-YFP protein) was similar to the simply labelled subunit G6PT-YFP (Figure 5a,b). After YFP fluorescence extinction under the conditions previously described (3.2), we showed an increase in cyan fluorescence, demonstrating the existence of a FRET phenomenon between CFP and YFP before bleaching (compare figure panels 5a with 5c). The ratio of the YFP/CFP fluorescence intensities was constant and not correlated with FRET efficiency. This observation illustrated that the labelling stoichiometry was 1:1 and that both fluorophores were never expressed independently (Figure 5e) [START_REF] Hink | Imaging protein-protein interactions in living cells[END_REF].

The mean intramolecular FRET efficiency of the CFP-G6PT-YFP protein was 27%. This result indicates that the G6PT protein adopted a wrapped conformation, so that FRET occurred between its two extremities [START_REF] Hink | Imaging protein-protein interactions in living cells[END_REF]. From these data, we estimated the distance between the C and N termini of G6PT to 5.8 nm (supposing the Förster radius for CFP/YFP pair is 4.9 nm [START_REF] Patterson | Forster distances between green fluorescent protein pairs[END_REF]). Some data [START_REF] Lei | Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse[END_REF][START_REF] Sloop | Specific Reduction of Hepatic Glucose 6-Phosphate Transporter-1 Ameliorates Diabetes while Avoiding Complications of Glycogen Storage Disease[END_REF] have argued for the existence of a direct influence of G6PC protein on G6PT transport function. We tested the hypothesis that the presence of unlabelled G6PC could induce a conformational change to the CFP-G6PT-YFP chimera detectable by a modification of its FRET efficiency. The co-expression of unlabelled G6PC with CFP-G6PT-YFP in HeLa cells did not modify the FRET efficiency of the doubly labelled G6PT (23.64 ± 6.29 vs. 25.61 ± 9.86%). These results suggest that G6PC did not modify the interaction between G6PT termini.

Effect of GSD-1 mutations on G6Pase subunit intracellular localisation and on G6PT intramolecular FRET.

Mutations identified in GSD-1 patients have been studied in vitro to assess their effect on protein stability and enzymatic activity [START_REF] Pan | Transmembrane topology of glucose-6-phosphatase[END_REF][START_REF] Pan | Transmembrane topology of human glucose 6phosphate transporter[END_REF][START_REF] Shieh | The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase[END_REF]. From these data, we selected mutations located in different parts of G6PC or G6PT which did not affect the stability of the proteins but annulled or strongly decreased their enzymatic activity (Table 1) [START_REF] Shieh | The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase[END_REF]. Our fluorescent chimera enabled us for the first time to test whether the decrease of G6Pase activity due to GSD-1 mutations was linked to a cellular mislocalisation of G6Pase subunits. Concerning G6PC, we introduced some GSD-1a mutations on the G6PC-CFP construct (E110K, A241T, G184R and G222R). We did not observe an effect of any of these mutations on the cellular localisation of G6PC-CFP. As an example, the E110K mutation, which induced a complete loss of G6Pase activity (Table 1) [START_REF] Shieh | The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase[END_REF], exhibited the same localisation pattern as the wild-type protein (Figure 6b). Concerning G6PT, we introduced some GSD-1b mutations in the CFP-G6PT-YFP construct (S54R, H301P, and A367T). We did also not observe an effect of any of these mutations on the cellular localisation of G6PT-YFP. Indeed, the S54R mutation, which induced a complete loss of G6Pase activity, or the H301P mutation, which kept about 25% of wild type G6Pase activity (Table 1) [START_REF] Shieh | The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase[END_REF], exhibited the same localisation pattern as the wild type CFP-G6PT-YFP construct (Figure 6c-h).

GSD-1b disease is characterised by the loss of G6Pase activity due to G6P transport deficiency [START_REF] Chou | The molecular basis of type 1 glycogen storage diseases[END_REF]. In addition to defects in glucose homeostasis, GSD-1b patients present symptoms of neutropenia and myeloid dysfunction [START_REF] Beaudet | Neutropenia and impaired neutrophil migration in type IB glycogen storage disease[END_REF]. These data are consistent with the idea that G6PT is not just a G6Pase partner but also a key protein for normal neutrophil function. Therefore, knowledge of G6PT structure-function relationships could be crucial for the understanding of both G6Pase function and GSD-1b pathophysiology. To identify whether the loss of function of G6PT observed in GSD-1b patients is associated with a spatial rearrangement between its C and N termini, we tested the influence of some GSD-1b mutations on the FRET efficiency of CFP-G6PT-YFP. However, the presence of the selected mutations did not affect the FRET efficiency of the CFP-G6PT-YFP construct (Figure 7). In summary, our results strongly suggest that the decrease or the loss of G6Pase activity in GSD1 patients was not linked to a mislocalisation of any G6Pase subunits or to an alteration of the interaction between G6PT termini.

Conclusion

In conclusion, this study constitutes an original focus on the intracellular distribution of G6PC and G6PT. We suggest that both proteins are strikingly colocalised in the intracellular membranes close to the nucleus. Moreover, G6PC, but not G6PT, is also present in vesicles throughout the cytoplasm and is subject to rapid motion within the cell. For the first time, our results demonstrated that G6PT C and Ntermini are separated by about 5-6 nm strongly suggesting that G6PT adopts within the ER membrane a wrapped conformation. Moreover, the intramolecular interactions are strong enough for the G6PT conformation be maintained either in the presence of G6PC or in the presence of mutations identified in GSD-1b patients.

Proprietary interest

None. FRET efficiency was calculated as previously described. Results are expressed as means ± SEM (n =3).

Table 1: GSD-1 mutations used in the study

GSD-1 mutations located in loops of G6PC and G6PT, with no effect on protein stability or G6Pase activity were chosen. Table indicates the amino acids targeted by the mutation, its localisation on the predicted model of the protein and its effect on G6Pase activity compared to wild type [START_REF] Pan | Transmembrane topology of glucose-6-phosphatase[END_REF][START_REF] Pan | Transmembrane topology of human glucose 6phosphate transporter[END_REF][START_REF] Shieh | The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase[END_REF].

Movie 1 (MOV file): HeLa cells were transfected with the G6PC-CFP construct. Confocal images of cyan fluorescence were detected with the emission band-pass filters 480/40 nm using an excitation wavelength of 420 nm. Images were acquired using a 63×/1.4 oil immersion objective every sec for over 70 sec.

Movie 2 (MOV file): HeLa cells were transfected with the G6PT-YFP construct. Confocal images of yellow fluorescence were detected with the emission band-pass filters 535/30 nm using an excitation wavelength of 514 nm. Images were acquired using a 63×/1.4 oil immersion objective every 0.4 sec for over 85 sec.

Supplemental material

Supplemental Figure 1: Amino acid sequences of the chimera proteins used in this study. Underlined texts correspond to the CFP or YFP sequences. Texts highlighted in grey correspond to G6PC or G6PC sequences. Nucleic acids coding for the stop codon of G6PC (A), G6PT (B), CFP (C) or YFP (D) and nucleic acids from the cloning vector located between G6PC and CFP (A and C) or between G6PT and YFP (B and D) were removed by site-directed mutagenesis. E. Nucleic acids coding for the nuclear localisation signal from simian virus large T-antigen contained in the pECFP-Nuc vector were removed by the cloning strategy.

Supplemental Figure 2: Analysis of the G6PC-CFP and G6PT-YFP interaction using FRET-FLIM. G6PC-CFP lifetimes measured by the TCSPC method in HeLa cells co-expressing G6PC-CFP and G6PT-YFP are represented before (a), and after (b) bleaching (see 2.4 for details). The bleached region is delimited by a white circle. Lifetime is indicated by the color bar.

  number Y15409) cloned at EcoRI and Sal I restriction sites (Bady and Mithieux unpublished results). Chimera proteins G6PC-CFP and G6PT-YFP with fluorophores at the C-terminus end were generated introducing g6pc and g6pt cDNA into pECFP-N1 and pEYFP-N1 vectors (Clontech) at EcoRI and Sal I restriction sites. Chimera proteins CFP-G6PC and YFP-G6PT with fluorophores at the N-terminus end were generated introducing g6pc and g6pt cDNA into pECFP-C1 and pEYFP-C1 vectors (Clontech) at EcoRI and Sal I restriction sites. Sequences encoding stop codons avoiding correct labelling were deleted by mutagenesis (QuikChange, site-directed mutagenesis kit, STRATAGENE). The resulting chimera contained no additional amino acids between the G6PC/G6PT and the CFP/YFP sequences (See supplemental figure 1 for amino acid sequences of the chimera proteins).

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Characteristics of the G6PC chimera. HeLa cells were transfected with the empty pSVK3 plasmid (mock) or with plasmids coding for the human wild-type G6PC or coding for G6PC-CFP. a. G6Pase activities assayed in the lysates of transfected HeLa cells. Results are expressed as means ± SEM (n=4).* P<0.05, significantly different from mock value (Student's t-test for unpaired data). b. Representative immunoblot for G6PC illustrating the sizes of the wild-type and chimera G6PC proteins. Molecular weight of the protein marker is indicated on the right of the figure. c,d. Representative confocal images of (c) G6PC-CFP in HeLa cells (d) endogenous G6PC in HepG2 cells. Bar = 10µm

Figure 4 :

 4 Figure 4: Photobleaching of yellow fluorescence associated with G6PT-YFP in HeLa cells. Representative images of G6PC-CFP (a,c) and G6PT-YFP (b,d) co-expressed in HeLa cells. The images were acquired immediately before (a, b) and after (c, d) bleaching (see 2.4 for details). The bleached region is delimited by a white circle. Bar = 10µm. e. Pixel intensities of CFP fluorescence acquired before and after photobleaching. Results represent the mean ± SD of eight independent experiments.

Figure 5 :

 5 Figure 5: Photobleaching of yellow fluorescence associated with CFP-G6PT-YFP. Confocal imaging of cyan (a, c) and yellow (b, d) fluorescence of CFP-G6PT-YFP expressed in HeLa cells. The images were acquired immediately before (a, b), and after (c, d) bleaching. The bleached region is delimited by a white circle. Bar = 10µm e. FRET efficiencies (y) (see 2.4 for calculation) for doubly labelled G6PT according to the ratio of corrected fluorescence intensities for CFP and YFP (x).

Figure 6 :

 6 Figure 6: Effect of GSD-1 mutations on G6Pase subunit localisation. Representative confocal images of wild-type (a) and E110K (b) G6PC-CFP in HeLa cells. Representative confocal images of cyan (left panels) and yellow (right panels) fluorescence of wild-type (c, d), S54R (e, f) or H301P (g, h) CFP-G6PT-YFP constructs in HeLa cells. Bar = 10µm.

Figure 7 :

 7 Figure 7: Effect of GSD-1b mutations on FRET efficiency of CFP-G6PT-YFP. FRET efficiency was calculated as previously described. Results are expressed as means ± SEM (n =3).

  Figure 5 Click here to download high resolution image

Table 1 :

 1 GSD-1 mutations used in the study

	Amino acid changes	Location	Effect on G6Pase activity
		GSD-1a mutations
	E110K	1 st Loop	Total inactivation
	A241T	3 rd loop	21.3 % of wild type activity
	G184R	5 th loop	Total inactivation
	G222R	6 th loop	4 % of wild type activity
		GSD-1b mutations
	S54R	1 st Loop	Total inactivation
	H301P	3 rd loop	24.2 % of wild type activity
	A367T	9 th loop	23.1 % of wild type activity

Table 1 ,

 1 Soty et al.

Table 1 Figure1 Click here to download high resolution image Figure2 Click here to download high resolution image Figure3 Click here to download high resolution image Figure4 Click here to download high resolution image

 1

Acknowledgements

The authors thank INSERM for funding their work, as well as CNRS (G.M.), INRA (A.G.-S.) and the French Ministry of Research (M.S., J.C. and S.C.) for funding their positions.


[12][13]

The vote for Hollande is not so much a radical desire for change as a possibly illusory desire to go back to the pre-crisis period. The socialists have meanwhile opened up a new approach to the economy. But 'racism from above' has led the way in this historic fight over what is normal François Hollande won the presidential elections with almost 52 of the votes. Since the François Mitterand years, he will be the first socialist president of the Republic. In his campaign he promised "change" to French voters. However I argue that rather than change, it is the tacit desire for going back to normal that determined the tone of the elections. The day after the elections, the French newspaper Liberation published on its cover page Hollande's picture and the caption that reads "Normal!" with an exclamation mark. What does the normal stand for? Does it mean that it was expected that he won the elections as the polls had predicted? Or does it mean that Hollande represents ordinary people and French ways, against the ostentatious liberal figure of Sarkozy?

The vote for Hollande translated in the first place the desire to get rid of Sarkozy. His aggressive style as a president, personalization of power, volatility in his decisions made him unpopular. In contrast with the super presidency of Sarkozy that irritated many French citizens, Hollande presented himself as a "normal President" meaning serious, consensual ("rassembleur" was his trademark in French) and a reliable statesman in conformity with the Republican State traditions. Many explained his success in terms of his capacity to follow in the footsteps of the legendary heritage of François Mitterand who embodied state power almost like a monarch. In this respect, we can interpret the "normal" as going back to Republican State traditions represented by the left.

During the pre-election television debate, Sarkozy attacked Hollande and implied that the aspiration for "normalcy" stood for mediocrity. To belittle his rival he reminded us that Mitterand was not an ordinary politician and that given the present context of economic crisis France needed more than ever an exceptional president, like himself, to lead the country. The fact is he already had his chance in the 2007 elections, coming to power with the same promise of changing, modernizing and liberalizing the French economy and its institutions. But the expected economic reforms did not succeed in consolidating French leadership in Europe. France lagged behind Germany in terms of economic growth, productivity, competitiveness and unemployment rates. During the campaign debates, Germany was the regular reference point as the successful economic model. On the other hand, the Socialist Party introduced the Keynesian model of economic growth as an alternative to the policies of austerity; proposing an economic programme that expands the state and public spending while creating employment and increasing the rate of income tax.

In denial?

In general, observers agree that little was said by anyone at all about the country's dire economic straits. French society does not seem to be prepared to acknowledge the gravity of the economic situation and wants to hold onto their rights and privileges as guaranteed by the social welfare state. Before the elections, the British weekly The Economist, spoke of France as "a country in denial" and sarcastically described the elections "as the most frivolous campaign" devoid of any serious engagement with the economic situation. So, we can interpret the vote for Hollande, not so much as a radical desire of change and implementation of reform, as a desire to go back to the pre-crisis period, hoping that this does not prove to be an illusion. The socialists, however, have opened up a new window for approaching the economy in an alternative way. They have weakened a dominant ideology that presented neo-liberal politics as an unquestionable article of faith, the only right way, as "normal".

The fact that the French are not fully facing up to the economic crisis does not mean that they are not anxious about their future. The rising popularity of the far right party illustrates very well the fertile ground for nationalist feelings and discourses of xenophobia. The economic crises facilitate the scapegoating politics targeting immigrants and Muslims, which singles them out as responsible for taking up jobs, abusing the welfare state, and invading French society with their religious visibility and backward cultural norms. With this anti-European, nationalist, and anti-immigrant Islamophobic politics, the female face of the far-right Party, Marine Le Pen achieved almost 20 percent of the votes and has become a key figure in the political arena. She has consolidated her leadership of her father's party, "Le Front National" but furthermore emerged as the potential leader of the right. Indeed, Marine Le Pen is the second winner of this campaign; she has succeeded in setting her political agenda and imposing it. Sarkozy, in order to attract the far right constituency, had no compunction in adopting her security discourse. But in doing so, he not only surrendered himself to the political agenda of the rival, but also gave legitimacy to radical right politics.

The normalization of far right politics and racisme d'en haut

The "normalization" of far right politics is the key to understanding the changes in the European political panorama and public life. For the last ten years, we have been witnessing a phenomenon not limited to France but shared throughout Europe, the proliferation of public figures and voices that signal the end of multiculturalism, criticize politically correct norms of public speech, and search incessantly for ways to rid themselves of taboos against racism.

A succession of public controversies around Islam -namely the headscarf of young students in the public schools, total veiling, the burkha in the streets, helal meat, street prayer -have preoccupied the public scene in most European countries. These debates end up by erasing the well established distinctions between right and left, and creating a consensus on the need to condemn and prohibit such religious and cultural practices. The values of secularism and feminism have been doggedly advanced as superior and distinctive values over and against those of Muslims living in Europe. The politics of tolerance and pluralism have been condemned not only by right wing politicians, but also by intellectuals from the left and secular backgrounds. Philosopher Jacques Ranciere has drawn attention to the changing forms of racism, not that racism supposed to be rooted among the underprivileged lower classes, but the one initiated and promoted from above, by state power boosted by the intellectuals. He named such racism "racisme d'en haut". More recently, the day before the Presidential elections, Alain Badiou published an article on "le racisme des intellectuels" in the newspaper Le Monde (5 may 2012), addressing his criticism to the intellectuals who reinvented the "Islamic threat" and defended the superiority of the western civilization -thereby contributing to the proliferation of anxious discourses on immigration, cultural racism and Islamophobia in France. He directly blames those intellectuals who facilitated this mental development aiding the ascension of fascism to power.

The rising popularity of these new faces of the right surfs over the dynamics of "normalizing" widely-shared anti-immigrant and anti-Islamic discourse. Secular-left and conservative-right join hands in making anti-Islamic and anti-immigrant politics appear quite "natural". As many say, "we should not diabolize the Front National", they are as legitimate as any political formation. One hears among the voters of Marine Le Pen that there is no shame in voting for a person who represents the feelings and anxieties of the people, and who is 'one of us'. Besides the new faces of the populist right are not like the first generation of patriarchal anti-Semitic mouthpieces; they speak against homophobia, they declare themselves feminist and they defend secularism. And they are not marginal. The more they turn the political agenda against Islam, the more they become central players in the system.

A normal future

Economic recession, anti-Islamic discourses and nationalist feelings are reshaping the public sphere in Europe. In such a context, what does Hollande's promise of being a normal president signify? There are several leitmotifs around 'the normal' that might be competing with each other. Going back to normal republican traditions of the left implies the defense of secularism, "la laicite", the community of the nation and egalitarian values among its citizens. But there is another move to the "normal"; it is the normalization of far right ideas, the way anti-migrant, anti-islam discourses appear natural and consensual.

How will the socialist party defend Republicanism, the politics of "laicite", and women's rights without converging with the new popular right? Anti-Islamic politics of the far right and economic recession are the two most important threats to European democracies. Can socialism be an alternative to these destructive waves? A French socialist victory bears a historical importance way beyond its national borders, for Europe in general.

Will going back to normal imply developing alternatives to economic crises, acknowledging a multicultural France (defending "France metisse" as only the leftist communist candidate Jean-Luc Melanchon managed to do), expanding democratic pluralism and embracing the European ideal? This may be way too optimistic. But at least the victory of Hollande gives us hope that far right ideas and politics can be avoided as aberrations. And that requires a new awareness on the part of European intellectuals.

For our coverage of the French elections see.... View the discussion thread. [START_REF]net%2Fnil%25C3 %25BCfer-g%25C3%25B6le%2Ffrench-elections-what[END_REF] $(document).ready(function(){ $("div#contentgrid").removeClass('grid-8'); $("div#contentgrid").addClass('grid-6'); }); About the author

  Philippe Marlière blog -Marlière Across La Manche[14] Patrice de Beer -France: towards a new right[15] and François Hollande and France: the big deal[16]

	Country or region: France
	Topics: Civil society
	Conflict
	Culture
	Democracy and government
	Economics
	International politics


Ahmed Ben Salah

Geneviève Cron

Nicolas Ragot

Thierry Paquet

Prediction of Selection Decision of Document Using Bibliographic Data at the National Library of France (BnF)

The selection process of the documents is a very important step in mass digitization projects. This is especially true at the BnF, where the digitization should include or not OCRization depending on the OCR results expected. Consequently, the selection task is very complex and time consuming due to the number of documents to be processed and the diversity of the selection criteria to consider. trying to improve and simplify this task by automation, we studied the relationship between bibliographic data and the selection decisions of documents. We used two statistical analysis : a factor analysis of correspondence and a multiple correspondence analysis. Our analysis has shown that, for example, the documents in format "4 or GR FOL" and edited "between 1961 and 1990" in Morocco are more likely to be "Selected". However, the documents in format "16 or 8" and edited "between 1871 and 1800 in English or Spanish have a greater chance to be "Not Selected".

Documents digitization process at BnF

Once the documents selected, they are sent to a digitization provider. For each scanned document, the provider builds a digi-

Introduction

Since 1992, the National Library of France has started a number of projects of mass digitization and since 2006, these programs include optical character recognition (OCR) of texts. The main objective of these projects is to preserve patrimonial documents and disclose their information to the public. These projects always begin by a selection step of documents which depends on physical and bibliographic criteria. This task is very complex due to, first, the time-consuming physical manipulation of documents ; second, the large number of documents in these projects and third the diversity of the selection criteria. Furthermore, our first study showed that only 10% of documents processed by the selection department at BnF are finally selected which make this task crucial. Physical aspects (such as quality of ink, Document opening, nature of paper....) are known as major impact factor on OCR efficiency. Unfortunatly, these data are not available since the document hasn't been manipulated by staff responsible for the selection process. This manipulation step by a human operator cannot be discarded. Consequently, the optimization of the selection should be done before and physical aspects criteria cannot be used easily. After several years of working in documents selection department, the staff in charge of selecting documents began to put some assumptions about the correlation relationship between some bibliographic data and the physical quality of documents. Trying to simplify and improve the task of documents selection, we took into account these hypothesis and we have studied the relationships between the bibliographic data and the documents selection decisions. In this respect, we present first in this article how is performed the selection at the BnF. Next, the digitization process itself is shortly presented. Finally, we provide the results of our analysis based on Correspondence Factor Analysis (CFA) and Multiple Correspondence Analysis (MCA).

Documents selection process

The documents selection process is a major step in the process of scanning documents. Indeed, a compromise as to be done to select the "interesting" documents considering the mass of documents available and several criteria. At the BnF, the selection process begins with an annual planning of documents according to their themes and to their intellectual values. Then, the departments of document selection do three tests :

-Test of copyright : the scanned documents at the BNF must be royalty free. This criteria is essential in the selection decision of document. -Test of the quality of bibliographic data : the bibliographic metadata of the documents must be correct complete and accurate. This criteria is also very important in the selection decision of document. -Test of the physical quality of the documents : the physical quality of the document must be evaluated to avoid sending to providers documents with too poor physical quality since OCR will not work properly. At the end of these tests, the documents selection decisions can be either "Not selected", "Selected for Raw OCR" or "Selected for High Quality (HQ)". Raw quality is obtained after the automatic processing of OCR systems, i.e. without human corrections. HQ is the corrected results of OCR systems to obtain an editorial quality. On these HQ digital documents, the BNF requires the final OCR rate to be higher or equal to 99.95%.

This selection task is very difficult and costly in terms of working time and staff number. Indeed, it requires great skill and a massive processing of documents. Moreover, a study showed that only 10% of documents processed by selection departments are selected (in Raw or HQ). This is the reason why the Bnf would like to improve this process to select more documents and at the same to save time and money (obtaining HQ documents cost more than Raw documents). Considering these objectives, optimal operations would affect the documents with a medium physical quality to the necessity of having a High Quality (HQ) digitization, and documents with very good physical quality to a digitization with Raw quality (see Figure 1).

FIGURE 1. The judgment of the selection decision of documents according

to their reachable OCR raw rate tal directory that contains the TIFF images of document pages and a "Refnum" file containing documents metadata. After scanning, the provider sends the digital documents to the BnF. The computer department supports the integration of these digital documents in the internal servers and monitors the file Refnum through automatic tools for verifying physical structure (number and order of pages, orientation, etc.). In parallel, the digitization department at the BNF provides bibliographic control of the metadata of digital documents. The department also controls the conformity of the quality of document images with the BnF's sepecifications. Depending on the type and the number of errors detected during these controls the digital document will be validated or rejected. All documents containing at least one major error such as error in ALTO 1 file structure will be rejected and all documents that contain more than 7 minor errors such as curved effect of the image of the page will be rejected At the end of this process the document will be either returned to the provider for reprocessing or available on Gallica 2 .

Bibliographic data analysis

To facilitate and improve the selection task, we intend to estimate the selection decision document before performing the selection task using only bibliographic data. So we tried to evaluate the relationships between these metadata and the possible selection decisions.

We used for this analysis a dataset of 20411 documents taken randomly from a database that contains 50000 documents obtained through an internal application used by selection documents department at the BnF. In this set, the amount of documents "Selected in HQ ", is very low compared to the number of documents "Not Selected " and "Selected in Raw".This fact skew our study. To overcome this problem, we merged both "Selected" classes into one. This leads to only 2 variables for the decision value : "Selected " and "Not Selected ".

A preliminary work guided us to focus on two factors involved in the selection decision of documents : the edition date and format of documents. The choice of these variables was also motivated by the observation of staff who are responsible for the documents selection that say :

-Old documents have more physical defects than new documents,

1. ALTO file is an XML file that contains the results of OCR systems. This format is managed by the Library of Congress http ://www.loc.gov 2. http ://www.gallica.bnf.fr -Small documents have poor quality of writing and are more difficult to digitized. Consequently, to conduct our study, we first applied a correspondence factor analysis (CFA) on two contingency tables :

-one table describing the link between the publishing date and the selection decision documents ; -one table describing the link between the format and the selection decision documents Then, in a second step, to have a more precise idea, we used a multiple correspondance analysis (MCA) on a data array that contains more qualitative variables.

Influence of edition dates and format on selection decision

The CFA is a statistical technique that analyses two qualitative variables intersected in a PivotTable. This method allows to study the correlation between modalities of each variable by referring to the model of independence of studied variables. According to [1][2], we begin our factorial analysis by the construction of the adjusted clouds of column profiles and line profiles. Then in the manner of Principal Component Analysis (PCA) and to explore the links between the two studied variables, we proceed to the determination of the factorial axes that maximize the inertia of the lines cloud and columns cloud. The simultaneous representation of profiles lines and profiles columns is possible due to the transformation equations from line profiles to columns profiles and vice versa. This representation simplifies a lot the interpretation of these connections since we can display the modalities of each variable on the same graph.

F s (i) = 1 √ λ s J ∑ j=1 f i j f i• G s ( j) (1) G s ( j) = 1 √ λ s I ∑ i=1 f i j f • j F s (i) (2)
where :

-F s (i) the coordinate of the profile i line on the axis of rank s. -G s ( j) the coordinate of the profile j column on the axis of rank s. -λ s the inertia of the cloud of lines (resp. cloud of columns) projected onto the axis of rank s in the values space of columns R j (resp. the value space of lines r i ).

Edition dates study

We begin by studying the relationship between edition dates and the selection decisions of the documents. For this, we applied a test of independence on the table 1 that crosses dates of editions with the selection decisions of document.

The critical probability of this test is very low 10 -16 which makes the interest of the CFA on these data beyond doubt. The two intersected variables are linked since the test of independence of χ 2 is different from zero (χ 2 = 359.703 = 0 ). Nevertheless, this relationship is very low since the intensity of the connection measured by the Cramer's V is low (V = 0.1370). Anyway, we will analyze the Table 1 by a CFA to determine the modalities that correspond to more selected documents than rejected ones. The maximum number of axes needed to represent the documents perfectly is equal to min {(card(lines) -1), (card(columns) -1)}.

Factor analysis of Table 1 gave two factorial axes. The percentage of inertia associated to the first axis is close to 100%. On the one hand, the terms of the variable edition dates "1871-1900 ", "1901-1930 " and "1961-1990 " have more weight (79%) in the construction of the first axis compared to the other edition dates.

On the other hand, we also find that the selection decision "Selected " has a significant contribution of 96% in the construction of the first axis (the modality "Not selected" contributes only for 3.97%). Figure 2 shows at the same time the modalities in lines and the modalities in columns on the same figure. This allows us to build links between the terms "date of edition" and the modalities "decisions selection of the document". According to this simultaneous representation, we notice that all the intervals of the edition date are close to the decision "Not selected ". This observation is due to the large number of documents rejected in each date of edition. Nevertheless, the representation of edition dates between "1781-1840 " and between "1931-1990 " are more likely to be selected than others. In the same way, documents published between 1841 and 1930 are much more rejected. However one should keep in mind that the intensity of these relationships are weak and we cannot use them alone as predicitive variables. This weakness cloud be easily explained by the fact that other criteria should be considered for the selection decision.

Document Formats study

In terms of bookbinding we often talk with the terms of format to describe the size of documents. According to internal documentation of the BNF, we use the format "in-folio (FOL) " when we fold the press papers once, "in-quarto (8) " in the case when we fold the press papers twice, "in-octavo (4) " in the case when we fold the press papers three times and "in-sixteen (16) " in the case when we fold the press papers four or more times. In the case where we do not fold the press papers we use the document format "in-plano (GR FOL) ". As we specified in the first part of this section, we want to study the relationship between document formats and the selection de- The test of independence applied on Table 2 showed that the two variables are linked since the value of χ 2 is greater than zero (χ 2 = 1313.304 ). The critical probability of this test is less than 2.2 × 10 -16 which makes the interest of the CFA on these data beyond doubt.

Even if the intensity of the connection between the two variables is weak V -Cramer = 0.25366 < 0.5, we have chosen to analyse our data by a correspondence factor analyse in order to identify the relationships between formats and their selection decision.

The factorial analysis of Table 2 has given two factorial axes. The clouds of rows and columns are well presented by the first factorial axis since it expresses around 100% of the total inertia. According to the Figure 3, the first factorial axis separates the two terms "Not Selected" and "Selected". We note also that the "not selected" documents are close to the origin of the first axis. In fact, the large numbers of the not selected documents compared with the number of selected documents influence the calculation of the independence model. On this figure, we can also visualize and interpret the connections that exist between the two variables. We notice that the documents in "16", "8" and "FOL" have a tendency to be not selected since these representations are close to the representation of modality "Not Selected". This proximity can be interpreted by the lack of selected documents in the categories of document format. Documents in format "GR FOL" and "4" have positive coordinates on the first axis. These formats have more selected documents compared to other formats. The calculation of the contributions of rows and columns in the construction of the factorial axes allows us to select the main points contributory in the construction of the principal axis. The modality "Selected" has an important contribution of 96.13% so we can say that the first axis represent perfectly the decisions of document's acceptance. The format "4" contributes decisively in the construction of the first axis. These document formats are a feature of selected documents. We can conclude, according to this analysis that document formats ("GR FOL" and "4" ) characterize the selected documents.

But, this proximity does not assert that all documents which possesses those format will be selected due, firstly, to the weakness of their relationship intensities and secondly to the complexity of the document selection decision which depends on several factors at the same time. This is the reason why we decided to operate a Multiple Correspondence Analysis.

Multiple correspondence analysis (MCA)

The MCA offer the opportunity to deal with many variables. In fact, it can be applied on complete disjunctive table that intersect individuals (documents) in lines with qualitative variables (Bibliographic data) in columns. The value of the elements x ik is 1 if the individual i has the modality k and 0 if not. The size of this table is equal to I × K with I is the number of documents and K is the number of modalities of our variable. The main focus of the MCA is the study of the relationships between modalities and variables. As in Principal Component Analysis (PCA) [1][2][3], we try to draw the links between variables. These links can be studied either two by two as in CFA or globally.

MCA method

According to [1], studying the similarities between modalities, implies to define the distance which separates them. Let two modalities k and k ′ assimilated to each group of individuals. One way to compare these two modalities is to count the individuals who have one or both of these modalities. As in the CFA, we should begin our analysis with the construction of the clouds of modalities and variables using the following formulas :

f ik = x ik I × J (3) f •k = I ∑ i=1 x ik I × J = I k (I × J) ( 4
)
f i• = K ∑ k=1 x ik (I × J) = 1 I (5)
Where :

-

x i j ∈ {0, 1} -I the number of documents -J the number of variables -i ∈ [1...I] -j ∈ [1...J]
The distance between two modalities of complete disjunctive table is calculated using the following formula :

d 2 χ 2 k, k ′ = I ∑ i=1 1 f i• f ik f •k - f ik ′ f •k ′ (6)
Then, as in PCA, we try to find the axes that maximize the projected inertia of the cloud of variables and modalities. If we use orthogonal axes, we can have plans that maximize the inertia of the cloud of variables and modalities. The eigen vectors are by definition orthogonal. Moreover, the eigenvalue λ s can also be interpreted as the inertia of the cloud projected on the axis of rank s. Therefore, the biggest eigenvalue represents the maximum inertia of our cloud of variables or modalities. To use this method, in our study, we constructed a data table which includes 5 variables we added to our CFA database some informations about the country [START_REF] It | Principal Component Analysis[END_REF] and language [START_REF] Rafik | Discriminant Multiple Correspondence Analysis[END_REF] of the documents because we know that the BNF's mission is to treat the French national heritage and especially the French documents. Then, theoretically, documents that contain these two criteria have more chance to be selected. The number of documents examined in this analysis is the same as in our previous studies (20 411 documents).

Results and discussion

We began our analysis by presenting the cloud of variables according to the first two factorial axes. Then, we have refined our analysis by a study of relationships between variables and we analyzed also the relationships between the terms of these variables to clarify our study. The variables can be plotted by calculating the correlation ratios between the coordinates of individuals on one axis and each of the variables. According to [1], if the correlation ratio between the variable j and axis s is close to 1, this means that individuals (documents) with the same modality (for this qualitative variable) have neighbor coordinates in this axis s. In our study, according to the plane representation of the cloud of the variables, we find, on the one hand, that variable Language, Country and Edition date are related to each of the first two axes (see, Figure 4). On the other hand we note that the qualitative variable Format is more related to the first axis than to the second one(see, Figure 4) According to [1], in MCA, the percentage of inertia associated to the first axis is lower than in PCA. This is because, in ACP for example, only linear combination are considered : this mean that only one axis can represent all variables if these are highly correlated with each other. Seemingly in MCA, we study much more general links : if we study two variables that have K j and K l modalities we need at least "min(K j , K l ) -1" dimensions to represent the relation between two variables.

3. The National Library of France uses the iso code in order to name the country of document http ://www.iso.org/iso/fr/french country names and code elements 4. The National Library of France uses the iso code in order to name the language of document http ://fr.wikipedia.org/wiki/Liste des codes ISO 639-2

In our study, we note that the decrease of the eigenvalues became regular from the third factorial axis. As a consequence, we will interpret here only the first two factorial axes although it is interesting to interpret the following axes. The simultaneous representation of modalities of the qualitative variables and individuals (Documents) is not highly efficient. In fact, we obtain a cluttered representation which makes our interpretation difficult and inaccurate (see Figure 5). Nevertheless, MCA statistics show a positive coordinates for modalities 4 and 16 of variable" Format" and a negative coordinate for modality 8. Furthermore, on the same axis, we also notice that the coordinate of "Selected " modality is positive whereas the coordinate of the modality "Not Selected " is negative. The automatic description of the second axis also shows that the coordinates of formats FOL and GR FOL are positive whereas the coordinates of formats 4, 8 and 16 are negative. Based on these results, we can say that the first axis represents the documents in 16 and 4 (resp. 8 ) and which are selected (resp. not selected) after the selection process of documents. We can also deduce that the second axis represents the documents in formats FOL and GR FOL (resp. 4, 8 and 16 ) that are not selected (resp. selected) by selection departments at the BnF. Often a multivariate analysis is completed by a univariate analysis to characterize some variables. In this step of our study, we constructed a χ 2 test of independence in order to evaluate the relationship between the variable of interest (in our case "selection state") and other variables. The more this test is significant, the more the modalities considered and the qualitative variables are related. According to [1], the critical probability (p-value) measures the significance of the χ 2 test . Therefore, the more the critical probability for the χ 2 test is smaller the more the assumption of independence is questionable and the more the qualitative variable characterizes the variable of interest. In our study and according to the Table 3 we notice that the variable "Format" is the most linked to the variable of interest "State of selection". Another way of interpreting links between variables is done by the study of links between the modalities of these variables.Then, we characterize in this part each modality of the variable of interest "Selection State" by the modalities of other variables. The "test value" is a tool that contributes to the exploratory and descriptive approach of large spreadsheets. To simplify our analysis, we divided our study in two steps. First, we focus on the analysis of the modality "Not Selected ". For this, we adapted the method presented in [1] which gives a table that contains qualitative variables sorted from the most to the least significant when the modality is over-represented (value-test is then positive) and from the least to the most significant when the modality is under-represented in the class (the value-test is then negative).

FIGURE 5. The simultaneous representation of modalities of bibliographic variables and documents

According to Table 4 5 , we find that 99.71% of documents in format "16 " are not selected ; 25.19% of the not selected documents are in format "16 " and 24.29% of documents processed in our study are in format "16 ". The critical probability of the test (5.54 × 10 -75 ) is low and the associated value-test is 18.32. Therefore, we can conclude that format "16 " characterizes very well the modality "Not Selected ". We also find that the formats "16 " and "8 " and the editions dates "1901-1930 " characterizes more the documents not selected. We remark also that contrary to what we thought at the beginning of our analysis the French documents characterize more the modality "Not Selected" than the modality "Selected". This result is logical since on the one hand the French documents are the majority of BnF collection (and then a huge part of them can be not selected), and on the other hand, the foreign documents are generally programmed in specials digitization projects in which the BnF tries to select most of the documents. As well to study the modality "Selected", we adopted the same previous analysis. Table 5 6 shows the results of this operation. According to these results, we found that 11.28% of documents in format "4 " are accepted ; 72% of documents accepted are in format "4 " ; 24.66% of documents processed in our study are in format "4 ". The critical probability of our test is (1.4 × 10 -179 ) and the associated value-test is (28.57). Furthermore, 19% of the documents in "GR 5. Cla/Mod : The percentage of documents that have the modality "i " and that is not selected ;Mod/Cla : The percentage of not selected documents that have the modality "i " ; Global : The percentage of documents having the modality "i " ; p-value : The critical probability ; V-test : The distance between the individual's average having the modality "i " and the overall average 6. Cla/Mod : The percentage of documents that have the modality "i " and that is selected ;Mod/Cla : The percentage of selected documents that have the modality "i " ; Global : The percentage of documents having the modality "i " ; p-value : The critical probability ; V-test : The distance between the individual's average having the modality "i " and the overall average FOL " are selected and 8.11% of selected documents are in format "GR FOL " ; 1.65% of documents processed in our study are in format "GR FOL ". The critical probability of this test is equal to (2, 601 × 10 -26 ) and the test-value associated is equal to (10.61).Therefore, we conclude that the format "4 " and "GR FOL " characterizes the selected documents. We notice also that 7.51% of documents printed between "1961-1990 " are selected and 51.33% of selected documents are prin- ted between "1961-1990 ". 26.41% of documents processed in our study are are printed between "1961-1990 ". The critical probability of our test is (6.3 × 10 -52 ) and the associated value-test is (15.16). As a conclusion, from the multiple correspondences analysis made in this section, we can conclude that for example the documents in format "4 or GR FOL " and edited "between 1961 and 1990 " in "Morocco(ma) " are more likely to be "Selected ". Ho- wever, the documents in format "16 " and edited "between 1901 and 1930 " in "English (eng) or Spanish (spa) " in "France (fr) " have a greater chance to be "Not Selected ".

Conclusion

In this paper we tried to estimate the selection decision made on BnF's documents. For this, we had studied the relationships between selection decision and bibliographic data. Our analysis has shown that there are relationships between bibliographic data (like Format, Edition date, country and Language) and selection decisions. For example, according to our analysis we have noticed that documents in format "4 " and edited "between 1961 and 1990 " in Morocco are more likely to be "Selected ". However, the documents in format "16 " and edited "between 1991 and 2021 " in English (eng) or Spanish (spa) " in "France (fr) " have a greater chance to be "Not Selected ". These relations still weak. In fact, others factors, as the intellectual value of the document and the topic of document digitization

FIGURE 4 .

 4 FIGURE 4. Representation of qualitative variables according to the two first factorial axes

Table 1 : Contingency table which crosses the edition dates of the document with the selection decisions of document

 1

		Selected	Not Selected
	1781-1810	17	230
	1811-1840	27	389
	1841-1870	34	1345
	1871-1900	85	4369
	1901-1930	59	3674
	1931-1960	134	2350
	1961-1990	405	4986

FIGURE 2. Simultaneous representation of edition date and documents selection decision

Table 2 : Contingency table which crosses the formats of the document with the selection decisions of document

 2

		Selected	Not Selected
	4	568	4466
	8	25	5265
	16	14	4944
	FOL	118	4673
	GR FOL	64	273

FIGURE 3. Simultaneous representation of Format and documents selection decision cisions. For this, we constructed Table

2

that intersects document formats with the selection decisions of documents.

Table 3 : Description of the variable "Selection State" by the other qualitative variables Critical probability

 3

	Format	4.341334e-283
	Country	1.214759e-80
	Date	3.869601e-72
	Langue	3.660244e-16

Table 4 :Description of "Not Selected" modality by the modali- ties of the others qualitative variables Cla/Mod Mod/Cla Global p-value

 4

	v.test

...... ...... ...... ...... ...... ......

Table 5 :Description of "Selected" modality by the modalities of the others qualitative variables (XXX : Language not men- tioned, XX : Country not mentioned) Cla/Mod Mod/Cla Global p-value

 5 , are involved in the selection decision of documents. Unfortunately, these data are not annotated in BnF databases. This makes the estimation of selection decisions of documents with bibliographic data difficult. Therefore if we want to provide a tool able to help the selection department of the BnF, we must enrich our data especially with physical and intellectual characteristics.

	v.test

...... ...... ...... ...... ...... ...... projects

Author Biography

Ahmed Ben Salah is a PhD student at the University of Rouen. His PhD is performed at the national library of France (BnF) on prediction of OCR rate and automatic control of OCR results. More generally, his research interests include data analyse and pattern recognition.