Juliette Bréhat

Mathieu Panel

Bijan Ghaleh

PhD Didier Morin
email: didier.morin@inserm.fr

Opening of mitochondrial

Keywords: Ferutinin, Mitochondria, permeability transition pore, cardiomyocytes cyclophilin D, mPTP: mitochondrial permeability transition pore, FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone

Introduction

The mitochondrial permeability transition is a critical event in cell death. It corresponds to an increase in the permeability of the inner mitochondrial membrane and is mediated by opening of the permeability transition pore (mPTP). mPTP contributes to cell injuries in a number of diseases and more particularly during myocardial ischemia-reperfusion [START_REF] Halestrap | A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection[END_REF]. Despite a considerable number of studies, the molecular structure of this channel remains unknown, potential constitutive proteins having been ruled out by genetic modulation. In recent years, experimental evidence suggest that ATP synthase participates to the formation of the pore [START_REF] Bonora | Molecular mechanisms and consequences of mitochondrial permeability transition[END_REF]. However, there is also a common agreement to consider that excess intramitochondrial Ca 2+ is a crucial factor to trigger mPTP opening [START_REF] Haworth | The Ca2+ -induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site[END_REF] and that cyclophilin D (CypD), a soluble protein located within the mitochondrial matrix, is a key regulator as it lowers the Ca 2+ threshold triggering mPTP formation [START_REF] Javadov | Mitochondrial permeability transition in cardiac ischemia-reperfusion: whether cyclophilin D is a viable target for cardioprotection?[END_REF].

The critical role of mPTP opening in cell death suggests that its inhibition may represent an effective therapeutic approach in several pathological conditions. Therefore, the identification of novel mPTP inhibitors should be considered with great interest but, currently, a convenient and reliable cardiomyocyte model is lacking to study potential inhibitors.

As Ca 2+ plays a major role in mPTP opening, we previously explored the effect of three Ca 2+ ionophores (A23187, ionomycin, and ETH129) as possible inductors of mPTP opening with the objective to develop such a model in primary cardiomyocytes where Ca 2+ fluxes have a fundamental role. We demonstrated that they were not suitable to induce mPTP opening in adult murine cardiomyocytes because their mechanism of Ca 2+ transport was not effective enough or they displayed effects independent from mPTP opening [START_REF] Panel | Ca2+ ionophores are not suitable for inducing mPTP opening in murine isolated adult cardiac myocytes[END_REF]. The Ca 2+ ionophore strategy remains, however, an interesting approach.

In the present study, we investigated the effect of ferutinin, jaeschkeanadiol phydroxybenzoate, for its Ca 2+ ionophore properties. Ferutinin was shown to mobilize Ca 2+ in cells, to increase mitochondrial Ca 2+ permeability and to induce mitochondrial Ca 2+ loading and depolarization [START_REF] Abramov | Influence of plant terpenoids on the permeability of mitochondria and lipid bilayers[END_REF]. Opening of the mPTP appeared as a possible mechanism of this depolarization [START_REF] Abramov | Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca 2+ ionophore on mitochondria in intact cells[END_REF], and several studies demonstrate that ferutinin possesses antiproliferative effects through the activation of the intrinsic apoptotic pathway and mPTP opening [START_REF] Macho | Calcium ionophoretic and apoptotic effects of ferutinin in the human Jurkat T-cell line[END_REF][START_REF] Poli | Antiproliferative effects of daucane esters from Ferula communis and F. arrigonii on human colon cancer cell lines[END_REF][START_REF] Gao | Ferutinin induces in vitro eryptosis/erythroptosis in human erythrocytes through membrane permeabilization and calcium influx[END_REF][START_REF] Reyhani Garmroudi | Ferutinin: A phytoestrogen from ferula and its anticancer, antioxidant, and toxicity properties[END_REF]. A recent study also showed that ferutinin induces mPTP opening in liver mitochondria [START_REF] Ilyich | Ferutinin Induces Membrane Depolarization, Permeability Transition Pore Formation, and Respiration Uncoupling in Isolated Rat Liver Mitochondria by Stimulation of Ca2+-Permeability[END_REF]. Unlike A23187 and ionomycin, which equilibrate Ca 2+ concentrations between cell compartments and the extracellular space in an electroneutral manner, ferutinin is an electrogenic Ca 2+ ionophore. This mechanism could be more appropriate to promote a physiological model of mitochondrial Ca 2+ overload [START_REF] Abramov | Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca 2+ ionophore on mitochondria in intact cells[END_REF].Therefore, using the calcein loading CoCl2 quenching technique [START_REF] Petronilli | Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence[END_REF], we investigated the effect of ferutinin on mPTP opening in primary cardiomyocytes isolated from adult mice and we examined its effect on isolated myocardial mitochondria.

Materials and Methods

Animals

All animal procedures used in this study were in accordance with the Directives of the European Parliament (2010/63/EU-848 EEC) and the recommandations of the French Ministère de l'Agriculture. Male C57BL/6J mice (8 to 10 week-old) were purchased from Janvier (Le Genest-St-Isle, France). CypD knock-out mice (Ppif -/-mice) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). Mice were housed in an air-conditioned room with a 12 h light-dark cycle and received standard rodent chow and drinking water ad libitum.

Isolation of cardiac mitochondria

Mitochondria were isolated as described previously [START_REF] Laure | Cardiac H11 kinase/Hsp22 stimulates oxidative phosphorylation and modulates mitochondrial reactive oxygen species production: Involvement of a nitric oxide-dependent mechanism[END_REF]. Briefly, hearts were scissor minced and homogenized in a buffer (220 mM mannitol, 70 mM sucrose, 10 mM HEPES, 2 mM EGTA, pH 7.4 at 4°C) using a Potter-Elvehjem glass homogenizer in a final volume of 10 mL.

The homogenate was filtered and centrifuged at 1000g for 5 min at 4°C. The supernatant was centrifuged at 10000g for 10 min at 4°C. The mitochondrial pellet was resuspended in 50 µL of homogenization buffer without EGTA and protein concentration was determined using the advanced protein assay reagent (Sigma catalog number #57697). In experiments requiring a lot of mitochondria, mitochondrial preparations from two to three hearts were pooled.

Measurement of mitochondrial respiration

Mitochondrial oxygen consumption was measured at 30°C with a Clark-type electrode (Hansatech Instruments Ltd, Norfolk, UK). Mitochondria (0.4 mg protein/mL) were incubated in a respiration buffer containing 100 mM KCl, 50 mM sucrose, 10 mM HEPES and 5 mM KH2PO4, pH 7.4 at 30°C. Respiration was initiated by addition of 5 mM glutamate/ 5 mM malate (state 2, substrate dependent respiration rate). Then, ATP synthesis was induced by adding 0.4 mM ADP (state 3, ADP-dependent respiration rate, V3). The rate of state 4 respiration was measured either after exhaustion of ADP (V4) or after addition of 1 µM of carboxyatractyloside (V4CAT) and the respiratory control ratio (state 3/state 4) was evaluated.

When tested, ferutinin was added 1 min after the beginning of state 2 respiration.

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (Ψ) was evaluated by the uptake of the fluorescent dye rhodamine 123, which accumulates electrophoretically into energized mitochondria in response to their negative-inner membrane potential. The respiratory substrates glutamate/malate (5/5 mM) and rhodamine 123 (0.2 μM) were added to 1 mL of respiration buffer into the cuvette maintained at 30°C. Fluorescence was monitored over time using a Perkin-Elmer SA LS 50B fluorescence spectrometer (excitation wavelength = 503 nm, emission wavelength = 527 nm). After 40 s of initial monitoring, mitochondria (0.2 mg/mL protein) were added and the effect of ferutinin on membrane potential was examined following addition of the compound at t = 120-150 s. When used, carboxyatractyloside (1 µM) was added before ferutinin. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 1 μM) was added to depolarize the mitochondrial membrane potential thus acting as internal reference.

Mitochondrial swelling assays

Mitochondrial swelling was assessed by measuring the change in absorbance at 540 nm using a Jasco V-530 spectrophotometer equipped with magnetic stirring and thermostatic control. Mitochondria energized with glutamate/malate (5 mM each) were incubated 30 s at 30°C in the respiration buffer before the induction of swelling with ferutinin.

In de-energized conditions, mitochondria were incubated 30 s at 30°C without substrate in the respiration buffer including 1 µM rotenone and 1 µM antimycin at pH 7.4 before inducing swelling with ferutinin. In both conditions, cyclosporine A (1 µM) was introduced at the onset of the incubation period.

In some experiments, respiration buffer was replaced by a sucrose-HEPES buffer containing 250 mM sucrose and 10 mM HEPES at pH 7.4.

Swelling experiments were analyzed by measuring the initial rate of swelling which was expressed as change in absorbance/s.

Measurement of intra-mitochondrial calcium

Intra-mitochondrial Ca 2+ was evaluated in isolated mitochondria using the non-fluorescent probe Rhod-2/AM, which is converted to fluorescent Rhod-2 via the action of esterases. Isolated mitochondria (≈ 15 mg/mL) were incubated under stirring with Rhod-2/AM (30 µM) for 30 min at 25°C in 500 µL of homogenization buffer devoid of EGTA. The suspension was then diluted 30-fold with the homogenization buffer at 4°C and after centrifugation (10 min, 10000g) the pellet containing loaded mitochondria was resuspended in 100 µL of homogenization buffer.

Rhod-2-loaded mitochondria (0.5 mg/mL) were incubated in the respiration buffer (1 mL) maintained at 30 °C under constant agitation. Intra-mitochondrial Ca 2+ was monitored over time by following Rhod-2 fluorescence using a Perkin Elmer LS50B spectrofluorometer (exciting wavelength 552 nm; emission wavelength 581 nm).

Measurement of intra-mitochondrial pH

Intra-mitochondrial pH (pHi) was evaluated in isolated mitochondria using the nonfluorescent probe BCECF-AM, which is converted to fluorescent BCECF via the action of esterases. Isolated mitochondria (15 mg/mL) were incubated under stirring with BCECF-AM (30 µM) for 30 min at 25°C in the homogenization buffer devoid of EGTA. The suspension was then diluted 30 fold with the homogenization buffer at 4°C and after centrifugation (10 min, 10000g) the pellet containing loaded mitochondria was resuspended in 100 µL of homogenization buffer. BCECF-loaded mitochondria (0.2 mg/mL) were incubated in the respiration or in the sucrose-HEPES buffer (1 mL) maintained at 30 °C under constant agitation. Fluorescence was followed using a temperature-controlled spectrofluorometer (Jasco FP-6300) using an excitation ratio technique (dual excitation wavelength 439 and 488 nm and fixed emission wavelength at 525 nm). The signals at 439 and 488 nm correspond to the isosbestic point and to the maximal sensitivity of the probe, respectively. When used, rotenone (1 µM), antimycin (1 µM) and cyclosporine A were introduced at the beginning of the experiment.

Measurement of mitochondrial potassium flux.

Mitochondrial potassium flux was assessed with the fluorescent probe PBFI [START_REF] Jezek | Reconstitution of the beef heart and rat liver mitochondrial K+/H+ (Na+/H+) antiporter. Quantitation of K+ transport with the novel fluorescent probe, PBFI[END_REF][START_REF] Costa | The direct physiological effects of mitoK(ATP) opening on heart mitochondria[END_REF]. Isolated mitochondria (≈ 15 mg/mL) were incubated under stirring with 30 µM of the potassium fluorescent indicator acetoxymethyl ester (PBFI, AM) in 500µL of homogenization buffer devoid of EGTA for 20 min at 25°C. Before incubation, PBFI-AM was mixed with the nonionic surfactant pluronic F-127 (2:1 ratio, vol/vol) to improve the aqueous solubility of the probe and thus its mitochondrial loading. After incubation, the suspension was diluted 30-fold with the homogenization buffer at 4°C, and after centrifugation (10 min, 10000 g) the pellet containing loaded mitochondria was resuspended in 100 µL of homogenization buffer. PBFI-loaded mitochondria (0.2 mg/mL) were incubated in the respiration buffer (1 mL) maintained at 30 °C under constant agitation. Fluorescence was followed using a temperature-controlled spectrofluorometer (Jasco FP-6300) using an excitation ratio technique (dual excitation wavelength 340 and 380 nm and fixed emission wavelength at 525 nm). The signals at 340 and 380 nm correspond to the maximal sensitivity and to the isosbestic point of the probe, respectively. When used, rotenone (1 µM), antimycin (1 µM) and cyclosporine A (1 µM) were added at the beginning of the experiment.

Isolation of primary adult mouse cardiomyocytes

Ventricular cardiomyocytes were isolated from mice by an enzymatic technique. The heart was retrogradely perfused for 15 min at 37°C with a stock perfusion buffer bubbled with 95%O2/5%CO2 containing 133 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 12 mM NaHCO3, 10 mM KHCO3, 10 mM HEPES, 30 mM taurine, 0.032 mM phenol red, 5.5 mM glucose, 10 mM 2,3butanedionemonoxime pH 7.4 to wash out blood.

After 2 min of perfusion, liberase (Blendzyme 10 mg/100 mL, Roche Applied Science, Mannheim, Germany), trypsin EDTA (14 mg/100 mL) and 12.5 μM Ca 2+ were added to the buffer. After approximately 8-9 min of perfusion, the heart was placed in the same buffer containing 10% bovine serum albumin pH 7.4 at 37°C to stop the digestion. Ventricles were then cut into small fragments and cells isolated by stirring the tissue and successive aspirations of the fragments through a 10 mL pipette. After 10 min, the supernatant was removed and the remaining tissue fragments were re-exposed to 10 mL of the same buffer.

Then, the cells were suspended in the same buffer and Ca 2+ was gradually added to 1 mM into an incubator at 37°C. Finally, the cardiomyocytes were suspended in culture medium M199 supplemented with 1% Insulin-Transferrin-Selenium. They were seeded on 35 mm Petri dishes (150000 cells/dish) precoated with 10 μg/mL sterilized laminin and incubated for 90 min before being used.

Measurement of mPTP opening, membrane potential, cell death and mitochondrial calcium in cells

mPTP opening was assessed by means of the established loading procedure of the cells with calcein acetoxymethyl ester (calcein-AM) and CoCl2 resulting in mitochondrial localization of calcein fluorescence [START_REF] Petronilli | Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence[END_REF][START_REF] Katoh | Diazoxide opens the mitochondrial permeability transition pore and alters Ca2+ transients in rat ventricular myocytes[END_REF]. Cells were loaded with 1 µM calcein-AM for 30 min at 37°C in 2 mL of M199, pH=7.4, supplemented with 1 mM CoCl2 after 20 min of incubation.

They were then washed free of calcein-AM and CoCl2 and M199 was replaced by Tyrode's buffer (NaCl 130 mM; KCl 5 mM; HEPES 10 mM; MgCl2 1 mM; CaCl2 1.8 mM, pH 7.4 at 37°C). To determine cell death, cells were co-loaded with 1.5 µM propidium iodide, which permeates only the damaged cells. When the mitochondrial membrane potential was monitered, cardiomyocytes were loaded with 20 nM tetramethylrhodamine methyl ester (TMRM) for 30 min at 37°C and then rinsed with Tyrode's solution. To monitor mitochondrial Ca 2+ , cardiomyocytes were loaded with 1.5 µM Rhod-2 AM (Thermofisher Scientific, Villebonsur-Yvette, France) for 30 min at 37°C and then washed free of Rhod-2/AM. The cardiomyocytes were placed into a thermostated (37°C) chamber (Warner Instruments Inc, Connecticut, USA) which was mounted on the stage of a IX81 Olympus motorized inverted microscope, were perfused with Tyrode's solution at a rate of 0.5 ml/min and were paced to beat by field stimulation (5 ms, 0.5 Hz).

Cardiomyocytes were imaged with the microscope equipped with a mercury lamp as a source of light for epifluorescence illumination and with a cooled CCD Hamamatsu ORCA ER digital camera. For detection of calcein fluorescence a 460-to 490nm excitation and a 510 nm emission filters were used. Propidium iodide and TMRM fluorescence were excited at 520-550 nm and recorded at 580 nm. For the detection of Rhod-2 a 525-to 550nm excitation and a 580-to 635nm emission filter were used. Images were acquired every 30 or 60 s within the first 30 min and then every 5 min after an illumination time of 25 ms (calcein) and 70 msec (propidium iodide, TMRM and Rhod-2). Images were analyzed using a digital epifluorescence imaging software (XCellence, Olympus, Rungis, France).

First, for each experiment, the change of fluorescence was observed from a single cardiomyocyte. Fluorescence was integrated over a region of interest (≈ 80 µm 2 ) and a fluorescence background corresponding to an area without cells was substracted. Then, the global response was analysed by averaging the fluorescence changes obtained from all the cardiomyocytes (20-30 cells) contained in a single field. For comparative purposes, the fluorescence intensity minus background was normalized according to the maximal fluorescence value (initial value for calcein, TMRM, and Rhod-2, final value for propidium iodide). The time necessary to induce a 50% decrease in calcein fluorescence ([Tcalcein50]) and to induce a 50% increase in propidium iodide fluorescence (time to 50% cell death [Tcell death50]) were determined.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 9.1.2 (Graph Pad Software, USA). Data were presented as the means ± standard error of mean (SEM). Statistical significance of differences between two groups was determined using an unpaired two-tailed t-test.

Significant differences between more than two groups were evaluated by one-way analysis of variance (ANOVA) analysis followed by a Tukey's post test if ANOVA produced a significant value of F (p < 0.05). Statistical parameters and significance are reported in the figures and the figure legends.

Results

Ferutinin displays calcium ionophoretic properties

First, we verified the Ca 2+ ionophoretic properties of ferutinin in isolated cardiac mitochondria.

Fresh isolated mitochondria were loaded with the Ca 2+ sensitive fluorescent dye Rhod-2 as its acetoxymethyl esters Rhod-2/AM. Rhod-2/AM is hydrolyzed by mitochondrial esterase and Rhod-2 is entrapped in the mitochondrial matrix space as reported for others acetoxymethyl ester dyes [START_REF] Lukács | Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2[END_REF][START_REF] Long | CORM-3, a water soluble COreleasing molecule, uncouples mitochondrial respiration via interaction with the phosphate carrier[END_REF]. This allows the monitoring of mitochondrial free Ca 2+ concentration. When mitochondria were energized by the addition of respiratory substrates (pyruvate/malate), the addition of Ca 2+ in the medium induced a rapid and concentration dependent increase in fluorescence. This effect was strongly inhibited by the presence of the Ca 2+ uniporter blocker ruthenium red, demonstrating the validity of the method (Fig. 1A).

Then, cardiac mitochondria were incubated in the same buffer in the presence of rotenone and antimycin to inhibit respiration and therefore the uniporter complex dependent Ca 2+ influx. In these deenergized conditions, ferutinin caused a concentration-dependent Ca 2+ entry into mitochondria, which was not inhibited by ruthenium red confirming the ionophoretic properties of the drug (Fig 1B). On the contrary, ruthenium red stimulates mitochondrial Ca 2+ uptake, which may be due to the blockade of the uniporter complex avoiding a Ca 2+ efflux [START_REF] Jurkowitz | Ruthenium red-sensitive andinsensitive release of Ca2+ from uncoupled heart mitochondria[END_REF] Then we verified the ability of ferutinin to cause mitochondrial Ca 2+ influx in isolated cardiomyocytes. Mitochondrial compartment of cardiomyocytes were loaded with 1.5µM Rhod-2/AM and then supplemented with increasing concentrations of ferutinin. Ferutinin

Ferutinin and mPTP opening in isolated adult cardiomyocytes.

We next investigated the ability of ferutinin to induce mPTP opening in isolated mouse adult cardiomyocytes. Cells were coloaded with calcein in the presence of CoCl2 and TMRM to monitor mPTP opening and mitochondrial potential, respectively. Figure 2A shows that the addition of 25 µM ferutinin decreased both calcein and TMRM fluorescence concomitantly in a time-dependent manner, which may be the signs of mPTP opening. Next, we verify if the mitochondrial calcein loss induced by ferutinin was associated with cell death. We coloaded mitochondria with calcein/CoCl2 and propidium iodide before adding ferutinin. As shown in Figures 2B and2C, the decrease in calcein fluorescence was associated with an increase in propidium iodide fluorescence and these effects were concentration-dependent. At 5 µM, the effect of ferutinin was weak: The loss of calcein fluorescence and cell death were only partial at 60 min.

To confirm the occurrence of mPTP opening in the effect of ferutinin, similar experiments were performed on cardiomyocytes isolated from CypD knock-out mice which are protected from mPTP opening [START_REF] Baines | Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death[END_REF]. CypD deletion slightly delayed both the loss of calcein and the increase in propidium iodide fluorescence but after 25 min, all the cells lost their calcein and died (Figures 3A and3B). Similar results were found at a lower concentration of ferutinin and in the presence of cyclosporine A, a well-known de-sensitizer of mPTP opening (Figure 3C).

It should be noted that the times necessary to induce a 50 % decrease in calcein (Tcalcein50) and a 50 % increase in cell death (Tcell death50) were not statistically different (Figure 3D). This suggests that mPTP opening is not the major contributor to the effect of ferutinin and that other mechanisms contribute to mitochondrial calcein loss, mitochondrial membrane potential disruption, and cell death in cardiomyocytes. To decipher these mechanisms, we next investigated isolated cardiac mitochondria.

Ferutinin and mPTP opening in cardiac mitochondria isolated from mice.

To investigate the effect of ferutinin on mPTP opening we examined whether ferutinin could induce mitochondrial swelling. As depicted in Figure 4A, ferutinin induced a concentrationdependent swelling in mitochondria energized with glutamate/malate as attested by the decrease in absorbance of the mitochondrial suspension. The swelling is strongly dependent on the ferutinin/mitochondria concentration ratio. Under 80 nmol ferutinin/mg mitochondrial protein, no swelling could be detected. For example, 25 µM ferutinin did not induce swelling at a mitochondrial protein concentration of 0.4 mg/mL (Figure 4A).

Cyclosporine A prevented the swelling induced by ferutinin, highlighting the role of the mPTP in the swelling process but the inhibition was only partial (Figure 4B) implying the intervention of other mechanism(s). Surprisingly, the swelling was obtained without Ca 2+ addition, a critical factor for mPTP opening. Drugs acting on Ca 2+ such as the Ca 2+ chelator EGTA or the mitochondrial Ca 2+ uniport ruthenium red had no effect on the swelling (Figure S1). This indicates that part of the swelling of the organelles is due to the formation of a nonspecific cyclosporine A insensitive permeability of the inner membrane.

Similar swelling induction and cyclosporine A inhibition were observed under de-energized conditions, that is, in the absence of substrate and in the presence of rotenone and antimycin that fully block the respiratory chain (Figure 4C). This confirms the non-necessity of Ca 2+ and indicates that mitochondrial membrane potential does not play any role in the swelling induced by ferutinin. Similarly, the absence of phosphate in the incubation medium (not shown) did not alter the swelling whereas the utilization of a minimal sucrose-hepes buffer (devoid of potassium) slowed down the rate of the swelling suggesting the involvement of potassium in the kinetic of the swelling process (Figure 4C).

3.4.Ferutinin inhibits oxidative phosphorylation and mitochondrial membrane potential

To further understand the mechanism by which ferutinin alters mitochondrial function, we studied the effect of increasing concentration of ferutinin on the rate of oxygen consumption in myocardial mitochondria energized with glutamate/malate. Ferutinin increased state 4 and decreased state 3 respiration rate, resulting in a total inhibition of the respiratory coefficient ratio at 25 µM ferutinin (Figure 5A). This effect was significant even at low µM concentrations. It was independent from mPTP opening as cyclosporine A was devoid of effect and the concentrations of ferutinin tested (6.25-25 µM) did not induce mitochondrial swelling at 0.4 mg/mL of proteins (Figure 4A). It should be noted that a clear difference was observed when state 4 respiration value was measured either after exhaustion of ADP (V4) or by addition of carboxyatractyloside (V4CAT). Carboxyatractyloside limited the increase in V4 value and thus the decrease in respiratory control ratio. The effect of carboxyatractyloside is clearly observed in figure 5B showing the basal rate of oxygen consumption by mitochondria energized with glutamate/malate. Whether introduced before or after ferutinin, carboxyatractyloside inhibited oxygen consumption induced by ferutinin.

Ferutinin stimulation of respiration was associated with a concentration-dependent decrease in the membrane potential monitored by the mitochondrial release of rhodamine 123 (Figure 5C). This effect was not altered in the presence of cyclosporine A (Figure S2) and was obtained in a Ca 2+ -free medium, ruling out the roles of mPTP and of a Ca 2+ ionophoretic property of the drug in this effect. Ferutinin behaved like the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a protonophore which enters mitochondria in the protonated form, discharging the pH gradient and destroying the membrane potential. Here again, carboxyatractyloside modulated the effect of ferutinin as it limited the release of rhodamine 123 at low ferutinin concentrations (Figure 5D).

Ferutinin modulates mitochondrial matrix pH.

To verify a potential protonophore effect of ferutinin, we studied its effect on matrix pH (pHi) using the BCECF method. This represents an interesting approach to study uncoupling agents as most of them recycle protons across mitochondrial inner membranes, disrupting proton gradient and decreasing pHi. As shown in Figure 6A, feeding the electron transport chain via complex I with glutamate/malate increased pHi as the electron transport chain extruded protons. The complex I inhibitor rotenone and FCCP abolished the gradient of protons and thus decreased pHi confirming the relevance of this method.

Addition of low ferutinin concentrations (6.25 and 12.5µM) were also followed by a slow matrix acidification confirming the protonophoric-like effect of the drug (Figure 6B). At higher concentrations, ferutinin, unlike FCCP, increased pHi with a marked effect at 50 µM (Figure 6B). This increase in pHi persisted under de-energized conditions, and was concentration dependent (Figure 6C) but was not observed when a minimal sucrose-hepes buffer devoid of potassium, which maintains an acidic matrix pH, was used (Figure 6D). In this minimal buffer, ferutinin induced alkalinization was partially restored by the selective addition of cations including Na + or K + in the medium (Figure 6D). This is in accordance with previous data showing that the acidic matrix pH maintained by nonrespiring mitochondria in sucrose can be raised by the mitochondrial uptake of cations counteracting the pH-driven proton diffusion potential [START_REF] Bernardi | delta pH induced calcium fluxes in rat liver mitochondria[END_REF]. Interestingly, cyclosporine A slightly inhibited the rate of alkalinization of the mitochondrial matrix induced by high ferutinin concentrations, suggesting the contribution of mPTP opening in this effect (Figure 6D).

Ferutinin induces potassium transport

As potassium influenced the effect of ferutinin on mitochondrial swelling and pHi, we measured changes in matrix potassium by loading mitochondria with the potassium selective probe PBFI. Energization of mitochondria with glutamate/malate generates the electrical membrane potential, which drives mitochondrial potassium influx [START_REF] Garlid | Mitochondrial potassium transport: the K(+) cycle[END_REF] (Figure 7A). In these conditions, addition of low ferutinin concentrations (6.25-25µM) was without effect (Figure 7A). Deenergized conditions, however, revealed a marked uptake of potassium into mitochondria elicited by the same concentrations of ferutinin (6.25-25µM). This effect is probably masked in energized conditions because PBFI is saturated by the entry of potassium generated by the membrane potential. Indeed, determination of mitochondrial potassium with PBFI exhibits a limited resolution for increases in matrix mitochondria because the mitochondrial concentration of potassium is high (≈ 150 mM) and PBFI displays a lower Kd value [START_REF] Costa | The direct physiological effects of mitoK(ATP) opening on heart mitochondria[END_REF]. Thus, any increase in mitochondrial PBFI fluorescence can only be relatively small. By contrast, high ferutinin concentrations (> 25µM) elicited a release of potassium from mitochondria whatever the experimental conditions. The rate of this release was slow down by cyclosporine A without reduction of the extent of the release (Figure 7B).

Discussion

It is now well-established that mPTP opening plays a key role in numerous diseases [START_REF] Boyenle | Targeting the mitochondrial permeability transition pore for drug discovery: Challenges and opportunities[END_REF].

Thus mPTP inhibition possesses a high therapeutic potential but the identification of novel inhibitors is limited by the undefined molecular identity of the pore and by the lack of cellular models able to evaluate the effect of the inhibitors. Although it is easy to monitor mPTP opening in isolated mitochondria, the evaluation of mPTP opening in cells is more complex since it is regulated by many factors. The development of the calcein loading CoCl2 quenching technique was an important step, allowing to investigate the regulation of the pore and to examine mPTP opening in living cells [START_REF] Bonora | Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques[END_REF]. However, we currently do not have an easy-to-use tool to induce mPTP in primary cardiomyocytes and to identify potential mPTP inhibitors, which is an important step to develop cardioprotective agents. In a previous work, we demonstrated that three Ca 2+ ionophores, which were used to trigger mPTP opening in different cell types, were not effective in primary cardiomyocytes [START_REF] Panel | Ca2+ ionophores are not suitable for inducing mPTP opening in murine isolated adult cardiac myocytes[END_REF]. The only experimental protocol we found suitable to induce mPTP opening in primary cardiomyocytes is a model of hypoxia-reoxygenation [START_REF] Panel | Ca2+ ionophores are not suitable for inducing mPTP opening in murine isolated adult cardiac myocytes[END_REF][START_REF] Obame | Cardioprotective effect of morphine and a blocker of glycogen synthase kinase 3 beta, SB216763 [3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], via inhibition of the mitochondrial permeability transition pore[END_REF][START_REF] Schaller | TRO40303, a new cardioprotective compound, inhibits mitochondrial permeability transition[END_REF][START_REF] Hansson | Differences in the profile of protection afforded by TRO40303 and mild hypothermia in models of cardiac ischemia/reperfusion injury[END_REF]. However, this model involves a complex and long protocol, which is inappropriate to easily screen and identify novel mPTP inhibitors.

Here, we examined the effect of ferutinin because this drug demonstrated Ca 2+ ionophoretic properties and was able to induce apoptosis in cell lines. Furthermore, it was suggested to trigger mitochondrial Ca 2+ overload and mPTP opening [START_REF] Abramov | Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca 2+ ionophore on mitochondria in intact cells[END_REF][START_REF] Ilyich | Ferutinin Induces Membrane Depolarization, Permeability Transition Pore Formation, and Respiration Uncoupling in Isolated Rat Liver Mitochondria by Stimulation of Ca2+-Permeability[END_REF]. Using Rhod-2, we confirmed the Ca 2+ ionophoretic effect of ferutinin since it induced a rapid elevation of matrix Ca 2+ in isolated cardiac mitochondria and cardiomyocytes. This is in line with the data observed in Jurkat T-cells where ferutinin mobilized Ca 2+ by both mechanisms, a release of Ca 2+ from intracellular stores and a cellular entry of extracellular Ca 2+ [START_REF] Macho | Calcium ionophoretic and apoptotic effects of ferutinin in the human Jurkat T-cell line[END_REF].

To assess whether ferutinin could represent an interesting tool to monitor mPTP opening in cardiomyocytes, we used freshly isolated murine adult cardiomyocytes, which represent a more physiological setting than cultured cells because they are fully differentiated and can be electrically paced. Using a direct measurement of mPTP opening by mean of the calcein/cobalt protocol, we observed a rapid and large calcein loss in cardiomyocytes challenged with increasing concentrations of ferutinin. Cyclosporine A and CypD deletion slightly delayed but did not prevent the loss of calcein and the associated cell death, indicating that mitochondrial depolarization and cell death is not only related to a CypDrelated pore. This is confirmed by the experiments performed in isolated myocardial mitochondria. The effect of ferutinin is more complex than a sole Ca 2+ ionophore effect since it is able to induce mitochondrial swelling and depolarization in a free Ca 2+ medium. Ferutinin acts by different mechanisms at mitochondrial level, dependent and independent of the mitochondrial membrane potential. At low ferutinin/mitochondria ratio, ferutinin displays protonophoric-like properties, lowering the mitochondrial membrane potential and limiting oxidative phosphorylation without mitochondrial swelling. Both parameters were partially restored by carboxyatractyloside suggesting the involvement of the adenine nucleotide translocase in the effect of ferutinin. Ferutinin may act on adenine nucleotide translocase to transfer proton inside mitochondria and the adenine nucleotide translocase blocking effect of carboxyactyloside would inhibit the depolarizing effect of ferutinin. This is in line with old but also recent observations that adenine nucleotide translocase can mediate proton transport across the inner mitochondrial membrane [START_REF] Panov | Adenine nucleotide translocase as a site of regulation by ADP of the rat liver mitochondria permeability to H+ and K+ ions[END_REF][START_REF] Bertholet | H+ transport is an integral function of the mitochondrial ADP/ATP carrier[END_REF].

The impairment of mitochondrial potential by ferutinin may also reflect the movement of potassium across the mitochondrial inner membrane since we showed that low ferutinin concentrations induced a potassium influx into mitochondria. This is compatible with the ionophoric properties of ferutinin, which is not uniquely selective for Ca 2+ but is able to increase, to a lesser extent, the permeability of mitochondrial membrane to monovalent cations [START_REF] Abramov | Influence of plant terpenoids on the permeability of mitochondria and lipid bilayers[END_REF][START_REF] Zamaraeva | Ionophoretic properties of ferutinin[END_REF].

At high ferutinin/mitochondria concentration ratio, ferutinin induced a sudden mitochondrial swelling, which is independent of mitochondrial membrane potential. This swelling is inhibited by cyclosporine A and is accelerated by the presence of potassium in the extramitochondrial medium. This is indicator of the participation of mPTP opening [START_REF] Halestrap | A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection[END_REF]. However, the mechanism by which ferutinin induces mPTP opening is different to those described so far since it occurs in absence of elevated intramitochondrial Ca 2+ concentration and of other factors that modulate its sensitivity such as oxidative stress or phosphate concentrations. In addition, cyclosporine A inhibition is only partial and extramitochondrial potassium is not essential, indicating the contribution of an additional mechanism.

The suddenness of the swelling, which is accompanied by a release of potassium in the extramitochondrial medium and a concomitant complete inhibition of mitochondrial function suggest that starting at a certain ferutinin/mitochondria concentration ratio, the interaction of ferutinin with the mitochondrial membrane might alter the topology of the membrane. This effect could reflect a structural reorganization of the mitochondrial inner membrane induced by the ferutinin-mediated increase in membrane permeability. This is in line with the results of Gao et al [START_REF] Gao | Ferutinin induces in vitro eryptosis/erythroptosis in human erythrocytes through membrane permeabilization and calcium influx[END_REF] showing that at high concentrations ferutinin is able to permeabilize the membrane of red blood cell ghosts in a way similar to digitonin. This mechanism will require further investigation.

Conclusion

In conclusion, this study shows that ferutinin is not a suitable tool to assess mPTP opening in our model of isolated cardiomyocytes. The direct measurement of mPTP opening by means of the calcein/cobalt protocol demonstrates that this is not the predominant factor leading to the depolarization of mitochondria and to cell death at the concentrations of ferutinin.

Ferutinin possesses other mitochondrial properties such as induction of swelling and mitochondrial uncoupling properties. These findings impede its utilization for investigating mPTP opening in isolated cardiomyocytes.

Legends for Figures

  induced a time-dependent increase in mitochondrial Ca 2+ confirming the ionophoretic properties of the drug (Fig 1C).

Figure 1 :

 1 Figure 1: Ferutinin induces mitochondrial Ca 2+ uptake in isolated mouse mitochondria and cardiomyocytes. Ca 2+ uptake was assessed by measuring the change in fluorescence in mitochondria and cardiomyocytes preloaded with Rhod-2. A: Representative experiment showing mitochondrial uptake of increasing concentrations of Ca 2+ in the absence or in the presence of 5 µM ruthenium red (RR). Mitochondria (0.4 mg/mL) were incubated in a respiration buffer and energized with glutamate/malate (G/M). Then increasing concentrations of Ca 2+ (40, 60 and 100 µM) were added in the absence or in the presence of RR. B: Representative experiment showing the effect of ferutinin in deenergized mitochondria. Mitochondria (0.4mg/mL) were incubated in the presence of rotenone. At t=25 s, 60 µM Ca 2+ were added. Then, increasing concentrations of ferutinin (FE) 25, 50 and 75 µM were added in the absence or in the presence of RR. C: Rhod2-loaded cardiomyocytes were incubated in Tyrode's buffer for 4 min and then ferutinin was added. Fluorescence values were obtained by averaging pixel intensities after background substraction in an area containing 25-30 cells and were normalized as a percentage of the initial value. Each trace represents the mean ± SEM of four to seven experiments. The ordinate of the bar graph is the value of Rhod-2 fluorescence observed at 12 min in the figure. p<0.05 versus 0 and 5 µM ferutinin. AU: arbitrary units.

Figure 2 :

 2 Figure 2: Effect of ferutinin on mitochondrial calcein, TMRM and propidium iodide (PI) fluorescence in isolated adult cardiomyocytes. Isolated adult cardiomyocytes were incubated in Tyrode's buffer at 37 °C and co-loaded with calcein, CoCl2 and TMRM (A) or calcein, CoCl2 and PI (B,C). After 5 min incubation ferutinin or vehicle was added to the medium. Fluorescence values were obtained by averaging pixel intensities after background substraction and were normalised as a percentage of the maximal value. Each trace represents the mean ± SEM of four to seven experiments.

Figure 3 :

 3 Figure 3: Effect of CypD deletion and cyclosporine A on ferutinin-induced mitochondrial calcein loss and propidium iodide fluorescence increase in mouse cardiomyocytes. A and B: Time course of calcein (A) and propidium iodide (PI) fluorescence (B) in WT and CypD-KO mouse cardiomyocytes after ferutinin exposure (arrow; 25 µM). Each trace represents the mean ± SEM of six experiments.

Figure 4 :

 4 Figure 4: Ferutinin induces mitochondrial swelling in isolated mouse cardiac mitochondria. A: representative experiments and corresponding bar graphs showing swelling assessed by measuring the absorbance of the mitochondrial suspension at 540 nm. Mitochondria (0.2 mg/mL (red curves) and 0.4 mg/mL (blue curves) were incubated in the respiration buffer including 5 mM glutamate/malate and increasing concentrations of ferutinin (arrow) were added at 40 s. Curves are representative of at least four independent experiments.

Figure 5 :

 5 Figure 5: Effect of ferutinin on mitochondrial oxygen consumption and membrane potential in isolated mouse heart. A: Ferutinin inhibits mitochondrial oxygen consumption in a concentration-dependent manner. State 3 (V3) and state 4 (V4) respiration rates were measured. V4 was evaluated either after exhaustion of ADP (V4) or after addition of 1 µM of carboxyatractyloside (CAT; V4 CAT ) and the corresponding respiratory control ratios (RCR and RCR CAT ) were evaluated. Each value represents the mean ± S.E.M. of at least four independent experiments. p<0.05 versus 0 µM ferutinin value for V3 ( §), V4( * ), V4 CAT ( †), RCR (+) and RCR CAT (#).

Figure 6 :

 6 Figure 6: Ferutinin modulates mitochondrial matrix alkalinization A: Mitochondrial pHi was studied by following BCECF fluorescence. Mitochondria were incubated in the respiration buffer until signal stabilization. Curve a: the addition of glutamate/malate (G/M) at 50 s generated membrane potential, thus matrix alkalinization. Addition of FCCP decreased pHi. Curve b: the addition of 1 µM rotenone from the beginning of the experiment inhibited the effects of G/M and FCCP. B: Effect of increasing concentrations of ferutinin on pHi. Experimental conditions were similar to A and FCCP (1 µM) or ferutinin (6.25, 12.5, 25 and 50 µM) were added at 250 s. C: Effect of increasing concentrations of ferutinin on pHi in deenergized conditions. Mitochondria were incubated in the respiration buffer in the presence of rotenone and antimycin. After 40 s increasing concentrations of ferutinin (12.5, 25 and 50 µM) were added. 0: no ferutinin addition. D: comparison of the effect of 50 µM ferutinin on pHi in the respiration (RB) and the sucrose-Hepes (SHR) buffers supplemented or not with NaCl 100mM, KCl 100mM or cyclosporine A (CsA) 1 µM.

Figure 7 :

 7 Figure 7: Effect of ferutinin on mitochondrial potassium flux. Mitochondrial potassium was studied by following PBFI fluorescence.

Figure S1 .

 S1 Figure S1.Ferutinin induces mitochondrial swelling is not inhibited by EGTA and ruthenium red in isolated mouse cardiac mitochondria. Swelling was assessed by measuring the absorbance of the mitochondrial suspension at 540 nm. Mitochondria (0.2 mg/mL) were incubated in the respiration buffer in the presence of rotenone (1 µM) and antimycin (1 µM). Ferutinin (50 µM) was added at 40 s. When used, EGTA (100 µM) and ruthenium red (RR, 5 µM) were introduced at the beginning of the experiment. Control: no addition of ferutinin. Curves are representative of at least four independent experiments.

Acknowledgements

Juliette Bréhat and Mathieu Panel were supported by doctoral grants (grants number 2018-057 and 2014-140, respectively) from the Ministère de l'enseignement Supérieur, de la Recherche et de l'Innovation.

Declaration of competing interest

The authors declare no conflict of interest.


Roberta Garcia Mello De Araújo

Marco Aurélio De Carvalho

Comparative Analysis of Methods for Identifying Opportunities for Reusing Solid Waste

Keywords: Systematic Method for Identifying Reuse Opportunities of Supporting Goods (SMIROSG), Product DNA, Waste Reuse, Waste Recycling, Design Research Methodology (DRM)

Waste produces a significant impact on the environment. According to the hierarchy of waste management, reuse should be preferred to material recovery and recycling, since less energy tends to be used. In order to prioritize waste reuse, the question of how to do it becomes important. There is no standard method aimed at finding reuse opportunities, and most of the published examples of reuse were identified through intuitive techniques, such as brainstorming, or even by chance. We have found two main systematic techniques: Product DNA and SMIROSG (Systematic Method for Identifying Reuse Opportunities of Supporting Goods). In this paper, we report a test involving both methods, focusing on an ordinary solid wastethe styrofoam tray. The results were analyzed, and improvements to the techniques were suggested. The main research methodology used to structure this study was the DRM (Design Research Methodology). The specific methods, Product DNA and SMIROSG were also used. We have found out that the SMIROSG method offers a greater scope of possibilities for reuse, although it is necessary to screen these possibilities, while Product DNA provides a more succinct range of opportunities for reuse, sometimes lacking more possibilities, however, all the alternatives offered are readily applicable, thus not requiring a new screening. Therefore, these are the aspects of each method in which improvements have been suggested. Given this, the requirements for a new method containing the best aspects of both SMIROSG and Product DNA were defined, aiming to eliminate the deficiencies presented in each method, improve the performance of the future method and enable an exponential increase of the effectiveness in identifying reuse opportunities.

Introduction

Every day, tons of garbage are produced, resulting in a growing environmental impact that harms the planet and that is a threat to life [START_REF] Hansen | EU waste policy and challenges for regional and local authorities[END_REF]. In this context, several policies and strategies were created for waste management, aiming to reduce the environmental impact caused by garbage and even reverse the damage already done. Among these strategies, we have the concept of the three R's [START_REF] Hansen | EU waste policy and challenges for regional and local authorities[END_REF], which consists of reducing, recycling, and reusing. Reuse is preferable to the other alternatives, due to its lower energy expenditure [START_REF] Saeed | Transitions and institutional change: The case of the Dutch waste subsystem[END_REF].

Although the issue of reuse is very relevant, when researching methods to identify opportunities for waste reuse, the literature presents only one specific systematic alternative, the Systematic Method for Identifying Reuse Opportunities of Supporting Goods (SMIROSG) [START_REF] Frazão | Systematically finding opportunities for product reuse the case of PET bottles[END_REF]. Another systematic method that can be easily applied for this purpose is Product DNA [START_REF] Dewulf | Directed variation of properties for new or improved function product DNA -A base for connect and develop[END_REF]. However, its steps are highly dependent on the researcher's experience, which makes Product DNA slightly less systematic than the SMIROSG method. There are also articles reporting the use of non-specific intuitive processes, such as brainstorming [START_REF] Frazão | Systematically finding opportunities for product reuse the case of PET bottles[END_REF] and serendipity [START_REF] Frazão | Systematically finding opportunities for product reuse the case of PET bottles[END_REF]. Other approaches that can support the identification of reuse opportunities are the Life Cycle Assessment [START_REF] Khasreen | Life cycle assessment and the environmental impact of buildings: a review[END_REF][START_REF] Yao | An integrated method of lifecycle assessment and system dynamics for waste mobile phone management and recycling in China[END_REF][START_REF] Zink | Comparative life cycle assessment of smartphone reuse: repurposing vs. refurbishment[END_REF][START_REF] Pini | Preparation for reuse activity of waste electrical and electronic equipment: Environmental performance, cost externality and job creation[END_REF], Industrial Symbiosis [START_REF] Frazão | Systematically finding opportunities for product reuse the case of PET bottles[END_REF][START_REF] Patricio | Enabling industrial symbiosis collaborations between SMEs from a regional perspective[END_REF][START_REF] Marconi | An approach to favor industrial symbiosis: the case of waste electrical and electronic equipment[END_REF][START_REF] Leigh | Industrial ecology, industrial symbiosis and supply chain environmental sustainability: a case study of a large UK distributor[END_REF], Reverse Logistics [START_REF] Kumar | Cradle to cradle: Reverse logistics strategies and opportunities across three industry sectors[END_REF][START_REF] Sellitto | Reverse logistics activities in three companies of the process industry[END_REF], and programming methods and decision models [START_REF] Bereketli | WEEE treatment strategies' evaluation using fuzzy LINMAP method[END_REF][START_REF] Ahmed | A comparative decision-making model for sustainable end-of-life vehicle management alternative selection using AHP and extent analysis method on fuzzy AHP[END_REF][START_REF] Mangun | Incorporating component reuse, remanufacture, and recycle into product portfolio design[END_REF].

Considering that SMIROSG and Product DNA are the only methods that have a systematic approach and include a practical tool easy to be applied by product development teams, we decided to investigate these methods in greater depth. Two papers present practical applications of SMIROSG: that of Verhaegen et al. [START_REF] Verhaegen | Systematically identifying reuse applications for supporting goods[END_REF] and that of Frazão et al. [START_REF] Frazão | Systematically finding opportunities for product reuse the case of PET bottles[END_REF]. The practical application of Product DNA is demonstrated in the article by Dewulf [START_REF] Dewulf | Directed variation of properties for new or improved function product DNA -A base for connect and develop[END_REF]. From the applications, it appears that both methods have great potential for reuse. The next questions are: how effective is each one? Are there possibilities for complementarity between the methods? Are there possibilities for improving these methods? Considering these research questions, a comparative analysis is necessary. To this end, it was decided to focus on a very common type of solid waste: the Styrofoam tray (Figure 1). This type of tray has many uses, but it is most often employed to store cold cuts and meats. The final destination of this waste is problematic since Styrofoam is known to be difficult to recycle [START_REF] Chandra | Real CostofStyrofoam.The Green DiningAlliance[END_REF].

.

Methodology

In this work, we use the Design Research Methodology (DRM) as the basic research structure. In addition to DRM, the methods to be compared (SMIROSG and Product DNA) are also applied.

Design Research Methodology (DRM)

DRM is defined as an approach and a set of methods and support guidelines that are used as a framework for doing research in the area of design [START_REF] Blessing | [END_REF]. DRM consists of four stages: Research Clarification (RC), Descriptive Study I (DS I), Prescriptive Study (PS), and Descriptive Study II (DS II). Figure 2 synthesizes the structure of DRM, with the methods and results obtained for each stage. It is not mandatory for a specific research project to include each stage, or to undertake all stages in equal depth. In some cases, literature alone will provide enough material for a particular stage. In other situations, a research project may focus only on one stage for an in-depth study, due to time constraints or because the project is part of a larger program. Figure 3 illustrates the seven existing study alternatives for applying DRM. Research and hypothesis questions, as well as the time and resources available, will determine the type of research to be undertaken. This paper describes a type 2 study since the methods analyzed are applied practically and their results are used for comparison and analysis between the results generated by each method. Thus, proposal number 2 was used, as shown in Figure 3. Hence, it is proposed to use the first three stages of DRM, with the conclusion of the study in the Prescriptive Study stage.

Therefore, in the Research Clarification stage, an extensive literature review was carried out, aiming at finding methods previously used to identify opportunities for reusing solid waste, with the purpose of delineating the research objectives and the desired results to be obtained. Besides, criteria are obtained that will be used as measures to evaluate the result of the study. The RC also has the function of providing a focus for the DS-I and drawing up a study plan to be carried out in the second phase of the research.

In the DS-I stage, the practical application of the Product DNA and SMIROSG methods was carried out in the case of the Styrofoam tray to obtain the necessary information for a better understanding of the existing situation and also to allow the performance of the comparison between the methods. With this, a basis is provided for the next stage, making it possible to start developing a support that addresses the factors that have the greatest influence on the success of the situation studied.

On the PS stage, once we obtained a greater understanding of the original situation, the description of the desired situation was corrected and elaborated, thus initiating the systematic development of the proposal to solve the problem. Therefore, the main contributions to be obtained in the PS stage are: a description of the points at which each method can be improved, aiming to enhance the identification of opportunities for reuse when using Product DNA or SMIROSG; and a description of the requirements for a new method for identifying reuse opportunities, which is derived from both Product DNA and SMIROSG but does not include the flaws presented in each of these methods and, consequently, has the best characteristics of each one, which will result in the most ideal method for identifying reuse opportunities. Figure 4 illustrates the DRM methodology used in the context of this paper.

Product DNA

Product DNA consists of a systematic innovation method that promotes a simple way of describing a product, and its main focus is to connect previously unrelated domains, with the aim of transferring existing knowledge to a new area or domain, thus aiming to provide innovative solutions. These connections that are created are based on properties and functions, and by listing such characteristics of a system, it is possible to distinguish one object from another. Through Product DNA, it is possible to categorize the products by their properties and functions [START_REF] Dewulf | Directed variation of properties for new or improved function product DNA -A base for connect and develop[END_REF]. A property is an attribute, a dimension, a trait by which objects or individuals can be distinguished. And the analysis of properties is the first part of the Product DNA work chain. In the second step, there is an analysis of the function, purpose, intention, or use of the material.

In its initial conception, Product DNA is a method in which each property of the material is analyzed and this property is modified, thus gaining a new or improved function for the material. Or, knowing the desired function, one must analyze which properties must be modified. This process allows identifying products that are related and technological domains that act as inspiration for the innovation of a product or system. Therefore, through the properties and functions of an existing system, new markets can be identified that require those properties and functions.

However, it was identified that the process present in the Product DNA for analyzing the properties and functions of a given material, with subsequent alteration of the properties in order to exercise new functions, would be of great use in identifying opportunities for reuse of the same material. This occurs because, once finding new functions for a product, it is possible to reuse it in innovative and creative ways. Therefore, despite not having been created for this purpose, Product DNA can help a lot in the process of identifying opportunities for reusing products.

The first step of the Product DNA method is to characterize the product along the lines of Dewulf's article. This characterization consists of the description of a material or object in relation to spectra of properties. The standard spectra considered are porosity, surface, flexibility, color, components, state, unity, transparency, information, automation, and senses. Once all the characterization of a given product is obtained, it is also possible to fit it into domains or compare it with other products.

Once such characterization is carried out according to the material's properties, its functions are then analyzed, taking into account the regular use of the material. Considering the current product functions and how they relate to each property, in the stage of identifying opportunities for reuse the procedure used is to analyze each property of the material and how that property can be modified or adapted to gain a new or improved function. Or it is also possible to proceed as follows: knowing the desired function, analyze which properties must be changed so that the product fits the specific function. In this way, new possible functions are found for the product, consisting of possibilities in which the material can be reused.

SMIROSG (Systematic Method for Identifying Reuse Opportunities of Supporting Goods)

The SMIROSG method aims at finding opportunities for reusing products [START_REF] Frazão | Systematically finding opportunities for product reuse the case of PET bottles[END_REF]. It was designed to focus on supporting goods, which are products created to contribute to the manufacture of other products [START_REF] Verhaegen | Systematically identifying reuse applications for supporting goods[END_REF], such as packaging. However, with the necessary adaptations, it can be used for any product.

When applying the SMIROSG method, the first step is to describe the product to be analyzed through a set of properties or qualitative attributes, obtaining a list of about 10 to 12 attributes in total. The next step to the definition of attributes is the formation of pairs of attributes to be searched in an online image search tool since the search for only one of the terms does not provide appropriate results. As the results found in the surveys subsequently pass through an analysis and screening process, in this step all the results obtained are considered.

The third step of the method is to register each product category that appears as result, keeping it related to the pair of attributes whose search resulted in the specific product. It is important, due to the large number of research pairs and product categories obtained for each pair, to keep the recorded information organized in an easily quantifiable way, as in an Excel spreadsheet.

Then, there is the analysis of the data obtained, in which the products are classified according to the number of times that each one appeared as a result of the researches, and then this information is recorded in a new table and a graph, for easier observation of the results. Products that appear more frequently can be expected to be the best paths for innovation in the area of reuse of the waste in question, but it is also possible that the most frequent products are those that have already been developed through the reuse of that specific waste [START_REF] Frazão | Systematically finding opportunities for product reuse the case of PET bottles[END_REF].

Comparison of Methods

After applying both methods and obtaining the respective results, an analysis of both results is made and a comparison between them is performed, thus following the study plan based on DRM, described in summary form in Figure 4. It is observed whether there were results that were obtained through one method, but they were not through the other, and vice versa. If this occurs, these results are then analyzed to find an explanation for the discrepancy noted. Besides, it is also analyzed whether there was any possibility of reuse that, although possible and applicable, was not found as a result of any of the methods used. With this, it is possible to draw a draft of the possible improvements to be applied in each method, in addition to the requirements for a future new method focused on identifying opportunities for reusing solid waste.

It is noteworthy that the research implemented in this article, having presented as its main objective the comparison between the Product DNA and the SMIROSG methods, as well as identifying which method is reported to be more effective, did not delimit a specific number of reuse opportunities intended to be found as one of its objectives. On the contrary, we tried to find the highest number of reuse opportunities in each method, thus exploring their maximum potential.

Since this article is focused on establishing a comparison between two possible methods to identify solid waste reuse opportunities, the main criterion used to identify which method would be the most appropriate for this application was precisely to establish a qualitative comparison between both methods, evaluating parameters such as the practicality of application and the number of results obtained. However, when applying one of those methods to identify opportunities for reuse of a given solid waste, it will be necessary to deal with the range of results obtained, and choose the most viable among them. Therefore, it is necessary to have a set of criteria that will help determine the most viable reuse opportunities. Concerning possible criteria to establish the most viable opportunities for reuse among those obtained as a result of the application of a certain method, it is possible to make use of some key criteria widely used on creative design projects, such as creativity, innovation, and quality of the obtained results.

Results

Applying the Product DNA Method to Identify Opportunities for Reusing Styrofoam Trays

For the first step of the Product DNA method, the EPS tray was described according to the following categories: porosity, surface, flexibility, color, components, state, unity, transparency, information, automation, and senses. The characteristics obtained are shown in the table below. It is important to note that it was considered that the categories of "Information", "Automation" and "Senses" did not apply to the context of the Styrofoam tray, therefore they were disregarded for the application of the method. Besides, it seemed pertinent to highlight some other properties of the material that relate to its functions, so the following characteristics were also taken into account during the application of Product DNA:

• Light;

• Low density;

• Low thermal conductivity;

• Inert; • Resistant to aging;

• Non-hygroscopic, insensitive to humidity;

• High mechanical resistance;

• Good shock absorber. Then, considering the regular use of Styrofoam trays, which is storing sliced cold cuts in markets, the functions of this material were then analyzed. The result of this step is shown in the table below. Describing the relationship between the functions and properties of the Styrofoam tray, we have that, since the tray has high mechanical resistance and is an effective shock absorber, it has the function of protecting against impacts, preventing any damage of the food contained in it. Regarding the property of low thermal conductivity presented in the EPS tray, this fact explains its function of acting as a thermal insulator, thus preventing the food contained within it from losing its ideal temperature too quickly. As a non-hygroscopic material, the Styrofoam packaging also has the function of protecting food from moisture. The typical lightness of the Styrofoam contributes to facilitating the transport of the products and their grouping and stacking. Also, the anatomy of the tray itself, flat with curved edges, has the function of storing food inside.

For the stage of identifying reuse opportunities, the surface property of the Styrofoam trays was first analyzed. The trays are flat with curved edges, however, changing this characteristic so that the material acquires a spherically shaped surface, it can be applied to the production of helmets. The material would then assume the main function of protecting against mechanical shocks, in addition to preventing mold and the proliferation of microorganisms, promoting thermal insulation, and increasing comfort due to the lightness of the material.

Still thinking about its surface, if it is altered so that the material presents a flat surface, thus removing the curved edges, it can then assume the function of decoration, the function of preventing mold and moisture in buildings, or even the function of enhancing thermal and acoustic insulation of buildings.

In the decorative function, it is possible to texturize walls with brick-shaped rectangles cut out of the trays, which can be fixed with an adhering material to the wall. In addition, this type of decoration with Styrofoam materials can also be painted, since EPS is compatible with several types of paint.

For the function of protecting buildings from mold and moisture, the material can be applied, in the form of flat plates, directly on the wall with contact glue, forming a protective layer. This layer is then coated with spackle or paint, making the styrofoam invisible. In this way, it is possible to prevent the proliferation of fungi and bacteria, eliminate mold and moisture and also promote greater thermal insulation.

Thinking of the function of enhancing the thermal and acoustic insulation of environments, it is possible to use layers of compacted EPS plates inside the walls of buildings, which also reduces the weight of the structure and the use of cement and wood, without impairing the resistance of the edification. Besides, there is also the application of compacted styrofoam sheets in the composition of thermoacoustic tiles, or "sandwich tiles", in which a metal tile is filled with Styrofoam inside, which reduces the structural weight and promotes thermal and acoustic insulation. Now analyzing the unit item, the Styrofoam trays have the plate itself as a unit. However, if this property is changed and the unit cell becomes small Styrofoam flakes, after processing the tray, such flakes can be applied with the function of constituting the concrete, thus replacing the crushed stone. The function of EPS in the so-called light concrete is precisely to reduce the apparent density of the concrete, promoting lightness, thermal insulation, and increasing the strength of the material. Besides, styrofoam also has the function of facilitating the handling of the concrete itself.

Still, in the context of Styrofoam flakes as a unit cell, the product can also be adapted to the role of plant substrate component. When used in this way, Styrofoam flakes mixed with soil have the function of contributing to the aeration of roots, making the soils less compacted, in addition to controlling erosion, increasing water retention by plants, and reducing the spread of microorganisms and insects.

Applying the SMIROSG method to identify reuse opportunities for Styrofoam trays

In the first step of the method, when describing the product to be analyzed through a set of properties or qualitative attributes, a total of 12 attributes were assigned to the Styrofoam tray. These attributes are shown in the table below. The next step to define the attributes is the formation of pairs of attributes to be searched in an online image search tool. The image search tool used was Google Image Search [START_REF]Google Image Search[END_REF], and a total of 51 combinations of attributes were made to be searched in pairs. For each search, the images provided in the results were annotated in an Excel spreadsheet, and after searching all 51 attribute pairs, an analysis was made of the frequency that each type of result appeared.

Table3

The following image illustrates the search process for an attribute pair. In this example, the attributes "Porous" and "Thermal insulation" were searched together. It is possible to observe that some of the results provided were: light concrete, styrofoam sheets, bricks, plumbing pipes, among others. Thus, from the 51 pairs of attributes surveyed, a total of 158 results was obtained. These results were transferred to a table and used to create a graph, as shown below. The table below presents a list of the most frequent image results throughout the surveys, separated into categories, and the number of times each item was noticed.

Table4.Image search results were separated into categories and frequencies in which they appeared.

Result Frequency

Thermal/Acoustic insulation panels 18

Shoes, sneakers, boots, and sandals 12 According to the results obtained through the application of the SMIROSG method, it was observed that the greatest possibility of reuse for Styrofoam trays is probably within the area of panels and plates for thermal and/ or acoustic insulation; in the construction sector with bricks, tiles and materials such as lightweight concrete; in the area of shoes; and the helmets sector. However, there is also the possibility of reuse within the other areas.

When comparing the results obtained using the SMIROSG method with the results obtained using the Product DNA method, it is possible to observe some similarities. Items such as insulation panels, bricks, tiles, helmets, walls, building blocks, concrete, and paving were obtained in both research methods. However, there are also some discrepancies between them, and the following table compares the performance of each method concerning some questions. As we can see, the SMIROSG method had a greater variety of results, presenting more opportunities for reuse at the end of its application. However, among the 19 results obtained, after analyzing these, only 10 were considered to have a realizable and therefore more credible application. On the other hand, the Product DNA method offered 7 opportunities for reuse as a result, but all of these are relevant and possible to be applied. Therefore, it is possible to conclude that, despite the wide range of results obtained through SMIROSG, they are very varied results that require further analysis and filtering, while Product DNA, despite having a reduced scope of results, does not require further filtration, delivering the results ready for use. Likewise, a reasonable amount of results obtained through SMIROSG was not obtained through Product DNA, however, most of these were not among the most relevant reuse opportunities. The result obtained by the Product DNA that SMIROSG failed to identify is the use of Styrofoam flakes as a component of plant substrate, which is a very relevant and important possibility to be included among the possibilities of reuse obtained in research.

Therefore, it is possible to infer that the use of Product DNA results in a more filtered range of possibilities, with all its results worthy of attention and consideration, while SMIROSG delivers a wide range of results, which still need to be filtered and passed through a selection of the best among them. However, SMIROSG also presents a method that has well-defined stages and allows easy application, which makes obtaining the results a more accessible process.

Regarding the analysis of the results as a whole, it is noteworthy that both methods have failed to provide handicrafts as a result, which is an area in which the possibilities for reusing Styrofoam trays are quite wide. In fact, an area in which the reuse of Styrofoam is widely applied is the samba school sector, since the cars used in parades in Brazil are mostly built from Styrofoam, and often trays and other Styrofoam packaging are reused on these parade cars. Some items that appeared among the results and deserve attention were the bricks and tiles "stuffed" with styrofoam, the light concrete, and the use for agriculture. These items in particular are worthy of attention because they are useful and easy to apply reuse possibilities, in addition to using Styrofoam in creative and innovative situations.

Requirements for the New Method

When analyzing the results from the application of the Product DNA method and those from the SMIROSG method, it is possible to outline a set of requirements for a new method whose focus would be the identification of opportunities for reusing solid waste. Therefore, the future method must incorporate the most effective and efficient aspects of both Product DNA and SMIROSG. It is also necessary that this method is constituted by a systematic approach, of easy application, reproduction, and documentation, whose implementation will help to organize creativity in order to identify innovative opportunities for reuse. Furthermore, the method should provide a large scope of results, while not requiring an extensive accumulation of data in the steps preceding its application. Besides, the future method must present a step-by-step process, thus being less dependent on the user's experience, that is simple to understand and teach, and that can be applied to identify opportunities for reusing any solid waste, without being focused only on "supporting goods" or in another specific solid waste category.

Conclusion

Product DNA, although wasn't developed with the intention of identifying reuse opportunities, presented an excellent performance in this function, with significantly positive results. However, due to the fact that the SMIROSG method has a more objective methodology and consists of better-specified steps, which do not depend on the researcher's experience, obtaining and analyzing the results is facilitated. In addition, the inclusion of the image search tool is a great way to use technology to our advantage, in an innovative and creative way. And the great asset of the SMIROSG method is precisely the search for images, which results in a greater variety of results and, consequently, delivers greater possibilities of reuse for the analyzed waste.

Therefore, the method that provided the greatest number of opportunities for reuse was SMIROSG. However, it is worth mentioning that the results obtained through the Product DNA corresponded to the results of the SMIROSG with the greatest potential for application in the reuse area, and that, despite having delivered fewer alternatives, the Product DNA was able to identify an opportunity for reuse in the agriculture area, an opportunity that SMIROSG failed to identify. Therefore, aiming at enhancing the identification of reuse opportunities, it may be more advantageous to make use of both methods concomitantly, which will make it possible to use what each has of best and help to cover all existing possibilities.

Regarding the possibilities of reuse that both methods failed to identify, there is the possibility that a method that relies on the interconnection of Product DNA with SMIROSG will resolve this issue. Also, it would be interesting to review the methodology of the methods to identify the reason for this failure, which would allow to implement some improvements to the methods and make them even more efficient.

Fig. 1 .

 1 Fig.1.Typical Styrofoam tray.

Fig. 2 .

 2 Fig.2.DRM structure, with the representation of the stages, basic means, and results.

Fig. 3 .

 3 Fig.3.Types of projects using DRM and their respective main focci.

Fig. 4 .

 4 Fig.4.Use of DRM in the context of the current study.

Fig. 5 .

 5 Fig.5.Search results for a pair of attributes.

Table 1 .

 1 Characterization of the Styrofoam tray according to the property categories presented by Dewulf.

	Category	Material Property
	Porosity	Porous
	Surface	Flat, with curved edges
	Flexibility	Rigid

Table 2 .

 2 Styrofoam tray functions definition.

	EPS tray functions
	Promote protection against impacts, shocks, and mechanical wear
	Thermal insulation
	Protection against moisture
	Storage
	Facilitate transportation
	Facilitate grouping

  .Styrofoam tray attribute set.

	EPS tray attributes
	Porous
	Lightweight
	Rigid
	Opaque
	Impact Resistant
	Wear-resistant
	Massive
	Thermal Insulation
	Aging Resistant
	Moisture Resistant
	Acoustic Insulation
	Chemical Resistant

Table 5 .

 5 Comparison between Product DNA and SMIROSG methods.

		The total amount	Number of most	Number of results
	Method	of different	relevant results	obtained exclusively in
		results obtained	obtained	the respective method
	Product DNA	7	7	1
	SMIROSG	19	10	10


Guillaume Guarino
email: guillaume.guarino@insa-strasbourg.fr

Ahmed Samet

Denis Cavallucci

Patent specialization for deep learning information retrieval algorithms

Keywords: Deep Learning, TRIZ, Patent, Contradictions, Summarization

Extracting information from patents using machine learning algorithms in the context of TRIZ still faces a major problem: the very limited amount of annotated data. Therefore, most approaches, whether for parameter, problem or solution extraction, are based on unsupervised learning algorithms such as Latent Dirichlet Analysis (LDA), or very small supervised learning algorithms such as Support Vector Machine (SVM) or Multi-Layer Perceptron (MLP), which are relatively inefficient. The use of Deep Learning is still relatively uncommon in the extraction of knowledge from patents despite the significant capabilities of these algorithms, particularly BERT. The objective is therefore to present a method for specializing Deep Learning's supervised algorithms on patents while using a very low volume of annotated data. The effectiveness of the method will be analyzed in a task of extracting contradictions from patents, which is a complex task that cannot be performed using unsupervised techniques.

Introduction

Patents contain a great deal of human knowledge, yet they are not widely used because of their large number and their legal nature which makes them difficult to read. Engineers are able to extract useful information from a patent but do not have the capacity to study thousands of patents simultaneously. This is why the use of machine learning algorithms seems to be a valuable option to quickly analyze large masses of data. However, supervised learning of neural networks requires a large dataset. Nevertheless, the analysis of patents for the elaboration of datasets is very time-consuming, which limits the number of annotated data. It therefore seems necessary to develop a method that would allow a high performance with a limited volume of starting data. Transfer learning consists in training a model on a given dataset, usually of large size, and then specializing it on another nearby task with very little data. In the case of automatic language processing, the main issue is the quality of generated representations from words, sentences or even the document as a whole. These representations take into account the context and the links between words and sentences. This analysis of the links between words and sentences is common to all NLP algorithms, which explains why these representations can be trained on data that are slightly different from those related to the target problem.

The extraction of contradictions from patents is a major research axis in the perspective of automating the search for a solution or at least assisting in the resolution of problems. Actually, being able to understand a problem is the first step in understanding the resolution process. In this article we will consider contradictions at the sentence level. This means that we will look for the sentences that contain the parameters of the contradictions without extracting these parameters. In fact, it is relatively easy to extract the parameters from a sentence. What is more challenging is to locate the contradictory sentences carrying these parameters. This selection of sentences will be considered as a summarization task in the rest of this paper. Labeling patents to extract contradictions is tedious and the dataset we have developed only contains a few hundred patents. The extraction of contradictions seems to offer a good ground for experimentation of transfer learning.

The main contributions of this paper are therefore:

-a transfer learning method to improve extractive summarization from abstract summarization learning.

-an application of transfer learning to contradiction extraction. The paper consists of the following sections. Section 2 presents a brief state of art about information retrieval, contradictions and parameters extraction from patent documents. In Section 3 we introduce our method and in Section 4 we detail the model's architecture. We then show some extraction results in Section 5. We finally conclude our work and show perspectives for future works.

2

Related work

TRIZ-based automatic analyses

The automatic extraction of contradictions from technical documents, and in particular from patents, is a known and central problem since it would allow the reconstruction of a new updated matrix. However, no prior art approach has succeeded in addressing this problem. Automatic content analysis with the prism of TRIZ is a common theme in the community whether it is through the classification of inventive principles [START_REF] Loh | Automatic classification of patentdocuments for TRIZ users[END_REF][START_REF] He | Grouping of TRIZ Inventive Principles tofacilitate automatic patent classification[END_REF][START_REF] Liang | Computer-aided classification of patents oriented to TRIZ[END_REF], the extraction of parameters [START_REF] Chang | Computerizedinnovation inspired by existing patents[END_REF][START_REF] Cascini | Computer-aided analysis of patentsand search for TRIZ contradictions[END_REF], the reconstruction of TRIZ matrices for targeted domains [START_REF] Berdyugina | Setting Up Context-SensitiveReal-Time Contradiction Matrix of a Given Field Using UnstructuredTexts of Patent Contents and Natural Language Processing[END_REF].

However, these methods often use simplifications such as the reduction of the number of inventive principles [START_REF] Loh | Automatic classification of patentdocuments for TRIZ users[END_REF][START_REF] He | Grouping of TRIZ Inventive Principles tofacilitate automatic patent classification[END_REF], the use of keywords or key phrases [START_REF] Liang | Computer-aided classification of patents oriented to TRIZ[END_REF][START_REF] Berdyugina | Setting Up Context-SensitiveReal-Time Contradiction Matrix of a Given Field Using UnstructuredTexts of Patent Contents and Natural Language Processing[END_REF] or assumptions on the structure of patents [START_REF] Cascini | Computer-aided analysis of patentsand search for TRIZ contradictions[END_REF] which makes these approaches unusable in practice on "new" contents.

Automatic summarization

Extractive summarization generally consists in a binary classification of sentences from a source document. A positive label is assigned to a sentence if it contains important information in the context of the targeted summary. A computation of sentences' representations is therefore necessary. These representations should contain enough context information to be sufficient for the decision mechanism (classifier) to assign a label to the sentence. Numerous automatic summarization approaches exist such as graph-based methods [START_REF] Page | The PageRank Citation Ranking: Bringing Order to theWeb[END_REF][START_REF] Kleinberg | Authoritative Sources in a Hyperlinked Environment[END_REF][START_REF] Mihalcea | Graph-based Ranking Algorithms for Sentence Extrac-tion Applied to Text Summarization[END_REF][START_REF] Litvak | Graph-based keyword extraction for single-document summarization[END_REF], Bayesian approaches [START_REF] Kupiec | A trainable document summarizer[END_REF][START_REF] Aone | A Scalable Summarization System Using RobustNLP[END_REF], with Markov models [START_REF] Conroy | Text summarization via hidden Markovmodels[END_REF] or CRF (Conditional Random Field). Neural networks have become popular since the development of recurrent models like LSTMs (Long Short-Term Memory) or GRU (Gated Recurrent Unit) because of their ability to model the context and dependencies between words and sentences [START_REF] Nallapati | SummaRuNNer: A Re-current Neural Network Based Sequence Model for Extractive Summariza-tion of Documents[END_REF][START_REF] Zhou | Neural Document Summarization by Jointly Learningto Score and Select Sentences[END_REF].

The Transformers [START_REF] Vaswani | Attention is All you Need[END_REF] allow to build richer representations than the recurrent networks because they are, by design, bidirectional. Nevertheless, the number of parameters is proportional to the squared input length which means that they can be used only for short texts. The main reason of their massive use is the ability to be pre-trained (BERT [START_REF] Devlin | BERT: Pre-training of Deep Bidirectional Transform-ers for Language Understanding[END_REF]), XLNet [START_REF] Yang | XLNet: Generalized Autoregressive Pretraining forLanguage Understanding[END_REF]). They will, therefore, be better for complex tasks without requiring large amounts of labelled data. These bidirectional encoders can be trained on all kinds of documents such as Wikipedia pages, articles or journals. These models learn word representations on different datasets before being applied to a specific domain. The huge amounts of data used for pre-training make these models very powerful [START_REF] Liu | Text Summarization with Pretrained En-coders[END_REF].

When abstractive becomes extractive summarization

Abstract summarization consists in generating a word-by-word summary from a starting document. These words may or may not be part of the original text. Extractive summarization consists in selecting key sentences in a source document. A key sentence is a sentence that contains important information about the purpose of the summary. In the case of contradictions, the goal will be to select the sentences containing the parameters. In the case of a generative summary, the goal will also be that the generated sentences contain the parameters of the contradiction.

The main difference lies in the amount of data needed and available for training. The extractive summarization is more easily trainable since it is in fact only a matter of scoring each sentence with its probability of belonging to the summary. However, it is very difficult, if not impossible, to find data that can be used to learn extractive summarization. On the contrary, public data such as patents or press or scientific articles can be easily exploited for abstracting. Indeed, very often, abstracts are available. These abstracts contain the key elements of the documents without being exact copies, word for word, of sentences from the documents. A model can therefore be learned to generate the abstract or summary of an article from it. Nevertheless, the abstract summary will encounter the problem of language. Indeed, even if the data are very abundant (in millions for accessible patents, in hundreds of thousands for press articles or scientific papers) the generation of realistic sentences remains the main problem. Abstract models can lack consistency and generate misunderstandings even if the keywords are actually present in the abstracts.

The goal of the method is to make the most of both extractive and abstractive techniques. The objective is to develop an extractive model to keep the exact statement of parameters or parameter descriptions in patents using a model that will have already been specialized on patent analysis via abstractive summary learning. This method will overcome the limited size of the extractive summary dataset.

A first abstractive model is trained to generate patent abstracts from their descriptions. This model consists of an encoder and a decoder. The encoder is in charge of extracting the salient information while the decoder exploits this information to generate the abstract. The encoder will therefore be able to build rich representations of the tokens of the input document. These representations will also be adapted to information selection which is exactly the goal of the final model. The encoder of this abstractive model will then be taken and integrated in an extractive model that will learn to select the sentences containing the parameters of the contradiction.

Architecture

The abstractive summarization model is presented in Fig. 1. The encoding is provided by BERT [START_REF] Devlin | BERT: Pre-training of Deep Bidirectional Transform-ers for Language Understanding[END_REF] which will generate a vector representation for each input token. BERT is a Transformer based on attention mechanisms to model bidirectional dependencies between tokens. A token will be influenced by the set of tokens that precedes it and will influence the set of tokens that follows it. This encoder has allowed results' improvements on various benchmarks in Natural Language Processing such as similarity, Question Answering or automatic summarization which will interest us here. The attention mechanisms allow for a precise selection of information in relation to the information sought and the context, and thus the representations generated are richer and allow for greater extraction precision. A decoder, also transformed, will then exploit the extracted information to generate a summary, in our case, the patent abstract. To generate the abstract, each output of the decoder will point to a word of the dictionary, which allows to reconstruct a whole text. At the beginning of the learning process, this text will be made of random words, and the more the algorithm learns, the more the choice of words and the construction of sentences will be refined. This model cannot be used directly for extractive summarization (selection of sentences containing the parameters of the contradiction). The first adaptation is the addition of special tokens for classification ([CLS]) at the beginning of each sentence. The representations associated with these tokens will be the representations of the sentences, and it will be these representations that will be classified as the first or second part of the contradiction (depending on whether they contain the improved or degraded evaluation parameter). The goal is therefore to adapt the abstract summary model so that it can contain almost the entire extractive model and thus be able to learn the extractive model on the abstract data. This adapted model is presented in Fig. 2. The representations of the tokens composing the sentences are not used anymore. The representations of the sentences then pass through additional layers of transformations to perfect the encoding before being decoded. This integration of the extractive model will definitely degrade the abstract summary since all the information is forced to be contained in the sentences' representations which are much less numerous. However, these representations will be richer and will allow an easier classification of the contradictions.

US10286848-20190514

Vehicles have been provided with various approaches to providing privacy, security, or additional storage in the vehicle. While some clipless privacy, security, or storage implements have been developed, these clipless implements can also be difficult to install or remove.

Fig. 2. Adapted model for extractive summarization

US09009869-20150421

When undertaking an activity causing sweating, a person can suffer from the effects of sweat dripping into his eyes. These other types of sweat headbands, to remain effective, must be remain in tight apposition to the forehead, which may require an uncomfortably tight fit of the headband around the wearer's head.

US09939370-20180410

Thus, distorted band shapes result not only from the interaction between reflectance and absorbance but from anomalous dispersion of the refractive index relating to other physical parameters, such as the resonant and non-resonant signal components at play in car there have been ongoing attempts to compensate for distorted band shapes as arising from the superposition of reflective components onto the absorbance features of infrared spectra. This process also accounted for multiple interference spectra; however, the definition of such interference spectra added subjectivity to the process.

US07383707-20080610

Therefore, although the locking apparatus of the prior art achieves at least an advantage of capable of simply locking and unlocking the glove box, the operating space of putting the finger between the operating handle and the glove door needs to partition in view of a relationship of pivoting the operating handle from the lower side to the upper side and therefore, the glove door is integrally formed with the containing portion in the recessed shape, however, since the containing portion is significantly projected to an inner side of the glove box, there is a concern of constituting a hindrance in putting small articles into and from the glove box, or restricting an effective space of the glove box.

We can see that most of the time, the parameters are extracted in the sentences. Nevertheless, the extracted sentences may still be long which might cause noise when the parameters will be extracted. In patent US07383707-20080610, the same sentence is chosen as first and second part of the contradiction as it contains both parameters.

It is also important to note that these two sentences alone give a real understanding of the invention. For example, in the patent US10286848-20190514, we understand that the issue is around an additional storage for a car while guaranteeing a flexibility on the installation which is effectively shown in the abstract (without notion of contradiction this time): A vehicle cargo area includes a retention structure having an upper portion, a vertical portion, an arcuate portion, and an angled portion. The vehicle cargo area further includes a storage tray having a coupling portion that removably couples to the retention structure by passing over the vertical portion and engages with the vertical portion such that the coupling portion is secured in the retention structure.

The notion of summary (in the sense of TRIZ) makes sense since in only two sentences one can extract the "meaning" of an invention, which can be particularly useful in a targeted search for information within the framework of a problem-solving process.

Conclusion

The amount of data required by deep learning algorithms makes it difficult to use these models in the context of TRIZ. A solution, called transfer learning, consists in pretraining models on other data before specializing them on their "TRIZ" task.

In this paper we presented a new transfer learning method for patent analysis that can be exploited for contradiction extraction. This method consists in integrating an extractive summary model in an abstractive summary model and training the extractive model on an abstractive task.

Contradiction extraction is a major issue for a massive and detailed understanding of the available inventions and thus of the invention process in order to automatically exploit them to find new inventive solution. It would be possible, for example, starting from an encountered contradiction, to propose patents that would allow this same contradiction to be solved.

The first results are encouraging on the extraction of contradictions even if the chosen sentences are not always the most relevant.

Future works include the extraction of parameters from the sentences chosen by this summarization model, the reconstruction of an updated version of the matrix and the generation of new solutions from patents.

Fig. 1 .

 1 Fig. 1. Abstractive summarization model