Juliette U Bertrand
email: juliette.bertrand@skinosive.com

Valérie Petit
email: valerie.petit@curie.fr

Zackie Aktary
email: zackie.aktary@curie.fr

Pierre De

L A Grange

Nadav Elkoshi
email: nadav.elkoshi@gmail.com

Pierre Sohier
email: pierre.sohier@curie.fr

Véronique Delmas
email: veronique.delmas@curie.fr

Carmit Levy

Lionel Larue
email: lionel.larue@curie.fr

Carmit Levy

Loss of Dicer in newborn melanocytes leads to premature hair greying and changes in integrin expression

Keywords: Cell migration, Gene regulation, Integrins, Melanocytes, Models, Mouse

Premature hair greying occurs due to the depletion of melanocyte stem cells (McSCs) in the hair follicle, which can be accelerated by stress caused by genetic or environmental factors.

However, the connection between stress and McSC loss is not fully understood. MiRNAs are molecules that control gene expression by regulating mRNA stability and translation, and are produced by the enzyme Dicer, which is repressed under stress. Here, using two mouse genetic models and human and mouse cell lines, we found that the inactivation of Dicer in melanocytes leads to a misplacement of these cells within the hair follicle, resulting in a lack of melanin transfer to keratinocytes in the growing hair and the exhaustion of the McSC pool. We also show that miR-92b, which regulates ItgaV mRNA and protein levels, plays a role in altering melanocyte migration. Overall, our findings suggest that the Dicer-miR92b-ItgaV pathway serves as a major signalling pathway linking stress to premature hair greying.

INTRODUCTION

The cycle of hair follicles consists of three phases: growth (=anagen), destruction (=catagen), and rest (=telogen) [START_REF] Fuchs | The tortoise and the hair: slow-cycling cells in the stem cell race[END_REF]. For hair to be properly pigmented, there must be coordination between the proliferation and differentiation of keratinocytes and melanocytes during the anagen phase (Yardman-Frank and Fisher 2021). During each anagen phase, melanocyte stem cells (McSCs) located in the bulge region become active and multiply into transient amplifying cells (=TACs) [START_REF] Sun | Dedifferentiation maintains melanocyte stem cells in a dynamic niche[END_REF]). These TACs move along the growing hair follicle and develop into pigmented dendritic melanocytes in the bulb. The pigmentation process depends on the synthesis, transport, and transfer of melanin from melanocytes to keratinocytes that are committed to differentiating into hair-producing cells [START_REF] Luciani | Biological and mathematical modeling of melanocyte development[END_REF]. This intricate process is carefully regulated by both cell autonomous and non-cell autonomous signals such as Wnt, Edn, Tgf-β, and Notch, which control the timing of differentiation and quiescence [START_REF] Chang | NFIB is a governor of epithelial-melanocyte stem cell behaviour in a shared niche[END_REF][START_REF] Moriyama | Notch signaling via Hes1 transcription factor maintains survival of melanoblasts and melanocyte stem cells[END_REF][START_REF] Nishimura | Key roles for transforming growth factor beta in melanocyte stem cell maintenance[END_REF][START_REF] Rabbani | Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration[END_REF]. There is currently limited knowledge about how TACs migrate towards the new hair bulb. It is possible that TACs may simply follow the growth of the hair follicle passively, or they may actively migrate towards the hair bulb in a directed manner, or perhaps a combination of both mechanisms occurs.

The cycle of pigmented hair is carefully controlled by a combination of genetic factors and external signals, such as stress from the environment [START_REF] Fialkowski | Identification of Gene Variants Associated with Melanocyte Stem Cell Differentiation in Mice Predisposed for Hair Graying[END_REF][START_REF] Harris | Ectopic differentiation of melanocyte stem cells is influenced by genetic background[END_REF]. Dicer is an enzyme of the RNase III family that plays a critical role in the precise regulation of gene expression through its involvement in the maturation of microRNAs (= miRNAs) [START_REF] Torrez | Structural Insights into the Advances and Mechanistic Understanding of Human Dicer[END_REF]. Both the activity and expression of Dicer are regulated by different types of stress, both in vitro and in vivo, across a variety of cell types, including trophoblasts, pre-adipocytes, and breast cells. For instance, in vitro, exposure to chemical and physical stressors like hydrogen peroxide, hypoxia, interferon α, phorbol ester, trichostatin A, and UV radiation has been shown to reduce Dicer levels [START_REF] Van Den Beucken | Hypoxia promotes stem cell phenotypes and poor prognosis through epigenetic regulation of DICER[END_REF][START_REF] Mori | Role of microRNA processing in adipose tissue in stress defense and longevity[END_REF][START_REF] Wiesen | Dicer is regulated by cellular stresses and interferons[END_REF]. In vivo, Dicer plays a role in mediating resilience to psychological stress by facilitating the pro-resilient effect of β-catenin in a mouse model of depression [START_REF] Dias | beta-catenin mediates stress resilience through Dicer1/microRNA regulation[END_REF]. Different types of stress can modulate Dicer levels and subsequently impact miRNA expression. For example, UV radiation controls the cellular stress response by regulating Dicer and various miRNAs [START_REF] Dynoodt | Identification of miR-145 as a key regulator of the pigmentary process[END_REF][START_REF] Emde | miRNAs at the interface of cellular stress and disease[END_REF]. miRNAs are generated in the nucleus by RNA polymerase II/III as stem-loop RNAs known as primary microRNAs (pri-miRs). The pri-miRs are then converted into precursor miRs and exported to the cytoplasm by Exportin-5. In the cytoplasm, Dicer, which belongs to the RNA-induced silencing loading complex (RISC RLC) complex, processes the precursor miRs into duplex miRs [START_REF] Torrez | Structural Insights into the Advances and Mechanistic Understanding of Human Dicer[END_REF]. These duplex miRs are then processed by RISC, and mature miRNAs are loaded onto Argonaut proteins that recognize and bind to target mRNAs, leading to the inhibition of their expression by either blocking translation or causing mRNA degradation [START_REF] Ha | Regulation of microRNA biogenesis[END_REF]. As a result, Dicer and miRNAs can regulate a vast number of genes. Dicer is crucial for mammalian embryonic development, and mice with germline Dicer inactivation die before E7.5 [START_REF] Bernstein | Dicer is essential for mouse development[END_REF].

The loss of Dicer in the developing melanocyte lineage during embryonic development resulted in a white adult mouse lacking melanocytes in hair follicles [START_REF] Levy | Lineage-specific transcriptional regulation of DICER by MITF in melanocytes[END_REF].

Similarly, the knockout of Dicer1 in zebrafish also results in abnormal pigmentation with a reduced number of melanocytes in the head and ventral trunk [START_REF] Weiner | Dicer1 is required for pigment cell and craniofacial development in zebrafish[END_REF]. The miRNA-17 cluster and BIM were identified as partially responsible for the lack of viability of primary human melanocytes in the absence of Dicer [START_REF] Levy | Lineage-specific transcriptional regulation of DICER by MITF in melanocytes[END_REF]). Several miRNAs have been found to play a role in the regulation of pigmentation and melanin transport, while others have been associated with melanoma cell migration, EMT, progression, and invasion in vitro [START_REF] Ozsolak | Chromatin structure analyses identify miRNA promoters[END_REF][START_REF] Rambow | Regulation of Melanoma Progression through the TCF4/miR-125b/NEDD9 Cascade[END_REF][START_REF] Rambow | New Functional Signatures for Understanding Melanoma Biology from Tumor Cell Lineage-Specific Analysis[END_REF][START_REF] Rambow | miR-330-5p targets tyrosinase and induces depigmentation[END_REF]. In this study, after evaluating the consequence of the lack of Dicer in mouse melanoblasts during embryonic development, we investigated the impact of Dicer inactivation on mouse melanocytes by specifically targeting Dicer at birth in the melanocyte lineage. Our results revealed that the absence of Dicer resulted in abnormal melanocyte localization in hair follicles and, premature greying of hair, due to the reduction of miR-92b and the increase of one of its targets, ItgaV.

RESULTS

Dicer conditional knockout during embryonic development reduces the number of melanoblasts.

To assess the effects of Dicer inactivation during the establishment of the melanocyte lineage, we conducted crosses between Dicer Flox22-23 mice, which carry loxP sequences in introns 21 and 23, and Tyr::Cre mice, and monitored the phenotype of the mutant mice as they aged (Supplementary Figure S1a). At birth, homozygous Dicer knockout mice (Dicer-cKO, "c" for constitutive) were pigmented, but notably less than control littermates (WT), indicating that Dicer may affect the growth of melanoblasts or affect its differentiation (Supplementary Figure S1b-d). Heterozygous mutant mice had no coat color phenotype (not shown). At 2 months of age, Dicer-cKO mice showed salt-and-pepper coloring on their back but were white on the belly (Supplementary Figure S1e1). By 6 months of age, Dicer-cKO mice were completely white (Supplementary Figure S1f). Hair greying appears to be a gradual process that depends on the number of original melanocytes at birth, as there are more melanocytes on the back than on the belly [START_REF] Luciani | Biological and mathematical modeling of melanocyte development[END_REF].

Previously, the inactivation of Dicer was performed using the same Tyr::Cre mice and another Dicer allele: Dicer Flox23 [START_REF] Levy | Lineage-specific transcriptional regulation of DICER by MITF in melanocytes[END_REF]. For both Dicer alleles (Dicer Flox23 and Dicer Flox22-23 ), the RNase III domain of this enzyme is inactive [START_REF] Harfe | The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb[END_REF][START_REF] Murchison | Characterization of Dicer-deficient murine embryonic stem cells[END_REF]. These studies demonstrated that mice gradually became fully white as they aged.

However, it is important to note that there are some differences in the coat color phenotype between Tyr::Cre; Dicer Flox23/Flox23 [START_REF] Levy | Lineage-specific transcriptional regulation of DICER by MITF in melanocytes[END_REF]) and the Tyr::Cre; Dicer Flox22-23/Flox22-23 mice used in this study. The Dicer Flox23 mice "were virtually white from birth, except for the tip of the initial hairs on the dorsal trunk". However, the Dicer Flox22-23 mice were slightly pigmented at birth and then had salt-and-pepper hairs before becoming fully white. We investigated the impact of Dicer inactivation on melanogenesis and melanin production by studying a mouse melanocyte cell line (Melan-a) and a human melanoma cell line (MNT-1). The knock-down of Dicer in both cell lines, resulted in a minor, yet significant increase in melanin production per cell, a slight reduction in the growth of the cells, but did not affect the mRNA levels of Tyrp1, Dct, and Tyr (Figure 1a-h). Further analysis revealed a significant decrease in the number of melanoblasts at E14.5 in the entire body of mutant embryos compared to wild-type (Figure 1gj). Therefore, the overall reduction in pigmentation in the Dicer-cKO mutant pups could be attributed to a decrease in the number of melanocytes rather than a decrease in melanogenesis.

Dicer plays a crucial role in pigmentation renewal

To further investigate the role of Dicer in regulating pigmentation, we created spatio-temporal inducible Tyr::CreER T2 ; Dicer Flox22-23/Flox22-23 mutant (Dicer-iKO) mice. Dicer was defloxed in the melanocyte lineage, including McSCs, TACs, and differentiated melanocytes (Mc), by Tamoxifen (=Tam) treatment at p1, p2, and p3. Defloxing was confirmed by PCR analysis of hair bulb DNA (Supplementary Figure S2a-c). There was no significant difference in coat color until p30 after Tam treatment of both wild-type (WT) and Dicer-iKO mice (Supplementary Figure S2d-f, Figure 2a). At p30, after the first hair cycle, the belly of Dicer-iKO mice was lighter than that of WT mice, but the color of the back was similar. At p60, after the second hair cycle, the belly of Dicer-iKO mice was even lighter, displaying salt-and-pepper hairs (Figure 2b-e). By p180, after the fifth hair cycle, the whitening of Dicer-iKO mice had significantly increased, with a higher number of white hairs than at p60. These results suggest that Dicer plays a direct role in pigmentation renewal and that its inactivation leads to gradual whitening of the hair coat.

The absence of Dicer in melanocytes leads to their improper localization in the hair follicle

We determined the location of WT and Dicer-iKO melanocytes in hair follicles at p60 by Immuno-histochemistry (IHC) using as a Dct marker. In WT mice, Dct-positive cells were found at the level of the bulge and bulb (Figure 3a). In Dicer-iKO mice, most hairs were black and Dct-positive cells were found at the bulge and bulb but can be mislocated as shown in Figure 3b. The majority of Dicer-iKO ventral hairs were white at this age; either the Dctpositive cells were poorly mislocated (Figure 3c-e) or they were absent (Figure 3f). These observations suggest that in the absence of Dicer, melanocytes may undergo cell death as previously shown by Levy and colleagues [START_REF] Levy | Lineage-specific transcriptional regulation of DICER by MITF in melanocytes[END_REF]) and/or melanocytes have a migratory defect.

Dicer knock down affects the level of integrins

To gain a better understanding of the effects caused by a reduction of Dicer in melanocytes, we knocked-down Dicer using specific siRNAs in normal C57BL/6 mouse melanocyte Melan-a cells. This resulted in a decrease of Dicer protein levels to approximately 40% compared to the control siScramble (siScr) after 24 hours of transfection (Figure 4a,b). Microarray analysis was used to examine gene expression, revealing that 280 genes were deregulated in siDicer cells compared to siScr (Figure 4c,d and Supplementary Table S1). Of these genes, 200 were upregulated and 80 were downregulated (p-value ≤ 0.05 and FC ≥ 1.5), which is consistent with decreased miR maturation and increased levels of their target RNAs. The top 20 most significantly enriched GO terms showed that seven were associated with genes related to "membrane", "cell surface", "cell-matrix adhesion", "cell migration", "focal adhesion", "integrin mediated signaling pathway", and "integrin complex", suggesting that Dicer regulates Melan-a cell adhesion and migration through integrins (Figure 4e). KEGG and REACTOME pathways of the deregulated genes revealed enrichment in functions associated with integrins, "ECM receptor interaction", "Focal adhesion", "Regulation of actin cytoskeleton", "Cell adhesion molecules", "Integrin", and "ECM proteoglycans" (Figure 4f,g). The volcano plot displayed upregulation of several mRNA integrins, including Itgav, Itga8, Itga9, Itgb5 along with the expected downregulation of Dicer (Figure 4d). We confirmed the increased the levels of Itgav, Itga8, Itga9, and Itgb5 after transfection of Melan-a cells with siDicer (Figure 4h,i, and Supplementary Figure S3a,b). In vivo, the amount of integrin αV and β5 in Dct-positive cells appeared to be higher in Dicer-iKO melanocytes compared to WT melanocytes (Figure 4j-m).

Dicer regulates the level of ItgaV through miR-92b

To dissect the molecular mechanisms by which Dicer regulates integrin expression and melanocyte migration in vitro, we performed a miRNome analysis by microarray on Melan-a cells transiently transfected with siDicer or siScr (Figure 5a, Supplementary Table S2). Twentyfour and 48 hours after transfection, 151 and 189 miRs were differentially expressed with p ≤ 0.05, respectively. After pooling the 24-and 48-hour data, a total of 327 miRs were differentially expressed. Amongst these, five are putative regulators of ItgaV, according to MirTarBase and miRnome: miR-92b-3p, miR-16-5p, miR-195a-5p, miR-322-5p and miR-761 (Figure 5b).

Transient transfection of Melan-a cells with miR-92b-3p mimics allowed the validation of the decreased of ItgaV mRNA and protein levels by RT qPCR and western blot analyses 48 hours after transfection (Figure 5c,d). A miRglo experiment confirmed that miR-92b-3p reduced the stability of luciferase mRNA when fused to the ItgaV 3'-UTR sequence, but not when the putative targeted sequence was mutated (Figure 5e). Thus, miR-92b-3p specifically targets ItgaV in mouse melanocytes.

Dicer regulates melanocyte migration through miR-92b/ItgaV

We investigated the role of ItgaV, Dicer and miR-92b in regulating melanocyte migration by analysing single-cell migration on non-coated plastic dishes. In Melan-a cells, the reduction of ItgaV with siRNA reduced cell velocity (Figure 5f [START_REF] Couchman | Expression of basement membrane components through morphological changes in the hair growth cycle[END_REF][START_REF] Fujiwara | The basement membrane of hair follicle stem cells is a muscle cell niche[END_REF][START_REF] Jahoda | Changes in fibronectin, laminin and type IV collagen distribution relate to basement membrane restructuring during the rat vibrissa follicle hair growth cycle[END_REF][START_REF] Tanimura | Hair follicle stem cells provide a functional niche for melanocyte stem cells[END_REF].

The role of integrins is of a particular importance because we know that the mobility and plasticity of McSCs is a key element of melanocytes renewal [START_REF] Sun | Dedifferentiation maintains melanocyte stem cells in a dynamic niche[END_REF]. TAC migration differs between mice and humans, with mice showing migration primarily to the bulb and humans to both the apex and bulb. This suggests that TACs undergo two directed migrations, which may be cell-autonomous or non-autonomous. In mice lacking Dicer, integrin levels are altered, resulting in increased melanocytes at the hair's apex. It is possible that differences in integrin expression between humans and mice contribute to the bidirectional movement of TACs observed in humans.

The salt-and-pepper phenotype observed in all mutant mice progressed over time, with an increase in the proportion of white hairs, which is unlikely due to inefficient Dicer inactivation, as we demonstrated the high efficiency of TAM induction at birth using S1). Kit is also one of the most up-regulated genes. Kit is well known to be involved in melanoblast migration during embryogenesis [START_REF] Jordan | A late wave of melanoblast differentiation and rostrocaudal migration revealed in patch and rump-white embryos[END_REF][START_REF] Nocka | Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W[END_REF], is a prerequisite for melanoblast migration in hair at birth [START_REF] Peters | Migration of melanoblasts into the developing murine hair follicle is accompanied by transient c-Kit expression[END_REF], and is involved in melanoma cell migration [START_REF] Bernex | Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos[END_REF]. Furthermore, while Kit is described as participating in the regulation of McSC differentiation [START_REF] Chang | NFIB is a governor of epithelial-melanocyte stem cell behaviour in a shared niche[END_REF], its involvement in the migration of normal adult melanocytes has never been demonstrated."

Previous studies have demonstrated the expression of several integrins in the melanocyte lineage but not Itga8, Itga9 and Itgb5. In the future, the role of Itga8 and Itga9 in the melanocyte lineage should be looked at more carefully, and especially after stress in various diseases that includes vitiligo. However, the level of these two integrins remain low compared to ItgaV and Itgb5 in melan-A cells (see supplementary Figure S3c). Melanoblasts and melanoma cells rely on integrins for their migration. Melanoblasts migrate along the dorsolateral axis and tightly regulate the usage of various integrins, including ItgaV, to establish the melanocyte lineage [START_REF] Beauvais-Jouneau | A novel model to study the dorsolateral migration of melanoblasts[END_REF][START_REF] Haage | Analysis of Integrin-Dependent Melanoblast Migration During Development[END_REF]. We were expecting that the migration of melanoblasts during embryogenesis would be enhanced since integrins and Kit, to the least, were induced in the absence of Dicer in Melan-a cells. The analysis of the location of melanoblasts in WT and cKO embryos at E14.5 revealed that proportionally there was slightly more melanoblasts in the ventral part in cKO embryos, but it was not significant.

In vivo, it has been shown that melanocytes and melanoblasts express a3b1, a6b1, and avb3 [START_REF] Beauvais-Jouneau | A novel model to study the dorsolateral migration of melanoblasts[END_REF][START_REF] Haass | Adhesion, migration and communication in melanocytes and melanoma[END_REF]. In culture, melanocytes produce a3b1, a5b1, a6b1, and avb3 [START_REF] Hara | Role of integrins in melanocyte attachment and dendricity[END_REF][START_REF] Scott | Stem cell factor regulates human melanocyte-matrix interactions[END_REF]. Additionally, melanoma cell lines and melanoma exhibit the expression of a5b1, a6b1, a5b1, a7b1, and avb3 [START_REF] Haass | Adhesion, migration and communication in melanocytes and melanoma[END_REF][START_REF] Pinon | Integrins: versatile receptors controlling melanocyte adhesion, migration and proliferation[END_REF]. Melanoma is the most aggressive form of skin cancer and is known for its rapid metastasis. Integrins play a direct role in various cellular processes involved in melanoma progression [START_REF] Buommino | Artemisinin reduces human melanoma cell migration by down-regulating alpha V beta 3 integrin and reducing metalloproteinase 2 production[END_REF][START_REF] Voura | Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells[END_REF]. The regulation of integrin activity depends on factors such as mRNA and protein levels, as well as the abundance of specific α-β dimers. In cancer, miRs have been shown to regulate integrin levels, but it remains unknown whether miR-92 can regulate ITGAV [START_REF] Verhoeff | Non-coding RNA regulation of integrins and their potential as therapeutic targets in cancer[END_REF]. Our analysis of TCGA data revealed significantly higher ITGAV mRNA levels in melanoma metastasis compared to primary melanoma (Supplementary Figure S4a). Additionally, we observed an inverse relationship between ITGAV mRNA and miR-92 in various human melanoma cells (Supplementary Figure S4b). These findings suggest that miR-92 may play a crucial role in the regulation of ITGAV during homeostasis and the proper movement of TAC/melanocytes in the hair follicle.

In summary, our study reveals to our knowledge previously unreported pathway and mechanism for hair greying that involves stress-induced reduction in Dicer levels and dysregulation of integrins, which are involved in the directed migration of TACs through their interaction with the extracellular matrix. This suggests a retro-control mechanism for the bulge cycle, and highlights the link between stress, Dicer, integrins and hair greying.

Single-cell migration assay. Forty-eight hours after transfection, cells were transfected 48 hours prior, trypsinization and seeded for 5h at low concentration prior imaging cells every 4 min for 12 h. Tracking of the cells and path analysis was performed using iTrack4U software [START_REF] Cordelieres | Automated cell tracking and analysis in phase-contrast videos (iTrack4U): development of Java software based on combined mean-shift processes[END_REF].

Quantitative real-time PCR. After preparing and quantification of RNA, real-time PCR was performed using primers listed in Supplementary Table S3. Mouse Gapdh housekeeping gene and the internal control using ""Ct methods. Differences between two groups were assayed using unpaired Student's t-test in Prism (Graphpad software). Western blot analysis (a,e) showed siRNA-mediated Dicer knockdown in Melan-a (a-d) and MNT-1 (e-h) cells . The melanin content (b,f) melanogenesis gene RNA level (c,g) and cell number evaluation (d,h) analyses were performed as previously described [START_REF] Gallagher | General strategy to analyse melanoma in mice[END_REF]. Scr = scramble. The number of melanoblasts was decreased in E14.5 Dicer-cKO (=Tyr::Cre/° ; Dicer Flox22-23/Flox22-23 ) mice compared to WT controls (i-l). The number of Dct::LacZ/° WT and mutant head and truncal melanoblasts was determined after betagalactosidase staining [START_REF] Berlin | General strategy to analyse coat colour phenotypes in mice[END_REF]. Each experiment was repeated at least three times, and statistical analysis was performed using t-test with ns indicating not significant, *p < 0.05, and **p < 0.01. Following hair growth synchronization, 10-μm skin sections of p60 Dicer-iKO and WT were stained for Dct (red, highlighted by white arrows) and DAPI (blue). Twenty-six hairs from three mutants and 25 hairs from three wild-type mice were followed. Among the wild-type hairs, all 25 were pigmented, and 24 exhibited a normal distribution pattern with the profile a; Dct+ cells are located in the dermal papilla of the bulb (Bb) and in the bulge (Bg). From the mutant hairs, six were pigmented and twenty were not pigmented. The pigmented hairs had two profiles: two Western blot analysis (a,e) showed siRNA-mediated Dicer knockdown in Melan-a (a-d) and MNT-1 (e-h) cells.

The melanin content (b,f) melanogenesis gene RNA level (c,g) and cell number evaluation (d,h) analyses were performed as previously described [START_REF] Gallagher | General strategy to analyse melanoma in mice[END_REF]. Scr = scramble. The number of melanoblasts was decreased in E14.5 Dicer-cKO (=Tyr::Cre/°; DicerFlox22-23/Flox22-23) mice compared to WT controls (i-l).

The number of Dct::LacZ/°WT and mutant head and truncal melanoblasts was determined after beta-galactosidase staining [START_REF] Berlin | General strategy to analyse coat colour phenotypes in mice[END_REF]. Each experiment was repeated at least three times, and statistical analysis was performed using t-test with ns indicating not significant, *p < 0.05, and **p < 0.01. Following hair growth synchronization, 10-μm skin sections of p60 Dicer-iKO and WT were stained for Dct (red, highlighted by white arrows) and DAPI (blue). Twenty-six hairs from three mutants and 25 hairs from three wild-type mice were followed. Among the wild-type hairs, all 25 were pigmented, and 24 exhibited a normal distribution pattern with the profile a; Dct+ cells are located in the dermal papilla of the bulb (Bb) and in the bulge (Bg). From the mutant hairs, six were pigmented and twenty were not pigmented. The pigmented hairs had two profiles: two had the profile a, and four had the profile b.

  ,g) and the reduction of Dicer, with siRNA, or the antagomir for miR-92b-3p increased cell velocity and induced ItgαV level (Figure 5h-j). Altogether, these results identify the Dicer-miR92b-ItgaV axis as a signalling pathway affecting melanocyte migration. DISCUSSION This study used conditional and inducible mouse mutants to demonstrate that Dicer is essential for hair pigmentation in adult mice by regulating melanocyte positioning and renewal, leading to the maintenance of melanocyte stem cells (McSC) in hair bulges. In particular, reduced Dicer levels altered miR-92b and αV integrin levels, which regulate melanocyte migration. Integrin αVβ5 contributes to proper melanocyte migration along hair follicles and that melanocytes may actively migrate along the growing hair follicle using their integrins repertoire. Dicer inactivation also induced McSC loss, possibly due to premature differentiation, death, and/or exhaustion. During the hair cycle, keratinocyte and melanocyte stem cells exit quiescence together, in response to Wnt, Bmp/Tgf-β, and Edn (Chang et al. 2013). As keratinocytes form hair follicles, TACs migrate to the hair bulb and differentiate into melanocytes. While proteins involved in cell adhesion, cytoskeleton remodeling, and cellular traction have been studied for melanocyte migration (Hamidi and Ivaska 2018; Petit and Larue 2016; Pinon and Wehrle-Haller 2011), little is known about TAC migration during the hair cycle. We propose that the expression of integrin αVβ5, which mediates cell adhesion to the extracellular matrix, affects TAC migration in vivo due to the diverse ECM components in various parts of the hair follicle

  Tyr::CreER T2 mice (Le[START_REF] Coz | Targeted Knockout of β-Catenin in Adult Melanocyte Stem Cells Using a Mouse Line, Dct::CreERT2, Results in Disrupted Stem Cell Renewal and Pigmentation Defects[END_REF][START_REF] Yajima | Spatiotemporal gene control by the Cre-ERT2 system in melanocytes[END_REF]. Premature hair greying in mice can be attributed to three potential causes: premature differentiation, cell death, or exhaustion of melanocyte stem cells(McSCs). The premature differentiation of McSC in the bulge was reported in mouse mutants lacking Notch1/2, Rbp-j, TgfbRII, β-catenin, or Col17a1[START_REF] Aubin-Houzelstein | Melanoblasts' proper location and timed differentiation depend on Notch/RBP-J signaling in postnatal hair follicles[END_REF][START_REF] Moriyama | Notch signaling via Hes1 transcription factor maintains survival of melanoblasts and melanocyte stem cells[END_REF][START_REF] Nishimura | Key roles for transforming growth factor beta in melanocyte stem cell maintenance[END_REF][START_REF] Rabbani | Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration[END_REF][START_REF] Tanimura | Hair follicle stem cells provide a functional niche for melanocyte stem cells[END_REF]). McSCs cell death was observed in the absence of Bcl2 and Dicer[START_REF] Levy | Lineage-specific transcriptional regulation of DICER by MITF in melanocytes[END_REF][START_REF] Nishimura | Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche[END_REF], while McSC exhaustion was observed in the absence of TgfbRII[START_REF] Koludrovic | Chromatin-Remodelling Complex NURF Is Essential for Differentiation of Adult Melanocyte Stem Cells[END_REF][START_REF] Nishimura | Key roles for transforming growth factor beta in melanocyte stem cell maintenance[END_REF]. The absence of Dicer resulted in mislocated pigmented Dct-positive cells and premature differentiation (Figure3). It is possible that, in the absence of Dicer, McSC die in a cell autonomous way and the combination of mislocation and premature differentiation/death exhausts McSCs. Abnormal localization of melanocytes and loss of McSCs may or may not be linked. On one hand, if the two events occur independently, Dicer/miR/Itg-dependent melanocyte migration explains their abnormal localization, and death is only associated with apoptosis as it was shown by Levy et al. 2010. On the other hand, both events may occur in a coordinated fashion. As the number of Mc localized in the hair bulb is lower than normal, we can imagine the existence of a feedback mechanism by which TAC/Mc signal McSCs to regulate their proliferation. In the Dicer mutant, where the number of TAC/Mc is lower, this feedback signaling is diminished, and the McSCsundergo excessive proliferation to compensate, and are therefore prematurely depleted. This second possibility does not mean that apoptosis does not occur. In all cKO-Dicer and iKO-Dicer mice, the whitening of the fur is occurring faster on the ventral side than on the dorsal side (Supplementary FigureS1and Figure2). This occurrence can be attributed to a lower presence of melanoblasts in the ventral region of the embryo, resulting in fewer melanocytes and McSC in that aera. This suggests that the exhaustion of McSC happens more swiflty on the ventral side due to its initially lower abundance.The transcriptome analysis of Dicer knockdown Melan-a cells demonstrated deregulation of multiple genes involved in cell adhesion and migration, consistent with in vivo observations. Integrins αV, α8, α9, and β5 (Itgav, Itga8, Itga9, and Itgb5) were among the upregulated genes at the RNA level, and their induction was confirmed in Dicer-iKO melanocytes in vivo and Dicer knockdown Melan-a cells in vitro. MiR-92b-3p was identified as a strong repressor of ItgaV. In humans MiR-92b-3p is known to decrease ITGAV and repress migration and invasion of esophageal squamous cell carcinoma cells in vitro in another context[START_REF] Ma | Up-regulated Dicer expression in patients with cutaneous melanoma[END_REF]. However, the role of MiR-92b in proliferation is not clear, it promotes proliferation during pre-eclampsia[START_REF] Li | Lin28B/miR-92b Promote the Proliferation, Migration, and Invasion in the Pathogenesis of Preeclampsia via the DKK1/Wnt/β-Catenin Pathway[END_REF]) but paradoxically inhibits proliferation and promotes apoptosis in glioma cells[START_REF] Song | miR-92b regulates glioma cells proliferation, migration, invasion, and apoptosis via PTEN/Akt signaling pathway[END_REF]. Moreover, performing similar analysis, we could show that (i) miR-155-5p decreased Itgb5 mRNA level, but not those of Itga8, Itga9, or ItgaV, (ii) miR-466i-5p decreased Itga9 mRNA levels 48 and 72 hours after transfection, but not those of ItgaV, Itga8, or Itgb5, and (iii) let7b-5p decreased Itga8 mRNA level.Knocking down Dicer in Melan-a cells led to changes in the expression of several genes involved in melanocyte migration, including integrins and ECM components such as Npnt and Col6a1 (Supplementary Table

  mRNA microarray. Sample preparation and hybridization were performed by the Genomic platform of the Institut Curie. Affymetrix Mouse ClariomD arrays were hybridized according to the recommendations of Affymetrix (Santa Clara, CA, United States) using the WT PLUS protocol and labelling and hybridization kits from Affymetrix. microRNA microarray. Sample preparation and hybridization were performed by the Genomic platform of the Institut Curie. The flashTag Biotin HSR RNA labelling kit (Affymetrix) was used to label 100 ng total RNA according to the manufacturer's protocol. Volcano plot methods. RNA-seq counts and phenotypic traits of the cohort: GDC TCGA Melanoma (SKCM), were downloaded from the UCSC'a Xena Browser at https://xenabrowser.net. Analysis was performed as previously described (Petit et al. 2019) pmirGLO luciferase assay. Melan-a cells were transfected with 50 nM miR-92b-3p mimic (Dharmacon), 250 ng various pmirGLO Dual-Luciferase miRNA target expression vector constructs (Promega), and 250 ng TK::Renilla (Promega). The negative control, without insert, did not contain any matching miR-92b-3p sequence. Details can be found in supplementary information. Statistics and reproducibility. Quantitative data were performed at least in triplicate for analysis and expressed as mean +/-SEM or mean +/-SD as indicated in the figure legends. The exact number of replicates, statistical test and significance are reported in the figure legends.

Figure 1 .

 1 Figure 1. Absence of Dicer reduces melanoblast numbers without altering melanogenesis gene levels.

Figure 2 .

 2 Figure 2. The knockout of Dicer at birth induces a salt-and-pepper phenotype. (a) Ventral and dorsal views of Dicer-iKO (=Tyr::CreER T2 ; Dicer Flox22-23/Flox22-23 ) and littermate WT controls. Melanin amount in Dicer-iKO and WT hairs from the dorsal (b) and ventral (d) parts at p60. No difference was observed between females (pink) and males (blue). n = 5 to 7. t-test analysis: ***p < 10 -3 , **p < 10 -2 . Black and white hairs on the back (c) and belly (e) of Dicer-iKO and WT mice from the same litter. Relative abundance of black and white hairs on a representative pair of mice is given. Scale bar: 20 μm.

Figure 3 .

 3 Figure 3. Abnormal location of melanocytes in Dicer-iKO hairs.

  had the profile a, and four had the profile b. The profile b corresponds to hairs with Dct+ cells in the bulb and some with abnormal localization. The not pigmented had four profiles: four hairs had the profile c (without Dct+ cells in the bulb but with Dct+ in the apex of the hair), six the profile d (below and on the side of the bulb), four the profile e (below the bulb only), and six the profile f (without any Dct+ cells were observed). Hair follicles were schematically represented by black and white rectangles for pigmented and non-pigmented hairs, respectively, and a black diamond for melanocytes (Mc). Scale bar: 25 μm.

Figure 4 .

 4 Figure 4. ItgaV is induced in cells knocked down or knockout for Dicer Melan-a cells were subjected to Dicer knockdown (n=3), and subsequent analyses were performed, including Western blot (a), RT-qPCR (b), and transcriptomic (c-g). A heatmap (c) and a volcano plot (d) show genes differentially expressed between siDicer and siScr conditions with p ≤ 0.05 and fold-change ≥ 1.5. In (d) upregulated and downregulated genes are shown as red and green dots. Gene ontology (e), KEGG (f), and REACTOME-pathway (g) analyses reveal an enrichment of pathways involved in cell adhesion and cell-matrix interactions (red). Validation of of the upregulation of integrin mRNA and protein levels by RT-qPCR (h) and western blot (i) analyses, respectively, 24 and 48 h after transfection with siDicer or siScr. n = 4. t-test analysis: ****p ≤ 10 -4 , **p ≤ 10 -2 , *p ≤ 5.10 -2 . au: arbitrary unit. (j-m) Ten-week-oldDicer-iKO and WT mice from the same litter were depilated on the left side of the back, and a 0.25cm 2 biopsy sampled 10 days later from the depilated area. Dct (red) and ItgαV (green) costaining (j,k) and Dct (red) and Itgβ5 (green) co-staining (l,m) were performed on 10-μm skin

Figure 5 .Figure 1 .

 51 Figure 5. ItgaV is targeted by miR-92b-3p. Melan-a cells were transfected with siRNA Dicer (si-Dicer) or control siRNA (si-Scr) prior miRnome analysis (a,b), with mouse mimic of miR-92b-3p (92b-3p) (+) or a mimic (-) control in the absence prior RT-qPCR (c) and western blot analysis (d) or presence of a miRgloluciferase construct (e), with si-ItgaV or siScr prior migration analysis (f) and western blot analysis (g), and with si-Dicer, si-Scr (-), mimic of miR-92b or a mimic control (-) prior migration analysis (h) and western blot (i) and mRNA (j) analyses. (a) Heatmap presenting the differentially expressed miR between both conditions at 24 and 48h with p ≤ 0.05 for a total of 327 miR.b) Venn diagram comparing the miRNA from miRnome to the mirTarBase list of putative miRNA that target ItgaV. Vinc =Vinculin. Three biological replicates were used for each condition. t-test analysis: ****p≤ 10 -4 , ***p ≤ 10 -3 , **p ≤ 10 -2 , *p ≤ 5.10 -2 , ns: nonsignificant. r.u. = relative unit.

Figure 2 .Figure 3 .

 23 Figure 2. The knockout of Dicer at birth induces a salt-and-pepper phenotype. (a) Ventral and dorsal views of Dicer-iKO (=Tyr::CreERT2 ; DicerFlox22-23/Flox22-23) and littermate WT controls. Melanin amount in Dicer-iKO and WT hairs from the dorsal (b) and ventral (d) parts at p60. No difference was observed between females (pink) and males (blue). n = 5 to 7. t-test analysis: ***p < 10-3, **p < 10-2. Black and white hairs on the back (c) and belly (e) of Dicer-iKO and WT mice from the same litter. Relative abundance of black and white hairs on a representative pair of mice is given. Scale bar: 20 μm.

  The profile b corresponds to hairs with Dct+ cells in the bulb and some with abnormal localization. The not pigmented had four profiles: four hairs had the profile c (without Dct+ cells in the bulb but with Dct+ in the apex of the hair), six the profile d (below and on the side of the bulb), four the profile e (below the bulb only), and six the profile f (without any Dct+ cells were observed). Hair follicles were schematically represented by black and white rectangles for pigmented and non-pigmented hairs, respectively, and a black diamond for melanocytes(Mc). Scale bar: 25 μm.

Figure 4 .Figure 5 .

 45 Figure 4. ItgaV is induced in cells knocked down or knockout for DicerMelan-a cells were subjected to Dicer knockdown (n=3), and subsequent analyses were performed, including Western blot (a),RT-qPCR (b), and transcriptomic (c-g).A heatmap (c) and a volcano plot (d) show genes differentially expressed between siDicer and siScr conditions with p ≤ 0.05 and fold-change ≥ 1.5. In (d) upregulated and downregulated genes are shown as red and green dots. Gene ontology (e), KEGG (f), and REACTOME-pathway (g) analyses reveal an enrichment of pathways involved in cell adhesion and cell-matrix interactions (red). Validation of of the upregulation of integrin mRNA and protein levels by RT-qPCR (h) and western blot (i) analyses, respectively, 24 and 48 h after transfection with siDicer or siScr. n = 4. t-test analysis: ****p ≤ 10-4, **p ≤ 10-2, *p ≤ 5.10-2. au: arbitrary unit. (j-m) Ten-week-old Dicer-iKO and WT mice from the same litter were depilated on the left side of the back, and a 0.25cm2 biopsy sampled 10 days later from the depilated area. Dct (red)and ItgαV (green) co-staining (j,k) and Dct (red) and Itgβ5 (green) co-staining (l,m) were performed on 10-μm skin sections. In Dicer-iKO, all Dct-positive cells displayed an increase of ItgαV or Itgb5. Scale bars: 10 µm.

Acknowledgements

We are grateful to N. Robil from GenoSplice for helping us in the microarray analysis. We thank the Institut Curie staff responsible for the animal colony (especially P. Dubreuil), the genomic (A. Rapinat, A. Vieillefon, and D. Gentien) and PICT-IBiSA imaging (C. Lovo) facilities. We thank G. Hannon and E. Heard for providing the Dicer flox mice. This work was supported by FRM and is under the program «Investissements d'Avenir» launched by the French Government and implemented by ANR Labex CelTisPhyBio (ANR-11-LBX-0038 and ANR-10-IDEX-0001-02 PSL). JUB had a fellowship from Canceropole IdF and FRM (FDT20160435269). PS had a fellowship from INSERM. CL was supported by the Curie Institute Rothschild-Mayent award.

Funding Acquisition: LL. Investigation: JUB, VP, and ZA Methodology: JUB, VP, PDLG, VD, and LL. Project administration: VD, ID, and LL. Resources: JUB, VP, ZA, PDLG, PS, VD, CL, and LL. Software: PDLG and PS. Supervision: VP and LL. Validation: JUB, VP, ZA, and VD. Visualization: JUB, VP, ZA, VD, and LL. Bertrand et al 20 Writing -original draft: JUB, VP, and LL. Writing -review & editing: JUB, VP, ZA, VD, PS, CL, and LL. ORCID Juliette U BERTRAND: 0009-0002-4672-7750 Valérie PETIT: 0000-0003-1645-9396 Zackie AKTARY: 0000-0002-7238-3245 Pierre de la GRANGE : 0000-0001-6361-1878 Nadav ELKOSHI : 0000-0003-4546-2922 Pierre SOHIER : 0000-0002-2341-6848 Véronique DELMAS: 0000-0001-7368-3664 Carmit LEVY: 0000-0003-2683-8779 Lionel LARUE: 0000-0002-2010-6107

Data availability. Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE128757. The accession number for the mRNA and miRNA data reported in this paper is GEO: GSE128757.

Materials and Methods

Detailed materials and methods can be found in the supplemental information.

Animal Models.

Transgenic mice used in this study were previously described [START_REF] Murchison | Characterization of Dicer-deficient murine embryonic stem cells[END_REF][START_REF] Mackenzie | Activation of the receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the mouse embryo[END_REF] [START_REF] Delmas | Cre-mediated recombination in the skin melanocyte lineage[END_REF][START_REF] Yajima | Spatiotemporal gene control by the Cre-ERT2 system in melanocytes[END_REF].

All animals were housed in a specific pathogen-free mouse colony at the Institut Curie. Animal care, use, and experimental procedures were conducted in accordance with recommendations of the European Community (86/609/EEC) and Union (2010/63/UE) and the French National Committee (87/848). Animal care and use were approved by the ethics committee of the Curie Institute in compliance with the institutional guidelines.

Embryo X-gal staining. E14.5 embryo were X-gal stained and imaged as previously described [START_REF] Berlin | General strategy to analyse coat colour phenotypes in mice[END_REF].

Tamoxifen administration. Tamoxifen treatment was performed following a previously published protocol [START_REF] Pitulescu | Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice[END_REF].

Verification of defloxing by PCR. Primers and programs are described in Supplementary

Tables S3,S4.

Quantification of melanin levels. Melanin was quantified in mouse hairs following a previously published protocol [START_REF] Berlin | General strategy to analyse coat colour phenotypes in mice[END_REF] [START_REF] Gallagher | General strategy to analyse melanoma in mice[END_REF].

Tissue processing, immunostaining, and microscopic imaging. Tissues were fixed, stained and visualized as previously described [START_REF] Berlin | General strategy to analyse coat colour phenotypes in mice[END_REF].

Cell culture, transfection, and treatments. Plasmids and sRNA (siRNA, mimics, and hairpin inhibitors) used for transfection in Melan-a cells line [START_REF] Bennett | A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth[END_REF] are described in Supplementary Table S5).

Western blotting. Cells were lysed into RIPA buffer. Twenty !g total protein was separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with various antibodies.

CRediT

Conflict of interest statement

We declare that we have no conflict of interest


M N Nsoe

E V Amba

N M Kameni

G P Kofa

K S Ndi

M Heran

G J Kayem

Biodegradation of Natural Rubber Wastewater in the Submerged Membrane Bioreactor by Pichia Guilliermondii and Yarrowia Lipolytica

Keywords: Biodegradation, Membrane bioreactor, Pichia guilliermondii, Rubber effluent, Yarrowia lipolytica

Despite the many services that natural rubber provides to humanity, its production generates significant quantities of polluted effluents which have a negative impact on health and the environment. In order to reduce this pollution and allow water to be reused after treatment, a synthetic effluent treatment trial was carried out in a submerged membrane bioreactor in the presence of two strains of yeast (Yarrowia lipolytica, Pichia guilliermondii) and biodegradation parameters (COD, NH4

+ , NO2 -, NO3 -) were followed. The Mohlman Index (MI), the particle size distribution, the Lowry method for proteins and the Dubois method for sugars have made it possible to characterize the sludge generated during biodegradation. It appears from these experiments that the reduction rate of COD was 98% and that of nitrification and denitrification 90%. There is a decreasing linear relationship between the MI and SS (Suspended Solids) with an R 2 of 95%. The distribution of the particle size of the sludge is tri-modal with a maximum of sludge having an average size of 1000 µm. The sludge formed resists filtration with a polysaccharide/protein ratio of 0.45.

INTRODUCTION

The increase in population and increasing use of industrial and agricultural resources are causing water shortage, which are leading to a decline in the quality and quantity of freshwater [START_REF] Fao | Irrigation avec des eaux usées Traitées[END_REF]. According to statistics, water consumption has increased by 20% over the last century, and 90% of wastewater is discharged without any treatment [START_REF] Agnès | Baromètre 2017 de l'eau, de l'hygiène et de l'assainissement[END_REF].

In the industrial sector, 70% of factory wastewater is untreated and discharged into the environment, which is more than 22% of surface water pollution [START_REF] Pollet | Caractérisation du colmatage et de l'hydrodynamique dans les bioréacteurs a membranes : Influence de la configuration du module et de l'aération[END_REF]. According to WHO statistics in 2014, 2.3 million people die every year due to water contamination from industrial water waste. In addition, more than half of the world's wetland and 20% of aquatic living things have also disappeared. In full expansion with rubber production increasing from 1.3 million tons in 1985 to over 9.9 million tons in 2012. Production of 1 kg of rubber generated 22.7 L of wastewater: which corresponds to a volume of 247.5 million cubic meters of water used [START_REF] Lmc Rubber | Rubber and tyre market report[END_REF], these studies by the following authors show that this wastewater has a negative effect on the health of the local population, who get their drinking water from rivers and streams where these effluents are discharged [START_REF] Nsoe | Impact assessment on the waste from the rubber factories in Cameroon[END_REF][START_REF] Nik | Membrane Bioreactor for the treatment of natural rubber wastewater[END_REF][START_REF] Arimoro | Impact of rubber effluent discharges on the water quality and macroinvertebrate community assemblages in a forest stream in the Niger Delta[END_REF][START_REF]Rapport mondial des Nations Unies sur la mise en valeur des ressources en eau 2017. Les eaux usées, une ressource inexploitée[END_REF]. At the environmental level, we are seeing the eutrophication of mangroves and the reduction of oxygen in rivers. This is causing the disappearance of more than 200 aquatic species in the Niger Delta [START_REF] Arimoro | Impact of rubber effluent discharges on the water quality and macroinvertebrate community assemblages in a forest stream in the Niger Delta[END_REF][START_REF] Swarna | Bioremediation of rubber processing industry effluent by Arthrobacter sp[END_REF]. Given the health and environmental concerns related to this compound, it's important to be aware of its potential risks. It is important to treat the effluents resulting from the coagulation of rubber latex before they are discharged into nature.

Lagooning and oxidation pits are the most commonly used processes to treat rubber industry effluents [START_REF] Mitra | Treatment of wastewater from rubber industry in Malaysia[END_REF][START_REF] Tekasakul | Environmental problems related to natural rubber production in Thailand[END_REF]. There are limits to these processes. In particular, it has a large footprint, requires a long residence time for contact between microorganisms and contaminant loads, emits odors, and lacks compliance with legal restrictions on waste. To overcome these problems, other processes such as biofiltration [START_REF] Ndi | Elimination de l'azote et des Sucres de l'effluent de Caoutchouc par Biofiltration[END_REF], electrolysis [START_REF] Vijayaraghavan | Electrolytic treatment of Standard Malaysian Rubber process wastewater[END_REF], coagulation/ aggregation [START_REF] Ozbelge | Removal of phenolic compounds from rubber-textile wastewater by physicochemical methods[END_REF], membrane bioreactor [START_REF] Nik | Membrane Bioreactor for the treatment of natural rubber wastewater[END_REF] and ultrafiltration [START_REF] Konieczny | Ultrafiltration of latex wastewaters[END_REF] are also being studied in the laboratory.

Among the many processes still under study, processes using strains of microorganisms with a high purifying power are promising. Studies focus on testing the biodegradation of wastewater by bacteria [START_REF] Swarna | Bioremediation of rubber processing industry effluent by Arthrobacter sp[END_REF][START_REF] Jai | Rubber processing industry effluent treatment using a bacterial consortium[END_REF][START_REF] Senthil | Microbial diversity in rubber industry effluent[END_REF][START_REF] Shruthi | Bioremediation of rubber processing industry effluent by Pseudomonas sp[END_REF][START_REF] Cherian | Microbial degradation of natural rubber latex by a novel species of Bacillus sp. SBS25 isolated from soil[END_REF], but very little on yeast.

However, the use of yeast for wastewater treatment shows that the latter achieve better results than bacteria. In fact, the use of yeast allows to reduce sludge production, increase nitrification yield and increase resistance to inhibition with nitrogen, metals and phenolic compounds [START_REF] Alam | Enhanced settleability and dewaterability of fungal treated domestic wastewater sludge by liquid state bioconversion process[END_REF]. In the treatment of rubber effluents, Ndi et al. [START_REF] Ndi | Elimination de l'azote et des Sucres de l'effluent de Caoutchouc par Biofiltration[END_REF] used two yeast strains isolated from rubber effluents with yields in excess of 80% in total nitrogen removal. However, the main disadvantage of using yeast is the source of additional contamination [START_REF] Prigione | Decoloration and detoxification of textile effluent by fungal biosorption[END_REF][START_REF] Pokrel | Treatment of pulp and paper mill wastes[END_REF][START_REF] Sakurai | Removal of lignin in a liquid system by an isolad fungus[END_REF]. To separate this filamentous sludge from the treated water, membrane bioreactors could be used satisfactorily, which would limit the pressure on water resources, comply with increasingly restrictive norms and laws, and most importantly, reuse this water. For this reason, the biological degradation tests of a rubber waste water as well as the characterization of the sludge obtained are examined in this work.

MATERIEL AND METHODS

Yeast Inoculum

The yeast strains used in this work were isolated from the effluents of a rubber industry thanks to Nsoe protocol [START_REF] Ndi | Evaluation of the duration of inoculation of a granular pozzolan biofilter from strains of indigenous bacteria and yeasts isolated from a rubber industry effluent[END_REF][START_REF] Nsoe | Isolement et caractérisation des levures endogènes à fort potentiel de croissance dans les effluents de caoutchouc, 20e Conférence annuelle du Comité Camerounais de Biosciences[END_REF]. The yeast characteristics are shown in Table 1. Yeast were cultivated on the following growing medium [START_REF] Nsoe | Biodegradation of Ammonium Ions and Formate During Ammonium Formate Metabolism by Yarrowia lipolytica and Pichia guilliermondii in a Batch Reactor[END_REF]: meat extract 1 g L -1 (Sherlan réf 07-075, Spain), yeast extract 2 g L -1 (OXOID code L21, England), peptone 5 g L -1 (Liofilchem réf 610038, Italy), and sodium chloride 5 g L -1 (Jeulin réf 107 115, France) at pH = 6.5.

Experimental Set-up: Membrane Bioreactor

The submerged membrane bioreactor pilot (Figure 1) studied consists of a plexiglass feed tank with a capacity of 40 L and 35 L of useful volume. A RENA brand compressor is used to inject air at the bottom of the reactor through a thin cylindrical PVC bubble diffuser; 8 cm diameter. The aeration cycles were fixed using a COGEX brand timer in order to have a maximum oxygen concentration of 6 mg.L

Preparation of Synthetic Rubber Wastewater

In order to get closer to the real rubber effluent, the preparation was made using the latex taken from a rubber tree field, and kept at pH 9-10 with ammonia to prevent its coagulation. The latex is coagulated in a reactor (1L) in which it is kept under mechanical stirring using a stirrer (EUROSTAR, digital IKA-WERKE, Allemagne). Latex pH is determined using a portable pH meter (Fisher Bioblock Scientific type pH 330i WTW82362 Wellhem,Allemagne). 2% formic acid is introduced at 5min intervals until rubber coagulation. The coagulated rubber is then removed and then dried.

The yellowish colored filtrate obtained represents the serum which is very concentrated. A dilution with a dilution factor of 9 is carried out in order to obtain pollutant concentrations close to those found in industrial effluents [START_REF] Ndi | Evaluation of the duration of inoculation of a granular pozzolan biofilter from strains of indigenous bacteria and yeasts isolated from a rubber industry effluent[END_REF]. The physicochemical parameters of the synthetic effluent are presented in Table 2.

Analytical Methods

Samples for analysis were taken on a regular basis at three locations. On entry into membrane bioreactor, the mixed liquor (The effluent inside the bioreactor during biodegradation) is taken halfway up the reactor and centrifuged at 5000 rpm, 4°C, 10 min to each sample and finally the treated effluent (permeate). The wastewater was analyzed for Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), using the standard techniques described by [START_REF] Nsoe | Biodegradation of Ammonium Ions and Formate During Ammonium Formate Metabolism by Yarrowia lipolytica and Pichia guilliermondii in a Batch Reactor[END_REF] (APHA, 1998). The turbidimeter used is of brand HACH RATIO/XR TURBIDIMETER (0-2000 NTU) de HACH Inc. (USA). Ammonium ions (NH4+) where titrated by a portable mini-photometer of mark HANNA Checker HC ® HI 733 Woonsocket RIUSE ROMANIE. Nitrates were measured using a brand HORIBA LAQUAtwin B-743. Nitrites using a branded mini portable photometer HANNA Checker HC ® HI 764 Woonsocket RIUSE ROMANIE. The SS and EMS are thus calculated from the following equations 1 and 2 [START_REF] Rodier | L'Analyse de l'eau[END_REF]: The proteins are assayed according to the method of Lowry [START_REF] Lowry | Protein measurement with the folin reagents[END_REF] modified by Frolund. The assay of the polysaccharides was carried out by the colorimetric method proposed by Dubois et al. [START_REF] Dubois | Colorimetric method for determination of sugar and related substances[END_REF]. The volume of sludge and the Mohlman index were measured according to the standard methods protocol 2700 D [START_REF]Standard methods for the examination of water and wastewater, 21st Ed[END_REF]. A MASTERSIZER type laser particle sizer (HYDRO 2000SM) from MALVERN Instruments (England) is used to determine the particle size distribution of the sludge. . On the other hand, in the supernatant inside the bioreactor, a reduction in COD is observed around 500 mg.L -1 . COD reduction is caused by the activity of yeasts which degrade organic matter. The residual concentrations of COD at the outlet of the BRM are also presented. An almost total COD reduction was observed (≥ 98%), regardless of the initial concentration at the bioreactor inlet. These results are similar to those found by Nik [START_REF] Nik | Membrane Bioreactor for the treatment of natural rubber wastewater[END_REF] who studied the treatment of a rubber effluent in a submerged membrane bioreactor with a COD reduction of almost 96%. This shows us that the yeast strains studied are capable of improving the treatment of rubber effluents. The elimination of nitrogenous compounds in wastewater by microorganisms goes through two main steps: nitrification and denitrification. Nitrification takes place in the presence of oxygen and denitrification takes place in the absence of oxygen. But in our system all the reactions take place in the presence of oxygen with high yields of more than 90%. This leads us to believe that the yeast strains used metabolize nitrogen compounds in the presence of oxygen without an anoxia phase. But we notice a low production of nitrite: This is in agreement with the work of Kurakov and Popov [START_REF] Kurakov | Nitrifying activity and phytotoxicity of soil microscopic fungi[END_REF] who demonstrated that the use of yeasts in the degradation of nitrogenous compounds led to a reduction or absence of the production of nitrites. On the other hand, a high production of nitrates is observed [START_REF] Imajo | Granulation of Anammox microorganisms in up-fl ow reactors[END_REF][START_REF] Schmidt | Aerobic and anaerobic ammonia oxidizing bacteria competitors or natural partners[END_REF][START_REF] Lotti | The effect of nitrite inhibition on the anammox process[END_REF].

RESULTS AND DISCUSSIONS

COD Reduction

Reduction of Ammonium ions, Nitrites and Nitrates in the Membrane Bioreactor

Evolution of pH

Observation of the curves in Figure 4 shows the evolution of the pH in the bioreactor. And at the output of the filter present. These curves show two phases: a first phase during which the pH decreases by 6 and reaches a minimum value at pH 5.6. The drop in pH may be the consequence of the assimilation of organic matter linked to the growth of biomass and the consumption of the nitrogen contained in the effluent. The assimilation by the latter of nitrogen in the form of ammonium ion is known to cause a drop in pH during the process [START_REF] Huberson | Evolution du pH Pendant la fermentation alcoolique de moûts de raisins : modélisation et interprétation métabolique[END_REF], the cause being the expulsion of protons into the medium for each mole of NH 4 ⁺ consumed because according to Metahri. [START_REF] Delrue | Modélisation du procèdé bioréacteur a membranes immergées : calage et validation du modele ASM 1 sur un site réel -étude des interactions boues activées, conditions opératoires et membrane[END_REF] nitrification leads to the production of H + protons which leads to a decrease in pH. On the other hand, in the second phase during which the pH increases from 5 to pH 7, the opposite phenomenon occurs: the H + protons are used by the microorganisms causing an increase in the pH due to the phenomenon of denitrification. These results allow us once again to affirm that the yeasts used are capable of assimilating formate and certainly of carrying out nitrification and denitrification in aerobiosis.

Relationship Between Turbidity and Suspended Matter

During operation of the pilot, the SS concentration and turbidity in the bioreactor were monitored. These turbidity trends in the bioreactor are also observed for SS as can be seen in Figure 6. According to Delrue, [START_REF] Massé | Bioréacteur à membranes immergées pour le traitement des eaux résiduaires urbaines[END_REF] and Massé, [START_REF] Tijhuis | Formation of nitrifying biofilms on small suspended particles in airlift reactors[END_REF], the decrease in turbidity or SS during the first phase can be explained by a period of adaptation of the yeasts to the new environment. As for the increase in turbidity and SS, this is caused by the development of biomass with secretion of exopolymers. With regard to the decrease in turbidity up to 47 NTU and SS 350 mg/L, this phenomenon can be explained by the fact that the molecular weight of the biomass becomes increasingly high. This will cause it to settle at the bottom of the reactor. In order to find a relationship between SS and turbidity, we plotted the graph of SS versus turbidity as shown in Figure 7.

Figure 7 shows that there is a linear relationship between suspended matter and turbidity during biodegradation, the equation of which is: NTU = 0.52SS -136.27 with an R 2 of 0.98. In order to determine if our sludge is settleable, we studied the biomass sludge index during biodegradation.

Relationship between Mohlman Index and Suspended Matter

Figure 8 shows the relationship between the Mohlman Index and suspended solids after 30 minutes of settling at each sample during biodegradation. We observed that the sludge index decreases with the increase in SS concentration according to a linear law (MI = -3.78*SS -582) with an R 2 of 0.96. The decrease in the sludge index, which is the volume occupied by one gram of sludge, translates the densification of the flocs and leads to a better capacity for thickening and better settleability of the sludge. This allows us to conclude that the sludge resulting from the combination of the two strains does not float. This is why knowledge of sludge particle size is necessary.

Floc Particle Size

The size distribution of the flocs formed was analyzed at the end of the experiment (Figure 9). The sludge resulting from the biodegradation of the synthetic rubber effluent shows a trimodal distribution with average sizes of 10 µm, 100 µm and 700 µm. This explains the observed settling of sludge in the study of the sludge index. In an expanded biomass process, the size of the flocs is an important parameter which conditions the nitrifying activity of the yeasts [START_REF] Boran | Floc size distribution and bacterial activities in membrane separation activated sludge processes for small-scale waswater treatment/reclamation[END_REF]. When the (hydrodynamic) conditions allow it, too high an increase in aggregates can simultaneously cause nitrification and denitrification in the same reactor. This may explain the reduction of nitrites and nitrates observed in the bioreactor. Similarly, in a study of the distribution of flocs and the influence of their size on the activity of microorganisms, in a membrane bioreactor, Boran et al. [START_REF] Mikkelsen | Physico-chemical characteristics of full scale sewage sludges with implications to dewatering[END_REF] concluded that the specific nitrification rate increases with size.

Determination of some physico-chemical parameters of sludge

Proteins and polysaccharides (Table 3) are analyzed at the end of the operation to assess their influence on membrane resistance From Table 3, the protein concentration is higher than the polysaccharide concentration by 2.23 times. These results are in agreement with those of Mikkelsen and Keiding [START_REF] Petros | Biomass Characteristics and Their Effect on Membrane Bioreactor Fouling[END_REF] who based themselves on sludge samples from different purification systems to demonstrate the influence of the quantity of proteins in the resistance to filtration. They explained that a high concentration of proteins in the flocs is essential for the formation of well-structured flocs, and therefore for a low resistance to sludge filtration. They concluded that the amount of protein is important for the formation of a well-structured floc. Along the same lines, Petros et al., [START_REF] Wilén | The influence of key chemical constituents in activated sludge on surface and flocculating properties[END_REF], linked sludge filterability and sludge resistance to the protein/polysaccharide ratio compared to the total concentration of EPS or the concentration of individual compounds. This better correlation is explained by the neutralization of charges between proteins and polysaccharides. Affecting the surface characteristics. This neutralization of the charges will lead to a reduction in the surface charge and consequently the flocculation capacity [START_REF] Who | Directives OMS pour l'utilisation sans risque des eaux usées, des excreta et des eaux ménagères[END_REF].

Characterization of the Effluent after Treatment

The effluent parameters obtained allow us to comment on the physicochemical quality of the water obtained and to conclude whether this water is treated and perhaps can either be reused for specific uses or be discharged into nature. The results obtained were compared to Cameroonian standards for industrial effluent discharges and to WHO and FAO standards for use in irrigation. With regard to Cameroonian discharge standards, all the parameters followed comply with Cameroonian standards, which proves that the use of submerged membrane bioreactors makes it possible to have water that can be discharged into nature without any risk of pollution. Moreover, by comparing the values obtained with the standards the use of treated wastewater for irrigation and watering [START_REF] Who | Directives OMS pour l'utilisation sans risque des eaux usées, des excreta et des eaux ménagères[END_REF][START_REF] Fao | Irrigation avec des eaux usées Traitées[END_REF], we find that this water can be reused in irrigation and watering. fields with a nutrient supplement.

CONCLUSION

Rubber wastewater discharged without treatment pollute the receiving waterways and the soil. The objective of our work was to follow some physicochemical parameters during the biodegradation kinetics of a synthetic rubber effluent in the presence of yeast strains. The COD was abated to more than 98%. Nitrification and denitrification proceeded in the presence of oxygen with a purification efficiency of more than 90%. There is a linear relationship between the sludge index and TSS with an R 2 of 0.96. The sludge granulometry is tri-modal with a maximum sludge size of 1000 µm. The sludge formed is resistant to filtration with a polysaccharide/protein ratio of 0.45.

According to the physicochemical data obtained, these waters can be reused in the rubber washing line; which makes membrane bioreactors an avenue to explore for the treatment of rubber effluents.

Figure 1 :

 1 Figure 1: Experimental set-up: Membrane bioreactor [27].

Figure 2 :

 2 Figure 2: Variation of the COD of the wastewater at the inlet of the bioreactor, inside the bioreactor and of the permeate as a function of time during biodegradation.

Figure 2

 2 Figure 2 presents the measurements of the chemical oxygen demand (COD) at the reactor inlet, in the bioreactor and at the outlet (after membrane filtration). Throughout the period of monitoring the functioning of the membrane bioreactor, the COD concentrations at the BRM inlet fluctuated between 3080 mg.L -1 and 2800 mg.L -1

Figure 3 :

 3 Figure 3: Variation of NH4 + , NO2 -, NO3 -as a function of time at (pH 6.28 ± 2°C).

Figure 4 :

 4 Figure 4: Variation of pH as a function of time during the biodegradation of the synthetic effluent in the bioreactor and at the outlet of the membrane filter 28 ± 2°C.

Figure 5 :

 5 Figure 5: Variation of turbidity as a function of time during biodegradation in the bioreactor and after filtration (permeate).

Figure 6 :

 6 Figure 6: Variation of SS in the bioreactor as a function of time during biodegradation.

Figure 5

 5 Figure5above shows us the evolution of turbidity as a function of time in the bioreactor and at the outlet of the bioreactor. In the bioreactor, we observe three phases. The first phase is characterized by the decrease in turbidity from 25 NTU to 20 NTU during the first 25 hours. The second phase characterized by the increase in turbidity in the bioreactor until the 150th hour then drops to a value of 47 NTU at the end of the treatment. On the other hand, at the outlet of the bioreactor after filtration, the turbidity is almost constant throughout the biodegradation up to a value of 1 NTU. These turbidity trends in the bioreactor are also observed for SS as can be seen in Figure6. According to Delrue,[START_REF] Massé | Bioréacteur à membranes immergées pour le traitement des eaux résiduaires urbaines[END_REF] and Massé,[START_REF] Tijhuis | Formation of nitrifying biofilms on small suspended particles in airlift reactors[END_REF], the decrease in turbidity or SS during the first phase can be explained by a period of adaptation of the yeasts to the new environment. As for the increase in turbidity and SS, this is caused by the development of biomass with secretion of exopolymers. With regard to the decrease in turbidity up to 47 NTU and SS 350 mg/L, this phenomenon can be explained by the fact that the molecular weight of the biomass becomes increasingly high. This will cause it to settle at the bottom of the reactor. In order to find a relationship between SS and turbidity, we plotted the graph of SS versus turbidity as shown in Figure7.

Figure 8 :

 8 Figure 8: Relationship between suspended solids (SS) and Mohlman Index (MI).

Figure 9 :

 9 Figure 9: Floc size distribution.

Figure 10 :

 10 Figure 10: showing the color of the effluent before (A) and after treatment (B).

Table 1 : Characteristics of Yeast Strains [26] Growth in selective media Code Yeast color Potentiel Zêta pH (5,7-7) Yeast shape Yeast size (µm) Acid medium Alkaline medium

 1

	Yarrowia lipolytica	Red	-25,3 à -37,8	Oval	0,3-150	yes	no
	Pichia guilliermondi	White	-27,8 à -30,5	Oval	0,2-100	yes	no

Table 2 : Physico-chemical Characteristics of Synthetic Rubber Effluent Parameters Values Cameroonian standard, 2005 [28]

 2

		5.9±2	5.5 pH 9.5
	Turbidity(UNT)	27.4	<10
	ConductivitY (µS/ cm)	1300	< 400
	organic matter (%)	13.26	/
	COD (mg O2/L)	3050	< 400
	Ammonium (mg/L)	153	< 20
	Color (EBC)	8.325	/
	BOD (mg/O2 /L)	1087	<100

Table 3 : Composition of Sludge (Protein and Polysaccharide)

 3

	Polysaccharide (mg/mg DM)	94 ± 3
	Protein (mg/mg DM)	210 ± 7
	Rapport polysaccharide / Protein	0.45

Table 4 : Physico-Chemical Characteristics of the Effluent before and after Treatment

 4

	Parameters	Unit	Waste water before treatment	Waste water after treatment	[28]	[1, 45]
	Turbidité	(NTU)	21	12	< 10	/
	pH	/	6.1	8.5	5.5<pH<9.5	6.5-8.6
	NH4+	(mg/L)	66.13	38	10	<100
	NO3 -	(mg/L)	8.5	12,5	20	<50
	BOD	(mg /L)	1087	17	<100	28
	COD	(mg/L)	3050	69	<400


Keywords: abandoned mine, multi-hazard, interaction, matrix, loop

Mining, natural and technological hazards can occur in a former mining site. The multi-hazard analysis becomes critical. This paper aims to establish the methodological basis for assessing the interactions between the main hazards identified in abandoned mines. The interactions between 57 hazards are analysed based on: theoretical aspects, feedback analysis and expert opinions. Interaction matrices and loops are used, helping to study the interactions between hazards.

INTRODUCTION, OBJECTIVE AND METHODOLOGY

The number of abandoned mines is continuously increasing in the world. Several hazards can affect former mining sites [START_REF] Isrm | Mine Closure and Post-Mining Management -International State of Art[END_REF]. Generally, several square kilometres of a mine site may be vulnerable to various types of hazards, including mining hazards, which may interact with each other. Multi-hazard assessment is mandatory in this case. Multi-hazard is frequently used to describe such a situation (Gill & Malamud, 2016).

This paper aims to present a first reflection to develop a methodology for abandoned mines which allows, in the long term, the identification and evaluation of the potential interactions between hazards. First, the methodology identifies residual mining, natural, and technological hazards. Then, three types of interactions were sought: mining hazards versus mining hazards, mining hazards versus natural hazards and mining hazards versus technological hazards. Interaction matrices and loops have made it possible to facilitate the analysis and visualisation of potential interactions. The potential retained interactions are the result of the following:

• the theoretical basis of phenomena,

• back analysis of the real-life case studies and,

• the feedback of the experts.

Three levels of interaction have been considered: no interaction or zero interaction potential; unlikely interaction(s) or low interaction potential and likely interaction(s) or high interaction potential.

Development of post-mining multi-hazard assessment methodology

Hippolyte Djizanne

DESCRIPTION AND ASSESSMENT OF HAZARDS

The hazards that may occur in a former mining site are grouped into three main categories: 19 mining hazards, 21 natural hazards and 17 technological hazards; see Table 3. For mining hazards, hazard qualification relies on predisposing factors. However, natural and technological hazard qualifications are based on the probability of occurrence or the severity of a hazardous event, meaning that a qualitative, semi-quantitative, or purely quantitative approach is sufficient for their qualification.

Mining hazards

The methodological guide for assessing mining hazards established by Ineris [START_REF] Ineris | Evaluation of mining hazards[END_REF] provides details on mining hazards listed in Table 3. Mining hazards can have several origins: ground movement hazards, self-heating hazards (only coal mines), hydrological and hydrogeological hazards, and gas release hazards. Induced seismicity, under certain conditions, after the cessation of mining operations also is considered in this article. However, this analysis does not consider the pollution hazard from an abandoned mine. A single mining hazard qualification depends on its intensity and the predisposition of the studied mine site. This assessment includes three intensity classes (limited, moderate, and high) and three predisposition classes (very insensitive, sensitive and very sensitive). Table 1 provides an example of the cross-referencing of predisposition and intensity, which results in assessing the mining hazard defined in three levels: low, moderate and high.

Natural hazards

Natural phenomena are increasingly well-known, studied and mapped at all territorial scales (Table 3). These natural hazards represent two groups: land-related and climate-related. Most atmospheric hazards are not considered in the remainder of this methodology as they have little or no interaction with mining hazards. Given the diversity of procedures available for natural hazard evaluation, this section presents a single evaluation method for flood hazard assessment. Indeed, the natural flood hazard of an area corresponds to its slow or rapid submergence when it is usually out of water. The hazard qualification corresponds to four levels: low, moderate, high, and very high, according to the water height and the dynamics linked to the combination of the water flow speed and the water rise speed (see Table 2).

Technological hazards

Technological hazards result from permanent, intense or repetitive human activity around the abandoned mine site. They correspond to thermal effects, toxic effects, overpressure effects, and structure-related hazards (failure of civil engineering structures). Four classes help to define the intensity of the technological hazards: indirect, moderate, severe and very severe (Table 4). The probability of occurrence of an event (phenomenon), industrial installations are classified into five classes (French ministerial decree of 09/29/2005) from E (a possible but improbable event) to A (regular event).

Intensity

Moderate Indirect

Probability of occurrence

> D 5E to D < 5E All (A, B, C)

Hazard level

Medium+. Medium Low Low

IDENTIFICATION AND ASSESSMENT OF PHYSICAL INTERACTIONS BETWEEN HAZARDS

No methodological framework for multi-hazard analysis is dedicated exclusively to post-mining [START_REF] Touili | Managing multiple hazards in urban areas: an integrated multi-hazard analysis model for overall resilience[END_REF]. Nevertheless, several methodological tools are available to study the interactions between natural hazards: the interaction matrix, the interaction loop, fault trees, multi-criteria analysis, and statistical vulnerability modelling, including temporal variability. The tools presented in this article are the interaction matrix and the interaction loop. The hazard interaction matrix displays the typology and potential of expert judgement interactions between source and target hazards. An interaction loop is a display tool which allows the typology and interactions between a source hazard, placed at the centre of the interaction loop and one or more target hazards which revolve around the source hazard.

Hazard interaction matrix

The qualification of the hazard interaction is as follows:

• The interaction between two hazards has a zero potential (white colour also means no interaction) when they cannot interact at the same place due to the absence of common factors qualifying the two hazards or their associated mechanisms. • The interaction between two hazards has a low potential (blue colour also means unlikely interaction) when the interaction is phenomenologically possible but not yet observed or when the changes caused are limited in scope. • The interaction between two hazards has a high potential (red colour also means likely interaction) when the hazard interaction has already been in the same area or when the changes caused are very significant.

Figure 1 presents the hazard matrices based on the theory, feedback, and experts. The matrix of mining hazards is 19x19, which can potentially allow 342 interactions; however, at this stage of the study, only 128 interactions are possible (blue and red), corresponding to 37%. Forty-eight interactions present a high potential occurrence (red), corresponding to 14%. The matrix of miningnatural hazards is 19x21, which can potentially allow 399 hazard interactions. However, only 54 hazard interactions (blue and red) are possible, corresponding to 14%. Fifteen interactions present a high potential occurrence (red), corresponding to 4%. The matrix of mining-technological hazards is 19x17, potentially allowing 323 (19x17) interactions. Two hundred ninety-one interactions are possible (blue and red), corresponding to 90%. One hundred seventy-nine interactions present a high potential occurrence (red), corresponding to 55%. Analysis of these matrices shows that interaction between source mine and natural hazards is less frequent than between mining hazards alone and that mining hazards interact mainly with flood hazards, whether mining or natural. The mine hazard can heavily impact technological hazards.

Mining-mining hazards

Mining-natural hazards Mining-technological hazards Figure 1. Hazard matrices based on the theory, feedback, and experts (red case: high potential interaction, blue case: low potential interaction, white case: no interaction).

Hazard interaction loops

The construction of the loops considers the source hazard at the centre of an interaction loop. The arrow from the loop centre to the target hazard can be split in the opposite direction, allowing a single or double direct interaction. The arrow colour represents the potential of the interaction. This mode of presentation, complementary to the interaction matrix, deals with one source hazard at a time; for example, Figure 2 presents interaction loops between a subsidence (SUB), a sinkhole (FON) and a generalised collapse (EFG) and the other hand mining, natural and technological hazards. For instance, subsidence can interact with eleven mining hazards, and eight mining hazards can interact with subsidence. Another example is the localised collapse mining hazard which can interact with all seventeen technological hazards. However, only two technological hazards can interact with the localised collapse mining hazard.

CONCLUSION

This paper proposes a methodological and representation basis for assessing interactions between hazards identified around former mines. After recalling the advantages of this multi-hazard analysis, the methodology consisted of identifying these hazards, reflecting the methods of individual evaluation, analysing the predisposition factors to identify by expert opinion and evaluating the potential of the possible interactions, and finally validating this methodology on concrete cases. The interaction identification is based primarily on hazard knowledge (nature of the event, predisposition or probability of occurrence and intensity). The three categories of hazards do not have the same evaluation methods. Among the tools used to display the results of the interactions, the authors retained the matrices and the loops as two complementary representation tools. Testing the method on former mining sites concerned by these interactions will be necessary, allowing better

  SUB AFC CRE FON EFG TAS GSP GSS RAV COU EBO CHT GAZ COM IME IMC IPB IBE GON AVA TAS GSP GSS RAV COU EBO CHT ERC SIS ERP INO FEU PRE CYC OUR risk and thus better preservation of the general interests identified around the abandoned mines.

Figure 2 .

 2 Figure 2. Interaction loops between subsidence (SUB), localised collapse (FON) and generalised collapse (EFG) mining hazards (red arrow: high potential interaction, blue array: low potential interaction).

Table 1 .

 1 Example of mining hazard assessment by cross-referencing predisposition and intensity for mining hazard assessment[START_REF] Ineris | Evaluation of mining hazards[END_REF].

	Intensity	Predisposition Very insensitive	Sensitive	Very sensitive
	Limited	Low	Low	Medium
	Moderate	Low	Medium	High
	High	Medium	High	High

Table 2 .

 2 Qualification of the flood hazard according to the height and speed of the water.

	Intensity	Water level	Dynamics
			Slow dynamics	Medium dynamics	Fast dynamics
	Limited	H < 0.50 m	Low	Moderate	High
	Moderate	0.50 < H < 1 m Moderate	Moderate	High
	High	1 m < H < 2 m	High	High	Very high
	Very high	H > 2 m	Very high	Very high	Very high

Table 3 .

 3 Summary of the mining, natural and technological hazards used in this multi-hazard analysis.

	AFF Gas explosions EXP	FON Slick fire (liquid) FEN	DIS Flare fire (gas or liquid) FET	GON Solid fire (combustible solids) FES	GSP Boil over (heavy hydrocarbons) BLO	GSS BLEVE (flammable liquefied gases) BLV	RAV Liquid product release with vaporisation RPL	ERC Gaseous product release RPG	COU Release of a liquefied gas RGL	EBO Fire with the decomposition of toxic products IPT	RSR CHT Release of radioactive substances	RME AVA Discharge of water bodies	MVT Land movement due to human activities SIS	EBC Tank burst (Pneumatic energy release) ERP	VCE VCE (Combustion of gases, vapours) FEU	BLV BLEVE (explosive vaporisation) TAS	ENA An explosion of solids (ammonium nitrate, pyrotechnics	INO			PRE	CYC	OUR	TEM
	SUB Settlement	AFC Localised collapse (sinkhole)	CRE Dissolution (e.g., gypsum, chalk or salt)	FON Clay shrinkage or settlement	EFG Deep landslide	TAS Shallow landslide	GSP Gullying, reptation (erosion)	GSS Coastal erosion	RAV Mudflow	COU Slump	EBO Rock or block fall	CHT Avalanche	COM Earthquake	GAZ Volcanic eruption	IME Forest fire	IMC Settlement, consolidation	IPB Lowland flooding, as opposed to torrential flooding	IBE Flooding by runoff and mudslides	SIS Flooding by rising groundwater	Flooding by marine submersion	Heavy precipitation	Cyclone	Hurricane	Storm
		Brittle subsidence	Crevasse	Localised collapse (sinking)	Generalised collapse	Settlement linked to mining works	Deep landslide	Shallow landslide	Gullying, reptation or erosion	Mudflow	Slump	Rock or block fall	Heating of veins or slag heaps	Mine gas	Modification of the groundwater	discharge regime	Modification of the regime of a river	Flooding of topographic low points	Flash flooding -submergence	Induced seismicity in former mines

Table 4 .

 4 Technological hazard assessment

	Intensity	Very severe
	Probability of occurrence	> D	5E to D	< 5E
	Hazard level	Very strong +	Very Strong	Strong +
	Intensity	Severe
	Probability of occurrence	> D	5E to D	< 5E
	Hazard level	Strong +	Strong	Medium+.

ACKNOWLEDGEMENTS

This work has received funding from the European Union's Research Fund for Coal and Steel (RFCS) under the European project "POMHAZ" grant agreement No 101057326.

Mining hazards (19) Code Natural hazards (21) Code Technological hazards (17) Code Subsidence or progressive subsidence