Justine Viet

David Reboutier

Serge Hardy

Salil A Lachke

Luc Paillard
email: luc.paillard@univ-rennes1.fr

Carole Gautier-Courteille

Carole Gautier- Courteille

Triveni Menon

Sreelaja Nair
email: s.nair@tifr.res.in

Modeling ocular lens disease in Xenopus

Keywords: Cataract, CRISPR, Pathology, GJA8, PAX6, DNASE2B

come

INTRODUCTION

The ocular lens is an avascular structure in the eye whose function is to refract and focus light on the retina [START_REF] Cvekl | Signaling and Gene Regulatory Networks in Mammalian Lens Development[END_REF] . A major property of lenses is their transparency, and lens clouding, or cataract, is the leading cause of blindness worldwide. While most cataracts are age-related, there exist congenital cataracts, which can be genetic [START_REF] Shiels | Mutations and mechanisms in congenital and age-related cataracts[END_REF] . Their incidence is 1 to 6 children per 10,000 births [START_REF] Foster | Cataract in children[END_REF][START_REF] Francis | The genetics of childhood cataract[END_REF][START_REF] Shiels | Molecular Genetics of Cataract[END_REF] .

Cataracts are cured by replacing opaque lenses by artificial intraocular lenses. Despite generally excellent outcomes of cataract surgery, post-operative complications can occur, such as capsule rupture and loss of vitreous humour, infections, endophthalmitis or glaucoma. At a longer term, posterior capsular opacification (secondary cataract) results from the proliferation of epithelial cells that remain in the capsular sac after lens removal [START_REF] Nibourg | Prevention of posterior capsular opacification[END_REF][START_REF] Wormstone | Posterior capsule opacification[END_REF] . The annual number of patients with complications is far from negligible considering the high frequency of cataract surgeries. Cataract therefore remains a major public health issue, and a better understanding of the pathophysiological mechanisms that cause it as well as the quest for innovative treatments are highly important [START_REF] Anand | Systems biology of lens development: A paradigm for disease gene discovery in the eye[END_REF] .

While there are several models for cataract research, they vary in their strengths and weaknesses. 2D cell culture models only recapitulate a tiny part of the molecular events leading to cataracts. A 3D cell culture model (lens organoid) was recently developed as a promising in vitro model [START_REF] Murphy | Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro[END_REF] . Animal models of cataract are the zebrafish "cloche" mutants, which tend to have lens defects suggestive of cataracts [START_REF] Goishi | AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens[END_REF] , and mammals like rodents, rabbits or dogs [START_REF] Chan | Animal Models of Ophthalmic Diseases[END_REF] . For ethical and cost reasons, these models cannot be considered for large-scale studies like drug screening.

Xenopus embryos have several advantages as models of human pathologies. They develop externally, and raising hundreds of Xenopus embryos is highly cost-effective. The high number of raised embryos ensures unequalled statistical power of the observations. Xenopus are tetrapods, hence evolutionary closer to humans than other small multicellular models like flies, worms, or even fishes. Xenopus is amenable to F0 reverse genetics, i.e. analyses of phenotypes of larvae developed from genetically modified embryos without any requirement for time-consuming crosses.

However, only a few articles deal with cataract in Xenopus. Knocking-down sparc with antisense morpholinos in Xenopus results in a high mortality due to defective cell-cell adhesion after gastrulation, but surviving tailbuds injected with a reduced amount of morpholino display poorly characterised lens phenotypes evocative of cataract [START_REF] Huynh | Knockdown of SPARC leads to decreased cell-cell adhesion and lens cataracts during post-gastrula development in Xenopus laevis[END_REF] . Knocking-down the orthologues of Developmental Dynamics mammalian cataract genes like TMEM114, CHRLD1, SIPAL3 or CELF1 produces eye phenotypes generally loosely related to cataract [START_REF] Maher | The cataract-associated protein TMEM114, and TMEM235, are glycosylated transmembrane proteins that are distinct from claudin family members[END_REF][START_REF] Pfirrmann | Molecular mechanism of CHRDL1-mediated Xlinked megalocornea in humans and in Xenopus model[END_REF][START_REF] Rothe | An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation[END_REF][START_REF] Siddam | The RNA-binding protein Celf1 posttranscriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development[END_REF] . Hence, whether ocular lens developmental defects, including cataract, can be modelled in Xenopus remains an open question. A prerequisite to modelling cataract is to have a good understanding of normal lens development. A stage series of Xenopus lens development was proposed decades ago [START_REF] Mcdevitt | Ontogeny and localization of the crystallins during embryonic lens development in Xenopus laevis[END_REF] . More recent articles investigate Xenopus lens development, but are limited to early stages up to Nieuwkoop and Faber stage 38-40 18,[START_REF] Ishibashi | Distinct roles of maf genes during Xenopus lens development[END_REF] . In another article, these early stages are compared with a much older stage 50 embryo, but intermediate stages are not described [START_REF] Mizuno | Lens regeneration in Xenopus is not a mere repeat of lens development, with respect to crystallin gene expression[END_REF] . Lens regeneration following lensectomy has also been extensively investigated in amphibians. In Xenopus, lens regeneration was divided into 5 stages characterized by precise cellular and molecular events, and much has been learnt from regeneration studies regarding lens development. However, lens development and lens regeneration are two different phenomena. During development, the lens placode originates from the preplacodal ectoderm whereas it results from cornea transdifferentiation during lens regeneration [START_REF] Henry | Cornea-lens transdifferentiation in the anuran, Xenopus tropicalis[END_REF][START_REF] Henry | Molecular and cellular aspects of amphibian lens regeneration[END_REF][START_REF] Servetnick | Lens induction in axolotls: comparison with inductive signaling mechanisms in Xenopus laevis[END_REF][START_REF] Vergara | Lens regeneration: a historical perspective[END_REF] .

Furthermore, gene expression patterns differ between lens development and regeneration [START_REF] Mizuno | Lens regeneration in Xenopus is not a mere repeat of lens development, with respect to crystallin gene expression[END_REF][START_REF] Malloch | Gene Expression Profiles of Lens Regeneration and Development in Xenopus laevis[END_REF] .

Hence, while Xenopus lens regeneration and development share many properties, the underlying cellular and molecular mechanisms are not necessarily the same. These observations call for completing the anatomical description of Xenopus lens development. We first describe lens morphogenesis during Xenopus laevis development. Next, to test if cataract can be detected in Xenopus larvae, we disrupt pax6, gja8 and dnase2b by CRISPR/Cas9-mediated genetic engineering. We selected these genes because they are associated with eye defects or cataract in mammals, and can potentially also be associated with lens defects in amphibians. We investigate the consequences of gene inactivation by combining morphological examination with histochemical analyses and functional vision assays. Disrupting these genes leads to eye phenotypes with variable severity that include lens defects that share several features of cataract in humans. We discuss the potential interest of genetically modified Xenopus larvae to screen molecules that may prevent or cure cataracts and to understand the molecular pathobiology of congenital cataracts.

RESULTS

Lens development in Xenopus laevis

Developmental Dynamics

We made a series of sections of Xenopus embryos to generate a developmental time series reference for normal lens development (Figure 1). During early stages of vertebrate development, the presumptive lens ectoderm thickens to form the lens placode, which invaginates [START_REF] Cvekl | Signaling and Gene Regulatory Networks in Mammalian Lens Development[END_REF][START_REF] Ogino | Transcription factors involved in lens development from the preplacodal ectoderm[END_REF] . In Xenopus, this results in an internalised poorly structured cell mass or lens rudiment 18,20 observed at Nieuwkoop and Faber stage 27 (Figure 1A). At stage 32, the presumptive lens is polarized with anterior cells differing in their aspect from posterior cells, and hence is now referred to as a lens vesicle (Figure 1B). At these stages, the apparent detachment of the lens rudiment or vesicle from the sensory ectoderm (Figures 1A-B) is probably artifactual.

In mammals, the posterior cells of the lens vesicle elongate to form primary lens fiber cells, while the anterior cells form an epithelial layer [START_REF] Cvekl | Signaling and Gene Regulatory Networks in Mammalian Lens Development[END_REF][START_REF] Ogino | Transcription factors involved in lens development from the preplacodal ectoderm[END_REF][START_REF] Rao | The role of the lens actin cytoskeleton in fiber cell elongation and differentiation[END_REF][START_REF] Wride | Lens fibre cell differentiation and organelle loss: many paths lead to clarity[END_REF] . This is comparable to Xenopus stage 38, when a layer of epithelial cells surrounds the primary fiber cells (Figure 1C). The primary fiber cells were reported to form between stages 35 and 41 18 . The primary lens fiber cells then progressively lose their nuclei and other organelles. Kariolysis is detected at Xenopus stage 41 (Figure 1D). The high magnification of stage 41 lens reveals the absence of nuclei in the centralmost fiber cells (Figure 1D').

In mammals, the anterior epithelium is the place of cell divisions. In the so-called transition zone in the equatorial region of the lens, epithelial cells exit the cell cycle, and initiate differentiation into secondary fiber cells which internalize. These secondary fiber cells progressively form rings around the primary fiber cells. This results in a lens nucleus made of the original primary fiber cells and a lens cortex made of the secondary fiber cells. The gradual loss of organelles and nuclei in the fiber cells, and their elongation along the antero-posterior axis corresponding to the light path, support lens transparency and light transmission toward the retina [START_REF] Cvekl | Signaling and Gene Regulatory Networks in Mammalian Lens Development[END_REF][START_REF] Ogino | Transcription factors involved in lens development from the preplacodal ectoderm[END_REF][START_REF] Rao | The role of the lens actin cytoskeleton in fiber cell elongation and differentiation[END_REF][START_REF] Wride | Lens fibre cell differentiation and organelle loss: many paths lead to clarity[END_REF] . This is essentially the same situation in stage 44 Xenopus larvae. However, while in mammals the epithelium is essentially anterior, it seems to cover the fiber cells both anteriorly and posteriorly in Xenopus (Figure 1E). At stage 47, only the epithelial cells that surround the lens have retained their nuclei. The inner fibers are oriented around the antero-posterior axis and the lens is in the form of many concentric layers (Figure 1F,F'). At stage 48 (Figure 1G), the lens has grown further and has the same structure as in adults 29 .

Developmental Dynamics

Strong alterations of eye development in pax6 crispants pax6 is a key transcription factor regulator gene in eye development [START_REF] Altmann | Lens induction by Pax-6 in Xenopus laevis[END_REF][START_REF] Chow | Pax6 induces ectopic eyes in a vertebrate[END_REF][START_REF] Halder | Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila[END_REF][START_REF] Onuma | Conservation of Pax 6 function and upstream activation by Notch signaling in eye development of frogs and flies[END_REF] , and TALENmediated inactivation of pax6 gene has been described both in X. laevis and X. tropicalis [START_REF] Nakayama | Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients[END_REF][START_REF] Suzuki | High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos[END_REF] . To demonstrate that eye phenotypes can readily be observed following CRISPR/Cas9 mediated gene engineering, we co-injected Cas9 nuclease with a single sgRNA against pax6 exon 5 in X. laevis embryos soon after fertilization. X. laevis is allotetraploid, and homoeologous genes (pairs of homologous genes found together in the same allopolyploid genome) are distinguished by their suffix ".L" and ".S" [START_REF] Session | Genome evolution in the allotetraploid frog Xenopus laevis[END_REF] . The region targeted by the pax6 sgRNA is fully conserved between pax6.L and pax6.S. We amplified and sequenced the targeted loci with homoeologous-specific primers.

Figure 2A shows the Sanger chromatograms obtained by sequencing the pax6.L and pax6.S loci amplified from one control and one sgRNA and Cas9-injected embryo. In the edited tadpole, several nucleotides are overlaid at each position around the cleavage site, revealing its mosaicism. We used the ICE (Inference of CRISPR Edits) software to infer the composition of this mixture of sequences [START_REF] Hsiau | Inference of CRISPR Edits from Sanger Trace Data[END_REF] . For each chromatogram, ICE returns a percentage of sequences with insertions-deletions (InDels), which is a proxy for the efficiency of genome editing. ICE analysis revelead genome editing efficiencies in several embryos to be close to 100%, both for pax6.L and pax6.S (Figure 2B).

As compared with control larvae (Figure 2C, upper panel), disrupting pax6 does not strongly modify the overall morphology of stage 41-42 larvae (Figures 2D-E, upper panels). However, eye morphologies of pax6 crispants are affected in the injected embryos. The affected eyes have different aspects, as shown in Figures 2D-E, middle panels. Overall, the number of tadpoles with defective eye morphology is close to 95% (Figure 2F). Sections reveal no lens and a highly disorganised and/or hypotrophied retina (Figures 2D-E, lower panels). To confirm these data, we designed another sgRNA against pax6 exon 5. Gene inactivation mediated by this second sgRNA leads to externally visible eye defects in 84% of the injected tadpoles (Figure 2F), confirming gene editing specificity.

We expected this abnormal eye structure to lead to blindness. To test this hypothesis, we set up a functional vision assay in Xenopus larvae. This assay relies on the preference of tadpoles for white when placed in a tank with both white and black sides [START_REF] Viczian | A simple behavioral assay for testing visual function in Xenopus laevis[END_REF] . Within a pool of 10 tadpoles, we Developmental Dynamics counted the number on the white side every minute for 10 minutes. We repeated this experiment for 8 independent pools. While the cumulative number of tadpoles on white would be close to 50 (corresponding to 50% of the time spent on white) if the tadpoles had no preference for the white side, we observed that control tadpoles spend around 80% of their time on the white side (95% confidence interval [67; 86]). pax6 crispants spend almost the same time on either the white or black background (95% confidence interval of the percentage of time spent on white [50; 59]) (Figure 2G). This strongly suggests that pax6 crispants are virtually blind, though a defective development of the central nervous system in embryos lacking Pax6 39 may be an alternative explanation to these observations.

Disruption of gja8 causes a strong cataract

In human, GJA8 encodes connexin 50, a transmembrane gap junction protein which supports the diffusion of small molecules between neighbouring lens fibers. Genetic variations of GJA8 cause congenital cataracts [START_REF] Shiels | A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract, on chromosome 1q[END_REF][START_REF] Yi | Epidemiology and molecular genetics of congenital cataracts[END_REF] , but can also be involved in age-related cataracts [START_REF] Yu | The impact of GJA8 SNPs on susceptibility to age-related cataract[END_REF] .

We tested if inactivating gja8 in X. laevis could model human cataracts by co-injecting Cas9 nuclease and a sgRNA directed against gja8 exon 1. This sgRNA is expected to target both gja8 homoeologous genes. We separately sequenced gja8.L and gja8.S loci after homoeologous locusspecific amplification to assess gene targeting efficiency. Sanger chromatograms confirmed the mosaicism of gja8 crispants (Figure 3A). ICE analysis revealed that gja8 crispants contain around 80% InDels at gja8.L and gja8.S loci (Figure 3B). To confirm the ICE data, we subcloned one gja8.L amplimere and we sequenced 13 subclones. The obtained sequences and their number of occurences are consistent with the ICE results (Figure 3C). Together, these data show that the gja8 sgRNA targets both gja8.L and gja8.S with a good efficiency.

The overall morphology of gja8 crispants is completely unaltered, compared with sibling buffer-injected embryos (Figures 3D,E, upper panels). However, the eyes of gja8 crispants have greyish appearance in the central region, which is not observed in control embryos (middle panels). This is highly reminiscent of cataract, where lens clouding impedes light diffusion and correct observation of the dark pigmented retinal epithelium. A large proportion (78%, 213/278) of gja8

Developmental Dynamics

crispants have such a cataract detected under a simple binocular magnifier (Figure 3F). Phenotype specificity was confirmed by injecting another sgRNA against gja8, which leads to a similar cataract phenotype albeit with a reduced efficiency (Figure 3F). Histological sections revealed that the morphology of the eye is essentially unaffected in gja8 crispants (Figure 3D-E, lower panels). The retina has all the cell layers found in control eyes.

However, the lenses are smaller in crispants, and the inner cells of crispant lenses retain their nuclei, demonstrating a defective karyolysis that should have occurred at this stage. DAPI staining of lens sections confirm nuclei retention in gja8 crispant lenses (Figures 3G-H). Phalloidin staining reveals that the actin fibers are aligned along the antero-posterior axis in control lenses (Figure 3G). In mammals, this organisation supports the elongation of lens fiber cells along the light path.

Interestingly, it appears abnormal in gja8 crispant lenses (Figure 3H). Hence, at least two features that challenge lens transparency provide a potential explanation for the development of cataract in gja8 crispants, namely, defective organisation of fiber cells along the antero-posterior axis and the abnormal retention of their nuclei in the central region of the lens. Mice or rabbits disrupted for Gja8 show a reduced size of the lens, an arrest of the differentiation of lens fiber cells, and a retention of nuclei [START_REF] Rong | Disruption of Gja8 (alpha8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation[END_REF][START_REF] White | Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts[END_REF][START_REF] Yuan | CRISPR/Cas9-mediated GJA8 knockout in rabbits recapitulates human congenital cataracts[END_REF] . Hence, our observations in Xenopus recapitulate the phenotypes observed in mammals.

We next asked if lens clouding affects the vision of the tadpoles. In the same functional vision assay as before, gja8 crispants have a preference for the white background, indicating that they are not blind (95% confidence interval of the percentage of time spent on the white side of the tank [58; 65]). However, the crispant embryos spend much less time on white than sibling control embryos (Figure 3I. p = 9.1x10 -4 , Wilcoxon test). Hence, the cataract caused by disrupting the gja8 gene reduces but does not abrogate the visual acuity of Xenopus tadpoles.

Weak cataract in dnase2b crispants

In mice, Dnase2b encodes a nuclease responsible of degrading DNA during lens fiber differentiation, and Dnase2b-KO mice develop cataract associated with nuclei retention [START_REF] Nishimoto | Nuclear cataract caused by a lack of DNA degradation in the mouse eye lens[END_REF] . We asked therefore if inactivating dnase2b in Xenopus also elicits cataract in tadpoles. While dnase2b.S

Developmental Dynamics

gene is annotated in Xenopus laevis 9.1 genome, it is not the case of dnase2b.L. BLAST search identified putative dnase2b.L exons 1 and 2, but the other exons are apparently absent. Hence, there is probably only one functional dnase2b homoeologous gene, dnase2b.S, while dnase2b.L may have evolved as a pseudogene. Nevertheless, we co-injected Cas9 enzyme with a mixture of sgRNA against the first exons of dnase2b.S gene and dnase2b.L possible pseudogene. As we failed to amplify this putative dnase2b.L exon 1, we only assessed the efficiency of dnase2b.S gene editing by Sanger sequencing (Figure 4A) and ICE analysis. Gene editing was moderately efficient, with roughly 50% of InDels (Figure 4B). We found no evidence for lens clouding comparable to what had been observed in gja8 crispants, and most larvae have no eye defect detectable by external observation. However, one fifth of the larvae (46/233) have a smaller eye, with a gap in the ventral part of the retina suggesting chorioretinal coloboma (Figures 4C-D). We assessed the vision of dnase2b crispants. They tend to avoid the dark side of the tank, demonstrating that they are able to distinguish white from black (95% confidence interval of the percentage of time spent on the white side of the tank [61; 71]). However, compared with control embryos, their preference for white is reduced (Figure 4E. p = 3.7x10 -3 , Wilcoxon test). We conclude from these observations that dnase2b crispants have poorer visual acuity, and microphthalmia and possibly coloboma albeit not in a fully penetrant manner.

We made a series of sections to understand the anatomical bases of reduced visual acuity. At stage 41 the posterior poles of both control and dnase2b crispant lenses are made of a layer of several nucleated epithelial cells. However, while this layer corresponds to almost half of the dnase2b crispant lenses, it is much thinner in control lenses (Figures 4F-G). The posterior layer of nucleated cells is also thicker in dnase2b crispant lenses at stage 45 (Figures 4H-I). The most likely explanation for these observations is that, while lens fiber differentiation in control lenses is accompanied by a progressive loss of nuclei, the kariolysis is delayed in dnase2b crispant lenses, resulting in an increased number of nucleated cells within the lens.

Toward screening anti-cataract compounds in Xenopus tadpoles

The above results show that cataract can be elicited in Xenopus tadpoles by genome engineering, and we tested if this model responds to potential anti-cataract compounds. The Developmental Dynamics chemical chaperone 4-phenylbutyrate (4PBA) mitigates cellular defects associated with a GJA8 mutation (Cx50D47N) in HeLa cells and in a mouse model. However, it does not reduce lens opacification [START_REF] Jara | Chemical chaperone treatment improves levels and distributions of connexins in Cx50D47A mouse lenses[END_REF] . We tested if this compound has a detectable effect on gja8 crispant tadpoles. In preliminary experiments, we found that a dose of 0.025 mM 4PBA produces no morphological defect and is apparently not toxic. This is consistent with a previous report in zebrafish where 4PBA was used in a model of dominant osteogenesis imperfecta [START_REF] Gioia | The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta[END_REF] . Indeed, in vision assays, treated and untreated control embryos behave identically (Figure 5, 2 left panels). We treated gja8 crispants with 4PBA for 48h. We found no difference between treated and untreated tadpoles regarding lens clouding in external examination. However, we observed a tendency toward a vision improvement upon 4PBA treatment (p = 0.067, Figure 5, 2 right panels). Hence, 4PBA, which showed a limited capacity to improve pathological issues caused by a GJA8 mutation in HeLa cells and mice, apparently has the same effect in Xenopus gja8 crispants. This suggests that cataract-prone Xenopus tadpoles can be used to test the efficiency of potential anti-cataract drugs.

DISCUSSION

We disrupted three genes in Xenopus laevis by CRISPR/Cas9, and we investigated the resulting eye phenotypes after a few days of development. pax6 crispants have highly severe eye phenotypes, consistent with previous reports [START_REF] Nakayama | Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients[END_REF][START_REF] Suzuki | High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos[END_REF] . gja8 crispants have lens-specific defects, with clouded lenses directly visible by external observations, and defective karyolysis and organisation of fiber cells in sections. Finally, dnase2b crispants have a wild-type appearance in external observation and a thickened layer of posterior epithelial cells in sections, suggesting a blockage of lens fiber cells differentiation. Interestingly, functional vision assays reflect this graduation. This suggests that these vision assays can prove useful to quantitatively assess cataract severity, for instance in a process of screening potential anti-cataract drugs. Because the molecular chaperone 4PBA was reported to have a limited capacity to mitigate some cellular defects associated with a mutation in GJA8 [START_REF] Jara | Chemical chaperone treatment improves levels and distributions of connexins in Cx50D47A mouse lenses[END_REF] , we tested the same molecule on gja8 crispants. We found that 4PBA may have a weak capacity to improve the vision of gja8 crispants. The weakness of 4PBA may be due to the differences in the nature of the GJA8 mutations between our study and the previous study (i.e. Cx50D47A). In any case, this data reinforces the notion that functional vision assays in Xenopus cataract-prone tadpoles are a novel and relevant tool in a process of anti-cataract drug development.

Developmental Dynamics

Indeed, no pharmacological treatment of cataract currently exists, but recent findings indicate that sterol-based compounds hold promise to prevent/treat cataracts [START_REF] Makley | Pharmacological chaperone for α-crystallin partially restores transparency in cataract models[END_REF][START_REF] Zhao | Lanosterol reverses protein aggregation in cataracts[END_REF] . These studies report that steroid compounds reduce the aggregation of mutant crystallin protein forming amyloid fibrils in vitro and in cultured cells, and reduce cataract severity in rabbit or mouse models. Other compounds could certainly be screened in preclinical approaches to isolate novel molecules with a potential to enter clinical trials against cataracts. A promising tool toward this goal is the "micro-lens" obtained in vitro by differentiation of human stem cells [START_REF] Murphy | Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro[END_REF] . However, this organoid does not recapitulate the normal environment or the barriers of the eye (especially the cornea) that may prevent molecules of potential pharmaceutical interest to access to the lens. Furthermore, obtaining a large number of genetically modified Xenopus tadpoles is cheap and rapid, and larvae are generally not submitted to regulations on animal tests. For these reasons, we think that the genetically modified cataract-prone Xenopus tadpoles that we describe here are a promising novel preclinical model to screen potential anti-cataract drugs.

Having shown that cataract can be readily observed in Xenopus will also permit to tackle a subset of poorly understood congenital genetic cataracts. Tens of genes were identified by genetic approaches in human as involved in congenital genetic cataract. Most of these genes encode crystallins, the major proteins of the lens that make a structure for refraction of light, connexins (like that encoded by the GJA8 gene), which exchange small molecules between cells, proteins of the cell membrane, the extracellular matrix or the cytoskeleton that set up the highly peculiar morphology of lens fibres, or transcription factors such as FOXE3, HSF4, MAF, and PITX3 2,5,51 . However, for some other genes, the link between the molecular functions of the encoded proteins and cataract remains elusive. Understanding the involvement of these genes in cataract can be achieved by constructing and characterizing new animal models inactivated for these genes. Indeed, the bioinformatics resource tool iSyTE has predicted several promising candidates that need to be validated in animal models [START_REF] Anand | Systems biology of lens development: A paradigm for disease gene discovery in the eye[END_REF][START_REF] Siddam | The RNA-binding protein Celf1 posttranscriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development[END_REF][START_REF] Anand | RNA sequencing-based transcriptomic profiles of embryonic lens development for cataract gene discovery[END_REF][START_REF] Kakrana | iSyTE 2.0: a database for expression-based gene discovery in the eye[END_REF][START_REF] Lachke | iSyTE: integrated Systems Tool for Eye gene discovery[END_REF][START_REF] Aryal | MS/MS in silico subtraction-based proteomic profiling as an approach to facilitate disease gene discovery: application to lens development and cataract[END_REF][START_REF] Dash | The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development[END_REF] . The cost-effective pipeline of cataract investigation in Xenopus that we describe here would help to uncover the genetic basis of these rare genetic cataracts as well as to identify/validate new cataract-linked genes.

Developmental Dynamics

EXPERIMENTAL PROCEDURES

Ethics statement

The animals were housed in the Xenopus facilities of the IGDR as approved by the French animal care agency (Direction des Services Vétérinaires). Experiments were carried out according to standard procedures following local ethics committee opinion and acceptance by the ministry of research (APAFIS 14829, 2018).

Gene editing in Xenopus laevis

CRISPR/Cas9-mediated gene editing in Xenopus laevis was essentially as described for Xenopus tropicalis [START_REF] Nakayama | Cas9-based genome editing in Xenopus tropicalis[END_REF] . We designed the sgRNAs (single guide RNA) with CHOPCHOP [START_REF] Labun | CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering[END_REF] . We prepared sgRNAs by in vitro transcription from templates obtained by PCR with a 3' primer common to all sgRNA templates [START_REF] Nakayama | Cas9-based genome editing in Xenopus tropicalis[END_REF]

Developmental Dynamics

We checked gene editing by lysing the embryos and sequencing the PCR products obtained with the following primers: The traces (chromatograms) were uploaded on the ICE server (https://ice.synthego.com/#/)

and ICE analysed with the default parameters.

Histological methods

For histological analyses, we fixed and embedded in paraffin wax embryos at the indicated stages. Horizontal sections were stained with Hematoxylin and Eosin. For histochemical analyses, we fixed embryos in 4% PFA in PBS for 1h, rinsed them in PBS and incubated them in 30% sucrose overnight at 4°C. Embryos were embedded in Tissue Teck and stored at -80°C. Transversal cryosections were performed and stained with Phalloidin and DAPI.

Functional vision assays

The functional vision assays were essentially as described [START_REF] Viczian | A simple behavioral assay for testing visual function in Xenopus laevis[END_REF] . We put independent pools of 10 embryos in a rectangular water tank with a black and a white side. We switched the sides and we counted the numbers of embryos on the white side one minute after switching the sides. We repeated this operation every minute for 10 minutes. The cumulative number of embryos on the

Developmental Dynamics

white is comprised between 0 and 100 and corresponds to the percentage of time spent on white.

We show it for 8-10 independent pools of tadpoles. We assessed the significance of the results with R, including the CI function in Rmisc (https://cran.r-project.org/package=Rmisc). Functional vision assays of 4PBA-treated tadpoles were carryed out on gja8 crispants and controls incubated for 48h, starting at stage 44, in 0.025 mM 4PBA in water (Sigma P21006) and allowed to recover overnight in pure water. F, Quantification of eye defects, in tadpoles previously injected with the same sgRNA against pax6 as above (sgRNA #1) or an alternative sgRNA against pax6 (sgRNA #2) G, Functional vision assay of stage 45 buffer-injected embryos and pax6 crispants (sgRNA #1). We put 10 tadpoles in a water tank with a black and a white side, we switched the sides and we counted the number of embryos on the white side after one minute. We repeated the switching-counting procedure 10 times for each batch of 10 tadpoles. The cumulative number of embryos on white (between 0 and 100) is shown here, for 8 independent pools of 10 tadpoles. The tadpoles spend more time on white if they are able to distinguish white from black, and the bias in favour of the white side reflects their visual acuity.

Developmental Dynamics

We injected embryos with Cas9 enzyme and a sgRNA targeted against gja8.L and gja8.S, and we allowed the embryos to develop until stage 47. A-B, Representative Sanger chromatograms of one sgRNA and one buffer-injected embryo and percentages of InDels in individual embryos, as in We injected embryos with buffer or Cas9 enzyme and a sgRNA targeted against gja8.L and gja8.S (sgRNA #1). We allowed them to develop until stage 44, and we incubated half of them for 48h in 4PBA. We led them recover overnight in water before testing their vision as in Figure 2G.

  and the following 5' primers: pax6, sgRNA #1 (Targetting exon5) TAATACGACTCACTATAGGAGACCGGATCGATCCGACCTGTTTTAGAGCTAGAAATAGCA pax6, sgRNA #2 (Targetting exon5) TAATACGACTCACTATAGGATCCGACCTCGGGCGATCGGGTTTTAGAGCTAGAAATAGCA gja8, sgRNA #1 (Targetting an exon annotated as exon 1 in gja8.L and exon 2 in gja8.S) TAATACGACTCACTATAGGTTTGAGGTGGGCTTTGTGGTGTTTTAGAGCTAGAAATAGCA gja8, sgRNA #2 (Targetting an exon annotated as exon 1 in gja8.L and exon 2 in gja8.S) specific sequences are in bold underlined and are flanked by the T7 promoter and a region hybridizing to the 3' primer. We dejellyed the embryos 15 minutes after fertilization, and we injected them up to 45 minutes after fertilization with 9.2 nl of a mixture of Cas9 enzyme (IDT DNA 1085059, 0.5 µg/µl) and sgRNA (0.2 µg/µl) in 12 mM HEPES (pH 7.5), 90 mM KCl. We allowed them to develop at 22°C following standard procedures and we staged them according to Nieuwkoop and Faber 58 .

  , sequencing with the forward primer; pax6.L GACATGTAAGGGGCTATGTGC and GGGATATTGTCGTTGGTACAGA (amplification), TTAATGCTACCTATAAACTA (sequencing); gja8.L (sgRNA #1) ATGAGCACTCGACTGTGATCG and GTTTGGACAGGGCCACCTAC (amplification), sequencing with the forward primer; gja8.S (sgRNA #1) TTTTGTGTGCAACACTCAGCAG and GGATGCTGATCTTCGCTCCTCC (amplification), sequencing with the forward primer; gja8.L (sgRNA #2) GATCGGTAGAGTTTGGCTCACT and GTTTGGACAGGGCCACCTAC (amplification), sequencing with the forward primer; gja8.S (sgRNA #2) GATCGGTAGAGTTTGGCTCACT and GGATGCTGATCTTCGCTCCTCC (amplification), sequencing with the forward primer; dnase2b.S TCTGACAGAAAACCGTGCCA and AGCGTTTGTGTTCTCCCCTT (amplification), sequencing with the forward primer.

.

  Figure 1. Eye development in Xenopus laevis. Representative sections of embryos staged according to Nieuwkoop and Faber 58 , hematoxylineeosine stained. A, Stage 27, 30 hours post-fertilization at 23°C. lr lens rudiment; oc optic cup. B, Stage 32, 40 hours post-fertilization. lv lens vesicle. C, Stage 38, 2 days 5 hours post-fertilization. le lens epithelium; plfc primary lens fiber cells; rpe retinal pigmented epithelium. D, Stage 41, 3 days 4 hours post-fertilization. D', Higher magnification of the lens shown in D, with interpretative diagram. E, Stage 44, 3 days 20 hours post-fertilization. lfc, lens fiber cells. F, Stage 47, 5.5 days post-fertilization. gcl, ganglion cell layer; inl, inner nuclear layer; oc, outer cornea; onl, outer nuclear layer; pr, photoreceptors; rpe retinal pigmented epithelium. F', Higher magnification of the lens shown in F. G, Stage 48, 1 week post-fertilization. All scale bars 200 µm except F' 90 µm.

Figure 2 .

 2 Figure 2. Eye phenotype of pax6 crispants. We injected embryos with Cas9 enzyme and a sgRNA targeted against pax6.L and pax6.S or buffer, and we allowed the embryos to develop until stage 41-42. A, We separately amplified the pax6.L and pax6.S loci from total embryos for Sanger sequencing. We show representative chromatograms of one sgRNA and one buffer-injected embryo. The sgRNA and PAM sequences are underlined and the cleavage sites are shown. B, The percentages of InDels in individual embryos were calculated from Sanger chromatograms with ICE 37 . C-E, from top to bottom whole embryo, higher magnification of the eye and histological section of the eye, scale bar 200 µm. C, Buffer-injected embryo. D-E, Two Cas9 and sgRNA injected embryos. Key for lens and retina layers, see Figure 1.

Figure 3 .

 3 Figure 3. Eye phenotype of gja8 crispants.

Figures

  Figures 2A-B. C, We subcloned the gja8.L amplimere from one gja8 crispant and we sequenced individual clones. We show the sequences and their number of occurences. ICE is the result of the ICE analysis carried out on the same embryo. D-E, from top to bottom whole embryo, higher magnification of the eye, and histological section of the eye, scale bar 200 µm. D, Buffer-injected embryo. E, Cas9 and sgRNA-injected embryo. Note the greyish lens. F, Quantification of cataract, in tadpoles previously injected with the same sgRNA against gja8as above (sgRNA #1) or an alternative sgRNA against gja8 (sgRNA #2) G-H, From top to bottom, DAPI staining, Phalloidin staining, and Merge of lens sections. A and P highlight the anterior and posterior pole of the lens. G, Buffer-injected embryo. H, Cas9 and sgRNA #1-injected embryo. I, Functional vision assay of stage 47 buffer-injected embryos and gja8 crispants (sgRNA #1), as in Figure 2G.

Figure 4 .

 4 Figure 4. Eye phenotype of dnase2b crispants. We injected embryos with Cas9 enzyme and a mixture of sgRNA targeted against dnase2b.S and a putative dnase2b.L locus, and we allowed the embryos to develop. A-B, Representative Sanger chromatograms of one sgRNA and one buffer-injected embryo and percentages of InDels in individual embryos, as in Figures 2A-B. Since we failed to amplify the putative dnase2b.L locus, only the results for dnase2b.S are shown. C-D, Stage 41 larvae previously injected with buffer (C) or Cas9-sgRNA (D). Insets are high magnifications of the eyes of the same larvae. E, Functional vision assay of buffer-injected embryos and dnase2b crispants, as in Figure 2F. F, Histological section of the eye of a buffer-injected stage 41 tadpole. G, Section of the eye of a Cas9 and sgRNAinjected stage 41 tadpole with microphthalia. H-I, Same as F-G with stage 45 tadpoles. Scale bars 200 µm.

Figure 5 .

 5 Figure 5. Effect of 4-phenylbutyrate on the vision of gja8 crispants.

ACKNOWLEDGMENTS

We thank the Biosit platforms (Rennes, France) for access to their facilities, and notably MRic (microscopy) and H2P2 (histology).

LP is supported by a grant from Bouygues and the Association Retina France, and a CNRS-PICS.

SAL is supported by NIH Grants R01EY021505, R01EY029770. 1) Wellcome Trust Department of Biotechnology India Alliance (Intermediate Fellowship to Sreelaja Nair). Grant Number 13X301 2) Tata Institute of Fundamental Research (Department of Atomic Energy Government of India). Grant Number 12P0127 1) Wellcome Trust Department of Biotechnology India Alliance (Intermediate Fellowship to Sreelaja Nair). Grant Number 13X301 2) Tata Institute of Fundamental Research (Department of Atomic Energy Government of India). Grant Number 12P0127 1) Wellcome Trust Department of Biotechnology India Alliance (Intermediate Fellowship to Sreelaja Nair). Grant Number 13X301 2) Tata Institute of Fundamental Research (Department of Atomic Energy Government of India). Grant Number 12P0127 1) Wellcome Trust Department of Biotechnology India Alliance (Intermediate Fellowship to Sreelaja Nair). Grant Number 13X301 2) Tata Institute of Fundamental Research (Department of Atomic Energy Government of India). Grant Number 12P0127 1) Wellcome Trust Department of Biotechnology India Alliance (Intermediate Fellowship to Sreelaja Nair). Grant Number 13X301 2) Tata Institute of Fundamental Research (Department of Atomic Energy Government of India). Grant Number 12P0127


D Lopez

S Thuillier

Y Grohens

Prediction of elastic anisotropic thermo-dependent properties of discontinuous fiber reinforced composites

Keywords: Discontinuous fiber composite, Thermoplastic matrix, Elastic properties, Temperature effect, Micromechanical prediction

published or not. The documents may come

Prediction of elastic anisotropic thermo-dependent properties of discontinuous fiber-reinforced composites

D Lopez, Sandrine Thuillier, Y.

Introduction

Thermoplastic polymers reinforced with discontinuous fibers have been widely used for several years to reduce weight and develop new designs at competitive prices. They are used in the automotive industry with usual fiber content in the range between 30 wt% and 50 wt%, e.g. a roof bar [START_REF] Klimkeit | Multiaxial fatigue life assessment for reinforced polymers[END_REF], a clutch pedal [START_REF] Bernasconi | Fatigue strength of a clutch pedal made of reprocessed short glass fibre reinforced polyamide[END_REF], a fuel rail (3), an instrument panel (4), a front-end module [START_REF] Schijve | Common errors and honest materials comparison for long glass PP materials[END_REF]. Polypropylene (PP) is commonly used with glass fibers because the composite exhibits interesting mechanical properties for a high performance over price ratio, low processing temperatures with a glass transition temperature Tg below ambient temperature [START_REF] Lopez-Manchado | Thermal and dynamic mechanical properties of polypropylene and short organic fiber composites[END_REF][START_REF] Ashori | Mechanical and thermo-mechanical properties of short carbon fiber reinforced polypropylene composites using exfoliated graphene nanoplatelets coating[END_REF] and also low water absorption [START_REF] Do | Effect of polypropylene on the mechanical properties and water absorption of carbon-fiber-reinforced-polyamide-6/polypropylene composite[END_REF]. Such automotive components must resist demanding service life conditions, e.g. elevated temperatures (a few tens of degrees) during a few hours and meet thermo-mechanical specifications. Virtual design is then a necessity to optimize the proper use of these composites, while avoiding overestimating the part thickness. The reliability of this virtual design, based on finite element simulations, depends on the modeling of the anisotropic thermo-mechanical behavior of these materials.

The mechanical properties depend not only on fiber length or aspect ratio, shape and thickness of mold cavity but also on orientation and dispersion of the fibers. Indeed, injection-molded composites reinforced with discontinuous fibers display a very complex spatial fiber distribution [START_REF] Lopez | Anisotropic Mechanical Behavior of an Injection Molded Short Fiber Reinforced Thermoplastic[END_REF][START_REF] Dray Bensahkoun | Prediction of the thermoelastic properties of an injection molded short-fiber composite -Thesis[END_REF][START_REF] Bernasconi | Effect of fibre orientation on the fatigue behaviour of a short glass fibre reinforced polyamide-6[END_REF][START_REF] Redjeb | Three dimensional numerical simulations of fiber orientation in injection molding[END_REF] and the anisotropy related to the fiber orientation must be considered [START_REF] Nguyen | Approche micromécanique pour la modélisation du comportement élasto-plastique des composites : application aux mortiers de résine[END_REF][START_REF] Notta-Cuvier | Damage of short-fibre reinforced materials with anisotropy induced by complex fibres orientations[END_REF][START_REF] Mortazavian | Effects of fiber orientation and anisotropy on tensile strength and elastic modulus of short fiber reinforced polymer composites[END_REF]. Mechanical characterization is often performed in tension. However, as the microstructure is heterogeneous and anisotropic, the orientation of the tensile direction is defined with respect to the injection direction but also to the fiber orientation (or microstructure). A first way is to inject samples directly in their final shape [START_REF] Launay | Cyclic behavior of short glass fiber reinforced polyamide for fatigue life prediction of automotive components[END_REF][START_REF] Gehring | Etude expérimentale et modélisation du comportement de polymères thermoplastiques renforcés: polypropylène chargé de fibres de chanvre[END_REF][START_REF] Arao | Mechanical properties of injectionmolded carbon fiber/polypropylene composites hybridized with nanofillers[END_REF] but only one fiber orientation and one tensile direction can be investigated. The second and main trend is to consider only the central area of a rectangular plate: samples are rotated around this point and tensile tests are carried out at different directions for a fixed microstructure [START_REF] Klimkeit | Multiaxial fatigue life assessment for reinforced polymers[END_REF][START_REF] Dray Bensahkoun | Prediction of the thermoelastic properties of an injection molded short-fiber composite -Thesis[END_REF][START_REF] De Monte | Influence of temperature and thickness on the off-axis behaviour of short glass fibre reinforced polyamide 6.6 -cyclic loading[END_REF][START_REF] Launay | Multiaxial fatigue models for short glass fiber reinforced polyamide -Part I: Nonlinear anisotropic constitutive behavior for cyclic response[END_REF]. However, only one sample is cut out of one plate.

Several specimen can be cut with the same orientation in a plate, thus neglecting the planar fiber orientation distribution over the plate [START_REF] Jain | A statistical treatment of the loss of stiffness during cyclic loading for short fiber reinforced injection molded composites[END_REF]. However, in accordance to numerical predictions [START_REF] Dray Bensahkoun | Prediction of the thermoelastic properties of an injection molded short-fiber composite -Thesis[END_REF], X-ray computed microtomography reveals that distribution of fiber orientation depends on the specimen location [START_REF] Notta-Cuvier | Coupled influence of strain rate and heterogeneous fibre orientation on the mechanical behaviour of short-glass-fibre reinforced polypropylene[END_REF]. Though only a few studies consider this distribution, it has a sensitive effect on elastic properties [START_REF] Barbosa | Analysis of the relationship between processing conditions -fiber orientationfinal properties in short fiber reinforced polypropylene[END_REF]. These results highlight the importance of not neglecting fiber orientation distribution over the specimen as a source of variability of the mechanical properties. Indeed, a very large dispersion in terms of stress at rupture in tension, from 40 MPa to 57 MPa, is recorded for the same load direction but for several sample locations, corresponding to an average orientation tensor component from 0.44 to 0.55 [START_REF] Notta-Cuvier | Coupled influence of strain rate and heterogeneous fibre orientation on the mechanical behaviour of short-glass-fibre reinforced polypropylene[END_REF] for a PP reinforced with glass fibers. The idea is then to represent the microstructure by using local orientation tensors [START_REF] Klimkeit | Multiaxial fatigue life assessment for reinforced polymers[END_REF].

Structural parts may support in service demanding thermo-mechanical conditions and the material behavior must be tested under similar conditions. Temperature effect on mechanical behavior is not commonly observed in literature for polymer matrix composites [START_REF] Mortazavian | Effects of fiber orientation and anisotropy on tensile strength and elastic modulus of short fiber reinforced polymer composites[END_REF][START_REF] De Monte | Influence of temperature and thickness on the off-axis behaviour of short glass fibre reinforced polyamide 6.6 -cyclic loading[END_REF][START_REF] Mouhmid | A study of the mechanical behaviour of a glass fibre reinforced polyamide 6,6: Experimental investigation[END_REF], however its influence must be considered for a variety of applications, e.g. fatigue [START_REF] Mortazavian | Fatigue behavior and modeling of short fiber reinforced polymer composites including anisotropy and temperature effects[END_REF]. Temperature ensures a predominant role in the material behavior mainly due to the viscosity of the matrix [START_REF] Şerban | Tensile properties of semicrystalline thermoplastic polymers: Effects of temperature and strain rates[END_REF], e.g. Young's modulus of PP decreases by 75% at 90°C compared to its value at ambient temperature [START_REF] Zhai | Tensile behavior of quasi-unidirectional glass fiber/polypropylene composites at room and elevated temperatures[END_REF]. The same decrease is recorded for composites, e.g. glass fiber reinforced polyamide 6-6 [START_REF] Mouhmid | A study of the mechanical behaviour of a glass fibre reinforced polyamide 6,6: Experimental investigation[END_REF] and PP reinforced by quasiunidirectional glass. In automotive applications, the typical temperature range is between -40°C and 130°C.

The temperature-dependent elastic behavior of an injection molded PP reinforced with discontinuous glass fibers is addressed in the present paper. Material description and experimental procedure, including sample preparation and cutting, X-ray computed micro-tomography and tensile test, are detailed in section 2. Section 3 is dedicated to experimental results on local fiber orientation, characterized by an average orientation tensor, and tensile test results under isothermal loading conditions. Finally, section 4 focuses on a micromechanical prediction of elastic properties regarding anisotropy and temperature effect, using Mori-Tanaka model.

Material and experimental procedure

Material

In this study, a glass fiber reinforced PP named PPGFL40 is considered, with a fiber content of 40 wt%. Rectangular plates of dimensions 179 mm x 240 mm, with a constant thickness of 2.75 mm, are injected in industrial conditions at Technocentre Renault. The injection speed is 30 mm.s -1 and the mold temperature 250°C.

A calcination method is chosen to verify the fiber content. Five samples of 2.75 x 10 x 20 mm 3 dimensions are cut from three different plates and heated up to 750°C during 30 min according to the standard NF EN ISO 1172. The average dispersion of fiber content between plates is less than 2.8% whereas samples from the same plate exhibit a maximum relative gap of 3.5%. The maximum relative gap reaches an acceptable 5% regarding the 40% aimed at during elaboration of the material. This weak dispersion of the fiber content shows a good control of the injection process. The initial fiber length is 11 mm with a diameter of 17 µm. These values are common ones for such a composite, e.g. [START_REF] Teixeira | Influence of injection molding on the flexural strength and surface quality of long glass fiber-reinforced polyamide 6.6 composites[END_REF][START_REF] Coulon | Flexural creep behaviour of long glass fibre reinforced polyamide 6.6 under thermal-oxidative environment[END_REF]. To determine the fiber length after injection, specimens are incinerated and then dispersed in a dilute solvent to separate the fibers. The fiber length is measured with an optical microscope on ten specimens showing almost a hundred fibers each and extracted from several areas. The resulting fiber length average is 1.5 mm and the median value is around 0.8 mm. The smallest value is around 0.3 mm when a maximum of 11 mm is sometimes reached, and therefore the composite should be qualified as semi-long fiber material [START_REF] Teixeira | Influence of injection molding on the flexural strength and surface quality of long glass fiber-reinforced polyamide 6.6 composites[END_REF][START_REF] Lafranche | A microstructural approach for modelling flexural properties of long glass fibre reinforced polyamide 6.6[END_REF]. Three temperatures representative of common industrial specifications are considered in this study: 23°C, 60°C and 85°C. Only positive temperatures are chosen, to represent desert-like conditions responsible for creep. Main properties of each constituent of PPGFL40 are specified in Table 1. Fiber elastic properties are considered unchanged in this range of temperature.

Glass fibers

PP matrix at 0° and 90° regarding the injection direction. Analysis is performed at 1 Hz in torsion mode and the temperature run from -30°C to 150°C at a heating rate of 2°C/min under a controlled sinusoidal strain of 0.15%. Evolution of the storage modulus G' and loss factor tan δ versus temperature is shown in Fig. 1 for unfilled PP and PPGFL40, for two samples.

𝑬 𝒇 [MPa] 𝝂 𝒇 r f [g.cm 3 ] 𝑬 𝒎 [MPa] 𝝂 𝒎 r m [g.cm 3 ] 23°C 60°C 85°C 72000
As the temperature rises, the storage modulus decreases continuously when the loss factor increases and two peaks can clearly be seen. The first one corresponds to the glass transition and occurs around 0°C. The second one, at around 90°C, corresponds to the beginning of melting. Incorporation of fibers reduces tan δ peak height by restricting the movement of macromolecules [START_REF] Rezaei | Effect of fiber length on thermomechanical properties of short carbon fiber reinforced polypropylene composites[END_REF]. Temperatures which are considered in this study are situated in a same relaxation state from 23°C to 85°C. Though the dispersion is higher for the composite, the reproducibility for both matrix and composite is satisfying.

Cutting pattern of tensile specimen

To cover the whole plate in an easy way, orientation intensity is observed by terahertz 2D imaging based on reflection and transmission of high frequency waves, located between infrareds and microwaves in the electromagnetic spectrum [START_REF]Terahertz Waves Technologies[END_REF]. Result is shown in Fig. 2(a); a value of 1 (red areas) represents a strong fiber orientation in a direction which is not known. Two methods are considered for the tension, i.e. aligning the tensile direction with the injection direction, at several locations in the plate and rotating the tensile direction. The cutting pattern is defined based on the results of Fig. 2(a) and is shown in Fig. 2(b). Only three main angles relatively to the injection direction are considered to limit the number of mechanical tests: 0°, 45° and 90°. It is possible to cut out seven samples in the same plate corresponding to five configurations, three in the longitudinal direction (named L1, L2 and L3) considering the plate symmetry [START_REF] Notta-Cuvier | Coupled influence of strain rate and heterogeneous fibre orientation on the mechanical behaviour of short-glass-fibre reinforced polypropylene[END_REF] and two in the transverse one (called T1 and T2). Two more configurations are taken in a different plate at 45° (45-1 and 45-2). This cutting pattern has the benefit to cover a large range of fiber orientations and to avoid a waste of materials. Specimen cutting is done by low speed machining and a cooling system and proper knife edge angle to avoid affecting material properties. Samples have a dog-bone shape with a gauge area of 50x10 mm 2 and dimensions according to the standard NF EN ISO-527-b [START_REF] Notta-Cuvier | Damage of short-fibre reinforced materials with anisotropy induced by complex fibres orientations[END_REF][START_REF] Mouhmid | A study of the mechanical behaviour of a glass fibre reinforced polyamide 6,6: Experimental investigation[END_REF]. They are stored several days in a controlled atmosphere room, with temperature and hygrometry rate regulated respectively at 23°C and 50%. Tensile tests are carried out at three temperatures under isothermal conditions, i.e. 23°C, 60°C and 85°C, representative of potential service life conditions. It should be emphasized that ambient temperature is not strictly imposed at 23°C, whereas it is regulated at higher values. Mechanical tests are carried out on an Instron electro-mechanical testing machine with a load cell of maximum capacity 10 kN. The distance between the fixed grip and the moving one is initially set at 108 mm. The local displacement is measured with an extensometer of 25 mm gauge length set in the middle of the sample. Tests are performed at several crosshead speed, corresponding to almost constant strain rates of 10 -5 , 10 -4 and 10 -3 s -1 . For each configuration, 3 to 5 tests are conducted at room temperature, to check the reproducibility. And, as it is very good, at least two samples are tested at higher temperatures. After pre-conditioning at the test temperature, the isothermal test is started. At least 30 min are required to reach the temperature of the test with the maximum rate of 5°C/min. To control the temperature, thermocouples are located on the sample and on the grips.

Cauchy stress and logarithmic strain are calculated from raw data and used to plot the graphs.

X-ray micro-tomography

The local microstructure is also experimentally investigated by means of X-ray micro-tomography, which is the most common non-destructive method used to observe fiber orientation [START_REF] Albrecht | Measuring fibre orientation in sisal fibre-reinforced, injection moulded polypropylene -Pros and cons of the experimental methods to validate injection moulding simulation[END_REF][START_REF] Dean | An invariant-based anisotropic material model for short fiberreinforced thermoplastics: Coupled thermo-plastic formulation[END_REF]. Only central areas of the tensile specimen are considered with sample dimensions of 10x20x2.75 mm 3 and cut out of two injected plates. Water jet cutting is used to avoid pulling out of fibers. Several 2D scans are taken with specific values given in Table 2 and 3D images are then built with a dedicated algorithm; image analysis is carried out by [START_REF]Novitom[END_REF]. To derive quantitative data, the average orientation tensor A [START_REF] Advani | The use of tensors to describe and predict fiber orientation in short fiber composites[END_REF] in the thickness of each specimen is calculated from X-ray micro-tomography analysis. The fibers appear in white and the matrix in grey in the reconstructed images and the voxel size is given in Table 2. The orientation vectors p i of the fibers are calculated with a method based on grey nuance gradient around fiber neighboring and using a dedicated tool developed by Novitom. Then, the second order orientation tensor is calculated based on orientation vectors p i for all voxels N of the fiber phase in the chosen frame, with equation 1. Only the components Axx and Ayy (the frame is defined in Fig. 2b) are given here because they correspond to the highest values.

𝑨 = 1 𝑁 % 𝒑 ! ⊗ 𝒑 ! " !#$ (1)

Results

Monotonic uniaxial tensile tests are performed at ambient and elevated temperatures for several fiber orientations, to highlight the fiber orientation effect and to check the reproducibility between two different plates.

Moreover, the local fiber orientation is characterized by the components of the orientation tensor, averaged over the sample thickness.

Fiber orientation

Fig. 3 shows the heterogeneity of the microstructure, characterized by a skin-core structure [START_REF] Teixeira | Influence of injection molding on the flexural strength and surface quality of long glass fiber-reinforced polyamide 6.6 composites[END_REF]. The fiber orientation is mainly composed of two skin layers with preferential orientation parallel to the flow direction, and a core region with orientation perpendicular to the flow and in the plane of the part, which is classical for injected parts.

Such a distribution is sometimes neglected, especially in industrial cases, to simplify the mechanical investigation and material behavior representation. As a consequence, an average orientation tensor (36) over the thickness is introduced in constitutive equations and this approach gives a reliable prediction of the elastic properties [START_REF] Launay | Multiaxial fatigue models for short glass fiber reinforced polyamide -Part I: Nonlinear anisotropic constitutive behavior for cyclic response[END_REF][START_REF] Andriyana | Mechanical response of a short fiber-reinforced thermoplastic: Experimental investigation and continuum mechanical modeling[END_REF][START_REF] Ogierman | A study on fiber orientation influence on the mechanical response of a short fiber composite structure[END_REF].

A similar approach is then considered in this study. Fig. 3(b) shows fibers mainly oriented in the injection direction corresponding to the L1 configuration contrary to Fig. 3(c) which displays a random orientation for the L3 one. This planar distribution of the fiber orientation is also highlighted in ( 4) for an industrial part in PP reinforced by 20 wt% of glass fibers. Experimental evaluation of the orientation tensor components is given in Table 3 for five configurations showing a stark contrast. Whatever the orientation, Azz is very small, of the order of 0.037. Moreover, all the nondiagonal components are very close to zero. Except for L1, which is a rather oriented configuration, all the other components are very close to 0.5. In addition, L1 is the only one to show a relative gap between Axx and Ayy superior to 50%. On the contrary, L3 and T1 exhibit a higher Axx value than Ayy. It can be seen that Ayy values for L1 and L3 are very different, with a relative gap around 39%, though they both correspond to tension in the injection direction.

However, for a PP reinforced by 30 wt% of glass fibers and similar plate thickness [START_REF] Notta-Cuvier | Coupled influence of strain rate and heterogeneous fibre orientation on the mechanical behaviour of short-glass-fibre reinforced polypropylene[END_REF] obtain close values, respectively around 0.7 and 0.615 (gap of 0.09). L3, T1, T2 and 45-2 exhibit all values close to 0.5 for both components. And the relative gap between these two components is of the order to 10%.

Configuration

Mechanical behavior in tension

Fig. 4 shows results of tensile tests performed up to rupture at room temperature for the different configurations.

Samples are cut out from two different plates obtained with similar process conditions and respectively named A and B. Testing results highlight a very good reproducibility for each orientation. A small dispersion is evidenced for each orientation and the relative gap on the stress level, calculated with respect to the minimum value, quantifies this dispersion. In the longitudinal direction, at 1.8% strain, a relative gap of 1% is calculated for L1, whereas they are equal respectively to 4.8% and 7.7% for L2 and L3. Despite their close location to the injection gate, at a strain of 1%, T1 has a relative gap of 0.2%, respectively 3.9% for T2. In addition, regarding 45-2, a gap of 3% is calculated at a strain of 1.5%.

L1, T1 and 45-1 show the highest reproducibility whereas L3 shows the lowest one. L3, which seems to be the most random configuration in terms of fiber orientation, gives the highest dispersion in stress level. Though clusters are sometimes visible on X-ray micro-tomography images, the fiber volume fraction exhibits a weak dispersion on the plate; however, the fiber length exhibits a wide range of variation, with non-negligible fiber curvature. The anisotropy of the mechanical behavior is rather strong, with a maximum stress level along the injection direction which can be divided by two for other configurations. L1 reaches the highest level of stress which is around 110 MPa. From L1 to L3, the stress level is reduced by 20 MPa. For each configuration, a strongly non-linear behavior is observed. It can also be seen that there is a clear distinction of the stress levels, without any overlapping of the curves. This validates the proposed cutting pattern and leads to a high reproducibility for each configuration, higher than results in literature [START_REF] Jain | A statistical treatment of the loss of stiffness during cyclic loading for short fiber reinforced injection molded composites[END_REF][START_REF] Notta-Cuvier | Coupled influence of strain rate and heterogeneous fibre orientation on the mechanical behaviour of short-glass-fibre reinforced polypropylene[END_REF]. Each configuration corresponds to a different average orientation tensor, with a particularly obvious difference for L1. Though the average orientation tensor is a local information, compared to the global information of the stress level, it seems that such a representation is relevant to distinguish the mechanical behavior.

Fig. 5 shows results of tensile tests performed for the same fiber orientations at 85°C. For more visibility and to limit the number of tests, the analysis at elevated temperature is shown for only five orientations which are L1, L3, T1, T2 and 45-2. Only two representative curves for each configuration are presented. Same trends regarding the mechanical behavior observed at 23°C are noticed at 60°C and 85°C. The weak dispersion of mechanical response is similar in temperature and is even enhanced for some orientations. Fig. 6 shows results of tensile tests for L1 and L3 at each temperature. It evidences the strong influence of the test temperature on the stress level. This effect does not depend on the fiber orientation. Indeed, similar decrease in term of stress level is observed with a maximum gap in between 20 and 30 MPa at the same strain level. The general trend is observed for other configurations. The influence of strain rate is investigated for three values, i.e. 10 -5 s -1 , 10 -4 s -1 and 10 -3 s -1 . Table 4 gives the initial slope of the stress-strain curve, referred as E0, at three strain rates for L1 and L2. They are calculated on the corresponding stress/strain curves in the strain interval from 0.05% to 2.5% according to the standard NF EN ISO 527-1 and the value is averaged on several samples. It increases slightly when the strain rate increases. However, this effect is rather weak, with an average increase around 3%, compared to the anisotropy effect. The evolution of E0 at three strain rates is also considered at 23°C and 85°C for L1, cf. Table 5. The maximum relative gap is of 7.9% at 23°C and almost three times higher at 85°C which shows the viscoelastic effect. Nevertheless, some overlapping is observed at both temperatures. In the following section, only anisotropy and thermal dependence are considered.

23°C 85°C

Strain rate (s -1 ) E0 min (MPa) E0 max (MPa) E0 min (MPa) E0 max (MPa)

Prediction of elastic properties

The initial slope E0 is derived from stress/strain curves and an average value is calculated on at least two samples for each orientation and each temperature. Fig. 7 shows experimental E0 of PP and PPGFL40 obtained for several temperatures. At 23°C for PP, a value of 1000 MPa is proposed by [START_REF] Zhai | Tensile behavior of quasi-unidirectional glass fiber/polypropylene composites at room and elevated temperatures[END_REF], similarly to [START_REF] Notta-Cuvier | Coupled influence of strain rate and heterogeneous fibre orientation on the mechanical behaviour of short-glass-fibre reinforced polypropylene[END_REF]. However, higher values of 1650 MPa and 2000 MPa are also obtained [START_REF] Barbosa | Analysis of the relationship between processing conditions -fiber orientationfinal properties in short fiber reinforced polypropylene[END_REF][START_REF] Parenteau | Characterisation and micromechanical modelling of the elasto-viscoplastic behavior of thermoplastic elastomers[END_REF], or also 566 MPa at 85°C for [START_REF] Eftekhari | Creep behavior and modeling of neat, talc-filled, and short glass fiber reinforced thermoplastics[END_REF] which are closer to the average values respectively of 1700 MPa and 587 MPa measured in this study (cf. Table 1). Such a variation for PP modulus is therefore significant and is probably due to crystallinity rate resulting from different injection parameters. For each specimen, E0 clearly decreases with the rising temperature. E0 is predicted with equation 1, proposed for PP and quasiunidirectional glass fiber/PP composites [START_REF] Zhai | Tensile behavior of quasi-unidirectional glass fiber/polypropylene composites at room and elevated temperatures[END_REF]. Based on this equation, a compromise is found for all configurations, except T2, by reducing the value of n from 2.3 for PP to 0.87 for the composite against 2.6. T2 is removed due to an inverse trend, i.e. E0 is close to T1 at a 23°C but much higher at 60°C and 85°C. Evolution of E0 for each configuration and temperature is also highlighted in Fig. 8. The highest dispersion is found at 23°C and mostly in the injection direction. Very close values of E0 for 45-2, T1 and T2 are found, which is consistent with results from literature [START_REF] Mortazavian | Effects of fiber orientation and anisotropy on tensile strength and elastic modulus of short fiber reinforced polymer composites[END_REF]. E0 significantly reduces from in-flow direction to perpendicular-to-flow direction at all temperatures and strain rates. At 23°C, the dispersion among all configurations is 33 % regarding the highest value.

For a similar material at 23°C, a dispersion of 50 % is obtained with specimen cut out from a 100 x 20 mm² plate and a gate in the middle of the longest edge [START_REF] Barbosa | Analysis of the relationship between processing conditions -fiber orientationfinal properties in short fiber reinforced polypropylene[END_REF]. At 60°C and 85°C, dispersion is about 42%. The cutting pattern proposed in this study, based on fiber orientation, thus minimizes the dispersion. At 23°C, T1 displays a higher E0 value than T2, but this trend is changed at elevated temperatures. Because T2 is close to the injection gate, skin-core effect and dispersion could explain these results. Configuration 45-2 displays E0 with the smallest value compared to other directions, whatever the temperature.

Moreover, from a technological point of view, these values obtained on samples cut out from a rectangular plate are compared to the ones measured on samples taken out of an industrial part (a front door made of the same material) in several areas and at different orientations to the injection direction. It is shown that they are representative of the industrial ones [START_REF] Lopez | Comportement d'un thermoplastique renforcé de fibres de verre soumis à des chargements thermomécaniques[END_REF], thus highlighting the interest of the cutting pattern to evaluate a relevant range of mechanical properties.

Mori-Tanaka micromechanical model is then used to predict elastic properties, based on the average orientation tensor, fiber and matrix properties, fiber length ratio and temperature [START_REF] Mori | Average stress in matrix and average elastic energy of materials with misfitting inclusions[END_REF]. This model is implemented in the software Digimat [START_REF]Digimat 2018 version 4.1.2. Linear and nonlinear multi-scale material software, e-Xtream engeneering, MSC softwate compagny[END_REF]. Such a model has become very popular, as it leads to reasonable predictions in linear elasticity with fiber volume fractions up to 30% and out of simplicity's sake. The ability of such a micromechanical model to predict the elastic anisotropic properties of a 35 wt% short glass fiber reinforced polyamide 66 was already checked, based on numerical orientation tensors [START_REF] Launay | Multiaxial fatigue models for short glass fiber reinforced polyamide -Part I: Nonlinear anisotropic constitutive behavior for cyclic response[END_REF]. Elastic properties in the configuration with fibers aligned to the injection direction are well described whereas some discrepancies are shown for other orientations.

In the present paper, the elastic tensor of the composite is calculated from i) the elastic tensors of the constituents, which is temperature-dependent for PP, ii) the fiber volume fraction and iii) the fiber shape factor. This factor, defined as the ratio of the fiber length over the fiber diameter, presents a large distribution coming from the relatively large variation of the fiber length. Therefore, three different values, i.e. 47, 36 and 25 are considered in this study. It should be stressed out that 47 is the value calculated from the median fiber length after the injection process. Fig. 8 presents a comparison between experimental values and numerical predictions for five configurations. It can be seen that the evolution of E0 at ambient temperature is correctly predicted, though slightly overestimated for a shape factor equals to 47, except for L3. The best compromise for the different configurations and temperatures is obtained for the shape factor of 47, though the intermediate value of 36 also leads to close predictions for T1 and 45-2 configurations.

For the L3 configuration, the orientation tensor component Axx is somehow surprisingly higher than Ayy, which can come from a limitation of the use of the average orientation tensor to fully represent the inhomogeneity of microstructure. For T1, T2 and 45-2, the two component values are close. Nevertheless, the evolution of E0 seems related not only to the component value but also to the gap between Axx and Ayy, this one being maximum for the strongly-orientated configuration L1 and of the order of 10% of the maximum component for T1, T2 and 45-2.

Mechanical properties of configuration L3 are clearly underestimated. A further trend should be to consider the full heterogeneity of the microstructure, without any average over the thickness.

Concerning the thermal dependence, the decrease of E0 comes only from the PP properties. Except for L3 and T2 configurations, the slight overestimation of the predictions at 23°C and 60°C increases significantly at 85°C. It could come from an overestimation of the Young's modulus of PP at this temperature; indeed, the presence of fibers and the injection conditions of the composite may affect the properties of the material, different when tested by itself or a composite.

It can be concluded that such an approach gives a realistic prediction of the anisotropic thermo-dependent elastic properties of the material, though some configurations necessitate taking account of the full heterogeneity, i.e. in the thickness, of the material.

Conclusions

This study is dedicated to the thermo-mechanical characterization of a discontinuous glass fiber reinforced PP with a mass fraction of 40%, with a focus on the prediction of the anisotropic thermo-dependent elastic properties.

Rectangular plates are injected using the same conditions as industrial parts. Samples are cut out following an original pattern that leads to a large range of stress levels and elastic properties, comparable to those recorded out of an industrial part. Tensile tests are performed at three different temperatures. It is shown that the dispersion on the mechanical properties is rather low compared to other patterns proposed in the literature. The initial slope of the stress-strain curve decreases with temperature by a factor of 1.5 between 23°C and 85°C. Predictions of the elastic properties with Mori-Tanaka micromechanical model are compared with experimental values, for different temperatures and configurations. The overall trend at ambient temperature is correctly described; however, the elastic properties of the configuration presenting a highly heterogeneous microstructure are underestimated.

Fig. 1 .

 1 Fig. 1. G' and tan δ obtained with DMA at 1 Hz between -30°C and 150°C for PP and PPGFL40.

Fig. 2 .

 2 Fig. 2. Fiber orientation intensity obtained by terahertz analysis on PPGFL40 plate (a) and cutting pattern for 0°, 90° and 45° samples, regarding the direction of injection, highlighted by the black arrows. Dotted lines correspond to areas observed by X-ray micro-tomography (b). The injection gate is located at the top of the plate.

Fig. 3 :

 3 Fig. 3: X-ray micro-tomography images (a) skin-core structure in the thickness (b) on the outer surface for L1 and (b) L3. Fibers are white while the matrix is in grey and porosities are in black. The injection gate is located at the top of the plate and the black arrows are parallel to the injection direction.

Fig. 4 .

 4 Fig. 4. Tensile test on PPGFL40 at 23°C and 10 -4 s -1 for several configurations. A and B stand for two different plates.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Tensile test on PPGFL40 for all orientations at 85°C, at 10 -4 s -1 and for five configurations.

Fig. 7 .

 7 Fig. 7. Variation of E0 for each fiber orientation at three temperatures and evolution predicted by the equation proposed in (27) for PP and PPGFL40.

Fig. 8 .

 8 Fig. 8. Experimental dispersion (full circle) on E0 for each configuration at three temperatures and a strain rate of 10 -4 s -1 . The three colors correspond to different temperatures. The measured variation is also highlighted with error bars. Predicted values with Mori-Tanaka's model, using three different shape factors, are also plotted. The cross, square and full triangle respectively corresponds to shape factor values of 47, 36 and 25.

Table 1 . Fiber and matrix properties at several temperatures (Renault data). Young's modulus, Poisson's ratio and density 𝑬 𝒊 , 𝝂 𝒊 , 𝝆 𝒊 subscript i=f or m for fiber or matrix respectively.

 1

	0.23	2.6	1720	792	587	0.24	0.946
	Dynamic Mechanical Analysis (DMA) tests are carried out with a viscoanalyser ARES G2 -TA Instruments at
	Technocentre Renault. Samples dimensions are 56 x 10 x 2.75 mm 3 . They are cut from injected plates in several areas

Table 2 . X-ray micro-tomography parameters.

 2

	X-ray source	Energy	Projections number	Break time	Voxel size	Analyzed volume
	ESRF, BM05	90 keV	4500	0.12 s	2.25 x 2.25 x 2.25 µm 3	3.0 x 8.8 x 19.3 mm 3

Table 3 . Experimental orientation tensor components for the five configurations with y the injection direction.

 3

	Axx	Ayy

10 -5 s -1 10 -4 s -1 10 -3 s -1

	L1	8127.54	8207.33	8460.94
	L2	6889.35	7139.82	7547.73

Table 4 . E0 (MPa) at 23°C obtained at several strain rates for L1 and L2.

 4

Table 5 . Maximal and minimal values of E0 at 23°C and 85°C obtained at several strain rates for L1.

 5

	10 -5	8025.50	8229.58	4319.06	4783.75
	10 -4	8185.54	8228.22	5154.26	5417.63
	10 -3	8259.78	8662.10	5178.00	5460.50

Pre-print, accepted for publication in Journal of Composite Materials (2019)

Acknowledgements

The authors wish to acknowledge support from RENAULT SAS and the French agency ANRT for the grant n° 2014/0614. Declaration of Conflicting Interests: The Authors declares that there is no conflict of interest


Łukasz Paśko
email: lpasko@prz.edu.pl

Pawel Litwin
email: plitwin@prz.edu.pl

Paweł Litwin

Methods of Data Mining for Quality Assurance in Glassworks

Keywords: big data, data mining, quality assurance, glass industry

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

Introduction

Today's manufacturing companies increasingly use different types of devices that generate data. This data may contain crucial information. An important area of modern enterprises' activities is discovering this information. Such task requires processing a large amount of data. Then the identified information is transformed into knowledge understood as information that has been confirmed and can be used to support decision-making [START_REF] Larose | Odkrywanie wiedzy z danych: wprowadzenie do eksploracji danych[END_REF].

The crucial stage of knowledge discovery process is data mining (DM). DM is defined as the automatic search for unknown dependencies and patterns hidden in the data. The detected information should then be presented to the user in an intelligible form, e.g. in the form of logical rules or visualizations [START_REF] Larose | Odkrywanie wiedzy z danych: wprowadzenie do eksploracji danych[END_REF].

In the research literature on production engineering, an increasing number of DM applications has been observed for several years. Examples include: scheduling and production planning [START_REF] Wang | Data Mining Based Approach for Jobshop Scheduling[END_REF], customer needs research [START_REF] Kittidecha | Application of Kansei Engineering and Data Mining in the Thai Ceramic Manufacturing[END_REF], machine failure prediction [START_REF] Ruschel | Establishment of Maintenance Inspection Intervals: An Application of Process Mining Techniques in Manufacturing[END_REF], improvement of the assembly process [START_REF] Knosala | Zastosowania metod sztucznej inteligencji w inzynierii produkcji[END_REF], supply chain management [START_REF] Akhtar | Manufacturing Services Classification in a Decentralized Supply Chain Using Text Mining[END_REF] or product design [START_REF] Giess | Investigating Manufacturing for Use within Design[END_REF].

Also in collaborative networks are places where DM methods can be used. A collaborative network has the potential to collect huge amount of data about its collaborative activities. For this reason, there is a need to extract significant patterns out from data and utilize them in a collaborative network. The authors of the paper [START_REF] Biuk-Aghai | An Integrative Framework for Knowledge Extraction in Collaborative Virtual Environments[END_REF] propose a framework for DM in the design of collaborative virtual environments.

Modeling of collaboration networks is also the topic of the work [START_REF] Tomasello | Data-driven modeling of collaboration networks: a cross-domain analysis[END_REF]. The aim of the paper is exploration of the structural and the dynamic features of collaboration to demonstrate that there is a unified modeling approach for reproduce these features in different domains. Proposed model allows understand collaboration patterns in two different domains: research and development alliances between firms, and coauthorship relations between scientists. In the work [START_REF] Zuo | Prediction of collaborative relationships by using network representation learning[END_REF], the authors draw attention to prediction of collaborative relationships in the form of collaboration platform where firms can address works to solve problems together. A similar topic is discussed in the paper [START_REF] Cox | Supply Chains and Power Regimes: Toward an Analytic Framework for Managing Extended Networks of Buyer and Supplier Relationships[END_REF], which presents an analytic framework for managing extended networks in supply chains.

The authors of the work attempt to demonstrate the suitability of using DM methods to ensure the quality of products and propose the methodology for analyzing industrial data in glassworks.

Data Analysis Methods Applications to Quality Assurance

Many applications of data analysis in manufacturing enterprises are related to the product quality. A large part of the research concerns the influence of input parameters of the manufacturing system on the quality of products. Usually the data analyzed includes parameters of the manufacturing process (MP), properties of materials used, employees (e.g. seniority, age), equipment and tools status (e.g. number of days since the last failure, number of hours worked), and the environment of the production system (e.g. atmospheric conditions, day of week).

Many publications using DM in the field of quality assurance come from the metallurgical industry. The aim of the research [START_REF] Mere | Estimation of mechanical properties of steel strip in hot dip galvanising lines[END_REF] was to use neural networks and multiple regressions to predict the quality of galvanized steel. Paper [START_REF] Krimpenis | Simulation-based selection of optimum pressure die-casting process parameters using neural nets and genetic algorithms[END_REF] presents models of neural networks that predict the influence of process parameters on the properties of products manufactured in a pressure die-casting process. The article [START_REF] Cser | Different Kinds of Neural Networks in Control and Monitoring of Hot Rolling Mill[END_REF] presents the use of Kohonen's neural networks to identify key factors affecting the quality of steel after the rolling process. Work [START_REF] Chang | Assessing quality performance based on the on-line sensor measurements using neural networks[END_REF] shows that data recorded with automatic sensors in the cutting process can be used to monitor the quality of products. In [START_REF] Cherian | Modelling the relationship between process parameters and mechanical properties using Bayesian neural networks for powder metal parts[END_REF], Bayesian neural networks were used to determine the appropriate parameters of the sintering process. In [START_REF] Hu | Hierarchical clustering methods for semiconductor manufacturing data[END_REF], grouping algorithms, similarity measures, and distance measures were used to improve production schedules. In work [START_REF] Chen | A novel manufacturing defect detection method using association rule mining techniques[END_REF], a method for determining the correlation between a sequence of machines and the quality of products is proposed, based on association rules.

All the above-mentioned works focus on a strictly defined field of industry and describe only selected cases of the use of DM in the context of product quality assurance. There are no solutions in the literature describing the use of DM techniques that comprehensively cover all stages of manufacturing.

In addition, it should be noted that the applications of DM in the area of quality assurance are mainly focused on the processing of metals, and the glass industry is an area in which there is a small number of studies [START_REF] Koksal | A review of data mining applications for quality improvement in manufacturing industry[END_REF].

The aim of this article is to present the concept of methodology for the analysis of large data sets from a production system in glassworks. The result of the research is expected to be the knowledge about the impact of MP parameters on the occurrence of defects in the products.

Problem Statement and Data Source Description

Analysis of industry data is a non-trivial task, because of the attributes that can be assigned to this data. The Big Data concept defines them as 3V: volume, velocity, variety. Volume refers to a large amount of data, velocity concerns the speed of data inflow and analysis, and variety indicates the heterogeneity of data. An additional difficulty is to determine the technique of extracting knowledge that will best suit to a given problem. These difficulties indicate the need to develop some general methods and guidelines that will be useful for researchers. A glassworks factory is an example of a company where large amounts of data are generated. There are 4 glass furnaces and 14 production lines in the analyzed glassworks. The daily production volume is over 5 million. The manufacturing is continuous, and as a result, data regarding process parameters are recorded 24 hours a day, 7 days a week. Data sets created in this way may be used when there are problems with ensuring high quality of products.

In the glassworks, at automatic quality control stations, an anomaly was recorded. The anomaly involves the occurrence of periods in which an increased number of defective products is observed. Periods of the increased number of defective products last from a few to over a dozen days. During this time, the number of defective products is about three times higher than the average. The abovementioned anomaly is that air bubbles occur in the product's head. The bubbles appearing on the surface of the seal are particularly dangerous; product with the defect becomes useless. It should be noted that with the use of the statistical data analysis methods, glassworks' employees were not able to determine the reason for the periods of increased defectiveness of the products. The aim of the research is to identify the parameters that are responsible for periods of increased number of products with the defect. Because the glassworks collects extensive datasets containing the values of hundreds of MP parameters, there was decided to find the reason of increased number of defective products using DM methods.

The quantity and dynamics of the collected data (values of most parameters are registered every second) justifies the use of artificial intelligence and machine learning methods. In addition, the research issue also includes the appropriate presentation of knowledge, so that it is understandable for decision-makers. The knowledge base, which is an integral part of the decision support system (DSS), should contain decision rules acquired by appropriate methods of induction.

Data from the production process monitoring system were recorded within a period of 27 days and can be divided into three groups: 1. The number of products with the defect recorded on three manufacturing lines. 2. Parameters characterizing the MP, concerning: operation of the glass furnace (glass level and temperature, electric and gas heating power); work of three forehearths (glass temperature and heating); cooling of moulds.

3. Meteorological data describing weather conditions outside the production hall, such as air temperature, atmospheric pressure and humidity. Data regarding the number of defective products are recorded at quality control stations located at the so-called cold end of the production line. Based on them, three variables were defined, which will be explained (output) variables during the test.

Parameters of the MP, together with meteorological data, will be used as explanatory (input) variables. The first device from the hot end is a glass furnace. Based on data obtained from sensors located in the furnace, 11 variables were defined, that describe the operation of the furnace and ventilation, as well as the level and temperature of the liquid glass. The next device in the MP is a forehearth, which receives liquid glass from the glass furnace. Glass moving through forehearth gets the right consistency and temperature. The acquired forehearth parameters refer to the air pressure, the position of the air supply valves and the temperature of the glass. At the end of forehearth, liquid glass is divided into portions (so-called gobs). A total of 139 parameters were registered in the forehearths. Gobs are moving to moulds that give them the shape of the product. Another group of acquired parameters describes the cooling of moulds, where nine parameters were registered. The number of registered parameters is shown in Table 1.

Table 1. The set of explanatory variables

The number of parameters used as explanatory variables

Research Methodology and Preliminary Results

In the paper [START_REF] Perzyk | Comparative Study of Decision Trees and Rough Sets Theory as Knowledge Extraction Tools for Design and Control of Industrial Processes[END_REF], it was shown that DM methods can be used to generate one of four types of information:

-logical rules based on IF (conditions) THEN (conclusion) construction; -relative significance of explanatory variables expressing their impact on the explained variable; -predicted values of explained variables; -results of grouping explanatory variables.

The analysis focuses on obtaining the four types of information, enabling the understanding of the relationship between the parameters of the production process and the number of defective products. Figure 1 presents the assumed research plan, leading to the automatic acquisition of knowledge from production data, and then to develop a decision support system in the quality assurance process. Data selection is aimed at choosing the right data that may potentially contain knowledge about the problem being studied. Preparation for analysis includes handling of missing data and outliers as well as generation of basic statistics describing the data. The principal component analysis (PCA) in the literature is most often used as a method of reducing dimensionality, i.e. reducing the number of explanatory variables.

Fig. 1. Proposed research plan

In the proposed approach, PCA plays a slightly different role -it is used for grouping observations. The expected results of PCA are the principal components, which are a linear combination of explanatory variables, and graphs presenting the view of observations (cases) on the plane of the principal components. The graphs will allow to identify groups (clusters) of observations. The clusters will help in the implementation of the next stage -the conversion of explained variables. An example of the application of this approach is the analysis of parameters obtained from one of the manufacturing lines. The eigenvalues of the 20 principal components are presented in the scree plot shown in Figure 2.

Fig. 2. A scree plot for 20 principal components

The selection of the principal components for further analysis assumes determining the division point at the place where the eigenvalues of subsequent components cease to decrease significantly. For the further analyzes the principal components number 1 to 4 are selected. It is worth noting that the percentage values of the four main components cover in total over 80% of the variability of the explained variables. This example shows the high usefulness of PCA in application to the reduction of the dimensionality of the analyzed dataset.

For each pair of four principal components, observations were projected onto the plane. The points shown in the charts are individual cases from the data set. Charts in which component No. 1 is present contain clear clusters of observations (Figure 3). It turns out that data cases belonging to cluster A are at the beginning of the graph, cases from cluster B are in the middle, and the final part of the chart is occupied by cases from cluster C. Therefore, the clusters found can be used to separate observation classes on the graph of the number of defective products, i.e. to convert a quantitative variable into a qualitative one. In addition to the "increased number of defective products" and "acceptable number of defective products" corresponding to clusters C and A, the PCA suggests also taking into account the third "predicting" class, covering a period of a few days before the sudden increase in the number of defective products.

The research carried out so far allows the first conclusions. The PCA showed the variables, which have the highest impact on the data set's variability. These are temperatures of liquid glass, whose values were measured in three zones of the forehearth. The variables are the main candidates for recognition as a cause of products' defects, because the clusters obtained from PCA are compatible with the variability of the number of defected products, what is depicted in the Figure 4.

Summary

The paper presents a DM application for acquiring knowledge from large data sets in the glassworks. The article proposes a proprietary research plan aiming to develop a decision support system in the quality assurance. Part of the plan, which was implemented, allowed for identification of the parameters of the production process responsible for the increased defectiveness of the products. Preliminary results have been positively verified in the glassworks. The implementation of the full range of research (Figure 1) will allow to build and test a DSS for quality assurance. Both the research problem and the proposed solution method can be successfully used in education in the field of Industry 4.0 and Big Data concepts. The plan of further work envisages extending the data analysis to a collaboration network including glassworks' suppliers (materials, industrial automation systems, energy) in order to obtain a more complete picture of the parameters affecting the glass MP.

Fig. 3 .

 3 Fig. 3. Observations for selected pairs of four principal components Particularly compact clusters of observations appear in the graphs of components No. 1 and 3, where they are marked with letters A, B and C.The next stage of the analysis is the conversion of explained variables. At this stage of the research, data cases belonging to clusters A, B and C were projected onto a chronological graph showing the number of products with the defect (Figure4).

Fig. 4 .

 4 Fig. 4. The diagram of the number of defected products with identified classes