Franck Chesnel
email: franck.chesnel@univ-rennes1.fr

Emmanuelle Jullion
email: emmanuelle.jullion@univ-rennes1.fr

Olivier Delalande
email: olivier.delalande@univ-rennes1.fr

Anne Couturier

Adrien Alusse
email: adrien.alusse@univ-rennes1.fr

Xavier Le Goff

Marion Lenglet
email: marion.lenglet@univ-nantes.fr

Betty Gardie
email: betty.gardie@univ-nantes.fr

Caroline Abadie
email: caroline.abadie@ico.unicancer.fr

Yannick Arlot-Bonnemains
email: yannick.arlot@univ-rennes1.fr

Xavier Le Goff
email: xavier.le-goff@univ-rennes1.fr

Yannick Arlot-Bonnemains

Mutation of the proline P81 into a serine modifies the tumour suppressor function of the von Hippel-Lindau gene in the ccRCC

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

Introduction

The von Hippel Lindau (VHL) gene is a tumor suppressor gene which deletion or invalidation by mutations induce multiple tumors including renal and central nervous system hemangioblastoma, (CNS), clear cell carcinoma (ccRCC) and pheochromocytoma. The most studied well-known function of pVHL is to be a substrate recognition component of an E3 ubiquitin ligase complex comprising Elongin B/Elongin C, Cullin 2 and Rbx1 [START_REF] Lewis | Role of the C-terminal α-helical domain of the von Hippel-Lindau protein in its E3 ubiquitin ligase activity[END_REF] to regulate the hypoxia-inducible-factors (HIF-α) transcription factors. The association of pVHL to HIF-1α and HIF-2α is oxygen-dependent and triggered through hydroxylation of HIF [START_REF] Maranchie | The contribution of VHL substrate binding and HIF1-α to the phenotype of VHL loss in renal cell carcinoma[END_REF] ; it results in ubiquitination and proteasome-dependent degradation of HIF-α.

Based on the pathologies, VHL disease is classified in two distinct types, Type 1 and Type 2, depending on the development of pheochromocytoma (Type 2) or not (Type1). Type 1 subtype describes typical phenotype VHL manifestation as hemangioblastoma and RCC. Type 2 is subdivided into three subtypes: patients with Type 2A and B develop pheochromocytomas and other VHL manifestations, with a low risk (Type 2A) or high risk (Type 2B) of RCC, patients with Type 2C develop only pheochromocytomas. Patients with ccRCC (kidney tumor of epithelial origin) are therefore mostly classified into Type 1 and 2B [START_REF] Nordstrom-O'brien | Genetic analysis of von Hippel-Lindau disease[END_REF]. Finally, a third class of patients (Type 3) is characterized by the development of erythrocytosis, the most described being the Chuvash polycythemia associated with the homozygous VHL-R200W mutation [START_REF] Zbar | Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan[END_REF] [START_REF] Lenglet | Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease[END_REF]. At the molecular level, Type 1 families are characterized with various mutations (missense, frameshift, nonsense, splice) as well as in frame deletion/insertions or partial/complete deletions of VHL gene, leading to a loss of pVHL function and the development of a large spectrum of cancerous tumors [START_REF] Nordstrom-O'brien | Genetic analysis of von Hippel-Lindau disease[END_REF]. On the other hand, VHL Type 2 families were mainly characterized by missense mutations (83.5%), with a low percentage of nonsense, frameshift, splice, in-frame deletion/insertions, or partial deletions.

With the exception of deletions which account for 30-40% of cases, most mutations cluster within two regions of high functional importance in pVHL protein, the Elongin C (amino acid residues 157-171) and HIF-α (amino acid (a.a) residues 91-113) binding domains which are highly evolutionarily conserved sequences [START_REF] Leonardi | Tosatto SCE Adding structural information to the von Hippel-Lindau (VHL) tumor suppressor interaction network[END_REF]. Missense mutations could be deleterious for the protein leading to its functional inactivation and resulting in HIF-α accumulation and the expression of HIF target genes involved in signal transduction pathways responsible of hyperproliferation and/or tumorization.

Structurally, the pVHL protein consists of two functional protein binding domains, the βdomain (comprised of residues 63-155 and 193-204) and the α-domain (residues .

Accepted manuscript / Final version

When analyzing the 3D structure of pVHL, three distinct pVHL interaction interfaces have been described engaging in exclusive interactions [START_REF] Minervini | Tosatto SCE Genotype-phenotype relations of the von Hippel-Lindau tumor suppressor inferred from a large-scale analysis of disease mutations and interactors[END_REF]. The α domain binds elongins and Cullin2 while the β sheet domain of pVHL extending from residues 63 to 157 binds and participates to the polyubiquitination and ultimately targets HIF-α for proteasomal degradation [START_REF] Ivan | The von Hippel-Lindau tumor suppressor protein[END_REF].

Among the 1230 mutations listed in the VHL database (http://umd.be/VHL) some mutants still remain biochemically uncharacterized [START_REF] Nordstrom-O'brien | Genetic analysis of von Hippel-Lindau disease[END_REF]. Referred families diagnosed with P81S mutation linked to low penetrance Type 1 von Hippel-Lindau disease and a mild phenotype [START_REF] Hes | Cryptic von Hippel-Lindau disease: germline mutations in patients with haemangioblastoma only[END_REF] [START_REF] Alosi | Management of Gene Variants of Unknown Significance: Analysis Method and Risk Assessment of the VHL Mutation p.P81S (c.241[END_REF]. A previous study investigating defects Type 2C associated mutant protein demonstrated at the molecular level the deleterious role involvement of pVHL L188V , pVHL P81S/L188V , pVHL V84L. [START_REF] Knauth | VHL Mutations Linked to Type 2C von Hippel-Lindau Disease Cause Extensive Structural Perturbations in pVHL[END_REF].

In this study, we were interested in a family with two members who showed clinical symptoms of VHL disease. One member (III.2) developed a ccRCC at the age of 37. The genetic analysis has revealed the presence of c.241C>T (P81S) germline mutation. The genetic study of the progeny evidenced the presence of this same mutation in children (IV. [START_REF] Zbar | Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan[END_REF][START_REF] Lenglet | Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease[END_REF].

The use of in silico algorithms (HOPE, Symphony) indicated a possible pathogenicity of P81S mutation but not the GVGD that assigned a low genetic risk. [START_REF] Alosi | Management of Gene Variants of Unknown Significance: Analysis Method and Risk Assessment of the VHL Mutation p.P81S (c.241[END_REF]. Most of the analysis of the effect of the P81S mutations, were performed in relation with an additional mutation in the gene or exposure to trichlorethylene [START_REF] Desimone | Pleiotropic Effects of the Trichloroethylene-Associated P81S VHL Mutation on Metabolism, Apoptosis, and Accepted manuscript / Final version ATM-Mediated DNA Damage Response[END_REF]. Most carriers with the only P81S mutation have no VHL-related manifestations [START_REF] Weirich | VHL2C Phenotype in a German von Hippel-Lindau Family with Concurrent VHL Germline Mutations P81S and L188V. The Jour[END_REF] . The clinical observations made for the above family raised the need to understand the involvement of the mutation P81S in the development of VHL pathologies. We have undertaken a structural, biochemical and cellular study to characterize the pathogenicity of this mutation and to conduct a medical follow-up for practitioners.

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Materials and methods

Site directed mutagenesis and plasmid construction

The plasmids pcDNA3.1-PPGK-FlagHA-VHL213 and -VHL213 P81S were generated as follows : The ORF of VHL variant 1 (excised from pCMV2c-VHL213, a generous gift from Dr A.

Buchberger, Würzburg, Germany) was subcloned in pcDNA3.1-FlagHA (a kind gift from Dr S. Rouquier, Toulouse, France) with murine phosphoglycerate kinase (PGK) promoter [START_REF] Hascoet | The pVHL172 isoform is not a tumor suppressor and up-regulates a subset of protumorigenic genes including TGFB1 and MMP13[END_REF].

P81S point mutation was introduced into the VHL cDNA inserted in pcDNA3.1-PPGK-FlagHA-VHL213 using a sequential two-step PCR approach with the high-fidelity PCR master polymerase (Sigma Aldrich, St Quentin-Fallavier, France). The oligonucleotides used to carry out the site-directed mutagenesis were as follows: P81Sfor, 5'-TGC AAT CGC AGT TCG CGC GTC-3'; P81Srev, 5'-GAC GCG CGA ACT GCG ATT GCA-3'. The resulting final amplicon was cut with BamHI and XhoI (Promega, Charbonnière les bains, France) and inserted into empty pcDNA3.1-PPGK-FlagHA (first linearized with the same restriction enzymes) with T4 DNA ligase (Promega).

All constructs were confirmed by Sanger sequencing, amplified in NEB5alpha bacteria (New England Biolabs, Evry, France) and purified using the Nucleobond Endofree plasmid purification kit (Macherey Nagel, Hoerdt, France) before transfection in mammalian cells.

Cell culture and transfections

The 786-O kidney cancer cell line, from the ATCC (LGC Standards), was cultured in RPMI-1640 (Life Technologies, Courtaboeuf, France) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37°C with 5% CO2. Stable cell lines were generated by transfecting 786-O cells with pcDNA3.1-PPGK-Flag-HA-VHL213P81S using Avalanche-Omni (EZ-Biosystems, College Park, USA) and selected with 500µg/ml G418 (Life Technologies).

The 786-O cells with pcDNA3.1-PPGK-Flag-HA-VHL213wt have been previously generated in the laboratory [START_REF] Hascoet | The pVHL172 isoform is not a tumor suppressor and up-regulates a subset of protumorigenic genes including TGFB1 and MMP13[END_REF].

Protein extraction and immunoblotting

Cells were lysed in RIPA extraction buffer, or directly in Laemmli loading buffer. Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose.

Membranes were probed with antibodies against VHL (1:1,000; clone JD1956 [START_REF] Chesnel | The von Hippel-Lindau tumour suppressor gene: uncovering the expression of the pVHL172 isoform[END_REF]), HA (1:4,000; Roche), Flag (1:2,000; Sigma-Aldrich), β-tubulin (1:2,000; Sigma-Aldrich), cyclin D1 (1:500; Cell Signaling Technology), cullin-2 (1:450; Life Technologies), Elongin-C (1:1,000; Bio Legend), HIF2-α (1:500; Novus Biologicals). Immune complexes were detected as previously described [START_REF] Chesnel | The von Hippel-Lindau tumour suppressor gene: uncovering the expression of the pVHL172 isoform[END_REF].

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Reporter assays

The parental 786-O or the stable 786-O-pVHL213 and 786-O-pVHL213-P81S cell lines were transfected with plasmids encoding Firefly-luciferase under the control of Hypoxia-Response-Elements (HRE-luciferase), and Renilla-luciferase for normalization. After a 24-h culture, luciferase activity was measured using a Dual-Luciferase®Reporter Assay System (Promega).

Wound healing assay

Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 until confluence in culture-inserts 2 wells (Ibidi-Cell in focus; Clinisciences, Nanterre, France). Cells were treated or not with 10µg/ml mitomycin C (Sigma-Aldrich). Cell migration was then recorded every 15 min for 24 hours using an Axiovert 200M microscope equipped with a LD Plan-Neofluar 20×/0.4 Ph2 lens and an AxioCam MRm camera under the control of the ZEN 2012 software (Zeiss). The wound area was measured using the Simple PCI6 software (Hamamatsu Photonics, Massy, France). The analysis of cell migration during the assay was performed with the MRI Wound Healing Tool (http://dev.mri.cnrs.fr/projects/imagejmacros/wiki/Wound_Healing_Tool) using Image J software.

Spheroid formation

Five thousand cells were plated in medium per well of a 96-well plate with round bottom previously coated with Poly(2-hydroxyethyl-metacrylate) (Sigma-Aldrich) and incubated for up to 4 days to allow spheroid formation. The spheroids were analyzed by microscopy (DMIRB-Leica) and the size calculated using Image J software.

Invasion tumor assay on Matrigel

The spheroids were embedded in a gel composed of a mixture of Matrigel (2mg/ml; Corning) in full medium RPMI-1640 supplemented with 10% fetal calf serum and 1%

penicillin/streptomycin at 37°C with 5% CO2. Cell migration away from the spheroids was recorded under a video microscope (Delta vision-MRIC-SFR-UMS-CNRS 3480-INSERM018)

for several hours.

Modeling

The pVHL213 model is derived from the protein databank structure PDB ID:1VCB completed by the i-tasser server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/).

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Results

Clinical evaluation

The family A with members suffering from VHL disease studied in the present study was recruited in the PREDIR center (France). A clear cell Renal Cell Carcinoma (ccRCC) was diagnosed in the proband (III.2) at the age of 37 and treated with radical nephrectomy. Genetic

Structural evaluation of the P81S mutation

The VHL tumor suppressor gene encodes a protein which contains two main structural domains:

Accepted manuscript / Final version

The proline 81 is located in the β-domain and included in a turn in the molecular structure (between the β strands #1and #2, Fig. 2, B andC). As many other proline residues of pVHL, Pro81 defines boundaries of secondary structure elements such as the β strands and could be essential for ternary structure formation (Fig. 2B).

Due to its localization, the P81S substitution might cause significant structural perturbations in the β domain organization [START_REF] Minervini | Tosatto SCE Genotype-phenotype relations of the von Hippel-Lindau tumor suppressor inferred from a large-scale analysis of disease mutations and interactors[END_REF]. Nevertheless, the reported link of the P81S substitution to Type 1 VHL disease remains enigmatic, given the lack of significant functional defects of the pVHL-P81S mutant protein in vivo [START_REF] Weirich | VHL2C Phenotype in a German von Hippel-Lindau Family with Concurrent VHL Germline Mutations P81S and L188V. The Jour[END_REF]. Such altered pVHL may still be able to exert some of its functions and/or may gain novel ones. Structural analyses suggest a modification of the β domain, but biochemical studies are required to clarify the alterations in biological functions.

Biochemical impact of P81S mutation on pVHL213 function(s)

To analyze the potential effect(s) of the mutation P81S on the function of pVHL, the Proline in position 81 was mutated into the polar uncharged amino acid, Serine. We stably transfected the Loss of pVHL activity might be the result of pVHL functional inactivation or degradation that is often observed in Type 1 pVHL mutants. We thus addressed the question whether the prediction of the stability of pVHL mutants may influence its functionality. As all clones obtained after selection expressed identical levels of the protein (Fig. 3A), we selected one of the three clones (E6). Tubulin visualization shown no obvious architectural differences were discriminant for the three cell lines (Sup Fig. 1). In order to control the stability of the mutant pVHL213-P81S protein, the different stable cell lines were treated with the protein synthesis inhibitor cycloheximide (CHX) for 3 and 7 hours. CyclinD1 expression confirmed the CHX treatment efficiency. Expression levels of pVHL in the 786-O-pVHL213-P81S stable cell line compared to that of wild type pVHL in the 786-O-pVHL213 were measured by immunoblotting (Fig. 3B andC). The stability of the pVHL213-P81S was not significantly changed. Indeed, evaluation of the amount of the pVHL213-P81S and pVHL213 showed that in both cases the Accepted manuscript / Final version remaining amounts of proteins after a 7-hour treatment with CHX consist in respectively 56% and 66% of the initial amount of endogenous protein (Fig. 3C, p=0, 396).

The pVHL tumor suppressor functions as a redox sensor, which controls stability of the hypoxia-inducible transcription factor family (HIFs) amongst other proteins.

The VHL gene product is the substrate recognition subunit of a multiprotein E3 ubiquitin ligase complex, in association with the scaffold protein Cul2, the ring box protein Rbx1 and the adaptor proteins, Elongins B and C. As most VHL mutations affect the oxygen-dependent regulation of HIF2-α cellular levels, we tested whether the mutant pVHL213-P81S participated to the E3 ligase complex to ubiquitinate and ultimately lead to HIF2-α degradation. In order to prevent HIF2-α proteasomal degradation that rapidly occurs upon polyubiquitination by pVHL and therefore allow its co-immunoprecipitation, the cells were first treated with the proteasome inhibitor (MG132) up to 5 hours before harvest and the proteasome inhibitor efficacy in preventing HIF2-α degradation was verified by western blotting (Fig. 4A).

Immunoprecipitation of flagged proteins expressed in 786-O-pVHL213 and 786-O-pVHL213-P81S was then performed. As observed on Fig. 4B, Cullin 2 was similarly co-precipitated by both pVHL213 and pVHL213-P81S proteins. Likewise, HIF2-α which associated with the pVHL213 was also detected with the immunoprecipitated pVHL213-P81S (Fig. 4B). The ratio We then evaluated the amount of HIF2-α in the different cell lines incubated in the presence of protein synthesis inhibitor (CHX) (Fig. 3B). In the absence of CHX treatment (t=0), the amount In contrast, no significant variation of HIF2-α was observed between when 786-O-pVHL213 and 786-O-pVHL213-P81S after hypoxic treatment (Fig. 3F).

To better understand the effect of the mutation P81S on the regulation of HIF, a functional study was performed using a Hypoxia Response Elements (HRE) dependent reporter assay [START_REF] Lenglet | Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease[END_REF] (Fig. 4D). The ability of pVHL213 and mutant pVHL213-P81S to downregulate firefly luciferase activity (reflecting HIF activity) was tested in 786-O cells and the results confirmed that pVHL213 as well as pVHL213-P81S displayed a similar luciferase activity, largely reduced compared to that in 786-O cells (Fig. 4D).

Altogether, our data show that the formation of the E3 ligase complex is not fundamentally altered when pVHL213 is mutated on Pro 81(to Ser) and the modest variations in HIF2-α (binding and stability) observed in the presence of pVHL213-P81S cannot account for the development of the pathology in a member of the family.

The P81S Mutation of pVHL213 modifies migration properties of renal cells

Dysregulation of the VHL gene is closely associated with ccRCC. Other pVHL, HIFindependent, functions have been shown to be required and help to explain why loss of pVHL leads to renal cancer. How the P81S mutation of pVHL might explain the development of the ccRCC for a 37-year-old patient in a HIF-independent manner was thereafter investigated. As malignant cancer cells exploit their intrinsic migratory ability to invade adjacent tissues and the vasculature, we have studied cell morphology and dynamics. Transformed cells often exhibit different growth characteristics than non-transformed cells. Therefore, in a first round of experiments, we investigated the migration/invasion properties of these different cell lines.

First, we set up a standard scratch wound healing assay (Fig. 5A). The migration of cells in the Accepted manuscript / Final version specific wound area was recorded every 15 min during 16 hours (= 1000 minutes), i.e. until the scratch area was fully repopulated by all of the tested cell lines (Fig. 5A). The images from the beginning to the end of a 16 hours incubation period were captured. The end of recording time was determined at 16h when the slowest moving cells (786-O-pVHL213 cells -dark line) were just about to fully fill in the scratch (Fig. 5B). The 786-O cells, which do not express wild type pVHL, covered the wound caesura after 7. The ability of cells to grow in a semi-solid medium was further recorded while testing the characteristics of malignantly transformed cells. The morphology of the spheroids obtained

Accepted manuscript / Final version with the 786-O cells was not changed when they were embedded in the Matrigel for 24 hours.

The appearance of the 786-O-pVHL213 aggregates deposited in the Matrigel was transformed and became more compact (Fig. 5C). In contrast, the 786-O-pVHL213-P81S spheroids appeared modified, the inner part of the spheroid became dense and compact whereas the peripheral cells escaped from the spheroids and migrated into the Matrigel (Fig. 6C). These observations strongly suggest that 786-O cells acquire invasive features upon expression of pVHL213-P81S.

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Discussion

In this study, we described the workup of a family carrying a germline missense mutation c.241C>T, p.Pro81Ser (P81S) with carriers' members : a 37-year-old adult diagnosed with a ccRCC and some family members carrying the mutations who were asymptomatic.

Moreover, the P81S in the VHL gene has been identified in 4 other French families in the PREDIR molecular genetics laboratories network (https://predir.org/View/accueil.aspx). One proband had a ccRCC at a young age (28 years), 3 others had pheochromocytoma or paraganglioma at older ages (68 and 70 years for two of them, age unspecified for the third case).

Other proline mutations have been reported in databases as well, including Proline in positions 2, 86, 97 and converted in non-polar residue (Leu, Ala), polar (Ser) or basic amino acid residues (Arg, His). Depending on the amino acid, the mutants were classified benign or as variants of uncertain significance (VUS). Patients bearing the Pro86Ala/Arg/Ser/His mutations and developing hemangioblastoma and kidney cysts were described as probably pathogenic [3][17].

The presence of the P81S mutation in various families were associated with low penetrance or mild phenotype together in the German VHL family with one affected member who developed central nervous system (CNS) hemangioblastoma and renal cell carcinoma as well as kidney and pancreatic cysts [START_REF] Nordstrom-O'brien | Genetic analysis of von Hippel-Lindau disease[END_REF]. Moreover, the substitution of the Proline 97 by a Leucine was described as pathogenic when associated with another mutation Leu166Pro [START_REF] Gnarra | Mutations of the VHL tumour suppressor gene in renal carcinoma[END_REF].

It was therefore of interest to discriminate the role of the mutant the P81S VHL pathology.

We employed a set of in silico predictions and in cellulo and in vitro methods along with structural analysis to investigate whether the single P81S mutation is involved in disease development or neutral.

Predictions for the impact of the P81S substitution in the VHL gene, obtained by in silico analysis, showed a discrepancy depending on the prediction tools. The different in silico algorithms used to analyze the mutants reported the difficulties to classify the P81S VHL mutation as it could come out as potentially pathogenic or as unclassified variant (www.umd.de). We should have replaced the Proline into an Alanine which is also a nonpolar and hydrophobic amino acid This modification brings great complexity to the structural and biochemical structure but it is consistent with the pathology. We are aware that serine not only changes the organization of the molecule in 3 dimensions and induces charge modifications that Accepted manuscript / Final version can generate new bonds. We focused on the patient's mutation P81S despite the fact that it combines the change in charge and structure.

In particular, replacement of proline by serine at residue 81 might disturb the native pVHL structure by destroying a rigid link known to be associated with proline and to favor the formation of hydrogen bonds with neighboring polar amino acids. However, as many other proline residues of pVHL, Proline 81 plays a role in defining the boundaries of secondary structure elements such as the β-strands composing the core of the protein. Indeed, among the Proline residues, several are delimiting the extremities of a β strand: Pro71 and Pro81 (start and end of β1), Pro95 and Pro99 (start and end of β3), the Pro102 and Pro103 pair (start of β4) and Pro146 and Pro154 (start and end of β 7) (Fig. 2C). The distribution of many of these specific amino acids along the pVHL sequence allows the chaining of short β-strands and a succession of turns organizing the β domain of the protein. It should nevertheless be emphasized that the in-silico prediction can only give an approximation for pathogenicity that need to be validated by in vitro and in vivo experimental validation.

Most frequently reports showed that Type 1-associated pVHL mutant proteins are most frequently reported as failing to assemble the E3-ubiquitin ligase [START_REF] Knauth | VHL Mutations Linked to Type 2C von Hippel-Lindau Disease Cause Extensive Structural Perturbations in pVHL[END_REF]. The risk relevance of this mutation was only identified in TCE-induced ccRCC or in association with other mutations [START_REF] Brauch | VHL mutations in renal cell cancer: does occupational exposure to trichloroethylene make a difference?[END_REF] [START_REF] Knauth | VHL Mutations Linked to Type 2C von Hippel-Lindau Disease Cause Extensive Structural Perturbations in pVHL[END_REF]. Moreover, previous studies have speculated that the impact of the P81S mutations might even be stronger in combination with the L188V mutation [START_REF] Weirich | VHL2C Phenotype in a German von Hippel-Lindau Family with Concurrent VHL Germline Mutations P81S and L188V. The Jour[END_REF]. Altogether, both mutations likely influence protein-protein interactions. Previous reports on structural studies of the P81S variant mentioned that this proline to serine substitution was not expected to cause significant structural perturbations on its own, but it has been proposed to affect the orientation of the neighboring residue Arg82, whose side chain forms the center of a hydrogen bond network stabilizing both the α/β domain interface of pVHL-Elongin C [START_REF] Stebbins | Structure of the VHL-ElonginC-ElonginB Complex: Implications for VHL Tumor Suppressor Function[END_REF].

The establishment of the stable cell line became essential to investigate the pathogenicity of pVHL213-P81S. As mentioned in previous work, the absence of native conformation could highly lead to a destabilization of the protein [11][21]. In addition, the B domain is implicated in substrates' recognition like the hypoxia induced factors alpha (HIFs-α, the A interface is important for the formation of the VBC complex [7][22]. Because of the critical location of P81S within the β-sheet domain of pVHL, we inferred that protein binding, including HIF-2α, may be impaired. In our hands, the stability of the protein pVHL213-P81S amount was not significantly different from that of wild type pVHL213, suggesting that the P81S mutation does not trigger a total protein unfolding as often observed for type I pVHL mutation.

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Moreover, the E3 complex was still formed with pVHL213-P81S and able to bind HIF2-α. The amount of HIF2-α in cells expressing pVHL213-P81S was detected mostly more related to the amount of the protein quantified in the 786-O cells. Therefore, the particular P81S mutation does not seem to abolish (or diminish) HIF binding, as one should expect with a type I mutation.

The role as hypoxia sensing component was conveniently used to explain some of the main VHL manifestations. However, it fails almost entirely in predicting the pathogenic risk of several mutations not directly connected with HIF-1α degradation. Defective HIF-1/2-α regulation is believed to be sufficient to induce development of hemangioblastomas and to be a key pathogenic event in the development of RCC but in the present study, the ability of pVHL213 P81S to down-regulate HIF, at least in normoxic conditions, suggest that other pVHL-HIF-independent functions may be affected to explain the development of the ccRCC.

Lastly, the experimental results did not explain why in the family described in this study one member developed a ccRCC. These observations reveal a preferentially non-canonical function of pVHL213-P81S. pVHL plays many roles, including a very important one in the formation of the extracellular matrix, epithelial differentiation and correct interaction between epithelial cells and the extracellular matrix [START_REF] Grosfeld | Interaction of Hydroxylated Collagen IV with the von Hippel-Lindau Tumor Suppressor[END_REF]. Moreover, deletion of the VHL gene can activate β-catenin [an important mediator of EMT [START_REF] Peruzzi | The von Hippel-Lindau tumor suppressor gene product represses oncogenic ␤-catenin signaling in renal carcinoma cells[END_REF]. The inactivation of pVHL in pheochromocytoma has led to the up-regulation of seven genes, such as CTGF, SDCBP, CYR61, COL3A1, COL1A1, COL5A2 and SERPIN1, which are closely related to cell proliferation, migration and differentiation [START_REF] Gao | Seven Novel Genes Related to Cell Proliferation and Migration of VHL-Mutated Pheochromocytoma[END_REF].

This naturally led us to test, in migration 2D and invasion 3D assays, the hypothesis that P81S mutation of pVHL would impact cell migration and/or invasiveness int the 2D or 3D assays. We indeed observed a more migrating potency of the 786-O-pVHL213-P81S. The speed of pVHL213-P81S cell invasion, the shape and the size of the spheroids, as well as the behavior of the cells from the spheroids, when included in Matrigel, supported the modified function of pVHL when the P81S mutation is present.

Other Prolines (P86 and P97) located in the β-domain have been shown pathogenic when mutated, while inducing hemangioblastomas and ccRCC [START_REF] Chen | Germline mutations in the von Hippel-Lindau disease tumor suppressor gene: Correlations with phenotype[END_REF]. Also in the B-domain , the Ser 72 of pVHL when mutated into proline was described as pathogenic, associated with hemangioblastomas and pancreatic cyst [27][28]. Functional interaction of pVHL with MetaDome and Mobidetails [30][31] web server which help to interpret DNA variants in the context of molecular diagnosis, we observe that the P81S mutant is classified as intolerant and certainly pathogenic (Supp Fig. 3). The development of kidney cancer in young subjects in the family studied and the detection of pheochromocytomas and angioblastomas in patients (http://www.umd.be/) strongly suggests a medical follow-up in the members of the impacted families. Further investigations will be necessary to define in more details the molecular pathway involved in cell transformation with such pVHL mutant.

Accepted manuscript / Final version

  analysis revealed the germline missense mutation c.241C>T, p.Pro81Ser (P81S) in the VHL gene (Reference sequence: NM_000551). A retrospective study of the potential presence of mutation in parents and descendants was undertaken. There were originally three patients with the P81S mutation in the VHL gene (II.8. Genetic testing of the descendants identified the P81S mutation in the genome of the two 8-and 13-years-old children. In addition, sequencing of the VHL gene of members of the background family showed the presence of the mutation for the sister of and the father who are respectively 65 and 38 years old (Fig.1II.1, III.1). Besides the proband's ccRCC, none of the carriers displayed any other signs of VHL-related manifestations including the 65-year-old father. The variant was identified in the index case and was declared as pathogenic. The hypothesis is that in the case of a spectrum tumour in a person carrying a VHL variant, the portal of entry here being renal, there is probably a loss of the 3p on the wildtype allele. Tumor material was not available to perform the analysis. Surveillance program, according to the French VHL guidelines (Centre PREDIR) (including Magnetic Resonance Imaging (MRI) of the Central Nervous System, MRI or Ultrasound of the Abdomen, ophthalmological examination, measurement of urinary metanephrines), was normal for all of the carriers. The single manifestation in the proband and the absence of clinical signs in the other mutation carriers questioned about the penetrance of the P81S germline mutation.

(

  β domain a.a 65-154 and α-domain-a.a157-193) held together by two linkers and a polar interface. (Fig.2A). Previous studies combining structural information and computational analysis have distinguished three distinct interfaces[START_REF] Leonardi | Tosatto SCE Adding structural information to the von Hippel-Lindau (VHL) tumor suppressor interaction network[END_REF], each interface refined motifs involved in specific protein-protein interactions. Moreover, five distinct pVHL interaction interfaces (A, B, C, D and E, Fig. 2A) are engaged in exclusive interactions with different interactors and may partially explain the pVHL binding plasticity. Interface A (a.a 154-189) is important for VCB complex formation and interface B (a.a 60-103) forms the HIF-1α binding-site while interface C (a.a 157-190) participates in the Cullin2 interaction.

  renal carcinoma cell line 786-O (VHL-/-) which does not express wild-type VHL with the plasmid pcDNA3.1-PPGK-Flag-HA-VHL213P81S to moderately express the pVHL protein with the mutation of interest. Three positive clones (namely E1, E6, and C7) were obtained and controlled for mutation by sequencing. The expression level of the protein pVHL was then analyzed in the different cell lines: parental 786-O, 786-O-pVHL213 (wild type) and the three clones corresponding to 786-O-pVHL213-P81S. The immune detection revealed a positive expression of the VHL protein in the three clones (Fig. 3A).

  HIF2-α/pVHL and Cul2/VHL evaluated on three distinct experiments revealed that HIF2α/pVHL ratio appears stronger in the 786-O-pVHL213-P81S than in the 786-O-pVHL213 cells but only after 2 hours (Fig. 4C p= 0,0313) and 5 hours (Fig. 4C p= 0,0041) of treatment with MG132 (n=5-Fig. 4C). This ratio is not significantly different in non-treated cells. (Fig 4C). The amount of Cul2 immunoprecipitated with the 786-O-pVHL213-P81S did not change notably from the amount of Cul2 detected in the 786-O-pVHL213. These first results show that the P81S mutation does neither diminished recruitment of E3-ligase subunit such as Cullin2 nor the binding of the major substrate HIF-2α.

  of HIF2-α was significantly diminished in the 786-O-pVHL213 cells as well as in 786-O-pVHL213-P81S cells, compared to the level detected in 786-O cell lines (p value = 0.00173 and 0.04156, respectively) (Fig.3D). No difference in HIF2-α level was observed between 786-O-pVHL213 and 786-O-pVHL213 P81S cells (pvalue : 0.685) (Fig.3 B and D). After treatment with CHX, the quantity of HIF2-α remaining in the pVHL213 and pVHL213-P81S cells represented 39% of the initial amount of the HIF2-α evaluated in the 786-O cells (Fig.3D). The level of Accepted manuscript / Final version immunodetected HIF2-α associated to pVHL did not seem to change in 786-O pVHL213-P81S cells as well as in 786-O pVHL213 (Fig.3D).To go further in understanding the role of the mutation towards the canonical function of pVHL, we also compared the amounts of the protein HIF2-α in the 786-O, 786-O-pVHL213 and 786-O-pVHL213-P81S cell lines under normoxia versus chemical hypoxia condition (19 hours of CoCl2 treatment). Under normoxic conditions, HIF2-α was lower in 786-O-pVHL213 cells (ttest, p=0.02616) as well as in 786-O-pVHL213-P81S cells (t-test, p=0.004998) in comparison to 786-0 (Fig.3E and F) . After treatment of the cells with CoCl2 for 19h, the quantity of immuno detected HIF2-α increased in both pVHL213 cells and pVHL213-P81S cell lines. In the presence of CoCL2, significant modification of HIF2-α amount was observed between 786-O cells and 786-O-pVHL213 (t-test, p=0.0365) and 786-O-pVHL213-P81S cells (t-test, p=0.0194).

  5 hours (Fig. 5B, grey line) whereas the 786-O pVHL213-P81S stably transfected cells close the caesura in 6 hours (Fig. 5B, dotted line). Therefore, comparison of the cells stably transfected with the pcDNA-VHL213 and those transfected with the pcDNA-VHL213-P81S showed modification of cell migration when VHL is mutated. The time-dependent changes of the open area due to cell migration were scored for each cell type as the time to repopulate 50% of the gap. The parental 786-O cells invaded 50% of the area after 202,9 ± 13,7 min versus 368 ± 48 min for 786-O pVHL213 (Fig. 5C) The 786-O cells expressing pVHL213-P81S covered 50% of the caesura in 162,4 ± 11 min. The statistical analysis of the experiments for each cell line revealed a significant difference in time of migration between 786-O and 786-O-pVHL213 (n=8 p .value 0.0006333 t test) and between 786-O-pVHL213 and 786-O-pVHL213-P81S (n=7 pvalue 0.0003016 -t-test) (Fig.5C). A slight difference of migration was observed when comparing the migration of between the 786-O and 786-O-pVHL213-P81S (n=6 pvalue : p 0,01342t test)(Fig.5C). Impact of pVHL213-P81S on aggregation properties of the transfected tumoral cells Multicellular aggregates of cells and tumor organoids have become preclinical model systems of great interest for studying tumor behaviors. Spheroids more faithfully reproduced the tumor macrostructure compared to classical 2D monolayers. As previously demonstrated, the spheroid formation assay was performed to determine the properties of 786-O cells to aggregate and the impact of wild type and mutated pVHL on this cell aggregation. The three cell lines 786-O, 786-O-pVHL213 and 786-O-pVHL213-P81S were tested for spheroid formation (Fig. 6A) and the expression of pVHL was controlled in the cell lines (Fig 6B). As previously shown, the 786-O cell line formed multicellular spheroids with highly uniformity in size and cell density[14]. Interestingly the 786-O-pVHL213-P81S cells formed a cluster of cells which structure differed from the structures observed with either the 786-O cells or the 786-O-pVHL213 cells. The contour of spheroids appeared as an intermediate shape between the one observed when prepared with the 786-O cells and the 786-O-pVHL213-P81S with dense cell-cell interactions and borders slightly widespread (Fig. 6A).

  The different 786-O-pVHL213-P81S clones obtained in our study showed identical behavior with respect to spheroid formation and migration into Matrigel (SupFig 2).The actin cytoskeleton undergoes dynamic assembly and disassembly during cell crawling, which regulates protrusion formation, focal adhesion assembly/disassembly, and contractile filament organization. Assembly of the actin cytoskeleton is essential for invasion and cell migration. Cell migration requires the formation of various structures, such as invadopodia and pseudopodia, which require actin assembly that is regulated by specialized actin nucleation factors. Cell migration is the result of a multistep and complex process initiated by the transformation of the actin cytoskeleton. The length of filaments varies greatly, the filaments are cross-linked into imperfect bundles and networks. The disruption of the actin cytoskeleton inhibits cell migration and adhesion[START_REF] Gordan | Hypoxia-inducible factors: central regulators of the tumor phenotype[END_REF]. The orientation of F-actin fibers (parallel and random distribution) was analysed in the different cell lines used in this study. The F-actin network was organized as dense and parallel fibers along the long axis of the 786-O pVHL213 cells while actin fibers were longer and less dense in 786-O cells (Fig.6D). In contrast, actin fibers were very short and mostly located near the membrane in cells expressing pVHL with the P81S mutation (Fig6D). In conclusion, the architectural network exhibited different organisation in the cells expressing or not pVHL213 or pVHL213-P81S.

  AURKA was shown to modulate microtubules stability and dynamics. Mutations of the Ser into Pro participate in tumor initiation/progression[START_REF] Martin | Identification of pVHL as a novel substrate for Aurora-A in clear cell renal cell carcinoma (ccRCC)[END_REF]. In the present situation, the P81S mutation might induce structural transformations that could affect the functions of the Accepted manuscript / Final version surrounding amino acids. This would therefore modify the non-canonical functions of the protein. The pVHL213-P81S drive morphological cell changes that might be induced by expression of protein involved in cell adhesion and migration expression. Conclusively, our results demonstrate that the P81S mutation changes the cellular tumor suppressor function of pVHL213 and confers to the cells a migratory and invasive behavior. Moreover, combining

Figures legends Figure 1 :Figure 2 :

 12 Figures legends

Figure 3 :

 3 Figure 3 : Characterization of the stable cell lines expressing the pVHL213-P81S A-pVHL expression in 786-O, 786-O-pVHL172 and 786-O-pVHL213 and 786-O-pVHL213 P81S cells (different clones : E1, E6 and C7) assessed by immunoblotting with the indicated antibodies VHL ( JD-1956-[15]) and GAPDH .B-Stability of pVHL was assessed in the different 786-O-pVHL213 and 786-O-pVHL213 P81S cell lines after treatment with cycloheximide for 3 and 7 hours. HIF-2α protein expression level was also evaluated by immunoblotting in the three cell lines 786-O, 786-O-pVHL213 and 786-O-pVHL213 P81S. Immunodetection of Cyclin D1 was performed as a control of the efficiency of treatment with CHX. GAPDH was used as a loading control. C-The histogram shows the quantification of the amount of pVHL (mean ±SEM; n=3) in 786-O-pVHL213 and 786-O-pVHL213 P81S. D-The histogram illustrates the amount of HIF2-α detected in the three 786-O, 786-O-pVHL213 and 786-O-pVHL213 P81S cell line before (0) or after treatment with CHX for 4 or 7 hours. The results are expressed as the mean ±SEM of three independent experiments (** p value :0.00173) and (*p=,0.04156). E-Effect of Hypoxia induced by incubation of the cells with CoCl2 during 19 hours on the amount of HIF2-α in the absence (or presence of pVHL213 or pVHL213 P81S. HIF2-α, VHL (and GAPDH as a loading control) were detected by Western Blot F-Quantification of the immunodetected amount of HIF2-α from five independent experiments (**t-test, p=0.02616 ; * t-test, p=0.004998 ; ns Non significant ).

Figure 4 :

 4 Figure 4: pVHL213-P81S is part of the E3 ligase complex A-786-O, 786-O-pVHL213 and 786-O-pVHL213 P81S cell lines were cultivated in the presence of MG132 for 2 or 5 hours. The amounts of VHL, Cul2 and HIF2-α were estimated following their detection by immunoblotting. GAPDH was used as a loading control to normalize the level of each protein B-Flag immunoprecipitation was performed with total protein extracts from the 3 cell lines and for each time of MG132 incubation. The proteins VHL, HIF2-α and Cul2 were then immunoblotted in the bound fractions. C-The ratio CUL2/VHL and HIF2-/VHL was calculated from five independent experiments. The histogram shows the quantification of the amount of CUL2/VHL (mean ±SEM; n=5) and HIF2-α/VHL in 786-O-pVHL213 and 786-O-pVHL213 P81S. Quantification of the immunodetected amount of HIF2-α /VHL from five independent experiments (**t-test, p=0.0041 ; * t-test, p=0. 0313 ; ns Non significant ). D-Functional HREdependent reporter assays were performed in 786-O cells (i.e. VHL negative cells that constitutively accumulate HIF-2α in normoxia. The results are expressed as relative Firefly luciferase activity with Renilla luciferase as an internal control. 1unit denotes the basal activity of endogenous HIF-2α using the HRE-luciferase reporter plasmid. The abilities of pVHL213 and pVHL213-P81S to downregulate Firefly luciferase activity (related to HIF activity) were compared.

Figure 5 :

 5 Figure 5: Expression of pVHL213-P81S promotes cell migration. A-The 786-O, 786-O-pVHL213 and 786-O-pVHL213-P81S cell were seeded in culture inserts. Analysis of cell migration by wound healing assay was then carried out with images taken every hour for 16 hours. B-Results were expressed as the percentage of wound closure at the indicated time points. The average corresponded to of 3 wells per cell line from 6 independent experiments is shown. C-Time needed for 50% closure was estimated for each cell line (mean ±SEM; 786-O and 786-O-pVHL213 (n=7 p-value 0.0006333 -t-test) 786-O pVHL213 -and 786-O-pVHL213-P81S (n=7 p-value 0.0003016 -t-test )7 86-O and 786-O-pVHL213-P81S (n=6 pvalue : p 0,01342t test).

Figure 6 : 3

 63 Figure 6: Tumorigenic effect of pVHL213-P81S in 786-O cells A-Morphology of 786-O, 786-O-pVHL213 and 786-O-pVHL213-P81S spheroids. Spheroid aspect was obtained after 48hours of incubation of cells on poly-Hema coated wells. Spheroids were imaged under Leica microscope. Scale bars: 100 µm. B-The spheroids were collected 48 hours post seeding and pVHLs expression was evaluated by western blot. GAPDH was used a loading control. C-The spheroids corresponding to the 3 different cell lines were loaded into Matrigel. The spheroid morphology was recorded during 24 hours. The picture showed the spheroids after 24 hours of incubation. D Actin staining, analyzed after Rhodamin-phalloidin

We thank the IGDR for all facilities. We would like to acknowledge S. Dreano (IGDR) for his contribution in plasmids sequencing. We acknowledge the ImPAcCell and MRic microscopy platforms at the SFR BIOSIT (CNRS UMS3480 Rennes). We thank the LNCC for financial support of this study. We thank L. Schmitt for its contribution to the revision experiments.

Additional Information

Authors contribution

Conceived and designed the experiments: FC YAB. Performed the experiments: FC ,AA AC EJ ML XLG .Analyzed the data : FC XLG BG YAB. Contributed reagents/materials/analysis data from patients: CA, BG. Wrote the paper: YAB.

Ethics approval and consent to participate:

No applicable

Competing Interests

No applicable

Accepted manuscript / Final version


Gilbert Teyssedre
email: gilbert.teyssedre@laplace.univ-tlse.fr

Jean-Luc L Franceschi

Christian Laurent

Cathodo-and electro-luminescence spectra in insulating polymers: a parallel approach for inferring electrical ageing mechanisms

HAL is

I. INTRODUCTION

Electroluminescence -ELis excited in insulating polymers by charge recombination and/or upon inelastic scattering of hot electrons. It provides a tool for defining critical transport regime leading to energy release in a range that is potentially harmful for the material [START_REF] Laurent | Optical pre-breakdown warnings in insulating polymers[END_REF]. Conditions of its occurrence can be investigated by a simultaneous analysis of electroluminescence and space charge distribution, giving access to the nature of the excitation process [START_REF] Leroy | Charge transport and dissipative processes in insulating polymers: joining experiments and model[END_REF]. Another related task is to investigate the nature of the emitting species and what they tell us. EL can sign reversible or irreversible electronic transitions (associated with physical and chemical reactions respectively) which has significance as regards electrical ageing and breakdown. It is therefore critical to be able to analyze the electroluminescence spectrum [START_REF] Teyssedre | Identification of the components of the electroluminescence spectrum of PE excited in uniform field[END_REF]. Up to now, we have used different forms of excitation to give a support for the EL spectra interpretation, being mild UV irradiation (featuring reversible transition), contact with a cold plasma (featuring chemical reaction and charge recombinationinduced luminescence) [START_REF] Teyssedre | Spectral analysis of optical emission due to isothermal charge recombination in polyolefins[END_REF] and chemi-luminescence (featuring degradation by oxidative processes) [START_REF] Tiemblo | Chemiluminescence spectral evolution along the thermal oxidation of isotactic polypropylene[END_REF]. If all these sources have allowed a better understanding, they never reproduced the EL spectrum in details.

In this study, we have installed an electron gun in the same equipment as that used for carrying out luminescence analysis with the other excitation means. Samples are irradiated with electrons having kinetic energies of up to 10 keV, i.e. able to break bonds of the polymeric chains (featuring therefore hot electrons effects). The so-called cathodoluminescence spectrum is acquired for different electron energies. Comparison is done with the electroluminescence spectrum of the same materials. It is shown that electron irradiation reproduces nicely the features of the EL spectrum, for a given material. These results allow to associate electroluminescence and electrical ageing in an implicit scheme, opening the way for defining safety limits for a given application.

II. EXPERIMENTAL SET-UP

A. Material -Samples

Measurements were performed on 25 µm-thick Polyethylene Naphthalate (PEN) films, on 100 µm thick films of Low Density Polyethylene -LDPE-, on 150 µm cross-linked polyethylene -XLPEslices from HVAC cables and on 1 mmthick epoxy samples. Samples were metallized only for EL measurements (30 nm thick gold electrodes unless otherwise specified). The difference in sample thickness is not important for cathodoluminescence -CLmeasurements as the energy of electrons used provide penetration in a very limited depth (order of 1µm).

B. Experiment environment

The experimental set-up used for light detection has been extensively described elsewhere [START_REF] Mary | Understanding optical emissions from electrically stressed insulating polymers : electroluminescence in Poly(Ethylene Terephthalate) and Poly(Ethylene 2, 6-Naphthalate) films[END_REF]. It is currently used for carrying out electroluminescence, photoluminescence, recombination-induced luminescence, or even luminescence during thermal oxidation or curing of materials, providing both integral light detection by means of a photomultiplier working in photon counting mode, and spectral resolution of the emitted light through a Charge-Coupled Device -CCDcoupled to a grating monochromator.

The new feature here is the introduction, in the same experimental arrangement, of an electron gun providing electrons of energy of up to 10 keV. The electron gun has been home-designed; a schema of it is shown in Fig. 1. Electron emission is made using a tungsten filament, the flux being dependent on the current (of the order of 1 A) flowing in the filament. As can be seen in Fig. 1, the filament itself is at high voltage and the anode is to the ground. In that way, it is expected that the electrons energy does not change to a great extent after their exit from the gun. The distance between the electron gun and the sample is about 40 mm. The axis of the electron gun is at about 50° to the normal of the sample plane. The arrangement is so for rendering possible light detection along the normal of the samples. All the experiments have been carried out under a vacuum of 5.10 -6 mb, at room temperature.

Except when otherwise mentioned, the electron current was about 0.5 µA in the experiments reported therein. The size of the spot is not precisely known (a few mm²). We have verified that the current is perfectly independent from the energy of the electrons for accelerating voltages in the range 1 to 10 kV, and so whatever the electron current.

The main difficulty encountered in these measurements was the incandescence of the filament with an emission in the wavelength range over 500 nm, depending of the current of the filament, cf. Fig. 2. As this emission is independent from the accelerating voltage, the baseline corresponding to the incandescence spectrum was recorded with low acceleration voltage (500 V or so) in which case the beam current is small and the light detected is constituted only of incandescence. This baseline is subtracted for correcting spectra. Note that the relative contribution of incandescence to spectra strongly diminishes when the beam current increases.

III. RESULTS AND DISCUSSION

A. PEN Fig. 3 shows CL spectra obtained for PEN for energies ranging from 2 to 10 keV (a) and at different times under a continuous irradiation with electrons of 10keV energy (b).

In (a), the fluorescence-to-phosphorescence ratio (430 nm to 585 nm/630 nm; for photo physical properties of PEN, see [START_REF] Teyssedre | Temperature Dependence of the Photoluminescence in Poly(ethylene terephthalate) Films[END_REF]) emission tends to increase when increasing the energy beam. When the beam is maintained constant for the same energy (Fig. 3b), the fluorescence contribution decreases significantly in time, shifts to the red and becomes broad. The 3 peaks observed in the phosphorescence region apparently do not evolve much, in respect to the others vs. energy beam or time.

The evolution in time of the CL spectrum has already been reported [START_REF] Teyssedre | Comparison between photo-, cathodo-, and electro-luminescence spectra of polyethylene naphthalate films and relationship with electrical aging[END_REF], notably what regards the emission in the region around 500 nm. In addition, we have observed emission in that range after UV-ageing of the material. The comparison of EL and CL spectra of Fig. 3c is even more stringent. The EL and CL spectra are in quite good agreement if one has in mind that in both cases, the shape changes with excitation conditions and with time. Both types of spectra are organized along the same spectral components and their features are clearly different from what is observed in photoluminescence in the phosphorescence region. We are very probably in conditions of heavy degradation in the two instances.

B. LDPE and XLPE

In the case of polyolefins also results are impressive. Fig. 4 shows the CL and EL spectra of LDPE (a) and XLPE (b). The CL spectra of the two materials are very similar and both resemble very nicely the EL spectrum.

Several bands are observed in these spectra and they appear correlated to identified features. The 410 nm/420 nm shoulder is associated with an emission involved in chemiluminescence of polyolefins [START_REF] Teyssedre | Spectral analysis of optical emission due to isothermal charge recombination in polyolefins[END_REF][START_REF] Tiemblo | Chemiluminescence spectral evolution along the thermal oxidation of isotactic polypropylene[END_REF]. Short oxidation times yield spectra which are relatively narrow and peak at about 410 nm, while the spectra of the CL emitted after a long oxidation time are broader and shifted to longer wavelengths. The 410 nm component is presumably reflecting the formation of C=O groups. The 500 nm/510 nm shoulder has been revealed in charge recombination process [START_REF] Teyssedre | Spectral analysis of optical emission due to isothermal charge recombination in polyolefins[END_REF] and is associated with recombination occurring on poly-enone sequences. These chemicals provide adequate recombination centers (as revealed by molecular modeling) and they emit in the long wavelength range of the optical spectrum because enone compounds have low lying triplet states. The 570 nm peak seems only obtained in CL and EL, pointing out to a common origin which could be a specific degradation path involved in hot electron processes.

Note that the spectra in CL are independent of the electron beam energy and the irradiation time (in the limit of 300s / 10keV). This seems consistent with the fact that the EL spectra do not show a great dependence on time under stress, although it has not been monitored for long ageing period (a few hours max).

Note also that the emitting species are due to the molecular network itself since LDPE and XLPE emission spectra are similar. Additives and/or anti-oxidant cannot be involved as they were not part of the formulation of the tested LDPE samples. Excitation energies range from 2 to 10 keV for CL. EL spectra were realized under 50Hz AC stress. Correction has been done for surface plasmon contributions.

C. Epoxy resin

Fig. 5 shows the CL and EL spectra of the epoxy sample. Again, the two kinds of excitation lead to very similar spectra. In CL, there is a dependency of the spectral shape on the energy beam with an increased ratio between the 450 nm/550 nm components with an increasing energy. In EL, only the main peak at 550 nm is clearly visible (although the shoulder at 450 nm can be distinguished) due to the low signal/noise ratio. Note that the epoxy is weakly emitting in EL when excited under similar conditions when compared to PEN or PE. Spectral acquisition is therefore a difficult task, all the more than the spectrum of Fig. 5 was acquired at low field (12kV/mm). Indium-Tin-Oxide electrodes were used in order to avoid the emission in the red due to the radiative decay of surface plasmons excited at the metallic electrodes (see [START_REF] Teyssedre | Identification of the components of the electroluminescence spectrum of PE excited in uniform field[END_REF][START_REF] Aubert | Monitoring electrically-active defects in silica-filled epoxy using light detection[END_REF]).

The photoluminescence features of the epoxy have been thoroughly studied by considering the PL of the base resin and hardener taken separately, of their mixture, and in the course of curing [START_REF] Gallot-Lavallée | Identification of photoluminescence features of an epoxy resin based on components features and curing effects[END_REF]. Most of the bands observed in the cured resin could be interpreted based on those found in the components taken separately. Some of the phosphorescence bands revealed in PL experiments are in the range of the EL and CL spectra reported in this study (up to 500 nm) but none are able to map the max of emission at 560 nm. However, when monitoring the spontaneous emission of the resin during curing (chemiluminescence path), we got a spectral shape with a maximum at 562 nm, very similar to CL and EL spectra. This chemiluminescence has been attributed to oxidation reactions of the hardener occurring during or after the curing [START_REF] Gallot-Lavallee | Curing and post-curing luminescence in an epoxy resin[END_REF].

D. Discussion

Electroluminescence spectra have been often questioned when obtained under uniform field configuration because voltages of several kV are needed to generate internal field up to 100 kV/mm. There is always the possibility of stray light due to plasma discharges in the surrounding if all the care has not been taken to avoid them. Another difficulty is to discard the light emitted by radiative decay of surface plasmons when using electrode metallized with standard metals (gold, aluminium, silver, etc.). Their contribution has to be avoided by using ITO electrodes (surface plasmons on Indium-Tin-Oxide electrodes radiate in the far IR region) or the spectra have to be corrected for this effect. From then, EL spectra can be interpreted. Along this approach, we have investigated different emission processes excited by different sources: charge recombination (that does not involve carrier kinetic energy), mild-UV photoluminescence (to map the physical pathway in which the excited states relax to the ground state of the same speciesreversibility), and chemiluminescence (to map the chemical pathway, usually when oxidation is taking place). We were never able to reproduce precisely the EL spectral shape using these sources. Only CL spectra fit nicely the EL spectrum shape. Moreover, although we had not the space to discuss all the results in details, we were able to link the main secondary components -appearing as shoulder in the EL spectra-with identified features of the emission involved in charge recombination-induced luminescence and in chemiluminescence for a given material. This leads to the conclusion that EL involves material degradation. The signatures of charge recombination and chemiluminescence are seen in the EL spectra thereby supporting the existence of these mechanisms in EL. They can be a direct consequence of hot electron impact with the creation of reactive species (which could react further with oxygen molecules always present inside the amorphous fraction of the polymer) and the generation of secondary electrons (which would then be trapped and available to recombine with trapped holes at low to moderate field without need to get an electronic avalanche). The extra spectral features revealed in CL, and that allow to fully understand the EL spectra, is likely linked with a specific pathway bound to electronic impact.

The use of a 10 keV electron beam energy could appear too far from the energy of hot electrons in polymers (< 10 eV) to be typical. However, one has to keep in mind that interaction between electron beam and matter leads first to a decrease of energy of the individual electron by X-ray emission (Bremsstrahlung) before they efficiently start their interaction with valence electrons. It follows a set of possible excitation mechanisms among which impact ionization at moderate kinetic energy (order of 15 eV), impact excitation (10 eV), quasielastic scattering with phonons (energy < 10 eV), and finally electron trapping at thermal energy. All these events are those that are thought to be important under electric field, whatever is the initial electron beam energy.

IV. CONCLUSION

By irradiating various materials with energetic electrons, we were able to reproduce their EL spectrum. It gives definitive evidence that EL under uniform field can be analyzed in terms of material properties. These results allow to associate electroluminescence and electrical ageing in an implicit scheme, opening the way for defining safety limits in terms of electric stresses applied to a material for a given application. New routes are also opened for understanding the nature of the reactive species and to develop improved formulation of polymer materials for high field application.

Fig. 1 .

 1 Fig. 1. Schema of the core of the electron gun. The diameter of the hole of the anode is 1mm. Anode (to ground)

Fig. 4 .

 4 Fig. 4. CL and EL spectra of LDPE (a) and XLPE (b).Excitation energies range from 2 to 10 keV for CL. EL spectra were realized under 50Hz AC stress. Correction has been done for surface plasmon contributions.

  Fig. 5. CL and EL spectra for epoxy resin. CL spectra are shown at two different irradiation energies. EL has been acquired under 50 Hz AC stress at a field of 12 kV/mm, using ITO electrodes.

			XLPE pirellicourant 110v
					 = 580nm
			2 keV
		1	4 keV			EL a.c.
			8 keV			45kV/mm
			10keV			37kV/mm
		(a.u.)
		Intensity
		0
		300	400	500	600	700	800
				Wavelength (nm)
		 = 570nm
	2 keV
	4 keV
	8 keV
	10keV	EL 45kV/mm a.c. stress
	Intensity (a.u.)
		Wavelength (nm)

stress Cite as: G. Teyssedre, J.L. Franceschi, C. Laurent, "Cathodo-and electro-luminescence spectra in insulating polymers: a parallel approach for inferring electrical ageing mechanisms", IEEE Int. Conf. on Electrical Insulation and Dielectric Phenomena (CEIDP), Vancouver, Canada, 14-17 Oct. 2007. Proc. CEIDP-07, pp. 824-827, 2007. DOI: 10.1109/CEIDP.2007.4451566


Diego Velazco
email: diego.velazco@supergrid-institute.com

Guy Clerc

Emmanuel Boutleux

François Wallart

Comparison of Redundancy Requirements for Modular Multilevel Converter Considering Manufacturer Reliability Inputs and Mission Profile

Keywords: Multilevel converters (MMC)», «HVDC», «Mission profile», «Lifetime», «Reliability»

During the design phase, target reliability values of componentsincluding semi-conductors -allow the computation of converter level reliability and redundancy requirements. This work proposes a more accurate method based on manufacturer lifetime models and mission profile evaluation. The new method is applied on a Modular Multilevel Converter.

I -Introduction

The growing needs of power transmission systems are pushing towards the rapid development of HVDC technologies. Moreover, HVDC has been recognized as a major contender for performing long-distance bulk-power transmission [START_REF] Bahrman | The ABCs of HVDC transmission technologies[END_REF], [START_REF] Páez | Overview of DC-DC Converters Dedicated to HVdc Grids[END_REF]. Among HVDC technologies, voltage source converters (VSC-HVDC) are considered the most suitable to interact with existing AC grids [START_REF] Gonzalez-Torres | Power system stability enhancement via VSC-HVDC control using remote signals: Application on the Nordic 44-bus test system[END_REF]. Nowadays, the most promising VSC-HVDC topology is the Modular Multilevel Converter (MMC) [START_REF] Marquardt | Modulares Stromrichterkonzept für Netzkupplungsanwendungen bei hohen Spannungen[END_REF], [START_REF] Allebrod | New transformerless, scalable Modular Multilevel Converters for HVDC-transmission[END_REF].

The MMC for HVDC applications is a three-phase converter as seen in Fig. 1 (a). Each phase comprises an upper and a lower arm. Each arm is composed of a stack of submodules (SMs) and an arm inductor. The SMs are connected in series in order to withstand the high voltage levels inherent to HVDC systems. Multiple SM configurations are proposed in the literature, but the two most popular are the half-bridge (HB-SM) and the full-bridge (FB-SM) configurations. Today, in practical applications, the HB-SM is the most used due to cost considerations. To guarantee the proper functioning of the converter, a large number of SMs must be installed in each arm. In the case of HVDC applications the number of HB-SMs is in the range of some hundreds per arm.

A HB-SM consists of a storage capacitor C, two IGBT T1 and T2 and two antiparallel connected diodes D1 and D2 as depicted in Fig. 1 (b). The different semiconductors allow the insertion, or the bypass of the capacitor and they will be active based on the command signals and the direction of the current. Given the criticality of energy outages and its potential repercussions, special care should be taken for assessing the reliability of HVDC technologies such as the MMC. According to the ENTSOE, improved reliability (e.g. decreasing 1-2 trips per year) and improved availability (e.g. decreasing 1-2 outages days per year) can bring significant cost savings to TSOs and / or society [START_REF] Entso-E | Improving HVDC System Reliability[END_REF].

Having such demanding reliability constraints, MMC arms are equipped with additional SMs for providing the redundancy required for attaining the availability requirements of an HVDC system. This work intends to shed a light on the estimation of the number of redundant SMs. In order to do so, two case studies will be compared: In the first case, the redundancy computation will use out-of-the-shelf reliability indicators from the manufacturer. In the second case, the redundancy will be computed using the mission profile of the converter.

In order to be able to consider the mission profile of the converter and obtain the number of redundant semiconductors, the thermal loading of power devices will be determined thanks to the methodology for lifetime estimation developed in [START_REF] Velazco | IGBT Lifetime Estimation in a Modular Multilevel Converter for bidirectional point-to-point HVDC application[END_REF]. This work will present the profile-based lifetime estimation methodology in Section II. Then Section III will depict the methodology for performing reliability predictions of the different elements of a SM. Section IV presents the methodology for translating the reliability calculations of a SM into redundancy requirements. Section V discusses the reliability predictions and redundancy estimations and gives a comparison on the two case studies. Finally, section VI gives the conclusions on this research.

II -Methodology for profile-based lifetime estimation

The methodology employed for considering the mission profile for the lifetime estimation of the power semiconductors [START_REF] Velazco | IGBT Lifetime Estimation in a Modular Multilevel Converter for bidirectional point-to-point HVDC application[END_REF] can be summarized in Fig. 2. It consists of 5 different stages: the modeling of the converter, the calculation of the losses of the semiconductor devices, the calculation of the thermal loading, the use of the rain flow counting algorithm [START_REF] Matsuichi | Fatigue of metals subjected to varying stress[END_REF] for organizing the thermal profiles and finally, the use of the lifetime models provided by the manufacturer as well as the Miners rule [START_REF] Miner | Cumulative Damage in Fatigue[END_REF] for estimating the lifetime consumption. Fig. 2: Flowchart of the lifetime estimation methodology An important step in the lifetime estimation methodology is the assessment of the temperature profile of the different semiconductors. This stage is performed considering two different timescales as seen in [START_REF] Velazco | IGBT Lifetime Estimation in a Modular Multilevel Converter for bidirectional point-to-point HVDC application[END_REF]. The thermal profiles obtained for the studied mission profile, which corresponds to an offshore wind farm (OWF), can be seen in Fig. 3. Then the thermal profiles are organized thanks to the rain flow counting algorithm as seen in the left portion of Fig. 4. Fig. 3: Thermal profile of T2 under the evaluated period for the studied mission profile One of the hallmarks presented in this methodology is the introduction of the model-based extrapolation method [START_REF] Velazco | IGBT Lifetime Estimation in a Modular Multilevel Converter for bidirectional point-to-point HVDC application[END_REF] for obtaining the B10 number of cycles to failure. The extrapolation method is necessary to be able to use the manufacturer lifetime models [START_REF] Kaminski | Load-cycling capability of HiPak IGBT modules[END_REF] for estimating the lifetime consumption in the evaluated period. The extrapolation method is constructed thanks to the Norris-Landzberg lifetime model [START_REF] Norris | Reliability of Controlled Collapse Interconnections[END_REF], yielded by expression [START_REF] Bahrman | The ABCs of HVDC transmission technologies[END_REF] and which results in the extrapolation grid shown in the central portion of Fig. 4.

𝑁 𝑓1 = 𝑁 𝑓2 ( Δ𝑇 2 Δ𝑇 1 ) 𝛼 ( 𝑡 2 𝑡 1 ) 𝛽 𝑒𝑥𝑝 (𝛾 ( 1 𝑇 1 - 1 𝑇 2 )) (1)
In ( 1), Nf1 represents the number of cycles to failure for condition 1 (resp. for Nf2), ΔT1 the temperature swing for condition 1 (resp. for ΔT2), t1 the cycle duration for condition 1 (resp. for t2) and T1 the temperature reference for condition 1 (resp. for T2). Coefficients α, β and γ can vary according to the evaluated conditions. The grid shown in the central portion of Fig. 4 allows for the calculation of the new coefficients α, β and γ for each cycling condition. These coefficients are computed for each mesh with least-squares algorithm considering the vertices of the mesh.

Fig. 4: Grid for model-based extrapolation method

The manufacturer provides different curves for performing the lifetime estimation of the different elements of the semiconductor devices such as the chip solder, the baseplate solder, and the bond wires [START_REF] Kaminski | Load-cycling capability of HiPak IGBT modules[END_REF]. The yearly B10 lifetime consumption, CLi,k for the k th element (chip solder, dbc solder or bondwire) and i th thermal cycle identified is obtained with (2). In (2), nbi is the number of cycles for a given thermal condition and Nfi,k is the number of cycles to failure for the same condition. Moreover, the expected time LFk for a 10% failure rate of the k th element of the semiconductors can be calculated with (3). In (3), Ncyc is the total number of thermal cycles in the evaluated period. Some of the lifetime consumption results for the evaluated profile can be seen in Fig. 5.

𝐶𝐿 𝑖,𝑘 = 𝑛𝑏 𝑖 𝑁 𝑓𝑖,𝑘 , 𝑘 ∈ {chip solder, dbc solder, bondwire} (2)
𝐿𝐹 𝑘 = 1 ∑ 𝐶𝐿 𝑖,𝑘 𝑁 𝑐𝑦𝑐 𝑖=1
, 𝑘 ∈ {chip solder, dbc solder, bondwire}

III -Methodology for reliability predictions

In this work the reliability predictions are performed considering two different case studies: manufacturer off-the-shelf reliability indicators and mission profile-based reliability estimations.

For the first case, the inputs considered are the failure rates of the different elements that compose a SM, such as the power semiconductors, the storage capacitor and the electronic board that drives the SM as done in [START_REF] Wylie | Analysis of the effects from constant random and wear-out failures of sub-modules within a modular multi-level converter with varying maintenance periods[END_REF]. The failure rates of these components, expressed in FIT are depicted in Table I.

Table I: FIT values for the SM elements [12]

Component FIT IGBT 100 Capacitor 300 Control Board 1200

Having the failure rates λ, of the elements of a SM the calculation of the failure rate of a SM λSM, can be calculated with (4) by adding the failure rates of the different elements of the SM and subsequently the reliability of a SM RSM(t) can be computed employing [START_REF] Allebrod | New transformerless, scalable Modular Multilevel Converters for HVDC-transmission[END_REF].

𝜆 𝑆𝑀 = 2 ⋅ 𝜆 𝐼𝐺𝐵𝑇 + 𝜆 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑜𝑟 + 𝜆 𝑏𝑜𝑎𝑟𝑑 (4) 𝑅 𝑆𝑀 (𝑡) = 𝑒 -(𝜆 𝑆𝑀 ⋅𝑡) (5)
It is also worth mentioning that for the case in which only off-the-shelf reliability indicators are employed, the reliability function is obtained employing an exponential distribution. This method does not consider wear-out failures. The resulting reliability distribution for this case can be seen in Fig. 6. The second case considers the mission profile experienced by the converter and employs the results obtained in section II as inputs. The profile evaluated in this work corresponds to a 1 GW converter installed in an offshore wind farm. After having calculated the lifetime consumptions of the elements of the semiconductors in the evaluated period and under the evaluated profile, the lifetime for a 10% failure rate is calculated by using the data from [START_REF] Kaminski | Load-cycling capability of HiPak IGBT modules[END_REF]. This result can be translated into a device level reliability function as depicted in Fig. 7.

Fig. 7: Methodology for calculating device level reliability indicators

As the manufacturer B10 lifetime models [START_REF] Kaminski | Load-cycling capability of HiPak IGBT modules[END_REF] are fitted using a two parameter Weibull distribution ( 6), [START_REF] Velazco | IGBT Lifetime Estimation in a Modular Multilevel Converter for bidirectional point-to-point HVDC application[END_REF] will allow the calculation of the failure rate describing the cumulative density function CDF, for the elements of the semiconductors. In [START_REF] Velazco | IGBT Lifetime Estimation in a Modular Multilevel Converter for bidirectional point-to-point HVDC application[END_REF], the CDF Fk(LFk) = 0.1. It is worth noting that different shape factors β were evaluated for the semiconductor devices in this study, as this parameter represents the variance of the instantaneous failure distribution of the studied elements. Having calculated the individual CDF for the elements of the semiconductors, the unreliability for the semiconductor devices Fdev(t) can be obtained with [START_REF] Matsuichi | Fatigue of metals subjected to varying stress[END_REF].

𝐹 𝑘 (𝑡) = 1 -𝑒 -(𝜆 𝑘 •𝑡) 𝛽 , 𝑘 ∈ {chip solder, dbc solder, bondwire} (6)
𝜆 𝑘 = - [𝑙𝑛(1-𝐹 𝑘 (𝐿𝐹 𝑘 ))] 1/𝛽 𝐿𝐹 𝑘
, 𝑘 ∈ {chip solder, dbc solder, bondwire}

𝐹 𝑑𝑒𝑣 (𝑡) = 1 -∏(1 -𝐹 𝑘 (𝑡)) , 𝑑𝑒𝑣 ∈ {𝑇1, 𝐷1, 𝑇2, 𝐷2} (7)
Once the unreliability function of all the semiconductors is obtained, the unreliability of the SM can be obtained with [START_REF] Miner | Cumulative Damage in Fatigue[END_REF]. In [START_REF] Miner | Cumulative Damage in Fatigue[END_REF], m represents all the different elements of the SM such as the semiconductor devices T1, D1, T2 and D2, as well as the capacitor and electronic board. The failure distribution of the capacitor and the electronic board are calculated with an exponential distribution, as done in the previous case study (off-the-shelf reliability indicator). With the failure distribution of a SM, its reliability function can be deduced with [START_REF] Kaminski | Load-cycling capability of HiPak IGBT modules[END_REF]. Fig. 8 depicts the reliability functions of the elements of a SM. The results in Fig. 8 were obtained with β = 2.5.

𝐹 𝑆𝑀 (𝑡) = 1 -∏(1 -𝐹 𝑚 (𝑡)) , 𝑚 ∈ {𝑇1, 𝐷1, 𝑇2, 𝐷2, 𝐶𝑎𝑝𝑎𝑐𝑖𝑡𝑜𝑟, 𝐵𝑜𝑎𝑟𝑑} (9)
𝑅 𝑆𝑀 (𝑡) = 1 -𝐹 𝑆𝑀 (𝑡)

Fig. 8: Reliability of a SM and its composing elements considering the mission profile (β=2.5)

In Fig. 9, the resulting reliability functions of the SMs for the two case studies can be appreciated. The profile-based case study considered different shape factors β for the reliability computations. The values considered are 1, 1.5, 2, 2.5, 5 and 10 as depicted in Fig. 9.

Fig. 9: Reliability functions of a SM for the studied cases It can be seen from Fig. 9, that manufacturer off-the-shelf data yields the more pessimistic reliability prediction when compared to the profile-based reliability estimation. The results considering the mission profile are notably dependent on the thermal conditions chosen for the study. The lifetime models employed depend greatly on the thermal profile experienced by the semiconductors and more particularly on the variation of the experienced thermal cycles ΔT.

For the purpose of this study and in order to obtain realistic results, a maximum junction temperature of 90°C was considered for the semiconductor devices as seen on Fig. 3. This requirement was then used for dimensioning the thermal characteristics of the heatsink associated to the semiconductors. If a higher maximum junction temperature would have been considered, the profile-based reliability calculations would yield significantly lower and worse results.

Additionally, it can be said from Fig. 9 that, given the thermal constraint imposed to the semiconductors, the shape factor β in the range [1.5 10] doesn't have a great influence on the final reliability computation of the SM. Moreover, since the reliability function for the capacitor and the control board are the same in all the configurations, the final SM reliability estimation is mostly dependent on those elements, as they have considerably higher failure rates than the semiconductors.

IV -Methodology for redundancy estimations

Knowing the reliability distribution of a SM allows the calculation of the reliability function of an MMC arm. In order to make a realistic evaluation of the arm reliability a Markov chain was developed for this purpose as depicted in Fig. 10. Along with the Markov chain, Monte Carlo simulations were performed for assessing multiple converter lifetimes. For the purpose of the simulations a target mission time of 40 years was considered.

Consequently, different redundancy levels were tested. For each redundancy level, 1000 simulations were carried out. The minimum number of SMs to be installed in an arm should guarantee a reliability of 99.5 % by the end of the target mission time of the converter. Maintenance is also considered for the simulations. At each maintenance period, if the number of failed SMs is smaller than the number of redundant SMs, all the failed ones are replaced. However, if at any point throughout the simulations the amount of failed SMs is bigger than the number of redundant SMs, then the converter has failed that test. A maintenance intervention every two years was considered for the Monte Carlo simulations. The arm reliability is approximated to the number of arm survivals for a given redundancy level. This methodology allowed for the calculation of the redundancy requirements of the MMC for the two case studies. These results can be seen in Fig. 11.

Fig. 11: Reliability of an MMC arm vs number of redundant SMs

As mentioned previously in this section, the chosen criteria for determining the number of redundant SMs required for an MMC arm is to guarantee a reliability of 99.5% throughout its operating life. Thus, the number of redundant SMs required for each of the evaluated cases can be seen in Table II.

V -Discussion on the results of the reliability predictions and redundancy estimations

The number of redundant SMs reflects the reliability functions calculated in section III. The results depicted in Table II and Fig. 11 indicate that using the manufacturer reliability indicators yields the worst-case scenario for redundancy dimensioning. As mentioned in section III, the thermal constraints imposed to the converter have a significant impact on the redundancy requirements obtained in the profile-based redundancy calculations.

The results seen in Table II show that the shape factor β used for calculating the reliability distributions of the semiconductors has almost no impact on the final redundancy estimations. This is mainly due to the fact that the failure rates of the storage capacitor and the control board of the SM are much higher than the failure rates of the semiconductors in both case studies.

In a Weibull distribution, the shape factor β allows the modeling of the wear-out of the studied element when β>1. The reason why there is not a noticeable loss of reliability in spite of the different β evaluated, is that the acceleration in the reliability decline happens much after the target mission time of the converter. An example concerning the reliability of the chip solder of IGBT 2 can be seen in Fig. 12.

Fig. 12: Reliability of chip solder of IGBT 2 for different β

The reliability functions seen on Fig. 12 are obtained considering a fixed time span for a loss of reliability of 10%, which corresponds to the B10 definition in [START_REF] Kaminski | Load-cycling capability of HiPak IGBT modules[END_REF]. The fixed time span for the loss of reliability is the result of (3) in the lifetime estimation methodology described in section II.

Another noticeable result from this research is the estimation of the number of failed SMs at each maintenance period. This result allows the calculation of the approximated amount of SMs required for a proper functioning of an MMC arm throughout all the target mission time of the converter (40 years). These calculations were possible thanks to the Monte Carlo simulations and the Markov chain depicted in Fig. 10. In Fig. 13(a), the number of failed SMs for the profile-based redundancy calculations can be seen. The results correspond to a shape factor β = 2.5. Consequently, in Fig. 13 In Fig. 13(a), it can be seen that the maximum number of failed SMs at a given maintenance intervention is equal to the number of redundant SMs installed. If the number of failed SMs would have been higher, the MMC arm wouldn't have survived and the whole converter would have stopped working. The average number of failed SMs per maintenance period is 11, but it can have a large variance as evidenced on Fig. 13(a).

It can be seen from Fig. 13(b), that the total number of SMs employed throughout the operating life of the converter is indeed much higher than the minimum number of SMs required for the arm functioning. The studied MMC arm requires only 400 SMs to operate, it requires 23 redundant SMs to guarantee a reliability of at least 99.5% by the end of its target mission time and in total, it will require 631 SMs in average. This means that the converter arm requires more than 200 additional SMs than the ones with which it started working, which by no means is a negligeable amount and can have a great impact on the OPEX of the converter. Moreover, an MMC has six arms, so the total number of SMs employed in the whole converter throughout its target mission time can ascend to 3800 SMs in average, a very large amount when compared to the 2400 SMs, required for MMC operation. A comparison between the total number of SMs employed for all the cases can be seen in Fig. 14.

Fig. 14: Comparison between the number of employed SMs

As mentioned previously, the shape factor β employed for the profile-based redundancy calculations does not affect notably the number of redundant SMs (Fig. 11). However, when β=1 a slight impact on the total number of SMs employed can be seen in Fig. 14. The average number of employed SMs when β=1 is 645, compared to 631 for the other profile-based redundancy estimations where β>1. It is worth reminding that when β=1, the Weibull distribution behaves as an exponential distribution. As it was shown in Fig. 12, with β>1 an acceleration in the decline of the reliability distribution is expected. However, this decline happens much after the target mission time of the converter, that means that before this decline the reliability distributions that have a higher shape factor β will have higher reliability values, as it was seen in Fig. 12.

Concerning the total number of the employed SMs that is determined by the manufacturer off-the-shelf reliability indicators, it can be clearly seen in Fig. 14 that this input yields the worse results. The exponential distribution was used to model the reliability function in this case. As previously seen on section III, the reliability function obtained with the manufacturer inputs is the more pessimistic and the number of total employed SMs reflect this condition. The average number of SMs employed with this information ascends to 663, which is bigger than the number of employed SMs coming from the profilebased methodology (645 for β=1 and 631 for β>1).

VI -Conclusion

In this research, the methodology for lifetime estimation and reliability predictions were presented. Additionally, the methodology for performing redundancy computations for an MMC arm was introduced. Two case studies were treated in this paper, one uses off-the-shelf reliability indicators and the other uses the mission profile experienced by the converter. The profile-based results were strongly influenced by the thermal constraints imposed to the converter.

There is a direct correlation between the reliability function of the SM and the number of redundant SMs to be installed to attain a target reliability by the end of the operating life of the converter. Moreover, this correlation is also reflected on the total number of SMs to be employed throughout the operating life of the converter. It was seen that, in terms of the number of redundant SMs required, the more pessimistic results came from the manufacturer off-the-shelf reliability indicators, whereas the profilebased calculations offered similar results for all the evaluated shape factors β.

When analyzing the total number of SMs employed throughout the operating life of the MMC, the methodology based on manufacturer reliability inputs yielded the poorest results as expected from the reliability predictions. The results obtained with the profile-based methodology show two different behaviors. When β=1 the number of employed SMs is larger than when β>1. This is mainly due to the fact that the shape factor influences on the reliability decline after the operating life of the converter.

Finally, it can be said that the study of the number of failed and employed SMs opens up many perspectives for future research. The authors have already started working on some, such as the choice of alternative maintenance strategies concerning the replacement of healthier SMs in order to optimize the number of redundant SMs required or the total number of SMs employed throughout the active life of the MMC.

The methodologies for lifetime estimation and reliability predictions developed in this work, can be generalized to other converter topologies.

Fig. 1 :

 1 Fig. 1: (a) MMC topology, (b) Half-bridge submodule

Fig. 5 :

 5 Fig. 5: Lifetime consumption results (a) Long-term timescale (b) Medium-term timescale

Fig. 6 :

 6 Fig. 6: Reliability of a SM and its composing elements considering off-the-shelf reliability indicators

Fig. 10 :

 10 Fig. 10: Markov chain for calculating the availability of the converter for a target mission time

  (b), the number of SMs employed during the operating life of the converter arm can be seen. (a) (b) Fig. 13: (a) Number of failed SMs per arm at each maintenance intervention (1000 Monte Carlo tests), (b) Number of total SMs employed during the operating life of the converter arm

Table II : Number of redundant SMs for a 40-year target mission time and 99.5% reliability

 II

	Case Study	Manufac-turer data	Mission profile, β=1	Mission profile, β=1.5	Mission profile, β=2	Mission profile, β=2.5	Mission profile, β=5	Mission profile, β=10
	Redundant SMs	26	24	23	24	23	24	24

Acknowledgements

This work was supported by a grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" Program (ANE-ITE-002-01).