Pierre Voisin
email: pierre.voisin@univ-poitiers.fr

Marianne Bernard

Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo

Keywords: retina, rhodopsin, transcriptional regulation, cyclic AMP Running cyclic AMP-dependent rhodopsin gene transcription

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Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo

INTRODUCTION

During retinal development, the neuroblasts generated in the inner leaflet of the optic cup give rise to all retinal neurons (photoreceptors, horizontal cells, bipolar cells, amacrine cells, ganglion cells) and to Müller glial cells [START_REF] Cepko | Cell fate determination in the vertebrate retina[END_REF][START_REF] Hatakeyama | Retinal cell fate determination and bHLH factors[END_REF]. In all vertebrate classes, the retinal cell types are generated in a conserved chronological order that may be reminiscent of conserved molecular determinants in retinogenesis [START_REF] Yang | Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis[END_REF]. Ganglion cells are the first to differentiate, from a cohort of neuroblasts that exit mitosis early in development. Then horizontal cells, amacrine cells, photoreceptors, bipolar cells and Müller glial cells are generated, in that order, from neuroblasts that accomplish their final mitosis increasingly late in development [START_REF] Levine | Soluble factors and the development of rod photoreceptors[END_REF][START_REF] Yang | Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis[END_REF][START_REF] Adler | Have we achieved a unified model of photoreceptor cell fate specification in vertebrates?[END_REF]. There is however considerable overlapping in this chronological sequence and the contextual signals that neuroblasts encounter at the exit of mitosis appear to play a major role in cell type determination. This is demonstrated by cell lineage studies indicating that the two sister cells of a final mitosis often differentiate into distinct cell types [START_REF] Cepko | Cell fate determination in the vertebrate retina[END_REF][START_REF] Belecky-Adams | Correlations between terminal mitosis and differentiated fate of retinal precurseur cells in vivo and in vitro: analysis with the "windowlabeling" technique[END_REF][START_REF] Yang | Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis[END_REF]. A better understanding of this developmental signaling has been gained with the identification of diffusible molecules that regulate photoreceptor differentiation (i. e. taurine, activin, ciliary neurotrophic factor, basic fibroblast growth factor, sonic hedgehog ; Levine et al., 2000, for review) and even the balance between rods and cones (i.e. retinoic acid and thyroid hormone ; [START_REF] Kelley | Ligands of steroid/thyroid receptors induce cone photoreceptors in vertebrate retina[END_REF]. Some of these signals would appear to act early-on in the cell fate decision cascade (i. e. sonic hedgehog ; [START_REF] Levine | Sonic hedgehog promotes rod photoreceptor differentiation in mammalian retinal cells in vitro[END_REF] while others may act at terminal steps of photoreceptor differentiation, for example to implement the spectacular amplification of opsin gene expression that characterizes all photoreceptor subtypes (i. e. retinoic acid ; [START_REF] Kelley | Retinoic acid promotes differentiation of photoreceptors in vitro[END_REF]. There is however considerable interspecies variability in the activity of the identified factors and a unifying model of photoreceptor differentiation signaling is still wanted [START_REF] Levine | Soluble factors and the development of rod photoreceptors[END_REF][START_REF] Adler | Have we achieved a unified model of photoreceptor cell fate specification in vertebrates?[END_REF].

In the chicken retina, photoreceptors differentiate from neuroblasts that exit mitosis between embryonic day 4 (E4) and E7, with a peak at E5 and E6 (Belecky-Adams et al., 1996). In this species, the diversity of photoreceptors, based on opsin gene expression, comprises rods and four types of cones : red-sensitive, green-sensitive, blue-sensitive and violet-sensitive [START_REF] Bruhn | Development of the pattern of photoreceptors in the chick retina[END_REF][START_REF] Adler | Have we achieved a unified model of photoreceptor cell fate specification in vertebrates?[END_REF]. Red-sensitive and green-sensitive cones are the first to express their respective opsins, at embryonic day 14 (E14) ; followed by rods at E15, blue-sensitive cones at E17 and violet-sensitive cones at E18 [START_REF] Bruhn | Development of the pattern of photoreceptors in the chick retina[END_REF][START_REF] Cailleau | Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursors cells[END_REF]. The long time lag between exit of mitosis and opsin gene expression would appear to give contextual signals plenty of time to act upon photoreceptor differentiation. As an experimental approach to this question, in vitro differentiation of photoreceptors can be obtained from retinal precursor cells isolated from chicken embryos around E7 [START_REF] Adler | A model of retinal cell differentiation in the chick embryo[END_REF]. This experimental system has yielded valuable information on the regulation of photoreceptor-specific gene expression, including phototransduction proteins, melatonin-synthesizing enzymes and circadian transcription factors [START_REF] Iuvone | Cyclic AMP-dependent induction of serotonin N-acetyltransferase activity in photoreceptor-enriched chick retinal cell cultures: characterization and inhibition by dopamine[END_REF][START_REF] Adler | Microenvironmental regulation of visual pigment expression in the chick retina[END_REF][START_REF] Cailleau | Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursors cells[END_REF][START_REF] Chaurasia | Circadian clockwork machinery in neural retina: evidence for the presence of functional clock components in photoreceptor-enriched chick retinal cell cultures[END_REF]. The photoreceptors that differentiate in vitro from chicken retinal progenitors are mostly red-sensitive cones, with about 1/10th green-sensitive cones [START_REF] Bradford | Roles of cell-intrinsic and microenvironmental factors in photoreceptor cell differentiation[END_REF]. Red-sensitive opsin gene expression is actually accelerated in this culture system as compared to the in vivo situation [START_REF] Adler | Microenvironmental regulation of visual pigment expression in the chick retina[END_REF], apparently in response to an unidentified serum factor [START_REF] Cailleau | Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursors cells[END_REF]. In addition, the proportion of green-sensitive cones can be increased by ciliary neurotrophic factor, and that of rods by staurosporine [START_REF] Xie | Green cone opsin and rhodopsin regulation by CNTF and staurosporine in cultured chick photoreceptors[END_REF]. A better understanding of rod differentiation may be of special interest because this cell type appears particularly prone to degeneration, either in response to excessive light exposure [START_REF] Lavail | Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light[END_REF], or as a result of mutations affecting either the structure or the expression levels of rhodopsin and other phototransduction proteins [START_REF] Farrar | On the genetics of retinitis pigmentosa and on mutation-independent approaches to therapeutic intervention[END_REF]. Therefore, identifying the signaling pathways that favor rhodopsin gene expression may identify targets for future therapies. The mode of action of staurosporine being difficult to explore, due to its inhibiting a large number of protein kinases [START_REF] Meggio | Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2[END_REF], we sought to identify a more physiological means of activating rhodopsin gene expression in developping chicken rods.

Previous studies had indicated that cAMP-dependent protein kinase A (PKA) increased rhodopsin gene promoter activity in transgenic zebrafish [START_REF] Yu | Mitogenassociated protein kinase-and protein kinase A-dependent regulation of rhodopsin promoter expression in zebrafish rod photoreceptor cells[END_REF], while other studies had shown that adenylate cyclase-coupled receptors regulated rhodopsin mRNA levels in salamander rods in vitro [START_REF] Alfinito | Dopamine D4 receptor-mediated regulation of rod opsin mRNA expression in tiger salamander[END_REF][START_REF] Alfinito | Adenosine A(2a) receptor-mediated inhibition of rod opsin mRNA expression in tiger salamander[END_REF]. The observed effects were of modest amplitude (30-50%), however, we reasoned that these studies had been performed on mature rods, where rhodopsin gene transcription is set at a very high rate, so that cAMP had little chance to further increase this rate. Studies on embryonic retinal cells with low levels of rhodopsin gene expression had a better potential to unmask its regulation by cAMP. In the present report, we describe a strong stimulation of rhodopsin gene transcription by cAMP in retinal precursor cells of chicken embryo.

MATERIALS AND METHODS

Animals

Fertilized chicken eggs (Gallus domesticus) were purchased from Rumulo C ie (Quinçay, France). The eggs were incubated at 37°C in an atmosphere of 70% humidity. Embryos were staged according to [START_REF] Hamburger | A series of normal stages in the development of the chick embryo[END_REF]. The embryos were sacrificed by decapitation at different Hamburger and Hamilton's (HH) stages: HH28 (embryonic day 5.5 : E5.5), HH29 (E6), HH30 (E7) or HH34 (E8). Embryonic retinas were immediately transferred in culture medium, and processed for cell dissociation and culture, as described below. Adult chickens (postnatal day 15: P15) were purchased from Lecorne C ie (Lencloître, France). They were anesthesized by an intraperitoneal injection of chloral hydrate and sacrificed by decapitation. Tissues from P15 chickens were frozen in liquid nitrogen and processed for RNA extraction. Animal care was conducted in accordance with the standard ethical guidelines (European Community Guidelines on the Care and Use of Laboratory Animals; and French Agriculture and Forestry Ministry guidelines for handling animals, decree 87848, license E 86-040) and approved by the local ethical committee.

Embryonic retinal cell cultures

Primary cultures of embryonic chicken retinal cells were prepared as previously described [START_REF] Adler | Preparation, enrichment and growth of purified cultures of neurons and photoreceptors from chick embryos and from normal and mutant mice[END_REF], with slight modifications [START_REF] Bernard | Photoreceptor-specific expression, light-dependent localization, and transcriptional targets of the zinc-finger protein Yin Yang 1 in the chicken retina[END_REF]. Neural retinas, free of pigment epithelium, were dissociated by trypsin-DNase digestion and multiple pipetting.

Unless otherwise indicated in the figure legends, 12.10 6 cells were seeded at high density (1.25x10 6 /cm 2 ) in non-coated 6-well plastic plates (NUNC, Illkirch, France) and cultured for 3 days (37°C, 5% CO2, constant darkness) in medium 199 supplemented with 100 U/mL penicillin/streptomycin, 2 mmol/L glutamine (all from Sigma-Aldrich, Saint Quentin-Fallavier, France), 0.1% bovine serum albumin (BSA) (Sigma-Aldrich) and 10% newborn calf serum (NCS) (Invitrogen, Cergy Pontoise, France).

Pharmacological treatments

Cells were allowed to adhere to culture dishes for 3 h prior to the addition of drugs. Forskolin and 3-isobutyl-1-methylxanthine (IBMX) were dissolved in dimethyl sulfoxide (DMSO) (all from Sigma-Aldrich) and an equal volume of DMSO was added to the controls. N 6- monobutyryl cyclic AMP (mB-cAMP), N 6 ,2′-O-dibutyryl cyclic AMP (dB-cAMP), 8-(4-Chlorophenylthio) cyclic AMP (8CPT-cAMP), 8-Bromo cyclic AMP (8Br-cAMP), 8-Bromo cyclic GMP (8Br-cGMP), sodium butyrate (all from Sigma-Aldrich), 8-Bromo-2'-O-methyl cyclic AMP (Me-cAMP) and N 6 -Benzoyl cyclic AMP (Bz-cAMP) (both from BioLog, Bremen, Germany) were dissolved in double-distilled water and controls received an equal volume of water. Me-cAMP and Bz-cAMP were chosen as selective activators of the guanine nucleotide exchange protein directly activated by cAMP (EPAC) and of protein kinase A (PKA), respectively, based on their previously published pharmacological profiles [START_REF] Christensen | cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension[END_REF]. Plasma membrane depolarization was induced by replacing 1/3 rd the volume of culture medium by 150 mmol/L KCl and controls received an equal volume of 150 mmol/L NaCl.

Cell viability assays

Cell viability under different culture conditions and pharmacological treatments was assessed using the CellTiter96  reagent (Promega, Charbonnières, France) and confirmed by trypan blue exclusion. Data are expressed as percent of the viability measured 6 h after plating.

RNA extraction

Total RNA from cultured embryonic retinal cells and from adult chicken tissues was extracted as previously described [START_REF] Cailleau | Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursors cells[END_REF]. RNA pellets were resuspended in diethyl pyrocarbonate (DEPC)-treated water (Invitrogen). RNA concentrations were measured by the optical density at 260 nm (OD260) and adjusted to 0.25 µg/µL with DEPC-water. We verified that the RNA yield (mean ± sem, n = 48 ) was similar in the control cultures (12.06 ± 0.57 µg/dish) and in the forskolin-treated cultures (12.64 ± 0.72 µg/dish), thus confirming that pharmacological treatment did not affect cell viability.

Real time RT-PCR

Total RNA (1µg) was reverse transcribed with Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Promega) and random hexamers (GE Healthcare Europe, Saclay, France), at 37°C for 2 h. The RT reaction was terminated on ice by a 1/5 dilution in doubledistilled water. Real time PCR was performed on a LightCycler  (Roche Applied Science, Meylan, France) as previously described [START_REF] Cailleau | Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursors cells[END_REF]. The primer pairs used in this study are listed in Table S1 and were validated as follows: they gave a single PCR product as verified by melting curve analysis, agarose gel electrophoresis and DNA sequencing, and the distribution of the PCR sigmoids was linear (r ≥ 0.99) over 5 logs of template concentration with an efficiency of 1.85 to 1.98. We verified that 2-to 10-fold dilutions of the RNA introduced into an RT-PCR reaction caused the expected critical cycle increments. Reverse transcripts copy numbers were calculated from standard curves established with known quantities of each template. Results are expressed as number of copies of a target template per 10 6 copies of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) template. Congruency of the results obtained by real time RT-PCR and by northern blot was verified (Fig. 1). Statistical analyses were performed with the two-tailed Student's t-test.

Northern blot analysis

Total RNA (10 µg) from cultured embryonic retinal cells was electrophoresed and northern blotted as previously described [START_REF] Bernard | Transcripitonal regulation of hydroxyindole-O-methyltransferase in the chicken pineal gland: day/night changes and long term effects of light and darkness[END_REF]. Rhodopsin and GAPDH cDNA probes were produced by RT-PCR (using the primer pairs indicated in Table S1) and labelled by random priming [START_REF] Feinberg | A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity[END_REF]) with [-32 P]dCTP (3000 Ci/mmol).

Following hybridization, the northern blot was exposed overnight on the screen of a Storm 820 (GE Healthcare, Saclay, France) and densitometric analysis was performed with the ImageQuant software (GE Healthcare).

Promoter-reporter assay

The chicken rhodopsin promoter [START_REF] Wiley | Characterization of the chicken rhodopsin promoter: identification of retina-specific and glass-like protein binding domains[END_REF] was PCR amplified from chicken genomic DNA with the primers listed in Table S1 and Before transfection, retinal cells from E8 embryos were seeded at high density in polyornithine-coated plates and cultured for 24h either in control conditions or in the presence of forskolin (10 -5 mol/L) and IBMX (10 -4 mol/L). They were then cotransfected by the CaPO4 method, with 150 ng of the normalizing plasmid pRL-TK (Promega) and either with 1.5 µg of pGL3-pRhodo1.6kb or with 1.5 µg of a promoterless, negative control plasmid (pGL3-Basic), as previously described [START_REF] Bernard | Photoreceptor-specific expression, light-dependent localization, and transcriptional targets of the zinc-finger protein Yin Yang 1 in the chicken retina[END_REF] and further cultured for 48h with or without forskolin and IBMX. Firefly and Renilla luciferase activities, were assayed using the Dual-Glo TM luciferase assay system (Promega). Light emission was measured on a GloMax TM 20/20 luminometer (Promega). Transfection efficiency was normalized to the firefly/Renilla luciferase activity ratio.

RESULTS

1) Regulation of rhodopsin gene transcription by cAMP-increasing agents

Embryonic chicken retinal cells dissociated at E8 expressed very low levels of rhodopsin mRNA (400 to 2000 cDNA copies per million copies of GAPDH cDNA), similar to the concentrations observed in non-photosensitive tissues (Fig. 1A). The concentration of rhodopsin mRNA remained at the same low values after 3 days of culture in control medium (Fig. 1A). Addition of IBMX (100 µmol/L) or forskolin (10 µmol/L) to the culture medium increased rhodopsin mRNA levels 5-fold and 20-fold, respectively (Fig. 1A and Table S2).

Addition of both drugs together caused a 50-fold rise in rhodopsin mRNA concentration (Fig. 1A and Table S2). This response was highly reproducible, although its amplitude could vary from 10-to 50-fold, as illustrated in Table 1. The rise in rhodopsin mRNA levels elicited by forskolin and IBMX was directly detectable on northern blot. Rhodopsin mRNA was detected as a major band of 1.6 kb and a minor band of 3 kb, in agreement with previous reports [START_REF] Okano | Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments[END_REF][START_REF] Adler | Microenvironmental regulation of visual pigment expression in the chick retina[END_REF][START_REF] Cailleau | Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursors cells[END_REF]. Densitometric analysis of the northern blot confirmed a 60-fold increase in rhodopsin mRNA levels (Fig. 1 B). Cell viability was not consistently affected by the cAMP-elevating agents (Fig. 1C). Transient transfection of a rhodopsin promoter-reporter construct into embryonic chicken retinal cells indicated that the rhodopsin promoter was very weak in control cultures (only 3-fold above a promoterless plasmid) and its activity increased 16-fold in the presence of forskolin and IBMX (Fig. 2). A result confirming that rhodopsin mRNA levels reflect cAMP-dependent activation of transcription.

2) cAMP-dependent activation of photopigment gene transcription is rod-specific.

As reported earlier [START_REF] Adler | Microenvironmental regulation of visual pigment expression in the chick retina[END_REF][START_REF] Cailleau | Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursors cells[END_REF], cultured retinal cells contained high levels of red cone opsin mRNA, nearing half the concentration of GAPDH mRNA (Table 1). The concentration of green cone opsin mRNA was about 1/10 th that of red cone opsin (Table 1). The mRNA concentrations for blue cone opsin and violet cone opsin were in the very low range (Table 1). In contrast to rhodopsin, the concentrations of transcripts encoding the different cone opsins were not increased by forskolin and IBMX, as confirmed in five independent experiments (Table 1). Depending on experiments, the cone opsins either failed to respond to cAMP-increasing agents or responded with a 50-80 % decrease in mRNA levels (Table 1). These results point to a rod-specific effect of cAMP.

3) Rhodopsin gene response to cAMP-increasing agents in different culture conditions Previous studies have indicated that in vitro differentiation of retinal precursor cells can be altered by changes in cell density, plastic coating, or serum supplementation [START_REF] Levine | Soluble factors and the development of rod photoreceptors[END_REF][START_REF] Haque | Diurnal regulation of arylalkylamine N-acetyltransferase activity in chicken retinal cells in vitro: analysis of culture conditions[END_REF][START_REF] Cailleau | Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursors cells[END_REF]. This prompted us to investigate how these changes in culture conditions might affect the rhodopsin gene response to cAMP-increasing agents. As illustrated in Fig. 3A, forskolin increased rhodopsin mRNA levels to the same extent in retinal cells cultured at either high or low cell-density, on coated or uncoated plastic, as well as in serum-free or serum-supplemented medium (see Table S2 for details). A result confirming the robustness of the rhodopsin gene response. As illustrated in Fig. 3B, cell viability was not consistently affected by either forskolin or culture conditions.

4) Time course and dose-response to forskolin

Forskolin caused a significant 4-fold increase in rhodopsin mRNA levels after 12h (Fig. 4A).

The concentration of transcript continued to increase at 24h and 48h, then plateaued-out 30-fold above control values after 72h (Fig. 4A). In a 72h experiment, a significant 8-fold increase in rhodopsin mRNA levels could be observed at 1 µmol/L forskolin and the stimulation reached 45-fold at 100 µmol/L forskolin (Fig. 4B and Table S2).

5) Effect of membrane depolarization

Plasma membrane depolarization induced by 50 mmol/L KCl caused a 5-fold increase in rhodopsin mRNA concentration (Fig. 5). IBMX, which caused a 5-fold increase in rhodopsin mRNA levels by itself, strongly potentiated the effect of KCl to reach rhodopsin mRNA levels 45-fold above control values (Fig. 5 and Table S2).

6) Effect of cyclic nucleotide analogs through protein kinase A

The effects of cyclic nucleotide analogs confirmed a cAMP-dependent increase in rhodopsin mRNA levels. The order of potency was mB-cAMP > dB-cAMP ≈ 8Br-cAMP > 8CPT-cAMP (Fig. 6A). This order of potency appeared to match the degree of specificity of these analogs for PKA [START_REF] Christensen | cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension[END_REF]. This prompted us to examine the effect of analogs specifically designed to activate either PKA or EPAC. The PKA-selective analog N 6 -Bnz-cAMP efficiently increased rhodopsin mRNA levels, whereas the EPAC-selective analog 8Br-2'O-Me-cAMP was without effect (Fig. 6B and Table S2). Similar results were obtained in 3 independent experiments. We verified that neither 8Br-cGMP (1 mmol/L), nor sodium butyrate (1 mmol/L) had any effect on rhodopsin mRNA levels (data not shown). It was also verified that a representative cAMP analog did not affect cell viability (Fig. 6C).

7) Development of the rhodopsin gene response to cAMP-increasing agents

Previous studies have indicated that the developmental stage of donor embryos had a strong impact on the differentiation of retinal cells in vitro [START_REF] Belecky-Adams | Correlations between terminal mitosis and differentiated fate of retinal precurseur cells in vivo and in vitro: analysis with the "windowlabeling" technique[END_REF][START_REF] Ivanova | Melatonin synthesis in the retina: circadian regulation of arylalkylamine N-acetyltransferase activity in cultured photoreceptor cells of embryonic chicken retina[END_REF]. In accordance with this, we observed that cells obtained at Hamburger and Hamilton (H.H.) stages 28 and 29 (E5.5 and E6) gave very weak responses of the rhodopsin gene to forskolin (Fig. 7), whereas cells obtained at H.H. stages 30 and 34 (E7 and E8) displayed full responses (Fig. 7 and Table S2). A result indicating that a critical event in the regulation of the rhodopsin gene takes place between E6 and E7.

8) cAMP-dependent regulation of transcripts related to rod-specific phototransduction

To investigate a possible role of cAMP as a general activator of rod differentiation, we examined the regulation of other genes of the rod-specific phototransduction cascade. Rodspecific transducin alpha subunit (Tr) mRNA levels were increased by forskolin and IBMX to the same extent (10-to 50-fold) as rhodopsin mRNA levels (Table 2). The concentration of mRNA encoding the rod-specific regulatory subunit of phosphodiesterase-6 (Pr) was reproducibly increased 4-to 10-fold by forskolin and IBMX (Table 2). The levels of transcripts encoding the rod-specific catalytic subunit of phosphodiesterase-6 (P) and rodspecific arrestin, displayed less reproducible responses to forskolin and IBMX (Table 2).

Nevertheless, a 3-to 10-fold rise in mRNA concentration could be observed for these genes, in 5 out of 7 experiments (Table 2). These results suggest a pleiotropic effect of cAMP on rod-specific transcription.

DISCUSSION

The present study describes a robust cAMP-dependent regulation of rhodopsin gene transcription in cultured retinal cells from chicken embryo. The potency of this cAMPdependent mechanism is underscored by the fact that it proved sufficient to activate rhodopsin gene transcription in the absence of serum-derived growth factors.

Despite many studies on photoreceptor differentiation in this model system, cAMP-dependent regulation of the rhodopsin gene had eluded detection until now. This response does not appear to reflect an accidental dysregulation of the rhodopsin gene in unspecified cells, because it was accompanied by the activation of four other genes of the rod-specific phototransduction cascade, with a strong correlation between rhodopsin and Tr regarding the amplitude of the response. The different amplitudes observed for the other rod-specific gene responses argue against a diffuse effect of cAMP on rod survival, as this would have resulted in an equal bias for all the rod-specific markers. Similar cell viabilities and RNA yields in control and forskolin-treated cultures also argue against pro-or anti-apoptotic effects of cAMP, in contrast to previous studies on cardiomyocytes and hippocampus [START_REF] Ding | A positive feedback loop of phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to cardiomyocyte apoptosis[END_REF][START_REF] Mioduszewska | Inducible cAMP early repressor (ICER)-evoked delayed neuronal death in the organotypic hippocampal culture[END_REF]. In addition, the cAMP effect appeared highly cell-type specific, as it failed to stimulate transcription of the cone opsin genes. Therefore, our data would rather suggest that cAMP is a significant second messenger in a signaling pathway that specifically activates rod differentiation. This is in keeping with multiple examples of a role of cAMP at specific steps of neuronal differentiation, including dopamine beta-hydroxylase gene activation in catecholaminergic neurons [START_REF] Ji | The cAMP pathway regulates both transcription and activity of the paired homeobox transcription factor Phox2a required for development of neural crest-derived and central nervous system-derived catecholaminergic neurons[END_REF], morphological maturation of hippocampal neurons and cerebellar granule cells [START_REF] Fujioka | Activation of cAMP signaling facilitates the morphological maturation of newborn neurons in adult hippocampus[END_REF][START_REF] Obara | The requirement of Ras and Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in cerebellar granule cells[END_REF], neurite outgrowth in pheochromocytoma cell lines (Shi et al., 2006 and refs therein), upregulation of neuronal genes in stem cells [START_REF] Tondreau | Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells[END_REF].

Because previous studies aimed at identifying signals that stimulate rod differentiation have yielded contrasted results in different species [START_REF] Levine | Soluble factors and the development of rod photoreceptors[END_REF], it will be important to determine whether the cAMP effect described herein can be extended to other species.

Previous studies on cAMP-dependent regulation of the rhodopsin gene in zebrafish, salamander and mouse cannot directly compare with the data presented herein, because they were performed on cells that already expressed appreciable levels of rhodopsin [START_REF] Alfinito | Dopamine D4 receptor-mediated regulation of rod opsin mRNA expression in tiger salamander[END_REF][START_REF] Alfinito | Adenosine A(2a) receptor-mediated inhibition of rod opsin mRNA expression in tiger salamander[END_REF][START_REF] Yu | Mitogenassociated protein kinase-and protein kinase A-dependent regulation of rhodopsin promoter expression in zebrafish rod photoreceptor cells[END_REF][START_REF] Da Silva | Interleukin-4 blocks proliferation of retinal progenitor cells and increases rod photoreceptor differentiation through distinct signaling pathways[END_REF]. Further studies in different species should be performed at adequate embryonic stages to determine whether cAMP-dependent activation of rhodopsin gene transcription is a conserved mechanism of rod differentiation in vertebrates .

Previous attempts to stimulate rod differentiation in cultured retinal cells from chicken embryo have indicated that staurosporine increased rhodopsin mRNA levels, as well as the number of rhodopsin mRNA-expressing cells [START_REF] Xie | Green cone opsin and rhodopsin regulation by CNTF and staurosporine in cultured chick photoreceptors[END_REF][START_REF] Bradford | Roles of cell-intrinsic and microenvironmental factors in photoreceptor cell differentiation[END_REF].

The lack of specificity of staurosporine, which is known to act as a general inhibitor of protein kinases [START_REF] Meggio | Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2[END_REF] and as an apoptosis-inducing agent [START_REF] Posmantur | Characterization of CPP32-like protease activity following apoptotic challenge in SH-SY5Y neuroblastoma cells[END_REF] makes it difficult to relate its effect with that of cAMP. However, this possibility cannot be ruled out, because one report on peripheral blood mononuclear cells indicated that staurosporine enhanced both receptor-mediated (prostaglandin) and direct (forskolin) activation of adenylate cyclase [START_REF] Buc | Regulation of adenylyl cyclase activity in human peripheral blood mononuclear cells: effects of protein kinase inhibitors and of a calcium ionophore[END_REF].

Because cAMP appears potentially relevant as a physiological regulator of rod differentiation, one obvious challenge raised by our findings is to elucidate the signaling pathway upstream and downstream of this second messenger. As a first step downstream of cAMP, it appeared important to determine whether this response was mediated by PKA or by EPAC, because both pathways have been shown to regulate gene expression and neuronal differentiation [START_REF] Kim | Activation of protein kinase A induces neuronal differentiation of HiB5 hippocampal progenitor cells[END_REF][START_REF] Shi | A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation[END_REF][START_REF] Obara | The requirement of Ras and Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in cerebellar granule cells[END_REF][START_REF] Gakhar-Koppole | Depolarization promotes GAD 65-mediated GABA synthesis by a post-translational mechanism in neural stem cell-derived neurons[END_REF]. Our experiments clearly decided in favour of PKA, based on the effects of PKA-selective versus EPAC-selective analogs and on the potency order of the more commonly used analogs [START_REF] Christensen | cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension[END_REF][START_REF] Connolly | 8-(4-Chlorophenyl)thio-cyclic AMP is a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA)[END_REF].

A further step down the signaling pathway will be to examine whether the rhodopsin gene promoter is directly activated by the phosphorylated form of the cAMP response elementbinding protein (phospho-CREB). We already obtained evidence in promoter-reporter experiments that a 1.6 kb promoter of the chicken rhodopsin gene is sufficient to drive a 16fold cAMP-dependent increase in transcription. Analysis of this sequence with the TESS program [START_REF] Schug | Using TESS to predict transcription factor binding sites in DNA sequence[END_REF] revealed three incomplete matches to the cAMP response element that will deserve further analysis.

The coordinated effect of cAMP on five rod-specific transcripts is of interest as it suggests a pleiotropic effect on rod differentiation. Preliminary in silico investigations revealed putative cAMP response elements in the 5'-flanking regions of chicken Pr, P and Tr genes, but not in the rod arrestin promoter. The possibility that coordinated activation of rod-specific transcription reflects the action of an intermediate regulatory gene should also be considered.

In contrast to mammals, where Nrl has been identified as a potent activator of rod differentiation [START_REF] Kumar | The bZIP transcription factor Nrl stimulates rhodopsin promoter activity in primary retinal cell cultures[END_REF][START_REF] Mears | Nrl is required for rod photoreceptor development[END_REF], a rod-specific transcription factor remains elusive in chicken. In the absence of Nrl, a possible candidate in chicken would L-Maf, whose timing of expression in rod precursor cells appears compatible with the activation of rhodopsin gene transcription [START_REF] Ochi | Temporal expression of L-Maf and RaxL in developing chicken retina are arranged into mosaic pattern[END_REF]. Regulation of L-Maf expression by cAMP has not been reported so far and would be worth studying.

Analysis of the signaling pathway, upstream of cAMP, warrants further studies aimed at identifying the adenylate cyclase-coupled receptors that regulate rhodopsin mRNA levels.

Hopefully, this may open the way to pharmacological treatment of retinopathies characterized by low levels of rhodopsin gene expression [START_REF] Farrar | On the genetics of retinitis pigmentosa and on mutation-independent approaches to therapeutic intervention[END_REF]. Another interesting possibility, however, is that a rise in cAMP levels might be driven in vivo by depolarization of rod precursor cells, as was observed experimentally with high KCl concentration. Recently, retinal waves of depolarization spreading through the population of developping retinal ganglion cells have been shown to increase cAMP levels via activation of the Ca ++ /calmodulin-activated adenylate cyclase, leading to cAMP-dependent regulation of ephrin-A5 signaling and refinement of axonal projections [START_REF] Ravary | Adenylate cyclase 1 as a key actor in the refinement of retinal projection maps[END_REF][START_REF] Rodger | cAMP regulates axon outgrowth and guidance during optic nerve regeneration in goldfish[END_REF][START_REF] Dunn | Imaging of cAMP levels and protein kinase A activity reveals that retinal waves drive oscillations in second-messenger cascades[END_REF][START_REF] Nicol | cAMP oscillations and retinal activity are permissive for ephrin signaling during the establishment of the retinotopic map[END_REF]. The possibility of a similar mechanism taking place in rod precursors to trigger the cAMP-dependent activation of phototransduction genes would appear both attractive and testable.

It is generally agreed that photoreceptor differentiation occurs in two chronologically well separated steps: first, acquisition of competence soon after the exit of mitosis (from E4 to E7 in chicken) ; then, about one week later, activation of photoreceptor-specific gene transcription [START_REF] Belecky-Adams | Correlations between terminal mitosis and differentiated fate of retinal precurseur cells in vivo and in vitro: analysis with the "windowlabeling" technique[END_REF][START_REF] Bruhn | Development of the pattern of photoreceptors in the chick retina[END_REF]Cepko, 1996, Levine et al., 2000 ;[START_REF] Adler | Have we achieved a unified model of photoreceptor cell fate specification in vertebrates?[END_REF]. Previous studies have shown that retinal precursors isolated at E6 are capable of activating the red opsin gene when cultured for 4 days in vitro [START_REF] Adler | Microenvironmental regulation of visual pigment expression in the chick retina[END_REF]. A result indicating that red cone precursors are competent for red opsin gene activation as early as E6. In contrast, the results presented herein clearly show that rod precursors are not competent for cAMP-dependent rhodopsin gene activation at E6 and that they acquire this competence about one day later. The molecular mechanism of this developmental switch in competence is open to further analysis. One attractive hypothesis is that it might reflect chromatin remodeling at the rhodopsin locus. This would deserve investigation by means of chromatin immunoprecipitation protocoles. Together, our data appear compatible with a scenario in which some retinoblasts become competent for rod differentiation as early as E7, provided they encountered a signal that raised cAMP levels. In vivo, this signal (receptor ligand or membrane depolarization) would be delivered only around E15, to activate rhodopsin gene transcription (and maybe other rod-specific gene transcription). This new point of view on rod differentiation opens perspectives of research aimed at elucidating the mechanism of competence acquisition, the nature of the signal that raises cAMP levels in rod precursor cells and the relevance of this scenario to the in vivo situation. E8 chicken retinal cells were seeded at low density (0.3x10 6 /cm 2 ) or at high density (1.25x10 6 /cm 2 ) in plastic plates that were either non-coated (NC), or coated with poly-Dlysine (PL, 5 µg/cm 2 ), or with poly-ornithine (PO, 20 µg/cm 2 ). Cells were cultured for 3 days in serum-supplemented medium or in serum-free medium supplemented with 0.5% BSA (no serum), either in control conditions (C: 0.1% DMSO) or with 10 -5 M forskolin (F). A.)

Rhodopsin and GAPDH mRNA levels were quantified by real time RT-PCR and expressed as thousand copies of rhodopsin (Rh) template per million copies of GAPDH template (mean  SEM n = 3). B.) Cell viability as measured with CellTiter96  .

  cloned upstream of the firefly luciferase reporter gene, in the pGL3-Basic plasmid (Promega). Recombinant clones were verified by DNA sequencing (Big Dye reagent and ABI 3130 Genetic Analyzer, Applied Biosystems, Courtaboeuf, France).

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Cyclic-AMP-dependent activation of the chicken rhodopsin gene promoter.E8 chicken retinal cells were cultured either in control conditions (0.2% DMSO) or with 10 -5 mol/L forskolin and 10 -4 mol/L IBMX (F+I). The cells were transiently transfected with 1.5

Figure 3 .

 3 Figure 3. Influence of culture conditions on the rhodopsin gene response to forskolin.

Figure 4 .

 4 Figure 4. Time-course and dose-dependence of the rhodopsin response to forskolin.A.) E8 chicken retinal cells were cultured for 12 to 72 h with 10 -5 mol/L forskolin (Fsk: full

Figure 5 .

 5 Figure 5. Effect of membrane depolarization.

Figure 6 .

 6 Figure 6. Effects of cAMP analogs on rhodopsin mRNA levels.

Figure 7 .

 7 Figure 7. Development of the rhodopsin gene response to cAMP-increasing agents.

Table 1 .

 1 cAMP-dependent activation of photopigment gene transcription is rod-specific. Retinal cells from E8 chicken embryos were cultured either in control conditions (0.2% DMSO) or with 10 -5 mol/L forskolin (Fsk) and 10 -4 mol/L IBMX. mRNA levels were quantified by real time RT-PCR. The table summarizes the results of 5 individual experiments on triplicate samples. Results are expressed as number of copies of a given template per million copies of GAPDH template (mean  SEM, n = 15 ; **p<0.0001, *p<0.05, Student's ttest). The column on the right shows the fold-change observed in each individual experiment, with the median value underlined.

		Control	Forskolin+IBMX	Fold-changes in 5 individual experiments
		(copies/10 6 GAPDH)	(copies/10 6 GAPDH)	(median underlined)
		mean  sem	mean  sem
		(n = 15)	(n = 15)
	Rhodopsin	785  106	19 264  1 290 **	10x ; 23x ; 25x ; 32x ; 50x
	Red Opsin	502 169  49 220	326 880  70 461	0.15x ; 0.58x ; 0.82x ; 0.84x ; 1.2x
	Green Opsin	40 157  7 260	27 240  6 322	0.38x ; 0.57x ; 0.58x ; 0.65x ; 1.2x
	Blue Opsin	79  20	50  14	0.3x ; 0.6x ; 0.68x ; 0.94x ; 1x
	Violet Opsin	2 385  595	857  238 *	0.18x ; 0.33x ; 0.35x ; 0.47x ; 0.58x

Table 2 .

 2 cAMP-dependent regulation of transcripts related to rod-specific phototransduction. The column on the right shows the fold-increase observed in each individual experiment, with the median value underlined.

	Retinal cells from E8 chicken embryos were cultured either in control conditions (0.2%
	DMSO) or with 10 -5 mol/L forskolin and 10 -4 mol/L IBMX (F+I). The transcripts encoding
	GAPDH, rod-specific transducin alpha subunit (T rod), rod-specific phosphodiesterase-6
	gamma subunit (PDEγ rod), rod-specific phosphodiesterase-6 beta subunit (PDE rod) and
	rod-specific arrestin were quantified by real time RT-PCR. The table summarizes the results
	of 7 individual experiments on triplicate samples. Results are expressed as number of copies
	of a given template per million copies of GAPDH template (mean ± SEM, n = 21 ;
	**p<0.001, *p<0.01, Student's t-test). Control	F+I	Fold-increases in 7 individual experiments
		(copies/10 6 GAPDH)	(copies/10 6 GAPDH)	(median underlined)
		mean  sem	mean  sem
		(n = 21)	(n = 21)
	T
	(rod)	487  56	12123  1560 **	9x ; 13x ;20x ; 34x ; 41x ; 42x ; 48x
	PDEγ
	(rod)	784  59	4465  473 **	3.7x ; 3.8x ; 4.5x ; 4.8x ; 4.9x ; 6.9x ; 12x
	PDE
	(rod)	671  183	1616  259 *	1.5x ; 1.7x ; 2.8x ; 3.5x ; 4x ; 4x ; 5.6x
	Arrestin
	(rod)	278  30	1626  202 **	1.7x ; 2.3x ; 4x ; 6.7x ; 10x ; 10x ; 11x

ACKNOWLEDGEMENTS

This work was supported by the CNRS (UMR 6187), Fondation Langlois and Retina France.

We would like to thank Mrs L. Cousin for her technical help at multiple steps of this work and Dr. C. Chadéneau for her advice on the CellTiter96  assay. We appreciate the technical assistance of the University facilities: Service Commun de Séquençage (Mr D. Guyonnet) and Service Commun de Publication Assistée par Ordinateur (Mr J. Habrioux).


Anthony El Hajj
email: anthony.el-hajj@edf.fr

Eric Semail
email: eric.semail@ensam.eu

Abdelmounaim Tounzi
email: abdelmounaim.tounzi@univ-lille.fr

Darius Vizireanu
email: darius.vizireanu@edf.fr

Jalal Cheaytani
email: jalal.cheaytani@edf.fr

Detection of incipient faults in nine-phase machines: impact of the star winding configuration

Keywords: multiphase permanent-magnet synchronous machines, fault detection, vector space decomposition, decentralized d-q modelling, symmetrical and asymmetrical star winding configurations

Two approaches are commonly used for modelling and control of nine-phase (triple star) fault-tolerant machines with symmetrical and asymmetrical star winding configurations: The Vector Space Decomposition (VSD) and the decentralized d-q modelling. In this paper, it will be shown how the VSD approach used for machines with an Asymmetrical Star Winding Configuration (ASWC), unlike the decentralized d-q one used for machines with a Symmetrical Star Winding Configuration (SSWC), can be helpful for non-intrusive Fault Detection and Diagnosis (FDD) purposes. Supporting MATLAB/Simulink simulations are discussed.

Introduction

During the last decade, the use of multiphase Permanent-Magnet Synchronous Machines (PMSMs) has become more and more common in several domains including wind energy conversion. These machines have been gaining interest due to their high torque/power density, good efficiency with a variable speed, and fault tolerance leading to a higher functional reliability. Furthermore, in the case of wind energy conversion, they are mainly used at variable speed connected to the grid through converters and as direct-driven machines thus avoiding problems of the mechanical gearbox [START_REF] Cheng | The state of the art of wind energy conversion systems and technologies: A review[END_REF].

However, a multiphase PMSM, like any other machine, is prone to faults: stator faults (inter-turn short circuits, increased resistance connection) and/or rotor faults (demagnetization of permanent magnets, eccentricity, bearings, etc). The fault tolerance and the robustness with respect to incipient faults require nevertheless a detection of such defects in early stages to avoid any severe damage, to reconfigure the control, and to organize maintenance schedule. In this aim, several research works are conducted to find the most relevant indicators [START_REF] Foster | Detection of incipient stator winding faults in PMSMs with single-layer fractional slot concentrated windings[END_REF][START_REF] Haddad | A Voltage-Based Approach for Fault Detection and Separation in Permanent Magnet Synchronous Machines[END_REF][START_REF] Hu | Detection and Classification of Turn Fault and High Resistance Connection Fault in Permanent Magnet Machines Based on Zero Sequence Voltage[END_REF][START_REF] Huang | Robust Stator Winding Fault Detection in PMSMs With Respect to Current Controller Bandwidth[END_REF][START_REF] Hang | Fault Diagnosis of High-Resistance Connection in a Nine-Phase Flux-Switching Permanent-Magnet Machine Considering the Neutral-Point Connection Model[END_REF]. Most studies dealt with 3-phase PMSMs. However, there are some differences between the mathematical properties of 3-phase machines and multiphase ones that turn out to be useful for control [START_REF] Slunjski | General Torque Enhancement Approach for a Nine-Phase Surface PMSM With Built-In Fault Tolerance[END_REF][START_REF] Kestelyn | Vectorial Modeling and Control of Multiphase Machineswith Non-salient Poles Supplied by an Inverter[END_REF][START_REF] Levi | FOC: Field-Oriented Control[END_REF] and Fault Detection and Diagnosis (FDD) of multiphase machines [START_REF] Trabelsi | Real-Time Switches Fault Diagnosis Based on Typical Operating Characteristics of Five-Phase Permanent Magnetic Synchronous Machines[END_REF].

Two approaches are mainly used for modelling and control of multi-threephase machines: the VSD that is used for any number of phases [START_REF] Slunjski | General Torque Enhancement Approach for a Nine-Phase Surface PMSM With Built-In Fault Tolerance[END_REF][START_REF] Levi | FOC: Field-Oriented Control[END_REF][START_REF] Levi | Multiphase AC Machines[END_REF], and the decentralized d-q modelling, used only when the number of phases is a multiple of 3 [START_REF] Onsal | A New Nine-Phase Permanent Magnet Synchronous Motor With Consequent Pole Rotor for High-Power Traction Applications[END_REF][START_REF] Prieto-Araujo | Decentralized Control of a Nine-Phase Permanent Magnet Generator for Off-shore Wind Turbines[END_REF]. In this paper, it will be shown how the VSD, unlike the decentralized d-q modelling, can also be used for FDD purposes in the case of a 9-phase PMSM with an asymmetrical star winding configuration. More precisely, it will be shown how the projection of non-intrusive quantities (such as stator currents) onto the space defined by the Concordia transform with the VSD approach can be helpful for FDD. Indeed, both Concordia transforms, the one with the d-q modelling approach and the one with the VSD approach, define orthonormal subspaces but with different harmonic properties. With the decentralized d-q modelling approach, the Concordia transform defines several subspaces containing each the 1 st order harmonic with quite the same amplitude in both healthy and faulty states. However, with the VSD approach, the Concordia transform defines several subspaces of which only one subspace contains the 1 st order harmonic in the healthy state. Therefore, the emergence of this harmonic in the remaining subspaces in the faulty state can be very useful for FDD. These mathematical properties and the differences between the healthy state and a faulty case (increase of resistance connection) are studied in details in this paper. To the best of the author's knowledge, these differences have not yet been studied in the literature.

The rest of the paper is organized as follows. In Section 2, the different types of multiphase machines and their modelling are introduced. In Section 3, the effects of the same fault on two 9-phase PMSMs, one with a symmetrical star winding configuration (SSWC) and the other with an asymmetrical star configuration (ASWC), are compared. Conclusions and future works are provided in Section 4.

Types of multiphase machines and their modelling

Types of multiphase machines

Depending on the applications, the number of phases of a multiphase machine may vary. There are mainly two categories of multiphase machines [START_REF] Levi | FOC: Field-Oriented Control[END_REF][START_REF] Levi | Multiphase AC Machines[END_REF]:

-Machines with a prime number of phases (5, 7, 11, etc.). All phases are often connected to one neutral.

-Machines whose number of phases is a multiple of 3. The multiple threephase star aspect of these machines might be interesting for industrials. Each three windings that are separated by 120 • or 240 • constitute a three-phase star, usually with one isolated neutral per star. If a fault occurs on one phase, the whole star to which the faulty phase belongs should be isolated. For these machines, there are symmetrical and asymmetrical star winding configurations (Figure 1). The asymmetrical configuration, ASWC, corresponds to the case where the stars are phase-shifted by an angle γ in the electrical space. The symmetrical configuration, SSWC, corresponds to the case where the stars are not phase-shifted.

In this paper, two 9-phase PMSMs are studied: the first one is a 30-pole PMSM with a SSWC and the second one is a 32-pole PMSM with an ASWC (Figure 1). For both machines, 3 stars with 3 isolated neutrals are considered.

VSD and decentralized d-q modelling

As introduced previously, two approaches can be used for the modelling and control of multiphase machines: the VSD and the decentralized d-q modelling. The former approach is a general one, it can be used whether the number of phases is a multiple of 3 or not [START_REF] Levi | Multiphase AC Machines[END_REF]. In the absence of reluctance effect and with regular manufacturing for the stator, the inductance matrix in the new space is then diagonal and there is no need to consider cross-couplings between the different subspaces. The latter approach, i.e. the decentralized d-q modelling, is used only when the number of phases is a multiple of 3. Note that depending on the machine winding arrangement, the magnetic coupling between phases belonging to two different stars can or cannot be neglected. Cross-couplings should therefore be considered between the different subspaces to provide an adequate modelling.

For both modelling methods, a Concordia transform matrix C n (n being the total number of phases) that allows the projection of a vector quantity g n (such as current or voltage) from the initial space onto a new space is defined, it gives g αβ0

g αβ0 = C n • g n , g n = g 1 g 2 ... g n T (1)
In the decentralized d-q approach, there are n 3 two-dimensional subspaces (α j , β j ), j ∈ 1, 2, ..., n 3 and n 3 one-dimensional (zero-sequence) subspaces 0 j , one (α j , β j ) and one 0 j per three-phase star. Each (α j , β j ) mainly contains the 1 st order harmonic in the healthy state. Other harmonics appear in the faulty state. The value of the 1 st order harmonic being very high compared to the other harmonics, the disturbance generated by the fault in these subspaces is very small.

In the case of the 9-phase machine with a SSWC studied in this paper, the mutual inductances between the different phases are negligible (8 % of the selfinductance at most). The Concordia transform matrix (2) can therefore be written in a simple manner as the concatenation of three diagonal blocks of the 3-phase Concordia transform (the machine is considered as three independent stars)

C n = C 9sym =   C 3 0 3 * 3 0 3 * 3 0 3 * 3 C 3 0 3 * 3 0 3 * 3 0 3 * 3 C 3   , C 3 = 2 3     1 cos 120 • cos 240 • 0 sin 120 • sin 240 • 1 √ 2 1 √ 2 1 √ 2     (2)
g αβ0 = g α1 g β1 g 01 g α2 g β2 g 02 g α3 g β3 g 03 T (3)   g 1 g 4 g 7   =   g 2 g 5 g 8   =   g 3 g 6 g 9   =   g • sin θ e g • sin(θ e -120 • ) g • sin(θ e -240 • )   (4)
The harmonics mapping of this machine, in the healthy state, is given in Table 1. It shows that each of the subspaces (α 1 , β 1 ), (α 2 , β 2 ), and (α 3 , β 3 ) contains mainly the 1 st order harmonic. In the VSD approach, there are n-1 2 (α j , β j ), j ∈ 1, 2, ..., n-1 2 subspaces (planes) and one zero-sequence subspace (line) if n is odd. There are

n-2 2 (α j , β j ), j ∈ 1, 2, ..., n-2 2
planes and two zero-sequence lines if n is even [START_REF] Kestelyn | Vectorial Modeling and Control of Multiphase Machineswith Non-salient Poles Supplied by an Inverter[END_REF].

In the case of the 9-phase machine with an ASWC studied in this paper, there are 4 (α j , β j ) and one zero-sequence subspace 0. The harmonics mapping of this machine is summarized in Table 2. (α 1 , β 1 ) mainly contains the 1 st order harmonic, (α 2 , β 2 ) the 7 th one, (α 3 , β 3 ) the 3 rd one, (α 4 , β 4 ) the 5 th one, and the zero-sequence subspace (0) the 9 th one. The corresponding Concordia transform matrix (γ = 40 • ) is defined below (5) [START_REF] Levi | Multiphase AC Machines[END_REF]

C n = C 9asym = 2 9                 1 cos γ cos 2γ cos 3γ
... cos 8γ 0 sin γ sin 2γ sin 3γ ... sin 8γ 1 cos 2γ cos 2(2γ) cos 2(3γ) ... cos 2(8γ) 0 sin 2γ sin 2(2γ) sin 2(3γ) ... sin 2(8γ) 1 cos 3γ cos 3(2γ) cos 3(3γ) ... cos 3(8γ) 0 sin 3γ sin 3(2γ) sin 3(3γ) ... sin 3(8γ) 1 cos 4γ cos 4(2γ) cos 4(3γ) ... cos 4(8γ) 0 sin 4γ sin 4(2γ) sin 4(3γ) ... sin 4(8γ

) 1 √ 2 1 √ 2 1 √ 2 1 √ 2 ... 1 √ 2                 (5)
g αβ0 = g α1 g β1 g α2 g β2 g α3 g β3 g α4 g β4 g 0 T ( 6
)
g j = g • sin(θ e -(j -1) • γ), j ∈ {1, 2, ..., 9} (7)
The torque being generally generated by the 1 st order harmonic, the currents in (α j , β j ), j ∈ {2, 3, 4} are null in the healthy state, when g n is balanced. In a faulty state, g n is no more balanced and the 1 st order harmonic appears in these subspaces. The changes are more significant than those in (α 1 , β 1 ) or those in the (α j , β j ), j ∈ {1, 2, 3} subspaces of the decentralized d-q approach. It will be shown that these changes are relevant indicators for fault detection in multiphase machines.

Based on the considerations introduced above, the changes in the phase currents projected onto (α, β, 0) for a five-phase machine were used to identify Voltage Source Inverter (VSI) open-switch faults in [START_REF] Trabelsi | Real-Time Switches Fault Diagnosis Based on Typical Operating Characteristics of Five-Phase Permanent Magnetic Synchronous Machines[END_REF]. In the following section, the same approach is considered for internal machine faults.

Simulation results

To show the effectiveness of the proposed approach, simulations are carried out in MATLAB/Simulink for both multiphase machines studied in healthy and faulty cases. Both machines, whose main characteristics are given in Table 3, are with tooth-concentrated windings and surface-mounted permanent magnets. Therefore, the values of the mutual inductances are low. Furthermore, to perform the simulations, some conditions and assumptions are made. They are listed hereafter: A general control diagram is shown in Figure 2. As discussed in Section 2, there are 3 (α j , β j ) subspaces for the machine with a SSWC, each containing the 1 st order harmonic (Table 1). A rotation of θ e is applied to each (α j , β j ) and the rotating subspaces (d j , q j ), j ∈ {1, 2, 3} are obtained. There are therefore 6 PI controllers (d 1 , q 1 , d 2 , q 2 , d 3 , and q 3 ) in the control loop of the first machine. This corresponds to a decentralized d-q control strategy. On the other hand, there are 4 (α j , β j ) subspaces for the machine with an ASWC, containing mainly the 1 st , 7 th , 3 rd , and 5 th order harmonics respectively (Table 2). Rotations of θ e , 7θ e , 3θ e , and 5θ e are applied to (α 1 , β 1 ), (α 2 , β 2 ), (α 3 , β 3 ), and (α 4 , β 4 ) respectively and the rotating subspaces (d j , q j ), j ∈ {1, 2, 3, 4} are obtained. There are therefore 8 PI controllers in the control loop of the second machine. This corresponds to a VSD control strategy. One of the easiest faults to model is the increase of resistance connection. The stator resistance value of one phase (or several phases) is increased. This fault is common in electric machines. It can occur in any power connection. If not detected at its incipient stage, it leads to local excessive heating and increases losses [START_REF] Hang | Fault Diagnosis of High-Resistance Connection in a Nine-Phase Flux-Switching Permanent-Magnet Machine Considering the Neutral-Point Connection Model[END_REF][START_REF] Tani | Detection and localization of high resistance connections in quadruple three-phase induction motor drives[END_REF][START_REF] Gritli | Detection and Localization of Incipient High Resistance Connection for Asymmetrical Twelve-Phase Induction Motor Drives[END_REF].

I 2 I 1 Ω Θ Θ Θ Ω ω d j ,q j V d j ,q j ref I d j ,q j E d j ,q j

Comparison of currents in healthy and faulty states

Figure 3 shows the steady-state phase currents versus time plots when a resistor R add = 0.1Ω ≈ 2R s is added to phase 1 of the machine with a SSWC at rated speed/power conditions. Figure 4 shows i β j = f (i α j ), j ∈ {1, 2, 3} and i α 1 = f (t). The changes between healthy and faulty states are insignificant (less than 1%). This is due to the fact that the 1 st order harmonic current is projected onto each (α j , β j ), j ∈ {1, 2, 3} in both healthy and faulty states with quite the same amplitude. Therefore, the detection of incipient imbalance in the working space defined by the considered transformation C 9sym (2) appears as difficult.

Figure 5 depicts the steady-state phase currents versus time plots when a resistor R add = 0.1Ω ≈ 3R s is added to phase 1 of the machine with an ASWC at rated speed/power conditions. For the currents, the differences in this natural space are slightly higher than in the previous case (30-pole machine). The currents are higher or lower in the healthy state than in the faulty state. It depends on the fault location and the mutual inductances between the faulty phase and the remaining healthy ones. Figure 6 shows that the changes in i α j ,β j = f (t), j ∈ {2, 4} are much more significant. The currents in these two subspaces are very close to zero in the healthy state (dashed curves). When a fault occurs, they increase and their main component becomes the 1 st order harmonic (solid curves). These changes give elliptical loci i β j = f (i α j ), j ∈ {2, 4} (Figure 7). As shown in Table 2, the 1 st order harmonic is not projected onto (α 2 , β 2 ), (α 3 , β 3 ) or (α 4 , β 4 ) in the healthy state. When a fault occurs, this mapping is no more valid and all harmonics appear in all subspaces. More specifically, the 1 st order harmonics appears in (α 2 , β 2 ) and (α 4 , β 4 ). Note that no changes are observed in (α 3 , β 3 ) because of the 3 isolated neutrals that impose a null 3 rd order harmonic current in both healthy and faulty states [START_REF] Kestelyn | Vectorial Modeling and Control of Multiphase Machineswith Non-salient Poles Supplied by an Inverter[END_REF]. In simulations, the currents in (α 3 , β 3 ) are about 10 -10 (A) due to some numerical errors.

     i 1 + i 4 + i 7 = 0 i 2 + i 5 + i 8 = 0 i 3 + i 6 + i 9 = 0 (8)
I β = f (Iα)
By doubling the value of R add , the different loci i β j = f (i α j ), j ∈ {2, 4} get wider as shown in Figure 7. The fault R add = 0.1Ω is then removed from phase 1 and added to phase 2. It is observed that the directions of the loci i β j = f (i α j ) change (Figure 8). Finally, the fault is added simultaneously on phases 1 and 2. The widths and the directions of the loci are different from those in the previous cases. Hence, it can be concluded that the variations of currents when projected onto (α j , β j ) depend on the severity of the fault and its location. This shows that the chosen subspaces and quantities provide useful information for the elaboration of sensitive non-intrusive fault indicators. 3.2. Comparison of phase currents in the rotating frame (d j , q j ) in healthy and faulty states: machine with an ASWC In the rotating frame (d j , q j ), i d j and i q j , j ∈ {2, 4} are nearly null in the healthy state. Only the (d 1 , q 1 ) plane exhibits significant DC currents. The d 1 axis exhibits a null DC current, this is due to the MTPA control strategy. In the faulty state (R add = 0.1Ω, phase 1), the 1 st order harmonic appears in (α 2 , β 2 ) and (α 4 , β 4 ) subspaces. The rotation angle in (d 2 , q 2 ) being of -7θ e , 6 th and 8 th order harmonics appear in this subspace. Similarly, 4 th and 6 th order harmonics appear in (d 4 , q 4 ) (rotation of -5θ e ). The spectra of i d j and i q j , j ∈ {1, 2, 4} are shown in Figure 9.

In the following subsection, it will be explained why fault signatures appear in phase currents, despite the current control. To do so, the interests of both Concordia and rotation transforms should be recalled.

Fault signatures in a closed-loop system

With MTPA control for non-sinusoidal PMSMs, the currents in the machine can be written as a sum of multiple harmonic currents [START_REF] Slunjski | General Torque Enhancement Approach for a Nine-Phase Surface PMSM With Built-In Fault Tolerance[END_REF][START_REF] Trabelsi | Real-Time Switches Fault Diagnosis Based on Typical Operating Characteristics of Five-Phase Permanent Magnetic Synchronous Machines[END_REF]. The control with PI of such a quantity is difficult and torque ripples may appear. By applying the VSD, each of the main harmonics (1 st , 3 rd , 5 th , and 7 th order ones in the case of a 9-phase machine) is projected onto one (α j , β j ) subspace and only one. Hence, sinusoidal quantities are obtained in the different (α j , β j ) subspaces. To obtain constant quantities, one specific rotation per subspace (α j , β j ) should be applied. The rotation angle depends on the main harmonic projected onto the given subspace.

For the 9-phase machine with an ASWC studied in this paper, rotations of θ e , -7θ e , 3θ e , and -5θ e are respectively applied to (α 1 , β 1 ), (α 2 , β 2 ), (α 3 , β 3 ), and (α 4 , β 4 ). The subspaces of the rotating frame are noted (d j , q j ), j ∈ {1, 2, 3, 4}. The stationary quantities can then be controlled with simple PI controllers, as long as four harmonics (1 st , 3 rd , 5 th , and 7 th order ones) are sufficient for the characterization of the electromotive forces and the reference currents.

When a fault occurs, stator currents are no longer balanced and different harmonics appear in the different (α j , β j ) subspaces. The control being based on the healthy operating mode, the transformation matrices and the controller parame-ters are the same in both healthy and faulty states. Therefore, the projection of the phase currents onto the rotating subspaces results in oscillating quantities: DC component, 6 th and 8 th order harmonics in (d 2 , q 2 ) and DC component, 4 th and 6 th order harmonics in (d 4 , q 4 ). The signals in the feedback of the current loops are thus oscillating signals and the PI controllers, whose bandwidths are limited (90.7 rad.s -1 for the machine with an ASWC), do not compensate these oscillations, i.e. the fault signatures do not disappear. Note that if the current-controller bandwidth is increased, the effect of the fault on the currents is less significant, while the opposite trend is observed in the reference voltages [START_REF] Huang | Robust Stator Winding Fault Detection in PMSMs With Respect to Current Controller Bandwidth[END_REF]. The proposed fault detection strategy can therefore be applied on reference voltages.

Conclusions

In this paper, the impact of a resistance imbalance has been analysed for two nine-phase PMSMs with quite similar external characteristics but different winding arrangements. With the considered resistance imbalance, the impact on the phase currents, when plotted versus time, is almost invisible for both machines. However, the differences observed in the (α, β, 0) subspaces are significant. For the 9-phase machine with a symmetrical star winding configuration, the impact of a resistance imbalance in the space defined by C 9sym for decentralized d-q modelling is difficult to detect. On contrary, for the nine-phase machine with an asymmetrical star winding configuration, the impact of the imbalance in the space defined by C 9asym for VSD is quite visible. The detection of incipient resistance fault appears as easier for the asymmetrical configuration.

For future works, a diagnosis tool based on the relevant indicators presented above will be developed (as in [START_REF] Trabelsi | Real-Time Switches Fault Diagnosis Based on Typical Operating Characteristics of Five-Phase Permanent Magnetic Synchronous Machines[END_REF]). To do so, several fault types and several severities should be considered and simulated. The ability of the indicators to discriminate between two different faults should also be analysed and proven.

igure 1 :

 1 Phase distribution of both 9-phase machines: SSWC (on the left) and ASWC (on the right)

-

  The machines are represented by equivalent-circuit models -Saturation effects are neglected -The neutrals of the 3 stars are isolated from each other -The machines are controlled by VSIs -The 'IGBT/Diode' block from Simscape is used to model the different switches of the VSIs -A Field-Oriented Control (FOC) with Maximum Torque Per Ampere (MTPA) strategy is applied -The switching frequency is: f P W M = 10 kHz -The simulations are done for the generator operating mode -The DC bus voltage is constant, V DC = 650 V

Figure 2 :

 2 Figure 2: Current-control loop of a 9-phase PMSM based on Field-Oriented Control or FOC technique: j ∈ {1, 2, 3} for the symmetrical machine and j ∈ {1, 2, 3, 4} for the asymmetrical one

Figure 3 :

 3 Figure 3: (Machine with a SSWC) 9 stator currents versus time in healthy (dashed lines) and faulty (solid lines, R add = 0.1Ω) states

Figure 4 :

 4 Figure 4: (Machine with a SSWC) Comparison of stator currents in (αj, βj), j ∈ {1, 2, 3} subspaces in healthy and faulty (R add = 0.1Ω) states: Iα 1 = f (t) and I β = f (Iα)

Figure 5 :Figure 6 :

 56 Figure 5: (Machine with an ASWC) 9 stator currents versus time in healthy (dashed lines) and faulty (solid lines, R add = 0.1Ω) states

Figure 7 :

 7 Figure 7: (Machine with an ASWC) Comparison of stator currents in (αj, βj) subspaces in healthy and faulty states with a variation of the severity of the fault R add = 0.1Ω then 2R add = 0.2Ω : I β = f (Iα)

Figure 8 :

 8 Figure 8: (Machine with an ASWC) Comparison of stator currents in (αj, βj) subspaces in healthy and faulty states with a variation of the location of the fault R add = 0.1Ω on phase 1, R add = 0.1Ω on phase 2, and R add = 0.1Ω on phases 1 and 2: I β = f (Iα)

Figure 9 :

 9 Figure 9: (Machine with an ASWC) Comparison of phase currents spectra in (dj, qj), j ∈ {1, 2, 4} subspaces in healthy and faulty (R add = 0.1Ω, phase 1) states

Table 1 :

 1 Harmonics mapping of the machine with a SSWC, healthy state

	Subspace	Harmonics
	(α 1 , β 1 ) 1, 5, 7, 11, etc.
	(α 2 , β 2 ) 1, 5, 7, 11, etc.
	(α 3 , β 3 ) 1, 5, 7, 11, etc.
	0 1 , 0 2 , 0 3	3, 9, 15, etc.

Table 2 :

 2 Harmonics mapping of the machine with an ASWC, healthy state

	Subspace	Harmonics
	(α 1 , β 1 ) 1, 17, 19, etc.
	(α 2 , β 2 ) 7, 11, 25, etc.
	(α 3 , β 3 ) 3, 15, 21, etc.
	(α 4 , β 4 ) 5, 13, 23, etc.
	0	9, 18, 27, etc.

Table 3 :

 3 Parameters of both machines

	Parameter	Machine with a SSWC Machine with an ASWC
	Slots/Poles	36/30	36/32
	Number of phases	9	9
	Phase winding resistance R s 57 mΩ	33 mΩ
	Rated voltage (V)	335	335
	Rated power (kW)	55	55
	Rotation speed (rpm)	200	187.5
	Frequency (Hz)	50	50
	Torque (N.m)	2780	2780


Mamadou Lamine Beye

Luc Bimmel

Anthony Bier

Jérémy Martin
email: jeremy.martin@cea.fr

Multilayer busbars for medium voltage ANPC converter dedicated to battery energy storage systems

Keywords:

kV SiC power modules », «High power», «Medium voltage», «Energy storage system».

Introduction

The energy transition leads to speed up the mass use of renewable energies, in particular solar and wind energy instead of fossil fuels [START_REF] Jaeger-Waldau | PV Status Report 2019, EUR 29938 EN[END_REF]. However, to ensure the balance between electricity production and consumption, energy storage systems are combined with renewable energies generators [START_REF] Komatsu | Development of Large-Capacity Converter for Battery Energy Storage Systems[END_REF]. These storage systems must also meet requirements in terms of efficiency and grid support. European Talent project was proposed to increase the voltage of BESS from conventional low voltage racks [START_REF] Burger | For Solar Energy systems -ISE[END_REF] 1 kV-1.4 kV to medium voltage racks (2×1 500 V with middle point grounded) achieving a high efficiency (> 99 %) and reducing the quantity of required power components raw materials. The ANPC converter operating in outer switching modulation mode (OSMM) presents the main advantage to use only small switching loops in inverter or rectifier mode allowing a switching speed increase [START_REF] Jiao | High Power High Frequency 3-level Neutral Point Clamped Power Conversion System[END_REF]. This paper focuses on the ANPC converter design. The DC/DC converter analysis will be down by the authors in furthers articles.

Figure. 1: Medium Voltage three-phase ANPC converter for battery energy storage systems

Several studies carried out to set up laminated busbars which have made it possible to reduce the parasitic inductance of busbars from a few hundred nH to around ten nH [START_REF] Zhang | Three-Level Two-Stage Decoupled Active NPC Converter With Si IGBT and SiC MOSFET[END_REF][START_REF] Popova | Stray inductance estimation with detailed model of the IGBT module[END_REF]. All of these papers use multilayer busbars that often requires algorithm to determine busbars sizing. In [START_REF] Gui | Methodology of Low Inductance Busbar Design for Three-Level Converters[END_REF], authors bring an approach to build multi layers layers busbars from two layers busbars once assembled show interesting stray inductor reduction. The methodology presented, is applicable to an ANPC conversion PEBB using 3.3 kV power modules.

A mixed Si/SiC ANPC leg and its control principle are presented Fig. 2. A ANPC leg controlled in outer switch modulation mode consists of using a 3.3 kV IGBT power module operating at grid frequency (50 Hz) connected to the leg output while two SiC modules operating at the switching frequency (…kHz) are connected to poles. The implementation of the ANPC leg is done by an external assembly of 3×2L modules. This allows the manufacturer to take benefits of a wide choice of two-level power modules easy to replace. In order to build a 3MVA converter two 1.5 MVA ANPC PEBB will be connected in parallel. A switching frequency of 7 kHz seems an acceptable compromise to target an efficiency > 99% including output filter losses and DC/DC converter losses. An electro-thermal model was built under PLECS [8] to estimate the power modules losses from power modules datasheet. From switching and conduction characteristics of IGBT and MOSFET power modules, the simulation results show an efficiency range between 99% -99.6%. In the section 1, the design methodology of the busbars will be described as well as a Q3D [9] based comparative studies between different layouts possibilities. Finally, the conclusion of this paper and the future work are presented in section 2.

Bus bar design and comparison for one phase Leg

As explained previously, the use of busbars is to ensure the connection between the power modules and the DC capacitor of the converter maximizing capacitive effect. Equations 1 to 4 give the characteristics of classic busbars formed by two conductive layers separated by an insulating layer [START_REF] Gui | Methodology of Low Inductance Busbar Design for Three-Level Converters[END_REF]. LC: is the parasitic inductance of each active layers, which depends on the length (l), the width (w), the thickness of the layers (e), the insulation layer thickness (d), the vacuum permeability (µ0) and relative permeability (µr), Minimum busbars area . Mc is the mutual inductance between the two conductive layers; it depends on the same parameters as LC. When the direction of current flow is the same in the two conductive layers the mutual inductance is positive, conversely it is negative. In order to reduce the total inductance of the bus bar (LT) a design with a reverse flow direction is recommended. Figure 4, presents the flowchart of the design methodology.

! µ # $ . ln & ' ( ( ) * √, , (1)

Efficiency (%) S ( M V A)

wit hout filt er wit h filt er

DC bus Capacitor Bank selection:

The capacitor bank (Cbat = Cbus1 + Cbus2) is calculated to provide the peak Irms current to the AC output of the converter. Its value can be determined by using equation 5 [START_REF] Wen | Analysis and Evaluation of DC-Link Capacitors for High-Power-Density Electric Vehicle Drive Systems[END_REF] where (fs) is the switching frequency, (∆> . ) is the ripple voltage of the DC bus, fixed to 5%.

? @A- B √ CD #EF_EHI CJ F C∆K LM (5)
Based on this criteria and the converter parameter (Figure 4), the minimum value of ? @A-is 1.2 mF.

ANPC Leg busbars layout comparisons

Figure 5 shows 3 layouts possibilities for inverter legs prototyping using two layer busbars and 3.3kV 2L power modules. Layout 1 consists of placing all power modules on a same line, so the capacitors are placed in front of MOS 1 and MOS 2 power modules (Figure 5b). This placement shows the advantage of using only one heatsink for the 3 power modules. However, placing the IGBT module (IGBT 1) between the negative layer (N) and positive layer (P) reduces the mutual inductance between these two layers. Layout 2 consists of placing the capacitors (Cbus1 and Cbus2) in the center and power modules on the top and the bottom (figure 5c). One of the consequences of this placement is the use of one heatsink per power module; this can increase the costs/complexity of the converter. This placement would also increase busbar's surface area of layers, which may result in increased costs and volume.

Layout 3 consists in placing the MOS 1 and MOS 2 on the same plane line while IGBT 1 is placed back and between the two modules so as to form a triangle (Figure 5d). Cbus1 and Cbus2 are placed in front of the SiC power modules and MOS 1 and MOS 2. This type of placement makes it possible to reduce surface area of bus bar layers and therefore the material costs. In addition to this, to bring the MOSFET modules closer together has a positive impact because this increases the mutual inductor. Regarding the cooling of the power modules, depending on the technology used, two heatsinks will be necessary because of the non-alignment of the power modules. Based on these comparative configurations, it can be concluded that layout 1 and 3 seems to be the most interesting configurations from a power density and cost point of view. However, taking into account the ANPC short switching loop inductance (Lb = L0 + Lp) presented in Table 1, layout 3 is more interesting. These parasitic inductance values were obtained by extracting the 3D model of these different bus bars under Ansys Q3D.

Experimental test

After these comparative studies, the paralleling 3 leg of inverter (500 kVA) is chosen in order to obtain 1.5 MVA ANPC power converter. The triangular layout placement of the power modules was chosen (figure 5d) since this solution shows the lowest parasitic inductance loop and busbar's volume (table 1a). Figure 6 presents the 3D model of the PEBB, the prototype ANPC converter phase leg as well as the test bench which made it possible to carry out preliminary tests presented in this part. .@SQ6 ) is determined using equation 6 as well as the electrical signals of Q2a (Id and Vds) measured during the turn off (Figure 8b). Lm is the parasitic of the SiC power module, LC is the equivalent parasitic inductance of Cbus1, Lscrew, is the parasitic inductance of the screw connection and Lshunt the shunt parasitic inductance. These parasitic inductances can be obtained from datasheet or can be calculated by estimation from the mechanical geometry.

NAOP 2 QRS - Q.OT .@SQ6 UV WXX WYZ#F[WW\ - ] (6)

Table 2: Comparison with other works

The value of the short switching parasitic inductance loop of the busbar 2 N obtained is listed in Table 2. It is compared with other work in the same field for ANPC or NPC converter prototyping. The parasitic inductance obtained is lower than the case [START_REF] Wang | Development of a compact 750KVA threephase NPC three-level universal inverter module with specifically designed busbar[END_REF] with a NPC converter, the dimensions of the converter are larger (1 000 × 700 × 500 mm) compared to the Talent converter. However, the study in [START_REF] Gui | Methodology of Low Inductance Busbar Design for Three-Level Converters[END_REF] shows a smaller parasitic inductance. This example uses different kinds of power modules (HT -3 000 series), with a lower DC busbar voltage (1 kV) than the one in Talent project (3.6 kV).

Conclusion

In this paper, a busbar design comparison for a high power and medium voltage ANPC converter has been presented. To validate the methodology, a comparative study is carried out on several types of placement for the power modules around the busbars and the capacitors. Simulation results show that the triangular configuration of power modules reduces the most parasitic inductance of the switching loops. The analysis of the switching waveform obtained by the experimental results shows a total parasitic inductor of 14.5 nH lower as the simulated value (20.68 nH). Next investigations will focuse on the SiC power modules switching losses measurements. Then gate resistors will be adjusted in order to increase the MOSFET switching speed in order to benefits the selected busbars and reduce the switching losses.

Fig. 2 :

 2 Fig. 2: Principle of operation of an ANPC leg (OSMM)

Fig. 3 :

 3 Fig. 3: a) Half-Bridge switching energy characteristics, b) Free-Wheeling diode forward characteristics (MOSFET), c) Free-Wheeling diode forward characteristics (IGBT), d) Efficiency curves for 3 MVA three-phase ANPC converter (7 kHz)

  2. 10 . . 2.303 log 0.5 0.2235

Figure. 4 :

 4 Figure. 4: Busbars algorithm design and ANPC converter characteristic

Figure. 5 : 2 :

 52 Figure. 5: Layout presentations, a) ANPC leg, b) Layout 1: line placement of power modules, c) Layout 2: Opposition placement of MOS 1 and MOS 2, d) Layout 3: triangular placement of power modules

Figure. 6 :

 6 Figure. 6: Inverter leg of the PEBB, a) 3D solid edge model, b) PEBB inverter leg prototype and c) the test bench of the DPT To evaluate the parasitic inductance of the bar bus, double pulse tests (DPT) are carried out. The electrical schematic of the DPT is shown in Figure 8a. The total inductance of the ANPC short switching loop inductance NAOP 2 N QRS -Q.OT

Figure. 7 :

 7 Figure. 7: Evaluation parasitic inductance ( N and 2 ) using Double Pulse Test of the PEBB, a) electrical scheme of the DPT, b) Vds and Id of Q2a using obtained by DPT

Table 1 : Parasitic inductances comparison, inverter leg busbars

 1

Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 864459.