Introduction:

When the first full sequences of the genomes of RNA viruses became available, it was noticed that some plant viruses had features very similar to those of animal, including human, viruses now classified within the family Picornaviridae [START_REF] Argos | Similarity in gene organization and homology between proteins of animal picornaviruses and a plant comovirus suggest common ancestry of these virus families[END_REF][START_REF] Franssen | Homologous sequences in nonstructural proteins from cowpea mosaic virus and picornaviruses[END_REF][START_REF] Goldbach | Molecular evolution of plant RNA viruses[END_REF][START_REF] Goldbach | Genome similarities between plant and animal RNA viruses[END_REF][START_REF] Goldbach | Evolution of plus-strand RNA viruses[END_REF]): (i) icosahedral particles about 30nm in diameter packaging a single stranded RNA genome; (ii) the genome 3' polyadenylated and linked to a 5'-terminal protein, VPg; (iii) proteins expressed as polyprotein precursor(s), processed to mature proteins by one or more a virus-encoded proteinases; (iv) similarities in the sequences of the domains encoding NTPase and putative helicase (Hel), proteinase (Pro) and polymerase (Pol) domains, and (v) with these domains arranged in the same order, Hel-(VPg)-Pro-Pol, along the polyprotein. Two main differences existed between these groups of viruses: (i) Some plant viruses were known to be bipartite, i.e. the genome is divided into two parts, each carried by a separate virus particle (Le Gall et al., 2005Gall et al., , 2005Gall et al., , 2005)), whereas animal viruses are invariably monopartite. (ii) Their capsids were composed of either one capsid protein (CP; about 55kDa in typical nepoviruses) or two smaller CPs (35 and 20 kDa in comoviruses and fabaviruses) subunits, whereas the picornaviruses had 3 major CPs (and sometimes an additional smaller one). In 3-D structure, however, the capsids of comoviruses and picornaviruses proved to be strikingly similar, since both were assembled from three distantly related jelly-roll domains arranged with pseudo T=3 (P3) icosahedral symmetry [START_REF] Hogle | Three-dimensional structure of poliovirus at 2.9 A resolution[END_REF][START_REF] Rossmann | Structure of a human common cold virus and functional relationship to other picornaviruses[END_REF][START_REF] Chen | Protein-RNA interactions in an icosahedral virus at 3.0 A resolution[END_REF][START_REF] Chandrasekar | The structure of tobacco ringspot virus: a link in the evolution of icosahedral capsids in the picornavirus superfamily[END_REF][START_REF] Tate | The crystal structure of cricket paralysis virus: the first view of a new virus family[END_REF] (Liljas et al. 59-84;[START_REF] Rossmann | Icosahedral RNA virus structure[END_REF]. This similarity was all the more remarkable because such a structure had never previously been observed.

Further complete genome sequences have revealed several more families that share many, or all, of these features. This has led to the now commonly used concept of "picorna-like viruses" and picorna-like "superfamily/supergroup" [START_REF] Franssen | Homologous sequences in nonstructural proteins from cowpea mosaic virus and picornaviruses[END_REF][START_REF] Goldbach | Molecular evolution of plant RNA viruses[END_REF][START_REF] Goldbach | Genome similarities between plant and animal RNA viruses[END_REF][START_REF] Goldbach | Evolution of plus-strand RNA viruses[END_REF]Gorbalenya and Koonin, 1993;[START_REF] Koonin | Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences[END_REF]. Figure 1 shows the genomic organisations of all the picorna-like viruses for which genome sequences are available. The list includes, not only the proposed members of the new order, but also more distantly related picorna-like families that, for reasons discussed below, are considered too divergent to merit inclusion. Until now no formal proposal has been made to the ICTV to create a higher order taxon. This proposal is to create an Order, named the Picornavirales, to comprise the following taxonomic groupings: Picornaviridae, Dicistroviridae, Marnaviridae, Sequiviridae, Comoviridae and the unassigned genera Iflavirus, Cheravirus and Sadwavirus.

Invariant properties of the proposed order:

It should be noted that not every member of the Picornavirales has been demonstrated to conform to all the taxonomic criteria listed below; for example, to confirm a jelly-roll protein structure "directly" would require the crystal structure to be determined. Nevertheless, direct evidence is available for a wide range of virus types, enabling such properties to be inferred in other cases from comparative sequence analysis, and other, evidence.

• Each virus particle comprises a non-enveloped capsid containing a single species of genomic RNA.

• The capsid, ~25-30 nM in diameter, is made up of 60 identical protomers, each protomer consisting of three similarly sized and distantly related domains each folded into an 8-stranded beta-barrel (jelly-roll fold). The capsid possesses T=1 icosahedral symmetry relative to protomers although the arrangement of the 3x60=180 constituent domains approximates to that of a T=3 icosahedron. The resulting pseudo-T3 (P3) symmetry is unique to members of this taxon. • The 5' terminus of the genome, where this has been identified, is covalently attached to a 3-4 kDa virusencoded protein (VPg). • The genomic RNA also serves as the virus messenger RNA (mRNA); i.e. there are no subgenomic mRNAs. • Each virus-encoded open reading frame (ORF) encoding multiple domains is expressed as a single polyprotein which is processed to the mature virus proteins exclusively by virus-encoded proteinase(s). • Conserved order of the non-structural protein domains (defined below) in polyprotein, Hel-VPg-Pro-Pol.

• Conserved order of the three structural domains in polyprotein, their locations in the capsid being as follows: the N-terminal and middle domains ("VP2" and "VP3", respectively, using picornavirus nomenclature) are located either side of the 3-fold icosahedral axis, the C-terminal domain ("VP1") at the 5-fold. Other properties which, while not invariant, are characteristic of the order:

• Each virus particle typically contains a single molecule of genomic RNA, although in some plant viruses with a bipartite genome two molecules of the small genome component can be encapsidated in a single virion.

• The genomic RNA is typically 3' polyadenylated, the sole exception being the genus Sequivirus (family Sequiviridae), where this feature appears to be absent [START_REF] Turnbull-Ross | Sequence analysis of the parsnip yellow fleck virus polyprotein: evidence of affinities with picornaviruses[END_REF]. • Viruses are typically monopartite (i.e. a single particle is infectious), the sole exception being several taxa of plant viruses (Comoviridae, Cheravirus, Sadwavirus), which have a bipartite genome. • Each virus RNA molecule normally encodes a single multidomain ORF, the sole exception being the Dicistroviridae where, as the name suggests, each molecule encodes two ORFs. • Individual domains of the capsid are normally cleaved proteolytically from each other to generate the three mature proteins typical of the Picornavirales. In the case of the family Comoviridae and (by analogy) probably the unassigned genus Sadwavirus, however, the processing of the CP precursor is not as extensive. This results in the jelly-roll domains remained fused in a single CP subunit: two in the large CP subunit (genera Comovirus and Fabavirus within the family Comoviridae; probably also unassigned genus Sadwavirus [START_REF] Iwanami | Nucleotide sequences and taxonomy of Satsuma dwarf virus[END_REF] or three in the single CP subunit (genus Nepovirus). • the Hel, VPg, Pro, and Pol domains of the eighth groups that make up the Picornavirales can be characterized as follows:

o Hel: a superfamily III helicase domain [START_REF] Gorbalenya | A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses[END_REF], with an "A" motif (GxxGxGK(S/T)), followed about 40 amino-acids downstream by a "B" motif (DD), followed about 30 amino-acids downstream by a "C" motif (KGxxxxSxxxxx(S/T)(S/T)N). This domain occupies the central part of 2C protein in the Picornaviridae and 2C-like protein in other viruses of the Picornavirales and it is involved in NTP-binding and hydrolysis (Mirzayan and Wimmer 176-87;Pfister and Wimmer 1611-19) o VPg: a 3-4 kDa VPg with little or no characteristic sequence features except for the presence near its N-terminus of an amino-acid with a free hydroxyl group to which the RNA 5' end is covalently linked by a phosphodiester bond o Between Hel and VPg: an hydrophobic domain which may, or may not, be cleaved proteolytically from the Hel moiety o Pro: a proteinase resembling trypsin-like serine proteases in structure and having substrate specificity resembling that of the prototype picornavirus 3C proteinase (3C-like proteinase) . It contains a catalytic triad of histidine, aspartate/glutamate and typically cysteine, in place of the canonical serine of "serine" proteases, which serves as the catalytic nucleophile [START_REF] Gorbalenya | Poliovirus-encoded proteinase 3C: a possible evolutionary link between cellular serine and cysteine proteinase families[END_REF][START_REF] Bazan | Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications[END_REF][START_REF] Gorbalenya | Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold[END_REF][START_REF] Allaire | Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases[END_REF][START_REF] Matthews | Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein[END_REF]. Interestingly, the only member of the family Marnaviridae to have been sequenced so far [START_REF] Lang | Genome sequence and characterization of a virus (HaRNAV) related to picorna-like viruses that infects the marine toxic bloom-forming alga Heterosigma akashiwo[END_REF] appears to possess a serine at this position (AEG, unpublished observation). The catalytic cysteine/serine is located near the C-terminus of the Pro domain and is generally followed 10-20 amino-acids downstream by a GΦH motif (where Φ is a polar residue) that appears important for substrate specificity [START_REF] Bazan | Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications[END_REF][START_REF] Cheah | Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases[END_REF][START_REF] Matthews | Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein[END_REF][START_REF] Hemmer | Functional characterization of the proteolytic activity of the tomato black ring nepovirus RNA-1-encoded polyprotein[END_REF][START_REF] Miyashita | Mutational analysis of the putative substrate-binding site of 3C proteinase of coxsackievirus B3[END_REF]. o Pol: a Type I Pol domain [START_REF] Kamer | Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses[END_REF][START_REF] Koonin | The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses[END_REF] with eight conserved motifs, motifs I (KxE), V (PSGΦΦΦTΦΦΦN(S/T)) and VI (YGDD) being the most conserved ones. The motifs V and VI are part of the catalytic center of this enzyme (Hansen, Long, and Schultz 1109-22) Phylogenetic evidence:

The taxa listed above as candidate members of the Order Picornavirales appear to form a monophyletic lineage when the sequence similarity of the non-structural proteins is used to reconstruct in a hierarchical clustering analysis (Figures 3 and5).

Divergent picorna-like viruses, not assigned to the order Picornavirales:

The level of structural, organisational and sequence divergence in the families Caliciviridae, Potyviridae and Hypoviridae were judged too high for them to be included in the order Picornavirales, for the following reasons: • The family Caliciviridae have several characteristics atypical of the Picornavirales, as follows: (i) Genome encodes only a single jelly-roll domain. As a result, the icosahedral capsid of 180 jelly rolls is only superficially picorna-like, it exhibits true T3 symmetry (i.e. the 180 domains are identical to each other), rather than P3 symmetry. (ii) Each jelly-roll domain is linked to second, outwardly protruding, domain that is not found among the members of the Picornavirales. (iii) A major divergence can be seen in their strategy of gene expression, which involves the use of sub-genomic mRNAs. For these reasons, it was judged appropriate, on balance, to adopt a somewhat restrictive set of taxonomic criteria that exclude the Caliciviridae.

• The family Potyviridae has the following major deviations (i) the members employ a single capsid protein unrelated to the jelly-roll domain that forms capsid exhibiting helical, rather than icosahedral, symmetry ; [START_REF] Shukla | The Potyviridae[END_REF] and (ii) they have a Hel domain of superfamily II rather than III found in the Picornavirales viruses ). • The members of the family Hypoviridae have the following specifics: they lack virions and 3C-like proteinase, have an atypical (double-stranded) RNA genome and a Hel domain of superfamily II, like the Potyviridae members with whom they phylogenically cluster.

Discussion:

The picornavirus supergroup comprises the eleven virus types listed in Fig 1, including three distantly related families, the Caliciviridae, Potyviridae and Hypoviridae, which differ markedly from the other eight. It should also be noted that there are other virus families, e.g. Sobemoviridae, Astroviridae and the order Nidovirales, which share certain properties with the Picornavirales viruses. The differences between these viruses and the Picornavirales are especially recurrent in relation to capsid proteins and virions (if any) they made. The caliciviruses appear the most picornavirus-like but, even in this case, there are major differences in the capsid organization, as well as the genome expression strategy, that separate this family from the other eight forming the Picornavirales (see above). As for the Potyviridae and Hypoviridae, these have deviated even more profoundly, including the lack of the icosahedral capsid. Despite these clear differences between the viruses included and not in the Order, we acknowledge that the choice of taxa to be assigned to the order Picornavirales fully depends on the criteria used. In our proposal, we have designed criteria using most conserved properties that characterize the prototype Picornaviridae and found that at least seven other virus groups pass this check. To include only one extra group, the closest Caliciviridae, in the Order, as was proposed before [START_REF] Koonin | Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences[END_REF] two major criteria related to the virion architecture and genome expression would have needed to relax. We feel that this revision of the criteria would significantly "dilute" the common basis of the Order and, thus, it may not be justified for a relatively minor order expansion. We hope that the creation of the new Order, as defined in this proposal, will facilitate research on picorna-like viruses and knowledge dissemination. We also hope that this proposal will trigger further insights that in due course may lead to an appropriate higher order classification for other viruses of the supergroup that left outside the Picornavirales.

Origin of the proposed order name

• Derived from the name of the family Picornaviridae, to reflect the common expression "picorna-like virus".

•

The etymology of "Picorna" derives from "pico" (small) and "RNA", to recall that these are small RNA viruses. This is a common property of all viruses in the proposed order. [START_REF] Koonin | Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences[END_REF]) and the corresponding regions from the other viruses included. The tree is based on maximum likelihood distances generated with TREE-PUZZLE. The sequence from the Carnation mottle virus (CarMV) was used as an outgroup. Support values based on 10,000 puzzling steps are shown above the branches. Bootstrap values (based on 1,000 replicates) for branches that are supported by >50% by neighbor-joining analysis are labeled below the branches (a dash indicates there was no corresponding branch in the neighbor-joining tree). The maximum likelihood scale bar is shown.

Figure 1 :

 1 Figure 1: Genomic organization of the picorna-like viruses. The abbreviations are Hel: Helicase; Pro: Proteinase; Pol: Polymerase; CP: Capsid Protein; CP: Virion Protein; MP: Movement Protein. The figure includes, not only the members of the proposed order Pixcornaviraless (boxed), but also the, more divergent, families not currently proposed for inclusion.

Figure 2 .

 2 Figure 2. Unrooted phenogram showing the relationships of the CrPV-like viruses to representatives of the Picornaviridae, Comoviridae, Sequiviridae and the floating genus Iflavirus. The phenogram was constructed from an amino acid similarity matrix of the replicase (RdRP) region of the non-structural proteins using the neighbour-joining method. A bootstrap analysis was performed and the percentage values are indicated at the branhing points. Viruses other than dicistroviruses included in the analysis, abbreviation ( ) and accession numbers [ ] are; Cowpea severe mosaic virus (CPSMV) [M83830], Encephalomyocarditis virus (EMCV) [M81861], Foot-and-mouth disease virus (FMDV) [X00871], Hepatitis A virus (HAV) [M14707], Infectious flacherie virus (IFV) [AB000906], Parsnip yellow fleck virus (PYFV) [D14066], Perina nuda virus (PnV) [AF323747], Poliovirus (PV) [J02281], Rice tungro spherical virus (RTSV) [M95497], Sacbrood virus (SBV) [AF469603]. Branch lengths are drawn to scale.

Figure

  Figure taken from the VIIIth ICTV report.

Figure 3 :

 3 Figure 3: Phylogenetic analysis of picorna-like RdRp domain protein sequences. CLUSTAL_X alignments were done with residues 1362-1619 of the HaRNAV polyprotein that represent the conserved regions I-VIII (defined in[START_REF] Koonin | Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences[END_REF]) and the corresponding regions from the other viruses included. The tree is based on maximum likelihood distances generated with TREE-PUZZLE. The sequence from the Carnation mottle virus (CarMV) was used as an outgroup. Support values based on 10,000 puzzling steps are shown above the branches. Bootstrap values (based on 1,000 replicates) for branches that are supported by >50% by neighbor-joining analysis are labeled below the branches (a dash indicates there was no corresponding branch in the neighbor-joining tree). The maximum likelihood scale bar is shown.

Figure

  Figure taken from the VIIIth ICTV report.

Figure 4 .

 4 Figure 4. Unrooted phenogram showing the relationships of the RdRps of the Nidovirales lineages with virus of the families of the "Picornavirus-like" supergroup, Tetraviridae and Birnaviridae. The most conserved part of RdRps from representative viruses in the Picornaviridae, Dicistroviridae, Sequiviridae, Comoviridae, Caliciviridae, Potyviridae, Coronaviridae, Ronivirida, Arteriviridae, Birnaviridae, Tetraviridae and unclassified insect viruses was aligned. The RdRps of Thosea asigna virus (TaV), Euprosterna elaeasa virus (EeV) and the birnaviruses were converted into the canonical ABC form before the analysis. Using an extended, gap-free version of the alignment containing 332 informative characters, an unrooted neighbor-joining tree was inferred by the ClustalX1.81 program. All bifurcations with support in > 700 out of 1000 bootstraps are indicated. Different groups of viruses are highlighted. Note that the relative positions of members of the families Arteriviridae and Roniviridae within the Nidovirales are not resolved in this tree. Virus families and groups, viruses included in the analysis, abbreviations ( ) and the NCBI protein (unless other specified) IDs [ ] are as follows: Picornaviridae: Human poliovirus type 3 Leon strain (PV-3L) [130503] and parechovirus 1 (HpeV-1) [6174922]; Iflavirus: Infectious flacherie virus (InFV) [3025415] and Unclassified insect viruses: Acyrthosiphon pisum virus (APV) [7520835]; Dicistroviridae: Drosophila C virus (DCV) [2388673]; Sequiviridae: Rice tungro spherical virus (RTSV) [9627951] and Parsnip yellow fleck virus (PYFV) [464431]; Comoviridae: Cowpea severe mosaic virus (CPSMV) [549316] and Tobacco ringspot virus (TRSV) [1255221]; Caliciviridae: Feline calicivirus F9 (FCV-F9) [130538] and Lordsdale virus (LORDV) [1709710]; Potyviridae: Tobacco vein mottling virus (TVMV) [8247947] and Barley mild mosaic virus (BaMMV) [1905770]; Coronaviridae: Human coronavirus 229E (HCoV) [12175747] and Berne torovirus (BEV) [94017]; Arteriviridae: Equine arteritis virus (EAV) [14583262]; Roniviridae: Gill-associated virus (GAV) [9082018]; Tetraviridae: Thosea asigna virus (TaV) [AF82930; nt sequence] and Euprosterna elaeasa virus (EeV) [AF461742; nt sequence]; Birnaviridae: Infectious pancreatic necrosis virus (IPNV) [133634] and Infectious bursal disease virus (IBDV) [1296811]. (Tree was modified fromGorbalenya et al., (2002), with permission).

Figure

  Figure taken from the VIIIth ICTV report.

Figure 5 :

 5 Figure 5: Unrooted phenogram derived from the RdRp domain of the viral non-structural proteins showing the relationships of representative picornaviruses, sequiviruses, dicistroviruses and the three members of the genus Iflavirus (IFV, PnV and SBV). Taxa used (with sequence accession numbers shown in brackets [ ]) were Cowpea severe mosaic virus (CPSMV) [M83830], Drosophila C virus (DCV) [AF014388], Encephalomyocarditis virus (EMCV) [M81861], Foot-and-mouth disease virus (FMDV) [X00871], Hepatitis A virus (HAV) [M14707], Poliovirus (PV) [J02281], Parsnip yellow fleck virus (PYFV) [D14066], Plautia stali intestine virus (PSIV), Rhopalosiphum padi virus (RhPV) [AF022937], and Rice tungro spherical virus (RTSV) [M95497]. A bootstrap analysis was performed and values obtained are shown next to the branching points. Branch lengths are proportional to distance.

FigureN

  Figure taken from the VIIIth ICTV report.

Figure 6 :

 6 Figure6: Hierarchical clustering based on the region between the Pro (CG) and the Pol (GDD) sequence motifs. The sequences were aligned using Clustal using the Neighbor-Joining approach, and p-distances are represented. For each sequence, the GenBank accession number, the virus acronym and a shortened description of the taxonomical status are given directly in the dendrogram. The outgroup used is a chimeric sequence reconstituted from the bovine trypsin C-terminus and the Tobacco mosaic tobamovirus RdRp N-terminus.


Sylvain Dupont

Yves Brunet

Mesoscale dispersal of maize pollen and implications for gene flow

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


Jérôme Brunet
email: brunet@lasmea.univ-bpclermont.fr

Alain Pauly

Christelle Varenne

Bernard Lauron

B Lauron Lasmea

On-board phthalocyanine gas sensor microsystem dedicated to the monitoring of oxidising gases level in passenger compartments

Keywords: on-board sensor, gas sensor microsystem, copper phthalocyanine, nitrogen dioxide, pollutant monitoring

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

Introduction

Besides the reduction of gases responsible for the greenhouse effect, the air quality improvement in industrialized cities by the decrease in urban pollutant concentrations is also a worldwide preoccupation. This growing awareness is enhanced by the fact that atmospheric pollution is not only an environmental problem but induces serious health hazards on humans, too. In urban context, all city-dwellers suffer from health problems induced by pollutants.

Nevertheless, we must distinguish two types of exposure: (1) the nonoccupational exposure which mainly affects pedestrians, cyclists, motorcyclists, car drivers, commuters, and street residents [START_REF] Chan | A study of bus commuter and pedestrian exposure to traffic air pollution in Hong Kong[END_REF][START_REF] Barrefors | Exposure to volatile hydrocarbons in commuter trains and diesels buses[END_REF][START_REF] Kingham | Assessment of exposure to trafficrelated fumes during the journey to work[END_REF][START_REF] Wijnen | The exposure of cyclists, car drivers and pedestrians to traffic-related air pollutants[END_REF][START_REF] Johnson | Recent advances in the estimation of population exposure to mobile source pollutants[END_REF][START_REF] Chan | The effect of commuting microenvironment on commuter exposures to vehicular emission in Hong Kong[END_REF][START_REF] Gómez-Perales | Bus, minibus, metro intercomparison of commuter's exposure to air pollution in Mexico City[END_REF][START_REF] Chan | Commuter exposures and indoor-outdoor relationships of carbon oxides in buses in Hong Kong[END_REF][START_REF] Fernandez-Bremauntz | Exposure of commuters to carbon monoxide in Mexico City -I. Measurements of in-vehicle concentrations[END_REF], and (2) the occupational exposure which concerns taxi, bus and truck drivers but also postmen or city sweepers [START_REF] Chan | Driver exposure to Volatile Organic Compounds, CO, Ozone and NO 2 under different driving conditions[END_REF][START_REF] Hertel | Exposure of bus drivers and postmen to air pollution from traffic in their working environment[END_REF][START_REF] Limasset | Assessment of bus driver's exposure to the pollutants of urban traffic[END_REF][START_REF] Son | Estimation of occupational and nonoccupational nitrogen dioxide exposure for Korean taxi drivers using microenvironmental model[END_REF][START_REF] Saenghirunvattana | Increasing carbon monoxide blood levels in Bangkok bus drivers[END_REF]. Let's notice that these professional groups are especially exposed because they are affected by urban pollution in nonoccupational context, too. Amongst all pollutants, nitrogen dioxide is a major problem in urban areas. This primary pollutant is highly concentrated in downtown and contributes to the formation of another toxic gas: ozone. Moreover, this gaseous species has very harmful effects on human health. Indeed, as low as 15 ppb, nasal irritations, eyes irritations and cough are associated to NO 2 exposure. For 30 ppb, hyperactivity of airway muscles appears. Above 80 ppb, some researches report an increased incidence of respiratory infections and sore throats [START_REF]Car Air Pollution: The Hidden Threat to Automobile Drivers: Report No. 4, An Assessment of the Air Quality Inside Automobile Passenger Compartments[END_REF]. The guidelines for nitrogen dioxide given by the World Health Organization (WHO) are 100 ppb for one hour and 75 ppb per day. In urban environment, these values can be greatly exceeded especially during unusual driving condition periods. So, for public protection, this gas needs to be continuously monitored.

To inform people about the quality of the air they breathe and to protect them from pollution episodes, local networks of air quality control have been set up like the AtMO network, in France. Gaseous and particular pollutants are monitored continuously by commercial gas analyzers implemented in fixed and air-conditioned stations at rural and urban places. These stations not being located on the roads, the concentrations measured for primary pollutants such as NO 2 are often lower than those really present in the traffic [START_REF] Chan | A study of bus commuter and pedestrian exposure to traffic air pollution in Hong Kong[END_REF][START_REF] Johnson | Recent advances in the estimation of population exposure to mobile source pollutants[END_REF][START_REF] Chan | The effect of commuting microenvironment on commuter exposures to vehicular emission in Hong Kong[END_REF][START_REF] Chan | Driver exposure to Volatile Organic Compounds, CO, Ozone and NO 2 under different driving conditions[END_REF][START_REF] Limasset | Assessment of bus driver's exposure to the pollutants of urban traffic[END_REF][START_REF] Son | Estimation of occupational and nonoccupational nitrogen dioxide exposure for Korean taxi drivers using microenvironmental model[END_REF][START_REF] Palmgren | Effects of reduction of NO X on the NO 2 levels in urban streets[END_REF]. So, to avoid any underestimation of the real exposure of commuters or drivers, it is judicious and more efficient to realize in-vehicle measurements.

Most studies dedicated to the determination of occupational and nonoccupational exposures to traffic-related pollutants such as NO X , CO, VOCs or hydrocarbons have been realized by means of passive samplers [START_REF] Wijnen | The exposure of cyclists, car drivers and pedestrians to traffic-related air pollutants[END_REF][START_REF] Chan | The effect of commuting microenvironment on commuter exposures to vehicular emission in Hong Kong[END_REF][START_REF] Hertel | Exposure of bus drivers and postmen to air pollution from traffic in their working environment[END_REF][START_REF] Son | Estimation of occupational and nonoccupational nitrogen dioxide exposure for Korean taxi drivers using microenvironmental model[END_REF] or commercial spectroscopic gas analyzers [8,10,12,]. Passive samplers evaluate gas concentrations for several hours or days of exposure and are not able to deliver real time results. So, they are not appropriated for permanent monitoring of pollutants. As for gas analyzers, their high dimensions, low mechanical vibration tolerance, high energy consumption and necessity to be maintained at wellcontrolled temperature make them not suitable as on-board pollutant monitoring systems.

Because of their low dimensions, low consumption powers, high level of mobility, electric output signal easily exploited by on-board electronic systems and ability to deliver real time response, gas microsensors are perfectly suitable for such an application. This article deals with an on-board and self-contained gas sensor microsystem for the continuous and real time monitoring of NO 2 concentration in passenger compartments.

Results under controlled experimental conditions illustrate the performance of our prototype.

Measurements in an urban bus under real driving conditions certify the possibility to use such a microsystem for this purpose.

Experimental

Specific methodology of measurement

The monitoring of oxidising gases in passenger compartments requires sensors with the best sensing performances, close to ideal ones, such as short response times, high resolution, low threshold, good reproducibility of results, and selectivity. To obtain such performances with phthalocyanine gas sensor devices, we have developed an original methodology of measurement. This method allows us to improve the reproducibility of results obtained and to take no notice of the too long response time these sensors suffer. This methodology optimized for ozone monitoring is detailed in a previous paper [START_REF] Brunet | A methodology of measurements for phthalocyanine gas sensors dedicated to the detection of ozone and nitrogen dioxide[END_REF]. To summarize, the principle of this methodology is based on the quantification of gas concentration by the kinetics of sensor response during short times of exposure, and on cyclic recalibration of sensor by a thermal cleaning process in purified air.

Figure 1 is a schematic representation of temperature, flow and conductivity evolutions during one cycle of our methodology. During the first step, the sensor is maintained at high temperature in clean air. The conductivity firstly increases due to the semiconductor effect and secondly decreases due to the thermal desorption of gaseous species already chemisorbed during the previous cycle. At the end of this step, the sensitive layer is "regenerated". During the second step, the sensor is still maintained in pure air but at lower working temperature.

The conductivity decreases due to the annihilation of intrinsic charge carriers by the negative temperature gradient. At the end of this second stage, the initial conductivity σ i is stabilized and measured. The third and last step corresponds to the exposure of the sensor to polluted air for a short time at low working temperature. Sensor conductivity slowly increases due to the creation of extrinsic charge carriers in the sensitive material by charge transfer between the oxidising chemisorbed gaseous molecules and the reduced phthalocyanine molecular units.

Interaction mechanisms between gases and this molecular semiconductor will be explained later. At the end of the cycle, the final conductivity under gas σ f is measured and a new cycle begins. The sensor response is thus defined as (σ f -σ i ). Obviously, responses and even performances are a function of delays and temperatures of each cycle. Optimization of all these working conditions will be discussed in the section 3.2.

Gas sensor microsystem

The complete microsystem constituted by the sensitive element, the fluid circuit and the electronic peripheral devices is represented by Fig. 2. The sensor is constituted by a thin film of copper phthalocyanine, this organic molecular semiconductor being judicious for this application because of its insensitivity to reducing gases which induces a partial selectivity to the structure. The layers are realized by thermal sublimation under secondary vacuum (P=10 -6 mbar) of copper phthalocyanine powder commercialized by Kodak. An automatic deposition control system (Model ADS-220 from Veeco) associated with a quartz oscillator regulates the deposition rate and controls the deposited thickness. This layer thickness is 300 nm and the deposition rate is maintained at 0.2 nm/s to achieve homogenous layers. During the process, the substrates are maintained at room temperature and no consecutive post-deposition thermal annealing is realized. Thus, the CuPc layers obtained are amorphous and exhibit better sensing characteristics than crystalline ones [START_REF] Collins | Thin film sensors : the role of defects and impurity sites in controlling sensor response and selectivity[END_REF][START_REF] Masui | Gas sensitive properties of copper phthalocyanine thin films[END_REF][START_REF] Hsieh | Response characteristics of lead phthalocyanine gas sensor : effects of film thickness and crystal morphology[END_REF][START_REF] Lee | Effects of heat annealing on the film characteristics and gas sensing properties of substituted and un-substituted copper phthalocyanine films[END_REF]. Sensitive thin layers are deposited onto interdigitated electrodes with accurate dimensions, screen-printed on the upper side of alumina substrates to determine their electronic conductivity. On the lower side, a screenprinted platinum resistor, with a predefined thermal coefficient, is used to regulate the sensor temperature during experiments. The dimensions of the sensing element are 3 mm × 5 mm. The sensor structure is polarized under 5 V d.c. and a data acquisition system (Model 2000 or 2700, Keithley) records a voltage as a function of the electric current passing through the sensitive layer, image itself of copper phthalocyanine conductivity. The conversion of current into voltage is realized by means of an electronic converter with adjustable gain. The structure temperature is controlled by a temperature regulator which can commute between two values by an external control signal.

To avoid gas adsorption on the inner walls, the measuring cell and the fluid circuit are realized in PTFE. Moreover, an exhaust pump sets the flow to 750 ml/min and is located downstream from the cell to avoid adding interfering species in the sensor environment. The commutation between polluted air and purified air is made by means of a PTFE solenoid valve, the purified air being obtained by an air flow through an active charcoal cartridge. At the sampling input, a dust filter can be added especially during experimental tests under real conditions. The solenoid valve, the temperature regulator and the data acquisition system are synchronously driven by a self-contained microcontroller unit developed at laboratory. Moreover, as microsystem performances are related to delays of each step and period, these parameters can be modified by experimenters in this unit. The best working conditions are argued in the following.

Infrastructure for tests under real conditions

After calibration under artificially polluted atmosphere in laboratory, this microsystem has been put in a bus of the network of Clermont-Ferrand (France) to realize on-board measurements and thus to validate this prototype. The experimental vehicle was a Heuliez GX 317 Compressed Natural Gas bus. The bus route is from the suburb of Clermont-Ferrand to the downtown, near the railway station district. The length of its route is approximately 7.6 km and the common delay of the journey for normal traffic conditions is approximately 40 min. The microsystem has been placed on the seat just behind the bus driver and the sampling input has been maintained at the respiratory level of passengers. During experiment, only the bus driver and four experimenters were present in the passenger compartment. To be in the same conditions as usual, the bus opens, waits for a few seconds and then closes its gates in the front of each bus stop. After each measurement, the global position of the bus is noted to correlate the concentration of pollutants deducted from microsystem response with traffic conditions and the nearest fixed station of air quality control.

Results

Target gas and interfering species: preliminary study

Nitrogen dioxide is a major pollutant of urban atmosphere but coexists with a few other gaseous species such as nitrogen monoxide, carbon monoxide, sulphur dioxide, ozone, volatile organic compounds (VOCs) and heavy metals. So, efficient NO 2 monitoring requires drastically selective gas sensors. Copper phthalocyanine is a molecule well-known to be highly reactive with oxidising gases and insensitive to reducing ones. So, phthalocyanine thin film conductivity is not very modified by carbon monoxide (CO) and nitrogen monoxide (NO). With sulphur dioxide (SO 2 ), none of previously published work has reported even a low reactivity with CuPc. Moreover, the development of electronuclear energy and the decrease in fuel sulphur rate have greatly contributed to the decrease in SO 2 concentration in atmosphere since several years, levels becoming very low. Electronic charge transfer between phthalocyanine and heavy metals being impossible, no conductivity variation can be correlated with heavy metals concentration variations. For VOCs and hydrocarbons, no response correlated with these compounds has been observed during previous experiments realized on phthalocyanine gas sensors. So, in our experimental context, ozone can be considered as the main interfering gas. is nine times higher than this relative to ozone. Ozone concentration remains very small (1 to 2 ppb) whatever the outdoor level. At the same time, NO 2 concentration depends from outdoor level. Moreover, for the same outdoor levels, the quantity of NO 2 in vehicle during the 10 min of air admission is higher than for O 3 . Ozone is quickly and completely decomposed in passenger compartments whereas nitrogen dioxide remains the predominant oxidising gas. Such a phenomenon has been already quantified [START_REF] Johnson | Recent advances in the estimation of population exposure to mobile source pollutants[END_REF][START_REF] Chan | Driver exposure to Volatile Organic Compounds, CO, Ozone and NO 2 under different driving conditions[END_REF][START_REF]Car Air Pollution: The Hidden Threat to Automobile Drivers: Report No. 4, An Assessment of the Air Quality Inside Automobile Passenger Compartments[END_REF].

If passenger compartments contribute to the dissociation of ozone, traffic density near the vehicle is another parameter leading to the same tendency. Indeed, in urban areas, a high proportion of nitrogen oxides are ejected by fuel engines as NO [START_REF] Carslaw | Development of an urban inventory for road transport emissions of NO 2 and comparison with estimates derived from ambient measurements[END_REF]. But, ozone reacts very easily with nitric oxide to produce nitrogen dioxide in accordance with the reaction:

NO + O 3 NO 2 + O 2 (1)
So, in traffic, O 3 is non-existent and NO 2 is the only oxidising gas.

As mentioned above, phthalocyanine-based gas sensors exhibit a partial selectivity towards oxidising species. Moreover, under our experimental conditions, i.e. vehicles moving on urban streets, ozone is quickly decomposed into nitrogen dioxide by nitrogen monoxide. So, the response of our gas sensor microsystem will be only correlated with the variations of invehicle nitrogen dioxide rate.

Sensor response under NO 2 : optimization of working conditions

Interaction processes between oxidising gases and phthalocyanine molecular units have been identified and discussed in previous papers [START_REF] Passard | Doping mechanisms of phthalocyanines by oxidizing gases: Application to gas sensors[END_REF][START_REF] Brunet | Improvement in real time detection and selectivity of phthalocyanine gas sensors dedicated to oxidizing pollutants evaluation[END_REF]. The first equilibrium involves physical sorption of oxidising molecules, competitive sorption with pre-adsorbed species and diffusion into the bulk of the layer. Consecutively, gaseous molecules can be chemisorbed to form negative ions and extrinsic charge carriers are created (holes) into the molecular semiconductor by charge transfer process. These charge carriers can be delocalized onto the other phthalocyanine molecular units of the layer leading to electronic conductivity by hopping process. Thus, interaction of oxidising molecules on phthalocyanine leads to the creation of holes at the surface and into the bulk, inducing the increase in thin layer electronic conductivity. Nevertheless, if interaction processes seem similar for ozone and nitrogen dioxide, sensor responses are different because of the lower stability of O 3 -anions as

compared to NO 2 - .
Consequently, if the working conditions have been optimized for the monitoring of O 3 in atmosphere [START_REF] Brunet | Improvement in real time detection and selectivity of phthalocyanine gas sensors dedicated to oxidizing pollutants evaluation[END_REF], some parameters must be improved for the efficient measurement of NO 2 .

As mentioned above, nitrogen dioxide anions NO 2 -being more chemically stable than O 3 species, desorption from the sensitive layer is more difficult and slower. So, to improve sensor cleaning just before pollutant exposure and to avoid the lack of reproducibility due to the irreversible part of chemisorbed species accumulation into the sensitive layer, the delay and the temperature of the thermal cleaning phase have been increased. However, for cleaning temperature above 180°C, we observe an irreversible decrease in microsystem sensor response even if NO 2 concentration remains constant. This drift can be explained by an irreversible hot oxidation of phthalocyanine molecules by chemisorbed NO 2 ones in material leading progressively to the deterioration of the sensitive thin layer. The time of the cycle is maintained to 15 min, this one corresponding to the period of measurement chosen by the national air quality control network. During this short delay, the evolution of pollutants is very slow and concentration variations remain very small. That justifies the detection of nitrogen dioxide in real time by our microsystem. Moreover, phthalocyanine gas sensors exhibiting very high sensitivities to oxidising gases, conductivity variations for short times of exposure remain high. Thus, the optimized working conditions for NO 2 are as follows: lower temperature (detection) = 80°C, higher temperature = 180°C, sensor regeneration time (t 0 ) = 12 min, sensor stabilization time (t 1 ) = 1 min, sensor exposure time (t 2 ) = 2 min, cycle period = 15 min.

To illustrate gas sensor microsystem performances, Fig. 5 represents sensor microsystem response versus nitrogen dioxide concentration in the range 0-100 ppb, a common range of urban atmosphere. Experiment has been made for monotone increasing ( ) and monotone decreasing ( ) concentrations, with a concentration variation step equal to 10 ppb. Moreover, for each NO 2 concentration, 4 consecutive measurements are realized. Results show that measurements are efficient and satisfying for all the concentrations of the range studied. The threshold of detection is very low and can be estimated to be a few ppb (approximately 2-3 ppb). The sensitivity is also high. The ratio is exactly the same between sensor microsystem response in mV and gas concentration in ppb (response is multiplied by 2 when gas concentration is twice). Moreover, the coefficient of proportionality between microsystem response and NO 2 concentration is 1 mV for 20 ppb with a signal/noise ratio very important.

For a monotone gas concentration variation, the reproducibility is excellent as illustrated by the superimposition of experimental points. Nevertheless, when NO 2 rate firstly increases and secondly decreases, a weak hysteresis phenomenon occurs: responses are higher for increasing concentrations than for decreasing ones. This induces particularly that the response to 60 ppb of NO 2 in increasing concentration is the same as that response to 50 ppb in decreasing one. Consequently, the microsystem resolution is lower than 20 ppb but remains greater than 10 ppb.

If the previous experiment is well adapted to quantify sensing characteristics, the fidelity of the microsystem for high concentration gradients cannot be determined. To estimate the reproducibility even for fast and high concentration variations of NO 2 (artificial pollution peaks), experiment has been realized with different concentration gradients. the accuracy of measurement is altered. This effect is not noticeable for fast decreases. The gas sensor microsystem response can be influenced by previous exposures but so fast NO 2 level variations are not often observed in rural or urban atmosphere. Thus, as illustrated by results obtained at laboratory, the working conditions mentioned above are the best ones for NO 2 monitoring in urban atmosphere.

Microsystem response variation law versus gas concentration: calibration curve

The main objective of this study being the continuous monitoring of NO 2 in air and not only the detection of thresholds, it is necessary to determine the relation between the microsystem response and the corresponding nitrogen dioxide concentration to inform drivers and passengers about the quantities they breathe in. To establish this variation law, a calibration curve has been realized with a 10 ppb concentration step. Figure 7 represents the response to NO 2 delivered by the gas sensor microsystem in the range 0-100 ppb. For each concentration 6 successive measurements have been made. The gain of the electronic current-voltage converter has been increased (multiplied by 200 as compared to Fig. 6) to obtain signal easily measurable by a simple voltmeter or available for signal treatment units, still with a high signal/noise ratio. In the first approximation, we could consider that the response is proportional to NO 2 concentration with a coefficient of proportionality to 0.012 V per ppb.

By applying a simple linear regression to the experimental points, the determination coefficient, also named regression coefficient, R² = 0.9931, R² being = 1 for a perfect correlation between a mathematical model and experimental points. Nevertheless, the best determination coefficient is obtained for a polynomial regression. Indeed, for Y = 3×10 -5 X² + 0.0098X, the determination coefficient is R² = 0.9992. So, the variation laws are as follows:

] b[NO ]² a[NO response 2 2 + = (2) or inversely: 2a / ) 4aresponse b² b (- ] [NO2 + + = (3)
with a = 3×10 -5 ; b = 9.8×10 -3 ; [NO 2 ] in ppb and response in V.

This last equation will be the mathematical relation giving the nitrogen dioxide concentration near the sensitive thin layer and the gas sensor microsystem response during experiments under real conditions.

Results in urban context

Results obtained at laboratory are confirmed under real using conditions by the implementation of this microsystem in a bus of the local bus network in Clermont-Ferrand, according to the conditions mentioned in section 2.3.

The results are illustrated in Fig. 8 which represents the NO 2 concentration variations in ppb deducted from the calibration curve of our microsystem versus the date and hour of the measurement. The variations are between 30 and 60 ppb, the middle of the gas concentration range studied. No specific trend is noticeable as a function of morning or afternoon. We can only observe that the higher concentrations appear at the beginning of afternoon.

The NO 2 level in urban areas strongly depends on traffic density [START_REF] Chan | A study of bus commuter and pedestrian exposure to traffic air pollution in Hong Kong[END_REF], the localization of vehicles in the city at the moment of measurement (downtown, suburb or industrial zone) [START_REF] Wijnen | The exposure of cyclists, car drivers and pedestrians to traffic-related air pollutants[END_REF] and driving conditions (flowing traffic, traffic jam, numerous traffic lights) [START_REF] Carslaw | Development of an urban inventory for road transport emissions of NO 2 and comparison with estimates derived from ambient measurements[END_REF]. These experimental details are essential for the best interpretation of results. Table 1 gives the local hour of measurement points, the nitrogen dioxide concentration deducted from the microsystem calibration curve, the localization of bus at the same time (road type) and the ambient NO 2 concentration delivered by the nearest station of air quality control network by means of a commercial chemiluminescent analyzer. We can notice from the table data that the lowest NO 2 concentrations have been recorded when the bus is parked on the campus where the traffic is lower. Nitrogen dioxide being essentially an automotive pollutant, its concentration is higher when the traffic is more important. Experimental results confirm this trend. Moreover, the higher concentrations are recorded in urban roads just after lunch. This increase could be explained by the rise in traffic due to the more important number of commuters driving from their houses or from restaurants to their work offices.

By comparison between NO 2 concentrations measured in passenger compartments and in ambient air by the nearest stations of pollutant monitoring, it is noticeable that all in-vehicle values are higher than those delivered by fixed stations. This seems to confirm that monitoring stations underestimate the real exposure of drivers and bus users to NO 2 because of their distance from driving ways.

To summarize, the satisfying results obtained under real conditions emphasize the strong correlation of urban NO 2 with traffic density and seems to confirm that the real exposure of drivers and commuters must be efficiently evaluated by an-board gas sensor microsystem.

Selectivity of gas sensor microsystem: discussion

As we have previously mentioned in this article, copper phthalocyanine thin layers exhibit a strong chemical reactivity with strong oxidising gases such as NO 2 and O 3 , which confers a partial selectivity to gas sensors using this semiconductor as a sensitive material. Moreover, under such real experimental conditions (in passenger compartments, on high traffic density, in urban areas), ozone levels are negligible and even zero as compared to NO 2 concentration.

So, under such working conditions, the gas sensor microsystem is drastically selective for nitrogen dioxide.

Nevertheless, we have demonstrated in previous works that for the same concentrations, the methodology of measurements provides higher sensor responses to O 3 than NO 2 , and even makes the sensor selective for ozone [START_REF] Brunet | Improvement in real time detection and selectivity of phthalocyanine gas sensors dedicated to oxidizing pollutants evaluation[END_REF]. Consequently, the gas sensor microsystem is selective for nitrogen dioxide only if ozone concentration remains equal to zero. The selective monitoring of NO 2 is not achieved especially if a bus is driving in the suburbs or in rural areas where with fair climatic conditions, ozone rates are often more important.

To reach the drastic selectivity for nitrogen dioxide under all conditions, one solution consists in removing completely ozone in the sensor environment, whatever ambient concentration, without changing nitrogen dioxide concentration. This can be realized by means of a selective gas filter. Amongst all the material investigated we can mention manganese dioxide MnO 2 , active charcoal, fullerene C 60 , stainless steel cuttings and indigo.

Only indigo can be considered as a real selective ozone filter. Experimental results on the filtering power of indigo powder towards NO 2 and O 3 are illustrated in Table 2. In the concentration ranges studied, O 3 is entirely eliminated and NO 2 preserved. Moreover, indigo powder being easily sublimed under vacuum to be deposited onto substrates and being insulating, this material is really suitable for the realization of an integrated selective ozone filter on semiconductor based sensors. Thus, the selectivity for NO 2 will be obtained by the implementation of an indigo filter in our microsystem. Two solutions could be considered: either by inserting an indigo cartridge in the fluid circuit, in the parallel fluid line of this set with an active charcoal cartridge (in Fig. 1, purified air = air without any pollutant and polluted air = air without ozone), or by using a phthalocyanine gas sensor with an integrated indigo filtering layer. The first very promising results obtained with this kind of structure will be the subject of a next paper.

Conclusion

The implementation of an on-board gas sensor microsystem is essential to determine the real exposure of drivers and passengers to NO 2 inside vehicles. After preliminary studies which establish that nitrogen dioxide is the only one oxidising species in vehicle passenger compartments, ozone being too unstable and quickly decomposed, we have optimized an original methodology to monitor NO 2 in real time by means of phthalocyanine-based gas sensors. This methodology allows determining gas concentration from the kinetics of sensor response during a short time of exposure. Working conditions such as temperatures and delays used are optimized for NO 2 as illustrated by experimental results. The high sensitivity, the high resolution (< 20 ppb), the low threshold (few ppb), the good reproducibility, and the real time detection obtained are in accordance with specifications relative to this application.

The variation law of microsystem response with NO 2 concentration is determined with accuracy and the calibration curve established. Measurements under real conditions, i.e. in an urban bus passenger compartment with usual driving conditions (cyclic gate opening/closing sequence) exhibit the possibility and the reliability of NO 2 monitoring by means of this microsystem, the results confirming the trends already reported. The results obtained clearly show the strong influence of traffic density on the sensor microsystem response. Moreover, the results highlight that the NO 2 concentrations deduced from in-vehicle measurements are always higher than those determined by the nearest fixed monitoring station of the local air quality control network. Ozone can be considered as the main interfering gas for the monitoring of NO 2 in urban atmosphere. This microsystem being sensitive to strong oxidising gases, the real selectivity for nitrogen dioxide will be achieved by removing ozone in the sensor environment. A way of improvement is finally proposed, especially using a real selective O 3 chemical filter added in the fluid circuit or integrated on sensor sensitive layers.

Figure captions

Table captions

Table1: Experimental conditions for measurements in a bus passenger compartment represented in Fig. 8. Simultaneously listed are the local hour of measurement recorded, the NO 2 concentration extracted from microsystem response and calibration curve, road type and NO 2 concentration of the nearest station of air quality control network, AtMO AUVERGNE.

Table 2: Quantification of the filtering power of indigo towards ozone and nitrogen dioxide.

Efficiency is defined as the percentage of O 3 or NO 2 removed by indigo powder in the concentration ranges of those measured in urban atmosphere.

  Figures 3 and 4 represent respectively NO 2 and O 3 concentration variations during two

Figure 6 represents

 6 the gas sensor microsystem response under optimized working conditions versus the time for various exposure sequences to NO 2 in the range 0-100 ppb. Microsystem responses ( , scale on the left) are delivered every 15 min and compared to the NO 2 concentration ( , scale on the right) measured by a commercial gas analyzer. For each concentration, 6 points of measurements are recorded. These results strengthen the low threshold, high sensitivity, and good reproducibility of successive measurements already emphasized by previous figure. For NO 2 concentrations between 0 and 80 ppb with a 20 ppb variation step, no common values are obtained for two different concentrations. The resolution is still < 20 ppb in this range. However, when the concentration quickly increases from 20 ppb to 100 ppb, we can observe a slow increase in sensor response, a few values being similar to ones relative to 80 ppb. The stabilization of response in this case is slow and

Fig. 1 :

 1 Fig. 1: Schematic representation of the methodology of measurement developed for

Fig. 2 :

 2 Fig. 2: Self-contained gas sensor microsystem for real time monitoring of oxidising gases in

Fig. 3 :

 3 Fig. 3: Comparison between NO 2 concentration variations in ppb measured by commercial

Fig. 4 :

 4 Fig. 4: Comparison between O 3 concentration variations in ppb measured by commercial UV

Fig. 5 :

 5 Fig. 5: Gas sensor microsystem response versus NO 2 concentration in the range 0-100 ppb.

Fig. 6 :

 6 Fig. 6: Gas sensor microsystem response ( ) for various NO 2 concentrations monitored by a

Fig. 7 :

 7 Fig. 7: Calibration curve of the gas sensor microsystem under NO 2 in the concentration range

Fig. 8 :

 8 Fig. 8: NO 2 concentration variations in a bus passenger compartment for common driving

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig. 3

Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

Fig. 6

 6 Fig. 6

Fig. 7

 7 Fig. 7

Fig. 8

 8 Fig. 8

Table 2

 2

Acknowledgements: Special thanks are given to the local air quality control network AtMO AUVERGNE for its help, the commercial analyzers lending and data from fixed stations of pollution monitoring. This research was supported by the mix association of transport of Clermont-Ferrand (SMTC) in the context of the EnviroBus project.

Biographies


Annabelle Le Parc

Joëlle Léonil

Formation of casein micelle in mammary epithelial cell

Formation of casein micelle in mammary epithelial cell

Casein micelle models

The intrinsic organisation and the mechanisms of casein micelle formation have not been fully established. The aggregation of the caseins appears to be initiated in the ER, and proceeds during their transport to the apical surface. To obtain information about the specific arrangement of the caseins in this structure and to characterize the interactions between the various components of the micelle, it is essential to study the dynamic of its elaboration within the secretory pathway.

During lactation, mammary epithelial cells synthesize and secrete huge quantities of milk specific proteins, lipids and lactose. The biogenesis of milk supramolecular structures, casein micelles and milk fat globules, is initiated in the ER. These components then follow independent intracellular trafficking pathways and milk supramolecular structures are released into the alveolar lumens of the mammary gland.

Milk Fat Globule Casein Micelles

  Annabelle Le Parc 1 , Joëlle Léonil 2 and Eric Chanat 1 1 INRA, UR 1196, Génomique et Physiologie de la Lactation, F-78352 Jouy-en-Josas, France. 2 INRA, UMR 1253, Science et Technologie du Lait et de l'oeuf, F-35042 Rennes, France.

  fraction of the immature and mature form of aS1-casein is present in a tightly membrane-associated form in the secretory pathway of mammary epithelial cells  Dimeric aS1-casein has a much higher affinity for membranes Interaction of aS1-casein with membranes of the secretory pathway might play a key role in casein micelle formation and/or exocytic trafficking in mammary


AutoBin v 0.9

 or GeneMapper (Applied Biosystems, USA). It can deal with unlimited number of loci and samples, with no consideration of the type of SSRs motifs. AutoBin helps the user to bin his data with visual alerts and format automatically the data for downstream analysis.

Input format:

Input data are in the form of four columns with fixed fields. As a consequence, data must always follow the example below:

Column A: Sample name Column B: Marker name Column C: Allele 1 in raw size Column D: Allele 2 in raw size Column headings must not be changed by the user. In some software, homozygous genotypes are missing one allele, so AutoBin automatically detects this problem and duplicates the first allele. Note that there are no limitations for the number of samples and loci, except the intrinsic limits of Excel. Names used for samples or loci are free.

Step by step execution of AutoBin

Step 1: By clicking the 'Format' button on sheet 'data', the number of samples and loci is automatically detected and genotypes for each locus are copied and pasted to a new sheet, with the locus name.

Step 2: By clicking the 'Binning' button on sheet 'data', for each locus, data are formatted from two to one column format (two lines per samples, on column by locus). Ascending sort of alleles by raw size is done automatically.

Step 3: By clicking the 'Validation' button on sheet 'data', for each locus, size difference between contiguous alleles is calculated. At this step, the user chooses two thresholds for gap detection. In red will appear strong gaps, in orange will appear moderate gaps for further checking. A conditional formatting of allele in raw sizes is done and the user can easily visualize the putative gaps (defined above). An arbitrary binning is proposed for each locus in column E. The user must check the binning with caution and if necessary modify it in column F (at least one value for each bin, which is copied automatically to other alleles).

Step 4: By clicking the 'Graph' button on sheet 'data', for each locus, a graph plot of cumulative length distribution is done, with automatic adjustment of vertical scale to omit missing data and optimize the interpretation. This step can be useful to visualize ambiguous samples.

Step 5: By clicking the "Format" button on sheet 'data', three output files are generated, for further analysis with STRUCTURE [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF] or GENEPOP [START_REF] Raymond | GENEPOP (Version 1.2) -Population-genetics software for exact tests and ecumenicism[END_REF]:

-STRUCTURE format (two lines by sample -one column by locus) -STRUCTURE format (one line by sample -two columns by locus) -GENEPOP format (one line by sample -two columns by locus)

From GENEPOP format, the user can reformat his data for commonly used population genetic software using CONVERT [START_REF] Glaubitz | CONVERT: A user-friendly program to reformat diploid genotypic data for commonly used population genetic software packages[END_REF].

IMPORTANT

At any time during the procedure, data can be erased by clicking the 'Erase Data' button. At any time during the procedure, graph can be erased by clicking the 'Erase Graph button.


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text:
J Jé

Lebrun Lebrun

Albert Kollmann Albert

Nelly Kollmann

Isabelle Wolff

Nelly Wolff

Isabelle Lamy

Christian Mougin Mougin

Morphological Responses to Cu and Zn in and Morphological Responses to Cu and Zn in Trametes versicolor Trametes versicolor

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

For that purpose, Trametes versicolor was cultured in liquid media exhibiting different levels of metal complexation. Three functional and two physiological traits were compared for their pertinence as metal exposure biomarkers. -Zn has no effect on the oxidase activities, whatever the complexation level of the used medium -Laccase and Mn-peroxidase activities are highly increased by Cu in low complexing media Aba5 and Aba2.5 -The growth of T.versicolor is not significantly affected by the essential metals whatever the medium, except with Cu at 1 mM and in Aba2.5 (data not shown) -The fungal morphology is not affected by Cu while Zn seems to induce a coalescence of mycelia but only at 1 mM and in low complexing media Aba5 and Aba2.5 (Fig. 2) The response of Trametes versicolor is specific to one essential metal and depends on metal speciation. We found that Zn leads to some morphological alterations at high concentration levels while Cu highly stimulates extracellular oxidase activities at environmental levels of metal contamination.

These enzymatic actors of soil functioning can be biomarkers of fungal exposure to Cu in soils. Furthermore, the specificity and sensitivity of fungal responses to metals confirm that fungal traits can be used as tools for metal ecotoxicity assessment.

Studies are now engaged to improve a quantitative extraction of enzymes from soils.

  Effect of metal speciation on the functional and morphological responses of soil fungi: case of Cu and Zn in Trametes versicolor Jérémie D. Lebrun, Albert Kollmann, Nelly Wolff, Isabelle Lamy, Christian Mougin To cite this version: Jérémie D. Lebrun, Albert Kollmann, Nelly Wolff, Isabelle Lamy, Christian Mougin. Effect of metal speciation on the functional and morphological responses of soil fungi: case of Cu and Zn in Trametes versicolor. 5. ISMOM International Symposium of Interactions of Soil Minerals with Organic Components and Microorganisms Commmission, Nov 2008, Pucon, Chile. J. Soil Sci. Plant Nutr., 8 key actors of soil functioning because of their ability to produce extracellular enzymes. Soil contamination by metals can alter these extracellular activities as well as fungal morphology. The impact will depend on metal bioavailability, which is linked to metal speciation. But the way the metal speciation influences the sensitivity of fungal responses is not well known, in particular in the case of essential metals Cu or Zn. Knowledge of metal speciation and its relationships with biological traits are important for the development of tools for metal ecotoxicity assessment, and could enhance the predictive interest in such fungal biomarkers. aims at establishing the influence of metal speciation on both specificity and sensitivity of fungal responses to essential metals.

-

  MEDIA and METAL SPECIATION -Use of three liquid media performed from Kirk medium (pH 5.5) differing only by their content in organic ligands : -Metal speciation was determined theoretically using a thermodynamic program (SOILCHEM) and validated experimentally using a Cu 2+ -specific electrode -Addition of copper in the different media yielded a good relationship: experimental vs theoretical [Cu 2+ ] values ; R 2 > 0.96 II. FUNGAL RESPONSES -Cultures of Trametes versicolor in the different media -Addition of Cu or Zn from 1 nM to 1 mM the 3 th day of culture -Exposure during one week to essential metals A. PHYSIOLOGICAL RESPONSES -Determination of dry biomass as a toxicity/tolerance indicator -Observations by scanning electron microscopy to characterize morphological impacts B. FUNCTIONAL RESPONSES -Measurements of 3 extracellular oxidase activities in liquid media: lignin-peroxidase ; Mn-peroxidase ; laccase Metals are mainly complexed by organic ligands in Lesa (>80%) -The presence of free metal species are favored in Aba5 and Aba2.5 (until 13% for Cu 2+ and 74% for Zn 2+ , in red Fig. 1)

Fig. 3 :

 3 Fig. 3: □ Control ■ Zn at 1 mM ■ Cu at 1 mM ; values ± SEM (n=9 ; *p<0.05)

Fig. 2 :Fig. 4 :Fig. 1 :

 241 Fig. 2: Scanning electron microscopy of T.versicolor mycelia cultured in the medium, Aba2.5 (Magnification: x 2000)