Linda J Kay

Kim Suvarna

Anne-Marie Scola

Amin Rostami-Hodjegan

Russell Chess-Williams

Peter T Peachell
email: p.t.peachell@shef.ac.uk

Influence of β 2 -adrenoceptor gene polymorphisms on β 2 -adrenoceptor expression in human lung

Keywords: asthma, human lung, ADRB2, β 2 -adrenoceptor, SNP ADRB2, β 2 -adrenoceptor gene BUP, beta upstream peptide

Background:

The aim of the present study was to establish whether polymorphisms, especially those within the promoter region, of the β 2 -adrenoceptor gene (ADRB2) influence β 2 -adrenoceptor expression in human lung.

Methods:

The density of β-adrenoceptors in human lung tissue (n=88) was determined by saturation binding using the radioligand, iodinated cyanopindolol. Discrimination of β 1 -and β 2 -adrenoceptors was determined using the highly selective β 1 -adrenoceptor antagonist, CGP20712A. Genotype was determined at 5 positions of ADRB2 previously reported as polymorphic. Potential influences of single nucleotide polymorphisms (SNPs) within the promoter region (-367, -47) and coding block (46, 79, 491) of ADRB2 on β 2 -adrenoceptor expression were investigated.

Results:

The density of β 2 -adrenoceptors was variable among the 88 lung preparations studied ranging from 17 to 177 fmol/mg protein (mean±S.E.M., 72±4 fmol/mg protein).

There was no influence of genotype on β 2 -adrenoceptor expression for any of the polymorphisms studied except at position 491. The polymorphism at position 491C>T, leading to a change from thr to ile at amino acid 164, is uncommon. Preparations genotyped as heterozygous (126±15 fmol/mg protein; n=5) expressed significantly (P = 0.0005) higher levels of β 2 -adrenoceptor than those that were homozygous (69±4 fmol/mg protein; n=83).

Introduction

A large number of studies has shown that the gene for the β 2 -adrenoceptor (ADRB2) is polymorphic [START_REF] Brodde | β 2 -adrenoceptor polymorphisms[END_REF]. Single nucleotide polymorphisms (SNPs) have now been found not only within the promoter region and the coding block [START_REF] Brodde | β 2 -adrenoceptor polymorphisms[END_REF] but also within the 3' untranslated region [START_REF] Hawkins | Sequence, haplotype and association analysis of ADRβ2 in a multiethnic casecontrol study[END_REF] of ADRB2. Additional studies have demonstrated that some of these polymorphisms may influence both receptor function and expression [START_REF] Brodde | β 2 -adrenoceptor polymorphisms[END_REF][START_REF] Green | Implications of genetic variability of human β 2 -adrenergic receptor structure[END_REF][START_REF] Kirstein | Autonomic nervous system Pharmacogenomics: a progress report[END_REF]. This could, potentially, impact quite heavily on how effectively agonists activate β 2adrenoceptors. This is of particular interest in the context of asthma therapy since β 2adrenoceptor agonists are the most commonly prescribed drugs used to treat asthma [START_REF] Waldeck | β-adrenoceptor agonists and asthma -100 years of development[END_REF].

Consequently, a considerable body of work has emerged geared toward establishing whether polymorphisms in ADRB2 influence the therapeutic benefits of β 2 -adrenoceptor agonists in asthma [START_REF] Taylor | Beta-adrenergic receptor polymorphisms and drug responses in asthma[END_REF]. β 2 -adrenoceptor agonists act in asthma primarily as bronchodilators relaxing airway smooth muscle but additional effects could include the stabilization of pulmonary cell activity [START_REF] O'connor | Non-bronchodilator effects of inhaled β 2agonists. Greater protection against adenosine monophosphate-than methacholineinduced bronchoconstriction in asthma[END_REF][START_REF] Nightingale | Differential effect of formoterol on adenosine monophosphate and histamine reactivity in asthma[END_REF]. One factor that could influence the effectiveness of β 2 -adrenoceptor agonists in the treatment of asthma is the extent of receptor expression in the lung.

Receptor density is known to influence both the potency and efficacy of agonists [START_REF] Macewan | Analysis of the role of receptor number in defining the intrinsic activity and potency of partial agonists in neuroblastoma x M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 18 glioma hybrid NG108-15 cells transfected to express differing levels of the human β 2adrenoceptor[END_REF]. This may be of especial importance in asthma since many of the β-adrenoceptor agonists used clinically, such as salbutamol and salmeterol, are partial agonists [START_REF] Taylor | Comparison of salmeterol and albuterol-induced bronchoprotection against adenosine monophosphate and histamine in mild asthma[END_REF]. Partial agonists are unable to attain maximal responses despite full receptor occupancy and responses are particularly dependent on the size of the functional receptor population [START_REF] Macewan | Analysis of the role of receptor number in defining the intrinsic activity and potency of partial agonists in neuroblastoma x M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 18 glioma hybrid NG108-15 cells transfected to express differing levels of the human β 2adrenoceptor[END_REF]. Should receptor expression be compromised, the response to a partial agonist will be further attenuated whereas a full agonist may continue to evoke maximal responses.

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Polymorphisms within the promoter region of ADRB2 have been associated with the regulation of receptor expression [START_REF] Mcgraw | Polymorphisms of the 5' leader cistron of the human β 2 -adrenergic receptor regulate receptor expression[END_REF][START_REF] Scott | Identification of novel polymorphisms within the promoter region of the human β 2 -adrenergic receptor gene[END_REF][START_REF] Drysdale | Complex promoter and coding region β 2 -adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness[END_REF][START_REF] Johnatty | β 2 -adrenergic receptor 5' haplotypes influence promoter activity[END_REF][START_REF] Westland | Limited impact of multiple 5' single-nucleotide polymorphisms on the transcriptional control of the human β 2 -adrenoceptor gene[END_REF]. In particular, a polymorphism at position -47 has been strongly linked to β 2 -adrenoceptor expression [START_REF] Mcgraw | Polymorphisms of the 5' leader cistron of the human β 2 -adrenergic receptor regulate receptor expression[END_REF]. Position -47 is found within an open-reading frame, just upstream of the start site for the β 2 -adrenoceptor, that encodes a putative 19 amino acid peptide that is sometimes referred to as beta upstream peptide (BUP) [START_REF] Parola | The peptide product of a 5' leader cistron in the β 2 adrenergic receptor mRNA inhibits receptor synthesis[END_REF]. The polymorphism (-47T>C) leads to a change in the terminal residue of BUP from cys to arg. BUP-arg has been reported to attenuate β 2 -adrenoceptor expression [START_REF] Mcgraw | Polymorphisms of the 5' leader cistron of the human β 2 -adrenergic receptor regulate receptor expression[END_REF]. In addition to position -47, position -367 within the promoter region has also been linked to influencing receptor expression as the polymorphism (-367T>C) interrupts a potential binding site for transcription factors [START_REF] Scott | Identification of novel polymorphisms within the promoter region of the human β 2 -adrenergic receptor gene[END_REF][START_REF] Johnatty | β 2 -adrenergic receptor 5' haplotypes influence promoter activity[END_REF].

An influence of polymorphisms on β 2 -adrenoceptor expression has been assessed in transfected cells and lymphocytes with equivocal outcomes [START_REF] Mcgraw | Polymorphisms of the 5' leader cistron of the human β 2 -adrenergic receptor regulate receptor expression[END_REF][START_REF] Scott | Identification of novel polymorphisms within the promoter region of the human β 2 -adrenergic receptor gene[END_REF][START_REF] Drysdale | Complex promoter and coding region β 2 -adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness[END_REF][START_REF] Johnatty | β 2 -adrenergic receptor 5' haplotypes influence promoter activity[END_REF][START_REF] Westland | Limited impact of multiple 5' single-nucleotide polymorphisms on the transcriptional control of the human β 2 -adrenoceptor gene[END_REF][START_REF] Lipworth | β 2 adrenoceptor polymorphisms: extended haplotypes and functional effects in peripheral blood mononuclear cells[END_REF]. The aim of the present study was to perform a comprehensive analysis of the influence of polymorphisms across ADRB2 on β 2 -adrenoceptor expression in human lung tissue.

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Materials and Methods

Buffers

Tris buffer contained (mM): Tris 50, NaCl 154, MgCl 2 .6H 2 O 10, EDTA 2. The pH of Tris buffer was titrated to 7.4.

Preparation of compounds

CGP20712A (2-hydroxy-5-(2-(hydroxy-3-(4((1-methyl-4-trifluoromethyl)-1-H-imidazol- 2-yl)-phenoxy)-propyl)-aminoethoxyl)-benzamide
) and (±)-propranolol HCl, were prepared daily as stock solutions (10 mM) in Tris buffer.

Lung tissue

Human lung tissue was obtained, anonymized, from surgical resections of patients with the approval of the Local Research Ethics Committee. Most of the patients were undergoing surgery for carcinoma. The majority of the patients were Caucasian (90%), and most were male (70%).

Radioligand binding

Membrane fractions were prepared from lung tissue by methods that have been described previously [START_REF] Nishikawa | Effect of short-and long-acting β 2adrenoceptor agonists on pulmonary β 2 -adrenoceptor expression in human lung[END_REF][START_REF] Chong | Pharmacological characterisation of the β-adrenoceptor expressed by human lung mast cells[END_REF]. The tissue (100 -150 mg) was homogenised in ice-cold Tris buffer using an Ultra Turrax homogeniser for 20 s followed by four strokes (x4) of a Teflon homogeniser. The homogenate was centrifuged (500 g, 10 min) and the supernatant was harvested and subjected to further centrifugation (40,000 g, 15 min) in an ultra-centrifuge (L80, Beckman). The pellet was washed and the high-speed centrifugation step repeated.

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The pellet was resuspended in Tris buffer and used in receptor binding assays. All procedures were carried out at 4 °C.

In saturation binding assays, the membrane preparations were assayed for βadrenoceptor binding sites using [ 125 I]-cyanopindolol. Membrane suspensions (100 µl) were incubated (1 h, 37 °C) using a range of radioligand concentrations (0.01563 -2 nM) in a total volume of 250 µl. Non-specific binding was determined by displacement with propranolol (1 µM). Specific binding, expressed as a percentage of the total binding, was 87±1% at a [ 125 I]-cyanopindolol concentration of 0.0625 nM. The subtypes of βadrenoceptor present in membranes were determined in competition studies with the use of the highly selective β 1 -adrenoceptor antagonist, CGP20712A [START_REF] Dooley | CGP20712A: a useful tool for quantitating β 1 -and β 2 -adrenoceptors[END_REF]. Membrane preparations (100 µl) were incubated (1 h, 37 °C) with CGP20712A (10 -14 -10 -4 M) in the presence of [ 125 I]-cyanopindolol (0.0625 nM) in a total volume of 250 µl. Although full displacement curves using an extensive range of CGP20712A concentrations were performed for some preparations (n=23), further studies indicated that the use of a single concentration (10 nM) of the antagonist was sufficiently discriminatory to determine the split of β-adrenoceptors present in lung membranes and this approach was used in the majority of instances.

Additions of ice-cold Tris buffer were used to terminate the reactions followed by rapid filtration through Whatman GF/B glass fibre filters. The filters were rapidly washed four times with 3 ml ice-cold buffer and the radioactivity remaining on filters measured in a Packard Cobra auto-gamma counter. All saturation binding experiments were performed in duplicate. All competition binding experiments were performed either in duplicate or quadruplicate. Twenty-five of the 88 lung preparations that were studied in

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these binding experiments were repeat assayed to ensure data reproducibility. These were, primarily, preparations that were at either end of the population's spectrum such as preparations that expressed very high or very low levels of β-adrenoceptors. Protein content of the membranes was determined by the method of Lowry et al [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF].

Genotyping

For genotypic analyses of the β 2 -adrenoceptor, genomic DNA was extracted from a small quantity of human lung tissue using a modification of the chloroform extraction and ethanol precipitation method described elsewhere [START_REF] Graham | The isolation of high molecular weight DNA from whole organisms or large tissue masses[END_REF]. The extracted DNA was amplified, using primers specific for ADRB2 (see Table 1), by polymerase chain reaction (PCR). The reaction constituents for PCR were, genomic DNA (70 -100 ng), dNTPs (200 μM of each), MgSO 4 (1 or 1.4 mM), Tris-SO 4 (pH 9.1; 60 mM), (NH 4 ) 2 SO 4 (18 mM), both primers (1 μM of each) and ELONGASE enzyme mix (1 μl) in a final volume of 50 µl. Conditions were essentially as described previously and involved 35 cycles of PCR [START_REF] Chong | Influence of genetic polymorphisms in the β 2 -adrenoceptor on desensitization in human lung mast cells[END_REF]. All PCR products were visualised with ethidium bromide staining on agarose gels.

PCR products were then subjected to genotypic analysis by either automated sequencing or restriction fragment length polymorphism (RFLP). PCR products generated from primer pair 1 (see Table 1) permitted determination of polymorphisms at nucleotide position -367 whereas products generated from primer pair 2 allowed determinations at positions -47, 46 and 79. PCR products subjected to automated sequencing (products of primer pairs 1 and 2) were first purified by either ethanol precipitation or using a QIAquick PCR purification kit before sequencing (Applied

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Biosystems, 373 Stretch). Alternatively, in order to determine the genotype at position 491, RFLP was performed on PCR products generated by primer pair 3 (Table 1) using the restriction enzyme MnlI according to methods similar to those described elsewhere [START_REF] Aynacioglu | Population frequency, mutation linkage and analytical methodology for the Arg16Gly, Gln27Glu M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 20 and Thr164Ile polymorphisms in the β 2 -adrenergic receptor among Turks[END_REF]. In order to confirm the genotypes determined by RFLP for position 491, all heterozygotes (n=5) and a random sample of homozygotes (n=9) were also genotyped by automated sequencing.

Materials

Materials were purchased from the following sources: QIAquick (Qiagen, Sussex, UK), Tris (Calbiochem, Nottingham, UK); propranolol (Sigma, Poole, UK); [ 125 I]cyanopindolol (New England Nuclear, Stevenage, UK); agarose, ELONGASE enzyme mix (Invitrogen, Paisley, UK); dNTPs (Promega, Southampton, UK); MnlI (New England Biolabs, Hitchin, UK). CGP20712A was kindly supplied as a gift from Ciba-Geigy (Basel, Switzerland).

Data analysis

Receptor densities (B max ) and radioligand affinity (K D ) were determined by non-linear regression analysis of saturation curves (GraphPad Prism, version 4). To determine whether β-adrenoceptor density varies among lung regions, repeated measures ANOVA was performed. To determine whether genotype influences β-adrenoceptor density either Kruskal-Wallis or Mann-Whitney tests were performed (Graph Pad Instat, version 4).

Hardy-Weinberg equilibrium was determined by means of χ 2 goodness-of-fit tests.

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Results

  β-adrenoceptor expression in human lung

The density of β-adrenoceptors in membranes generated from lung tissue was determined by saturation binding using the radioligand [ 125 I]-cyanopindolol (0.01563 -2 nM). The total β-adrenoceptor density was variable among the 88 lung preparations studied ranging from 19 to 180 fmol/mg protein (mean±S.E.M., 80±4 fmol/mg protein). Representative saturation binding data are provided (Fig. 1).

The density of β 2 -and β 1 -adrenoceptors was assessed by competition binding utilizing the highly selective β 1 -adrenoceptor antagonist CGP20712A (Fig. 2). These experiments demonstrated that of the total β-adrenoceptors, 91±10% were β 2adrenoceptors (n=88).

 2. β-adrenoceptor expression in different zones of human lung

The samples used in these studies were sub-pleural and taken from the periphery of either upper, middle or lower zones of lung following lobectomies. In order to determine whether the variability in β-adrenoceptor density and subtype distribution was due to the area of lung being sampled, binding assays were performed on tissue sampled from the periphery of upper, middle and lower zones, as well as deep, central regions of lung, following pneumonectomies (Table 2). There was no difference, statistically (P > 0.05), among regions of lung in the density (fmol/mg protein) of (a) total β-adrenoceptors, (b) β 2 -adrenoceptors, or (c) β 1 -adrenoceptors.

Influence of SNPs on β 2 -adrenoceptor expression

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In order to establish whether polymorphisms within ADRB2 influence receptor expression in human lung, the 88 lung preparations whose β 2 -adrenoceptor densities had been determined (see section 3.1.), were genotyped at 5 positions of ADRB2 (-367, -47, 46, 79 and 491) known to be polymorphic. For these positions, the frequency of the less prominent allele was either 0.40 or 0.41 with the exception of nucleotide position 491 in which only 5 heterozygotes and no homozygotes of the less frequent allele were detected (Table 3).

Position -47, within the promoter region, has been reported to influence β 2adrenoceptor expression [START_REF] Mcgraw | Polymorphisms of the 5' leader cistron of the human β 2 -adrenergic receptor regulate receptor expression[END_REF]. However, our data demonstrate that there was no influence (P > 0.05) of position -47 on β 2 -adrenoceptor expression in the 88 lung preparations that were studied (Fig. 3). Position -367 within the promoter region of ADRB2 has also been linked to influencing receptor expression [START_REF] Scott | Identification of novel polymorphisms within the promoter region of the human β 2 -adrenergic receptor gene[END_REF][START_REF] Westland | Limited impact of multiple 5' single-nucleotide polymorphisms on the transcriptional control of the human β 2 -adrenoceptor gene[END_REF]. However, in human lung this position had no influence on β 2 -adrenoceptor expression (Table 4).

The potential influence of coding block SNPs on β 2 -adrenoceptor expression in lung was evaluated (Table 4). None of the positions had an influence with the exception of position 491 (amino acid position 164) in which preparations genotyped as heterozygous expressed significantly (P = 0.0005) higher levels of β 2 -adrenoceptor than homozygotes (Fig. 4).

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Discussion

β 2 -adrenoceptor agonists continue to be significantly important drugs used to manage asthma [START_REF] Waldeck | β-adrenoceptor agonists and asthma -100 years of development[END_REF]. Therapeutically, β 2 -adrenoceptor agonists act as bronchodilators but additional benefits may include the stabilization of inflammatory cell activity [START_REF] O'connor | Non-bronchodilator effects of inhaled β 2agonists. Greater protection against adenosine monophosphate-than methacholineinduced bronchoconstriction in asthma[END_REF][START_REF] Nightingale | Differential effect of formoterol on adenosine monophosphate and histamine reactivity in asthma[END_REF]. As agonists that mediate effects through pulmonary β 2 -adrenoceptors, the actions of these drugs are likely to be influenced by the extent of β 2 -adrenoceptor expression in the lung.

In the present study, we found vast differences, as much as ten-fold, in the extent of βadrenoceptor expression among lung tissue specimens. Our intention was to determine whether this wide difference in the degree of receptor expression might be genetically influenced.

Polymorphisms within the promoter region of ADRB2, especially those at -47T>C (BUP-cys to BUP-arg) and -367T>C, have been associated with differences in β 2 -adrenoceptor expression in transfected cells [START_REF] Mcgraw | Polymorphisms of the 5' leader cistron of the human β 2 -adrenergic receptor regulate receptor expression[END_REF][START_REF] Scott | Identification of novel polymorphisms within the promoter region of the human β 2 -adrenergic receptor gene[END_REF][START_REF] Drysdale | Complex promoter and coding region β 2 -adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness[END_REF][START_REF] Johnatty | β 2 -adrenergic receptor 5' haplotypes influence promoter activity[END_REF][START_REF] Westland | Limited impact of multiple 5' single-nucleotide polymorphisms on the transcriptional control of the human β 2 -adrenoceptor gene[END_REF]. By contrast, a study using lymphocytes isolated from asthmatics demonstrated that there was no association between the polymorphism at -47 and β 2 -adrenoceptor expression [START_REF] Lipworth | β 2 adrenoceptor polymorphisms: extended haplotypes and functional effects in peripheral blood mononuclear cells[END_REF]. While lymphocytes were used in this study as a representation of lung β 2 -adrenoceptors, it is known that the behaviour and expression of β 2 -adrenoceptors can be influenced by the tissue or cell in which the receptor is expressed [START_REF] Hasegawa | Difference between lung and spleen susceptibility of beta-adrenergic receptors to desensitization by terbutaline[END_REF][START_REF] Hauck | β 2adrenoceptors in human lung and peripheral mononuclear leukocytes of untreated and terbutaline-treated patients[END_REF][START_REF] Chong | β-adrenoceptor reserve in human lung: a comparison between airway smooth muscle and mast cells[END_REF]. Nevertheless, findings from the present study are in accord with those reported for lymphocytes since there was no influence of position -47 on β 2 -adrenoceptor expression in human lung. Indeed, neither of the SNPs (-47 and -367) found up-stream from the coding block and that, conceivably, could have an influence on β 2 -adrenoceptor expression were associated with differences in receptor density in this study.

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A potential influence of SNPs within the coding block of ADRB2 was also considered. Thus, polymorphisms at positions 46 and 79, corresponding with amino acid positions 16 and 27 of the β 2 -adrenoceptor, were examined to establish whether any link existed with receptor expression. Positions 16 and 27 have been associated with differences in the degree of β 2 -adrenoceptor down-regulation [START_REF] Green | Influence of β 2 -adrenergic receptor genotypes on signal transduction in human airway smooth muscle cells[END_REF][START_REF] Green | Amino-terminal polymorphisms of the human β 2 -adrenergic receptor impart distinct agonist-promoted regulatory properties[END_REF] such that expression of a particular genotype at either of these two positions could, potentially, impact on the degree of receptor expression in lung. However, in the present study, there was no influence of either of these two positions on β 2 -adrenoceptor expression.

In addition, we also considered an alternative polymorphism, 491C>T, within the coding block corresponding with a change in amino acid at position164 of the β 2adrenoceptor from thr to ile [START_REF] Reihsaus | Mutations in the gene encoding for the β 2 -adrenergic receptor in normal and asthmatic subjects[END_REF]. In keeping with a number of studies [START_REF] Aynacioglu | Population frequency, mutation linkage and analytical methodology for the Arg16Gly, Gln27Glu M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 20 and Thr164Ile polymorphisms in the β 2 -adrenergic receptor among Turks[END_REF][START_REF] Reihsaus | Mutations in the gene encoding for the β 2 -adrenergic receptor in normal and asthmatic subjects[END_REF][START_REF] Büscher | β 2adrenoceptor gene polymorphisms in cystic fibrosis lung disease[END_REF][START_REF] Hall | β 2adrenoceptor polymorphisms and asthma from childhood to middle age in the British 1958 birth cohort: a genetic association study[END_REF], the appearance of the less common allele was very infrequent with only 5 preparations out of 88 genotyped as heterozygous at this position with none homozygous for the less common allele. Lung preparations genotyped as thr164ile expressed significantly greater levels, almost twice as many, β 2 -adrenoceptors as those genotyped as thr164thr. While interesting, these data were unexpected since no precedents exist in the literature for such an outcome. One possible explanation for the markedly greater β 2 -adrenoceptor density in lungs genotyped as thr164ile could be due to a compensatory mechanism. In transfected cells, agonists bind to the ile164-expressing β 2 -adrenoceptor with lower affinity and the receptor couples less efficiently to G-protein compared to the thr164expressing β 2 -adrenoceptor [START_REF] Green | A polymorphism of the human β 2 -adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor[END_REF]. This could mean that diminished constitutive or promoted down-regulation occurs of the ile164-expressing β 2 -adrenoceptor due to impaired activation of the protein kinases (protein kinase A and G-protein receptor

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14 kinase) that are thought to be involved in receptor desensitization [START_REF] Benovic | β-adrenergic kinase. Activity of partial agonists for stimulation of adenylate cyclase correlates with ability to promote receptor phosphorylation[END_REF][START_REF] January | β -Adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists[END_REF][START_REF] Kohout | Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization[END_REF]. Consequently, this could result in a relative increase in β 2 -adrenoceptors in preparations expressing the less frequent allele at position 164. While such a scheme is, admittedly, speculative, depressed desensitization of the ile164-expressing β 2 -adrenoceptor in transfected cells has been reported [START_REF] Green | A polymorphism of the human β 2 -adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor[END_REF].

Overall, these findings indicate that common polymorphisms in ADRB2 do not influence β 2 -adrenoceptor expression in human lung. This suggests that a direct genetic influence is not responsible for the wide variability in β 2 -adrenoceptor density observed and that alternative influences contribute to the variability. We explored the possibility that the underlying lung pathophysiology might influence β 2 -adrenoceptor expression by blinded histological review of the samples taken. Microscopic analysis of different lung tissue samples known to express a range of β 2 -adrenoceptor densities, was performed by a single observer (SKS). The analysis indicated minimal differences among samples in oedema, inflammatory cell infiltration, fibrosis and emphysema (see Fig. 5). These minimal differences are often seen in background resected lung tissues and are judged unlikely to have any material impact on β-adrenoceptor density.

One alternative factor that could potentially influence β 2 -adrenoceptor expression in lung is prior exposure to medications, in particular, to β 2 -agonists and corticosteroids.

Exposure to β 2 -agonists could lead to β 2 -adrenoceptor down-regulation [START_REF] Nishikawa | Effect of short-and long-acting β 2adrenoceptor agonists on pulmonary β 2 -adrenoceptor expression in human lung[END_REF][START_REF] Benovic | β-adrenergic kinase. Activity of partial agonists for stimulation of adenylate cyclase correlates with ability to promote receptor phosphorylation[END_REF][START_REF] January | β -Adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists[END_REF][START_REF] Kohout | Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization[END_REF][START_REF] Ferguson | Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling[END_REF] and corticosteroids have been shown to increase β 2 -adrenoceptor expression in a variety of test systems [START_REF] Lai | Dexamethasone regulates the β-adrenergic receptor subtype expressed by 3T3-L1 pre-adipocytes and adipocytes[END_REF][START_REF] Collins | β 2 -adrenergic receptors in hamster smooth muscle cells are transcriptionally regulated by glucocorticoids[END_REF][40][41]. In the present study, tissue was donated anonymously and drug histories were, therefore, unavailable. However, in an unrelated study in which 250 individuals had donated lung tissue for study and for whom details exist, 2% were on

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15 corticosteroids, 5% were on β 2 -agonists and 4% were on a combination of corticosteroids and β 2 -agonists. If an assumption is made that a similar proportion of the present study population was exposed to these drugs, it seems unlikely that exposure to corticosteroids and β 2 -agonists is a substantially confounding influence. Indeed, whether prior exposure to β 2 -agonists has a substantial effect on receptor density in the lung is a moot point since reports have shown that the pulmonary β 2 -adrenoceptor is unusually resistant to downregulation [START_REF] Hasegawa | Difference between lung and spleen susceptibility of beta-adrenergic receptors to desensitization by terbutaline[END_REF][START_REF] Hauck | β 2adrenoceptors in human lung and peripheral mononuclear leukocytes of untreated and terbutaline-treated patients[END_REF]42] In summary, the present study has shown that common polymorphisms both within the promoter region and the coding block of ADRB2 do not influence the expression of β 2 -adrenoceptors in human lung. By contrast, there was an association between the rare polymorphism at position 491 (amino acid 164) and β 2 -adrenoceptor expression. However, the possibility that other, recently identified SNPs in ADRB2, 5' and 3' of the region studied [START_REF] Hawkins | Sequence, haplotype and association analysis of ADRβ2 in a multiethnic casecontrol study[END_REF], might influence receptor expression cannot be excluded.

In conclusion, these findings indicate that common polymorphisms in ADRB2 do not influence β 2 -adrenoceptor expression in human lung. This suggests that a direct genetic influence is not responsible for the wide variability in β 2 -adrenoceptor density observed and that alternative influences contribute to the variability. [42] Bai TR, Mak JCW, Barnes PJ. A comparison of β-adrenergic receptors and in vitro relaxant responses to isoproterenol in asthmatic airway smooth muscle. Am J Respir Cell Mol Biol 1992;6:647-51.

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 6

10

 10

12

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  Mak JC, Nishikawa M, Barnes PJ. Glucocorticosteroids increase β 2 -adrenergic receptor transcription in human lung. Am J Physiol 1995;268:L41-L46.[41] Baraniuk JN, Ali M, Brody D, Maniscalco J, Gaumond E, Fitzgerald T et al.Glucocorticoids induce β 2 -adrenergic receptor function in human nasal mucosa. Am J Respir Crit Care Med 1997;155:704-10.

Figure 1

 1 Figure 1 Saturation binding curves of the radioligand [ 125 I]-cyanopindolol to human

Figure 2

 2 Figure 2 Distribution of β-adrenoceptor subtypes in human lung. Membranes were prepared from lung samples (n=88) and discrimination of β 2 -and β 1 -adrenoceptors was determined by competition of the radioligand [ 125 I]-cyanopindolol (0.0625 nM) with increasing concentrations of the highly β 1 -adrenoceptor-selective antagonist,

Figure 3

 3 Figure 3 Influence of position -47T>C on β 2 -adrenoceptor expression in human lung.

Figure 4

 4 Figure 4 Influence of position 491C>T on β 2 -adrenoceptor expression in human lung.

Figure 5 29 expressing β 2 -

 5292 Figure 5 Human lung histology. Twelve human lung preparations known to express high, low and intermediary levels of β 2 -adrenoceptor were examined histologically.

23 Table 1

 231 Primers used to amplify regions of ADRB2 by PCR

	Primer	Primers	Annealing	Fragment
	pair		conditions	size (bp)

Table 2 β

 2 -adrenoceptor density in different zones of human lung

			Zones
		Upper	Middle	Lower	Central
	β-AR density (fmol/mg protein)	80±13	90±16	75±10	114±18
	β 2 -AR (%)	92±1	95±4	95±2	94±2
	K D (nM)	0.05±0.01	0.04±0.01	0.04±0.01	0.05±0.01
	Tissue was sampled from peripheral regions of upper, middle and lower zones and from
	deep, central zones of lung following pneumonectomies. Membranes were prepared
	from these samples and β-adrenoceptor (β-AR) density was determined by saturation
	binding assays using the radioligand [ 125 I]-cyanopindolol (0.01563 -2 nM). The K D
	values for [ 125 I]-cyanopindolol, determined in different zones of lung, are provided in the
	table and were not significantly different, statistically (P > 0.05), from each other.
	Discrimination of β 2 -and β 1 -adrenoceptors was determined by competition of the
	radioligand with CGP20712A. There was no difference, statistically (P > 0.05), among
	zones of lung in the density (fmol/mg protein) of (a) total β-adrenoceptors, (b) β 2 -
	adrenoceptors, or (c) β 1 -adrenoceptors. Values are means±S.E.M. for seven experiments.

Table 3

 3 Allelic frequencies of polymorphic positions in ADRB2 (n=88)

	Position (aa)	SNP	amino acid	frequency
	-367	T	-	0.59
		C	-	0.41
	-47	T	cys	0.60
		C	arg	0.40
	46 (16)	G	gly	0.60
		A	arg	0.40
	79 (27)	C	gln	0.59
		G	glu	0.41
	491 (164)	C	thr	0.97
		T	ile	0.03
	Each polymorphic position was in Hardy-Weinberg equilibrium.

Table 4

 4 Influence of SNPs on β 2 -adrenoceptor expression in human lung

			β 2 -adrenoceptor density (fmol/mg protein)
	Genotype	-367	-47	46	79	491
	0	65±6	71±6	71±6	67±6	69±4
	1	84±6	77±6	74±6	79±6	126±15*
	2	63±6	61±6	71±8	63±6	-
	For each polymorphic position, homozygotes for the more common allele are represented
	by set 0 and for the less common allele by set 2 and for heterozygotes by set 1. Further
	information concerning alleles and allele frequencies can be found in table 3. None of
	the polymorphisms influenced β 2 -adrenoceptor expression except position 491 (amino
	acid 164) in which preparations that were heterozygous (n=5) at this position expressed
	significantly (*P = 0.0005) higher levels of receptor than homozygotes (n=83).

Acknowledgements

The authors are grateful to Mr J Edwards, Mr J Rao, Mr T Locke, Mr G Cooper and Mr D Hopkinson (Cardiothoracic Surgery), Dr SK Suvarna, Dr P Kitsanta and Dr C Layton (Histopathology) at the Northern General Hospital, Sheffield for their invaluable help in providing lung tissue specimens. The in-house sequencing facility and the assistance of Aileen McDermott, Helen Langsford and Dr Franco Di Giovine is gratefully acknowledged. This work was supported, in part, by Asthma UK.


K Wojciechowski

J Tobola

M Schmidt

R Zybala

Crystal structure, electronic and transport properties of AgSbSe 2 and AgSbTe 2

Keywords: thermoelectric materials, electronic materials, semiconductors, electronic structure, electrical properties

Undoped and p and n doped AgSbX 2 (X = Se and Te) materials were synthesized by direct fusion technique. The structural properties were investigated by X-ray diffraction and SEM microscopy. The electrical conductivity, thermal conductivity and Seebeck coefficient have been measured as a function of temperature in the range from 300 to 600 K.

To enlighten electron transport behaviours observed in AgSbSe 2 and AgSbTe 2 compounds, electronic structure calculations have been performed by the Korringa-Kohn-Rostoker method as well as KKR with with coherent potential approximation (KKR-CPA) for ordered (hypothetical AgX and SbX as well as AgSbX 2 approximates) and disordered systems (Ag 1- x Sb x X), respectively. The calculated density of states in the considered structural cases shows apparent tendencies to opening the energy gap near the Fermi level for the stoichiometric AgSbX 2 compositions, but a small overlap between valence and conduction bands is still present. Such electronic structure behaviour well agrees with the semi-metallic properties of the analyzed samples.

Introduction

The ternary chalcogenides AgSbSe 2 and AgSbTe 2 belongs to family of semiconductors with disordered NaCl cubic structure (s.g. Fm3m) in which silver and antimony occupy metal sublattice [START_REF] Geller | Ternary Semiconducting Compounds witch Sodium Chloride-Like Structure: AgSbSe 2 , AgSbTe 2 , AgBiS 2 , AgBiSe 2[END_REF][START_REF] Wernick | New semiconducting ternary compounds[END_REF][START_REF] Wolfe | Anomalous Hall Effect in AgSbTe 2[END_REF]. Alloys of both compounds either in single-crystal form or in thin-film form have received considerable interest owing to their optical and electronic properties. They are attractive phase-change (PC) materials used as a switching medium in rewritable optical memories [START_REF] Wang | Assessment of Se-based phase change alloy as a candidate for non-volatile electronic memory applications[END_REF][START_REF] Xu | Crystallization and C-RAM application of Agdoped Sb 2 Te 3 material[END_REF][START_REF] Zayed | Optical absorption behavior of AgSbTe 2 thin films[END_REF][START_REF] Soliman | Optical properties of thermally vacuum evaporated AgSbSe 2 thin films[END_REF][START_REF] Wand | Phase change properties of ternary AgSbSe 2 chalcogenide films[END_REF][START_REF] Patel | Effect of Substrate Temperature On the Crystalline of AgSbSe 2 Films[END_REF][START_REF] Abdelghany | Electrical conductivity and thermoelectric power of AgSbSe 2[END_REF][START_REF] Patel | Growth and Crystallization of AgSbSe 2 Films[END_REF].

Both compounds are also very interesting due to their thermoelectric properties (see Table1), especially very low thermal conductivity λ < 1 Wm -1 K -1 which is a consequence of their disordered structure. Cubic AgSbTe 2 is known as a good p-type thermoelectric material, but due to its thermodynamical instability below 360°C it slowly decomposes to secondary phases: α-Ag 2 Te and Sb 2 Te 3 , what limits its practical application. It is expected that alloying of AgSbTe 2 with other cubic compounds, such us: GeTe (so called TAGS alloys [START_REF] Rowe | Handbook of Thermoelectrics[END_REF]), PbTe and SnTe, can help to stabilize the NaCl structure. However, these alloys are also known to be complicated by their inhomogeneity, despite reports of complete solid solutions in phase diagram determinations [START_REF] Chen | Macroscopic thermoelectric inhomogeneities in (AgSbTe 2 ) x (PbTe) 1-x[END_REF]. Exceptionally high thermoelectric figure of merit ZT = 2, has been reported for (Ag 1-y SbTe 2 ) 0.05 (PbTe) 0.95 , which may involve the nanoscale second-phase inclusions. Authors believe that these inclusions are responsible for quantum effects increasing thermoelectric figure of merit ZT. Although, conflicting reports on the same materials claim only ZT of 1 or less. Chen et al. [START_REF] Chen | Macroscopic thermoelectric inhomogeneities in (AgSbTe 2 ) x (PbTe) 1-x[END_REF] have shown that these materials are multiphase on the scale of millimetres despite appearing homogeneous by X-ray diffraction and routine electron microscopy. Using a scanning Seebeck microprobe, authors found significant variation of Seebeck coefficient including both n-type and p-type behaviour in the same sample. It can not be excluded that complicated behaviour of the alloys can be result of the complex nature of their parent compounds.

A c c e p t e d m a n u s c r i p t

Despite that AgSbSe 2 and AgSbTe 2 based materials are widely investigated, the basic information about properties of pure compounds, e.g. the nature of the disorder or electronic structure is still not quite clear.

Experimental and theoretical details

The starting materials were prepared by direct fusion of stoichiometric amounts of their constituent elements (Ag, Sb, Se, Te) of purity 99.99% in sealed, evacuated silica tubes covered inside with thin layer of pyrolytical carbon. Tubes were heated in temperature controlled, cradled furnace at 700°C for 1 h, with continuous swinging to complete mixing and reaction. Tubes were gradually cooled to room temperature to obtain polycrystalline ingots. The materials were ground and hot-pressed in graphite dies (argon atmosphere, t = 20 min, p = 30 MPa, at T = 410 °C) and slowly cooled down with rate of 1 °C/min to the room temperature in order to avoid fracturing. Because AgSbTe 2 can undergo decomposition below 360°C some of AgSbTe 2 samples were heated in evacuated quartz ampoules to 450°C and rapidly cooled down by quenching in water, in order to obtain single-phased materials. The samples of 10 mm in diameter and about 20 mm in height, were cut with a diamond saw and polished.

The prepared materials were characterized by X-ray diffractometer (X'Peret Philips system, with filtered CuK α radiation) and scanning electron microscopy (JEOL JSM-840)

with an electron-probe microanalysis apparatus (EPMA). The lattice parameters were calculated from the experimental X-ray patterns using FullProf refinement program implementing Rietveld method. Mass densities were determined using the immersion technique with water as the liquid.

The electrical and thermal transport properties were measured over the temperature range from 300 to 600 K. The electrical conductivity was determined by four-probe AC technique.

The Seebeck coefficient and thermal conductivity measurements were conducted in radiation- Electronic structure computations have been performed using full potential Green function Korringa-Kohn-Rostoker method [START_REF] Yoneda | Temperature dependence of the figure-of-merit of[END_REF][START_REF] Bansil | Electronic structure and magnetism of Fe 3-x V x X (X=Si, Ga, and Al) alloys by the KKR-CPA method[END_REF][START_REF] Wojciechowski | Thermoelectric properties and electronic structure of CoSb 3 doped with Se and Te[END_REF] in ordered approximants of AgSbX 2 (see, Sec. 3.1) as well as hypothetical AgX and SbX. To account for chemical disorder in NaCltype Ag 1-x Sb x X alloys, the coherent potential approximation (CPA) has been incorporated.

The crystal potential of muffin-tin form has been constructed within the LDA framework, using von Barth-Hedin parametrization for the exchange-correlation part. For well-converged charges and potentials, the total, site-decomposed and l-decomposed density of states (DOS)

were computed applying k-space tetrahedron integration. The Fermi level was determined from the generalised Lloyd formula [START_REF] Stopa | Linear aspects of the Korringa-Kohn-Rostoker formalism[END_REF][START_REF] Kaprzyk | Green's function and a generalized Lloyd formula for the density of states in disordered muffin-tin alloys[END_REF]. The electronic properties of fully disordered system within the NaCl-type structure, corresponding to the chemical formula Ag 0.5 Sb 0.5 X A c c e p t e d m a n u s c r i p t

(

Results and discussion

Structural analysis

Scanning electron microscopy (SEM) observations with simultaneous EPMA analysis of the samples revealed that the resulting materials had an uniform microstructure and chemical composition. The AgSbTe 2 samples contained well-formed grains with sizes ranging from 5

to 50 µm (Fig. 1a) . The AgSbSe 2 samples had a glass-like texture without visible grain borders (Fig. 1b). The measured densities were found to be 98% of the theoretical value.

XRD analysis confirmed that obtained AgSbSe 2 samples were single-phased. In case of AgSbTe 2 only small amounts (appr. <3%) of secondary phase inclusions were observed.

The Rietveld method was used for refining of the structure parameters for the disordered NaCl structure model (Fm3m) as well as different models of ordered ABX 2 -type structures.

There were considered the most common superstructures of NaCl: e.g.

α- NaFeO 2 (R-3m), LiTbS 2 (Fd-3m) and γ- LiFeO 2 (I4 1 /amd).
The final results of refinement for the AgSbSe 2 material are presented in the Table 2.

Analysis of "goodness of fit" statistical parameters for the refinement results, such as: χ 2 , R wp , and Durbin-Watson statistics, show that all the models describe experimental data practically with the same precision and therefore they are indistinguishable by these criteria.

Moreover, in all calculated, theoretical X-ray diffraction patterns, intensities of characteristic superstructure reflections are lower than the experimental background signal. These results are a consequence of very similar (different only of about 5%) X-ray scattering factors of Ag and Sb for CuK α  radiation. Therefore, it seems that the nature of ordering in the AgSbSe 2 and AgSbTe 2 structure can not be easily investigated by classical X-ray diffraction techniques.

However, preliminary structural calculations show that such analysis would be possible by using neutron radiation method.

Transport properties

A c c e p t e d m a n u s c r i p t

Studies of temperature dependences of electrical conductivity revealed that AgSbSe 2 and AgSbTe 2 exhibit semimetallic/semiconducting behavior. The activation energies E a for both compounds, determined using an Arrhenius law, are about 0.03 eV and remain close to another result of 0.09 eV obtained from electrical investigations (Table 1). On the other hand, these data are much lower than values of direct band gap energy E In this model, the Seebeck coefficient α can be expressed as follows:

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - - = η η η α ) ( ) ( 2 0 1 F F e k B ( 2
)
The carrier concentration n can be expressed as follows:

) ( 2 4 2 / 1 2 / 3 2 * η π F h T k m n B ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ = (3)
where: k B is the Boltzmann's constant, η -reduced Fermi energy, F x -Fermi integral of order

x, m * -effective mass, T-absolute temperature.

A c c e p t e d m a n u s c r i p t

At first, we calculated the reduced Fermi levels from the experimental Seebeck coefficient data using Eq. ( 2), then determined electron effective masses from Eq. (3) using the calculated reduced Fermi energies and the experimental Hall carrier concentration values.

The calculated effective masses of carriers are significantly different: 2.7 for AgSbTe 2 and 0.7 for AgSbSe 2 , despite of comparable mobility (40-50 cm 2 s -1 V -1 ). Dissimilar effective masses m * can indicate some differences in band structure of both compounds near the Fermi energy.

The samples of AgSbSe 2 and AgSbTe 2 exhibit very low thermal conductivity (see Table 3). The measured value for AgSbSe 2 at room temperature is with very good agreement with literature data (Table 1), whereas thermal conductivity of AgSbTe 2 is higher than earlier reported value (0.62 W•m -1 K -1 ). The discrepancy can be result of different amount of secondary phase inclusions and larger electrical conductivity.

Total thermal conductivity λ of solids is the sum of carrier λ el. and lattice λ latt components:

latt el λ λ λ + = (7)
In order to explain impact of the electronic contribution on thermal conductivity, the Wiedemann-Franz-Lorenz law was applied and the values of thermal conductivity λ el were estimated:

σ λ LT el = ( 8
)
where L is the Lorenz number which may vary from 1.485 • 10 -8 V 2 K -2 for nondegenerated semiconductors to 2.443 • 10 -8 V 2 K -2 for metals and for strongly degenerated semiconductors.

However, above Lorenz number are not applicable for partially degenerated semiconductors. Therefore, an attempt have been undertaken to determine the Lorenz number L and the electronic part of thermal conductivity with the use of Fermi-Dirac statistics as follows:

A c c e p t e d m a n u s c r i p t

⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎭ ⎬ ⎫ ⎩ ⎨ ⎧ ⋅ + + - ⋅ + + = + + 2 1 2 2 2 ) ( ) ( 1 2 ) ( ) ( 1 3 η η η η r r r r B F F r r F F r r e k L ( 9
)
The Fermi level values, which were used in the above calculations, alike those of carrier effective masses, were estimated with the use of absolute Seebeck coefficient experimental data (Eq. 4.). The results of computations are presented in Table 3; they indicate that about 18% and 5% of the total thermal conductivity, for AgSbTe 2 and AgSbSe 2 respectively, can be contributed to its electronic part.

The thermoelectric figure of merit ZT which can be expressed as:

T ZT l el λ λ σ α + = 2 (10)
is commonly used parameter for evaluation of thermoelectric materials.

Maximal values of ZT were measured at temperature of 525 K and are equal about 0.2 and 0.1 for AgSbTe 2 and AgSbSe 2 respectively. However, optimization of carrier concentration (e.g. by doping) should enhance properties of both materials.

Electronic structure

Binary compounds AgX and SbX

The investigations of electronic structure characteristics in hypothetical AgX and SbX allowed to get more insight into formation of semimetallic properties in AgSbX 2 . From Fig. 4, presenting densities of states (DOS) in AgX and SbX, one learns that there is a deep valley (or a true energy gap) in the electronic spectra, above the 8 th or 18 th band in the case of SbX and AgX, respectively. Since the binary compounds possess the valence electron number (VEN) different from the aforementioned ones, i.e. VEN = 11 in SbX and VEN = 17 in AgX, they exhibit metallic ground state. Electronic structure of AgX consists of lower lying s-like states (containing 2 electrons), which comes essentially from X atom, and the block of presumably d-Ag and p-X states,

A c c e p t e d m a n u s c r i p t

enabling to accommodate 16 electrons. The semiconducting or semimetallic properties would be expected if all these bands are completely filled (VEN = 18). Since AgX possess 17 valence electrons (if accounting for s-X states), the Fermi level (E F ) is placed in upper region of the valence bands (VB). Consequently, AgX (VEN=17) can be viewed as one-hole compounds, since one electron must be added to reach the true energy gap (or a deep DOS valley).

Electronic structure of SbX consists of four successive blocks of bands. The lower lying two separate and narrow s-DOS peaks arrive from X (the lowest band) and Sb atoms. The higher lying two blocks of strongly hybridised p-states from X and Sb, are separated by a true gap or a deep DOS minimum seen in SbSe and SbTe, respectively. Hence, the semiconducting properties would be expected for VEN = 8 and then SbX (VEN = 9) systems can be viewed as one-electron compounds due to the Fermi level falling at the bottom of the conduction band (CB). From the analysis of hypothetical binary parent compounds, we see that there is a slight difference between selenides and tellurides concerning electronic states near E F , i.e. the presence of the energy gap separating VB and CB in AgSe and SbSe, whereas only a deep DOS minimum in AgTe and SbTe. This behaviour will have some consequences on differences in electronic structure of AgSbX 2 .

Ternary ordered AgSbX 2

Our computations of different crystallographically ordered approximants of AgSbX 2

(resulting in the X-ray diffraction patterns similar to that received for disordered NaCl-type structure, see Table 4) show that their ground state electronic properties are really very close.

In all considered cases, the Fermi level (E F ) was found either in the deep valley (semimetallic-like state) due to a small overlap of VB and CB, (Fig. 5) or it exhibits a pseudogap behaviour (VB and CB edges contact at E F ), depending on different atomic arrangements.

Thus, the question whether AgSbX 2 may form superstructures different from NaCl-type alloy

A c c e p t e d m a n u s c r i p t

should be addressed to the forthcoming neutron diffraction measurements as well as the accurate electronic structure computations including the analysis of the total energy. At the moment we focus more attention on electronic structure of the disordered model of AgSbX 2 , which may serve as a reference case for further consideration of possible atomic superstructures. Noteworthy, the fully disordered model of AgSbX 2 is widely accepted in the literature.

Fully disordered Ag 0.5 Sb 0.5 X

The appearance of semimetallic properties in the NaCl-type disordered AgSbX 2 is not so straightforward from the aforementioned analysis (see, Sec. 3.3.1) of the binary parent compounds (and predictions of rigid band model). The Fermi level was supposed to fall into the gap at VEN = 8 or VEN = 18 (accounting for d-shell), which can be expected when adding/removing one or three electrons to/from AgX or SbX, respectively (antimony possesses four electrons more than silver). In Ag 1-x Sb x X alloy, Ag 4d-states are noticeably present in the valence band but they are almost completely filled, which can be detected from almost vanishing d-contribution to total DOS above E F (Fig. 4). In Sb (Z = 51), due to more attractive crystal potential comparing to that of Ag (Z = 47), the 4d-states are practically expelled from the valence states (they form a semi-core levels) and only sp-states are present.

Hence, the onset of pseudo-gap on DOS can not be expected neither at VEN = 8 nor at VEN = 18, due to specific hybridization of different type of orbitals (d-orbitals of Ag and p-orbitals of Sb) in alloyed system. Fig. 5 illustrates the DOS evolution in Ag 1-x Sb x X, which can not be interpreted in terms of rigid band behaviour and a simple shift of E F with the variation of the number of valence electrons. Since the change in the relative Ag/Sb content strongly modifies the population of d-type Ag and sp-type Sb orbitals (and also their hybridization), one can establish the simple rule for appearance of the pseudo-gap at the Fermi level in Ag 0.5 Sb 0. and X -2 . The energy gap above the 7 th band in Ag 0.5 Sb 0.5 X was partly confirmed in view of accurate KKR-CPA computations (Fig. 6). However, our results rather indicate a pseudo-gap behaviour than the appearance of the band gap at E F in both compounds. Furthermore, the evolution of DOS in both series of compounds shows subtle differences, since Ag 0.5 Sb 0.5 Te exhibits slightly higher DOS value at E F than Ag 0.5 Sb 0.5 Se. This feature well corroborates with electronic properties detected in binary (true energy gap in selenides against a deep valley in tellurides). Interestingly, more 'metallic-like' character of Ag 0.5 Sb 0.5 Te (lower thermopower and electrical resistivity) with respect to Ag 0.5 Sb 0.5 Se was observed from our electron transport properties measurements.

However, accurate investigations of the Fermi surface properties (including computations of k-dependent electron group velocities and life-times [20]) are necessary to obtain more quantitative insight into the thermopower behaviour of these compounds.

Conclusions

We have studied the crystallographic and physical properties of AgSbSe 2 and AgSbTe 2 polycrystalline samples. Structural analysis using Rietveld refinement method shows that the nature of the disorder can not be handling by X-ray powder diffraction method due to similar scattering factors for Ag and Sb atoms. However, preliminary calculations show that such analysis would be possible by using neutron radiation method, what will be undertaken in further investigations.

Both compounds exhibit promising thermoelectric properties, in particular very low Besides, we suggest that the presence of low DOS near E F in AgSbX 2 allows easily tuning the Seebeck coefficient sign upon suitable doping/substitution. However, n-type AgSbX 2 compounds are expected to have smaller thermopower than the presented here p-type materials, as can be roughly detected on the KKR-CPA DOS.

A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t

  shielded vacuum probe by forcing variable heat flux across the sample and measuring corresponding linear variations of temperature differences and thermoelectric voltage in steady-state conditions. Heat flux, passing by the cold side of the sample, was measured by previously calibrated heat-flux sensor. The results were used for precise calculations of increased heat losses due to very low thermal conductivity of measured samples. The Hall coefficient R H was measured using low frequency (7 Hz) AC sample current of 50 to100 mA/mm 2 in a constant magnetic field of 0.705 T at the room temperature. Carrier concentrations were evaluated from the Hall coefficient R H , assuming a Hall scattering factor A equal to 1.0.

  3 to 0.71 eV) measured by optical method for intrinsic absorption region. It may suggest that results of electrical investigations correspond rather to activation energy of extrinsic charge carriers, but another explanation of discrepancies may lay in significant differences in microstructure of samples (thin layer vs. bulk polycrystalline material).The magnitude of the Seebeck coefficient of AgSbTe 2 at the room temperature is consistent with semimetallic behavior of this sample (68 µV K -1 ). The Seebeck coefficient for AgSbSe 2 is much larger (320 µV K -1 ) what can be an indication of more semiconducting-like character of this compound. Both materials exhibit p-type of conductivity and relatively large Hall concentration of carriers of 1•10 19 and 5•10 19 cm -3 for AgSbSe 2 and AgSbTe 2 respectively. The Seebeck coefficient and Hall carrier concentration data were used to estimation of effective mass of carriers m * , assuming a single parabolic band model with acoustic phonon scattering as a predominant carrier scattering mechanism.

5 XA c c e p t e d m a n u s c r i p t VEN=14.

 5t from the following relation : VEN = 0.5 x 11 [sd-Ag] + 0.5 x 5 [sp-Sb] + 6 [sp-Se], yielding The above-mentioned VEN rule is in line with the simple ionic condition leading to a saturation of chemical bonds and then an onset of the semiconducting state in AgSbX 2 system, if accounting for the expected valence states of the constituent atoms, i.e. Ag + , Sb+3

A c c e p t e d m a n u s c r i p t

  thermal conductivity and quite large, positive Seebeck coefficient. Temperature dependence of electrical conductivity points out semiconducting with a very narrow band (E g ~ 30 meV) or semimetallic properties of these materials. The AgSbSe 2 and AgSbTe 2 compounds exhibit p-type conductivity, what suggests that Fermi level is shifted on the valence bands edge.The electronic structure calculations in fully disordered as well as in different ordered models of AgSbX 2 confirm practically in all cases that their ground state properties are semimetallic due to a slight overlapping of conduction and valence bands near the Fermi energy. Interestingly, in AgSbSe 2 the calculated DOS value is about twice smaller than the corresponding value in AgSbTe 2 . All the DOS features well support electrical resistivity measurements exhibiting ρ weakly dependent with T (resulting in the activation energy gap close to zero in both systems) as well as the ρ values larger for AgSbSe 2 . Moreover, the measured RT thermopower, which is much larger in AgSbSe 2 (α = 320 µV K -1 ) than in AgSbTe 2 (α = 68 µV K -1 ), can be related to relatively stronger variation of valence-like DOS as well as lower N(E F ) in the former.

Fig. 1 SEMFig. 2

 12 Fig. 1 SEM picture of the fracture of a) AgSbTe 2 and b) AgSbSe 2 sample.

A c c e p t e d m a n u s c r i p tFig. 3 Fig. 4 Fig. 5 A c c e p t e d m a n u s c r i p tFig. 6

 3456 Fig. 3 Temperature dependence of the Seebeck coefficient values of AgSbSe 2 and AgSbTe 2 .

Table 1 Selected physical properties of AgSbSe 2 and AgSbTe 2 .

 1

	Parameter		AgSbTe 2	AgSbSe 2
	Space group		Fm3m	Fm3m
	Cell size [Å]	6.078 [1]	5.786 [1]
	Density [g•cm -3 ]	7.12 [1]	6.60 [1]
	Band gap [eV]	0.71 ** [6] 1.65 *** [6]	0.091 * [11] 0.34 * [10] 1.03 *** [7]
	Thermal [W•m -1 •K -1 ]	conductivity	0.64 [3]	0.82 [9]
	Electrical conductivity [S•cm -1 ]	130-150 [3]	154 [14]
	Seebeck coeff. [µV•K -1 ]	165-240 [3]
	Melting point [°C]	555 [2]	610 [2]
	* electrical measurements; ** direct gap -optical measurements;
	*** indirect gap -optical measurements

Table 2 . Results of Rietveld refinement for the disordered structure (Fm3m) and selected possible ordered systems of AgSbSe 2 .

 2

	Short-range order	s.g.	a [Å]	b [Å]	c [Å]	χ 2	R wp	DW-stat.
	disordered	Fm3m	5.7883	-	-	2.15	27.3	0.51
		Superstructures (short-range ordered structures)
	A1	R-3m	4.0933 4.0933 20.0475 2.25	28	0.48
	B1	Fd-3m	11.5766	-	-	2.15	27.3	0.51
	F1	I4 1 /amd	5.7883 5.7883 11.5766 2.25	28	0.48
	E1	Pmmn	5.7883	-	-	2.15	27.3	0.50

Table 3 . Thermoelectric properties of undoped AgSbTe 2 and AgSbSe 2 samples at room temperature.

 3 Elec. contr. of therm. conduct. λ el [W•m

	Parameter	AgSbTe 2	AgSbSe 2
	Cell size a [Å]	6.0816	5.7883
	Density ρ [g•cm -3 ]	7.15	6.60
	Band gap E g [meV]	26	30
	Electrical conductivity σ [S•cm -1 ]	332	79
	Seebeck coeff. [µV•K -1 ] (at 320 K)	68	320
	Thermal conductivity λ [W•m -1 •K -1 ]	1.15	0.81
	-1 K -1 ]	0.2	0.04
	Type of majority carriers	p	p
	Hall carrier concentration n [cm -3 ]	5.0 • 10 19	1.0 • 10 19
	Hall mobility µ [cm 2 s -1 V -1 ]	41	49.2
	Carrier effective mass m*/m 0	2.7	0.7
	ZT parameter (at 525 K)	0.2	0.1

Table 4 Results of computations for the disordered structure (Fm3m) and selected possible ordered systems of AgSbSe 2 .

 4

	Short-range		Band gap	Position of the
		s.g.
	order		E g	Fermi level E f
	disordered	Fm3m	semimetal	minimum of DOS
	A1	R-3m	semimetal	valence bands
	B1	Fd-3m	~0.1 eV	minimum of DOS
	F1	I4 1 /amd	semimetal	conduction bands
	E1	Pmmn	semimetal	valence bands

Acknowledgments

The work was supported by The EEA Financial Mechanism & The Norwegian Financial Mechanism, (grant No PL0089-SGE-00104-E-V2-EEA, 2007-2010).

A c c e p t e d m a n u s c r i p t


T Djouama

A Boutarfaia

M Poulain

Fluorophosphate glasses containing manganese

Keywords: fluorophosphate glasses, thermal properties, optical properties, IR transmission, physical properties

New fluorophosphate glasses based on MnF 2 , NaPO 3 and MF n (M = Zn 2+ , Sr 2+ , Mg 2+ , Ba 2+ , Li + , Na + and K + ) have been synthesised and characterized. Large vitreous areas were observed. Samples of 4 mm in thickness have been obtained. These glasses are easy to prepare and stable in ambient air. Depending on the composition and the nature of the M cation, glass transition temperature, Tg, lies between 230 and 314°C, crystallisation temperature, Tx is between 320 and 475°C. These glasses are pink coloured, and infrared transmission extends up to 4.5 m with extrinsic OH -absorption band at 3200 cm -1 and other bands around 2200 and 1600 cm -1 that relate to PO 4 tetrahedron vibration. Other physical properties including density, microhardness, Young modulus, thermal expansion and refractive index were investigated and correlated to composition.

Introduction

Numerous fluorophosphate glasses have been studied for their potential applications [1][2][3][4][5]. Most of them are based on alkali, earth alkali or trivalent polyphosphates with the general formula M(PO 3 ) n , with n = 1, 2 or 3. Among other features, they may have a good transmission in the UV spectrum [4] and host rare earth cations, leading to active optical devices.

This study focuses on fluorophosphate glasses containing manganese difluoride MnF 2 , both for fundamental studies and for special optical uses, taking advantage of fluorescence and magnetic properties of divalent manganese.

The starting point of our work is the glass formation in binary fluoride systems that combine MnF 2 with other fluorides [6]. While such glasses have been characterized, their stability against devitrification is limited as they require fast cooling rates, which does not allow thick samples to be obtained. The incorporation of sodium polyphosphate (Na(PO 3 )) n

A c c e p t e d m a n u s c r i p t

2 appears as a simple way to increase glass stability, as this has already been reported in the Na(PO 3 )-BaF 2 -MnF 2 ternary system [7]. This additive keeps a low temperature of melting, which limits fluorine losses that occur with the formation of gaseous POF 3 . While pure Na(PO 3 ) glass is somewhat sensitive to water, the incorporation of insoluble fluoride increases chemical durability, especially in wet atmosphere: sample surface remains clear even at high relative humidity.

The systematic investigation of the glass forming ranges in the ternary systems MnF 2 -NaPO 3 -MF n (M = Zn 2+ , Sr 2+ , Mg 2+ , Ba 2+ , Li + , Na + and K + ) has been carried out. The evolution of the physical properties versus chemical composition has also been implemented.

Experimental Starting Materials

Fluorides ZnF 2 , SrF 2 , MgF 2 , BaF 2 , NaF, LiF and KF are commercial products from ALDRICH and WWR, with a 99.9 % purity. Ammonium hydrogenofluoride NH 4 F.HF is from Riedel de Haen and from WWR (Prolabo).

Manganese difluoride has been synthesized in the laboratory, using MnO and NH 4 F.HF as a fluorinating reagent in a platinum crucible, and controlled by X-Ray diffraction.

Glass synthesis

Glasses are prepared at room atmosphere using the ammonium bifluoride processing [8]. The calculated amount of the starting materials is introduced in a long platinum crucible with an excess of NH 4 F,HF. This reagent acts as a fluorinating reagent to convert oxides in fluorides and also to protect the melt from atmospheric hydrolysis. After melting is complete, a clear liquid is obtained. Then this liquid is briefly heated at higher temperature for the fining step and cooled into the crucible to a temperature low enough to have larger viscosity without visible crystallization. Finally it is cast onto a brass mould and squeezed by another metallic plate. The resulting sample corresponds to a "quenched glass"; its thickness lies between 0.4 and 1 mm, and effective cooling rate may reach 1000 K.s -1 . Thicker samples are obtained when melt is cast between two brass walls that are heated at temperature close to Tg. A final annealing stage is carried out for 6 hours at temperature : T a = (Tg -10)°C, to remove residual thermal stresses prior to polishing.

Characterizations

Characteristic temperatures (Tg for glass transition, Tx for onset of crystallization and Tp for maximum of crystallization peak) were determined by differential scanning calorimeter (DSC) using a SEIKO DSC 220 set-up. The estimated error on the temperature is 2 K for glass transition and onset of crystallization which are obtained from tangents intersection and 1 K for the position of the crystallization peak. Samples, 10 to 20 mg in weight, were set in aluminum pans under N 2 atmosphere at 10 K min heating rate. Thermal expansion is measured using a TMA 2840 (TA Instruments) set-up, at 2 K/min heating rate, between 50 °C and 200 °C. Estimated experimental error is = ±2x 10 -7 K -1 . The densities were determined by gas pycnometry using He (Micromeritics Accupyc), with a precision of about 10 -4 . Microhardness (Vickers) is measured using a Matsuzawa MXT set-up with 50 g applied load for 10 seconds. A mean value is obtained after averaging several Infrared transmission spectra were recorded with a BOMEM Michelson Spectrophotometer in the 5000-2000 cm -1 range. Glass samples were either plates, 1 to 3 mm in thickness, or powdered glass in KBr pellets. UV transmission between 300 and 500 nm was studied using a Varian spectrophotometer Cary 5. In both cases, data were given directly in transmittance and/or absorbance. Refractive index in the visible spectrum, n D ( = 0.5893 m), was measured using an Abbe refractometer.

The chemical composition of glasses has been checked by element analysis using energy dispersion spectrometry EDS (Oxford Link ISIS) set-up incorporated into the ISM 6400 Jeol scanning electron microscope.

Results

Glass formation in the MnF 2 -NaPO 3 -MF n ternary systems.

Vitreous areas

Glass forming ranges have been investigated in ternary systems MnF 2 -NaPO 3 -MF n (M = Zn, Sr, Mg and Ba; Li, Na and K). Experimental results are gathered in Fig. 1a-g. Glass formation limits depend on cooling rate. Larger cooling rates are required when devitrification rate increases, that is for less stable glasses. Note that NaPO 3 itself may form a glass, and consequently makes the starting point for all vitreous areas. Manganese-containing glasses are pink and become darker at higher Mn content. Samples are stable at room atmosphere, except for a few compositions with high NaPO 3 concentration and MnF 2 content lower than 5 %.

Glass compositions

The chemical composition of the samples is defined by the relative amount of the starting materials. Taking into account the processing temperature, cations (Na, Mn, Zn, K, Sr, Ba) will not enter the gaseous phase. However there may be exchanges with atmosphere based on oxidation, hydrolysis and volatilization, especially HF and POF 3 molecules may form. Analyses have been carried out on some samples to compare nominal and real compositions. As reported in table 1 for a typical sample, the discrepancy between expected and observed values is limited. Complementary analyses emphasize that phosphorus and fluorine losses are not significant.

Thermal Properties

Glasses in the MnF 2 -NaPO 3 -ZnF 2 system

Glass stability versus devitrification may be estimated from the characteristic temperatures measured by DSC. While this assessment is not always accurate, it is widely acknowledged that the Tx-Tg difference between the temperature of glass transition and the onset of crystallization is strongly correlated to crystallization tendency [9]. Systematic measurements have been implemented on glasses from the MnF 2 -NaPO 3 -ZnF 2 system. Typical DSC curves appear in figures 2. The evolution of glass transition temperature Tg and thermal stability

Glasses in the 30 MnF 2 -50 NaPO 3 -20 MF n system

A second set of characterizations has been implemented on ternary fluorophosphate glasses with the general formula 30MnF 2 -50NaPO 3 -20MF n (M = Li, Na, K, Zn, Sr, Ba, Mg.) . Most of these glasses may be obtained as thick samples. Figure 4 makes the comparison between the characteristic temperatures and the stability range of these glasses. The lowest Tg are observed for lithium and zinc fluoride, while maximum stability is obtained with lithium, zinc and barium. Considering the earth alkali cations, the Tg value drops with increasing ionic radius, from 326 °C for Mg to 277 °C for Sr and 268 °C for Ba.

Glasses in the 30 MnF 2 -x NaPO 3 -y MF (M = alkali metal) systems

Another set of thermal measurements has been implemented to find the optimum alkali concentration to be incorporated in the MnF 2 -NaPO 3 binary system. While glass forming range is drawn in Fig. 1e-g, samples have been selected with the composition 30MnF 2 -(70x)NaPO 3 -xMF, (x = 10, 20, 30). As shown in figure 5, glass transition temperature and stability factor decrease with rising MF content, suggesting that optimum MF concentration is close to 10 %.

According to the confusion principle, glasses more stable against devitrification should be obtained by the addition of two alkali fluorides instead of one. A series of glass samples has been prepared to investigate this possibility according to the composition rule 30MnF 2 -40 NaPO 3 -(30-x)LiF-xKF. Corresponding thermal data that appear in table 2 confirm that these quaternary glasses are more stable than the basic ternary glass.

Note that Tg increases as K substitutes Li and most stable glasses (i.e. larger thermal stability range) correspond to 10 to 15 % KF content.

Other physical properties

Density

MnF 2 -NaPO 3 -ZnF 2 glass Two sets of samples have been prepared according to the general formula : xMnF 2 -(80-x) NaPO 3 -20ZnF 2 and xMnF 2 -(90-x)NaPO 3 -10ZnF 2 . From figure 6, it appears that density increases as MnF 2 substitutes NaPO 3 , and this evolution is roughly linear, density ranging from 2.80 and 3.36 g/cm 3 .

MnF 2 -NaPO 3 -MF (M = Li, K) glass

Alkali-containing glasses have been investigated. Incorporation of heavier alkali cations into MnF 2 -NaPO 3 -MF system leads to decreasing density. Experimental values are reported in table 2 for the 30MnF 2 -40NaPO 3 -(30-x)LiF-xKF glasses.

Thermal expansion

A c c e p t e d m a n u s c r i p t 5

As reported in table 3, the coefficient of thermal expansion (CTE) of glasses in the MnF 2 -NaPO 3 -ZnF 2 system is close to 188 x 10 -7 K -1 and variations resulting from composition changes are very small: neither the NaPO 3 /MnF 2 nor the ZnF 2 /MnF 2 substitutions influence significantly the CTE. But we can say that it increases slightly with the size of the third cation (Zn, Sr, Ba, K). The largest value is observed with the glass containing BaF 2 . These CTE values are fairly high and compare to those of fluorozirconate glasses.

Mechanical properties

The microhardness "Hv" evolution versus MnF 2 content is shown in figure 7 for a set of samples with the formula: xMnF 2 -(80-x)NaPO 3 -20ZnF 2 . There is a linear correlation between microhardness and MnF 2 concentration. These values are smaller than those observed for standard fluoride glasses [10].

For the alkali-containing glasses, microhardness increases with the ionic radius of the alkali cation. Indeed, in the glass 30MnF 2 -40NaPO 3 -30MF, the Hv value grows from 179 to 208 kg/mm 2 when potassium replaces lithium.

Sound velocity has been measured in a series of samples with variable manganese content. Young modulus and Poisson ratios are deduced from these measurements. Results are reported in table 4 and show that these moduli are larger at high MnF 2 content. Young modulus ranges between 39 an 60 GPa, which is close to the values measured for fluoride glasses [START_REF] Parker | Fluoride Glass Optical Fibres[END_REF].

Chemical durability

Samples are stable at room atmosphere. However, some compositions may be sensitive to high relative humidity, for example the 80NaPO 3 -20ZnF 2 glass and more generally samples with high NaPO 3 concentration. The surface corrosion that occurs may be the result of water condensation. A set of qualitative tests has been implemented on various samples, based on immersion in deionised water at 20 °C for 48 hours. The general trend is that corrosion layer is smaller when manganese fluoride content increases. NaPO 3 , KF and NaF enhance water attack. For example, using the 30MnF 2 -50NaPO 3 -20ZnF 2 composition, the weight loss of a glass sample, 2.6 g in weight and 7 cm 2 in outer surface, is only 1 mg.

Optical properties

Refractive index

Refractive index has been measured for samples of variable composition in the MnF 2 -NaPO 3 -ZnF 2. system. As shown in figure 8a, it increases from 1.50 to 1.52 when MnF 2 substitutes NaPO 3 . By comparison, alkali-containing glasses have a lower refractive index. For example, we measured n D = 1.516 for 30MnF 2 -50NaPO 3 -20ZnF 2 glass while it is only 1.480 for the 30MnF 2 -50NaPO 3 -20KF glass. Higher values are obtained with lithium as this is shown in figure 8b for the 30MnF 2 -40NaPO 3 -(30-x)LiF-xKF glasses. Divalent manganese induces optical absorption in the UV-visible spectrum. This is exemplified by figure 9 that shows the transmission spectra of samples containing an increasing amount of Mn 2+ . Binary NaPO 3 -ZnF 2 glass is colourless and the UV cut-off is below 300 nm. The UV absorption edge is shifted towards longer wavelength as MnF 2 concentration increases. Curves show one shoulder at 350 nm and two absorption bands located around 400 and 500 nm that correspond to transitions of Mn 2+ ions in octahedral sites [START_REF] Feuerhelm | [END_REF]13].

Optical transmission

b) Infrared transmission

Bulk samples

The transmission spectrum of the 30MnF 2 -50NaPO 3 -20ZnF 2 glass has been recorded between 100 and 5000 cm -1 and is reproduced in figure 10. The thinner sample (dashed line) evidences the complex structure of the absorption bands. The main features that emerge from this figure are: -The absorption band around 3200 cm -1 that originates from hydroxyl groups.

-An intense band at 2200 cm -1 that defines the transmission limit of the sample.

-Another band around 1600 cm -1 .

Powdered samples Infrared transmission of powdered samples of the same glass in KBr pellets is shown in figure 11. The spectrum is limited to 400 cm -1 , as KBr is no more transparent beyond this frequency. The comparison with bulk glass shows that the absorption observed between 1500 and 2500 cm -1 corresponds to the first overtones of the fundamental absorption bands of the PO 4 tetrahedra [14,15].

Discussion

As one could expect, sodium polyphosphate makes an efficient stabilizer of the fluoride glasses based on MnF 2 . Thick samples can be obtained, which facilitates mechanical and optical measurements.

The evolution of the physical properties versus composition logically relates to the intrinsic features of the chemical elements and also to glass structure. Main elements may be summarized as follows:

1) Phosphorus forms tetrahedra PX 4 in which X = O or F, predominantly O.

2) Manganese and zinc form MnX 6 octahedra (M = O or F) and ZnF 6 octahedra respectively [16]. 3) Sodium is considered as a network modifier, and its coordination number is large in the solid state: from 6 in NaF to 12 in NaWO 3 [START_REF] Wells | Structural Inorganic Chemistry[END_REF]. 4) Bond strength of P-O (110 kcal/mol) bond is larger than that of Mn-O, Zn-O and Na-O bonds [START_REF] Sun | [END_REF]. 5) Ionic radii [19] of Mn 2+ and Zn 2+ are close: 83 and 72 pm respectively. 6) The structure of the polyphosphate glasses is reported to consist of long (PO 3 ) n chains in which tetrahedra -labelled Q 2 in NMR studies -have 2 bridging and 2 non bridging oxygens. These chains bring negative charges that are balanced by the positive anionic charges (e.g. Na + ). Due to the low connectivity of the vitreous network, Tg is usually low: 260 °C in vitreous NaPO 3 [20].

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m a n u s c r i p t 7 7) Vibrational spectroscopy, especially infrared transmission, has been used to provide structural information about fluorophosphate glasses. Studies confirm that fluorides lead to the depolymerization of the polyphosphate chains [21][22][23][24]. Fluorides are known to lower network connectivity by creating terminal P-F bonds that take place of P-O-P bridges. 8) In manganese fluoride glasses, the vitreous network is made from MnF 6 octahedra sharing corners [16]. The connectivity is large to account for the chemical formula: in crystalline MnF 2 , each corner is shared by 3 octahedra, while all fluorine anions are bridging for a MnF 3 network. Non bridging fluorines appear when the network formula is MnF 3+x . This large connectivity accounts for the high Tg of these glasses, for example 377 °C for the 70MnF 2 -30BaF 2 binary glass [6].

The evolution of the glass transition temperature versus composition is consistent with these statements. Its value depends on the factors that rule the sensitivity of the structure to thermal motion, namely bond strength and network connectivity. The structure of these glasses may be described as a hybrid network constructed from the association of PX 4 tetrahedra and MnF 6 octahedra. In these materials, glass transition temperature is close to 250 °C. In a general way it decreases as NaPO 3 concentration increases (figure 3). Note that Tg of the 80NaPO 3 -20ZnF 2 glass is lower than that of pure NaPO 3 . The Tg decrease exemplifies the depolymerisation of the PO 3 chain by fluorine that creates non bridging anions through the formation of PO 3 F tetrahedra. As MnF 2 content increases, the concentration of modifying cations (Na + ) decreases and connectivity increases, which logically results in higher Tg. When MnF 2 substitutes NaPO 3 , the evolution of Tg is nearly linear versus MnF 2 content up to 30 mol% (fig. 3b). Beyond this value Tg increases faster, which suggests that there is a structural change beyond this limit. This could correspond to the formation of Mn(O,F) 6 octahedra including the non bridging oxygen of the PO 4 tetrahedra. Anyway it also indicates the transition from a chain structure to a tridimensional network. Note also that, for the same nominal composition, glass transition temperature is higher with large and heavy alkali cations (figure 4) and Tg increases as KF substitutes LiF (table 2). The similar trend has been observed in various halide systems [25][26][27].

While introduction of NaPO 3 clearly improves resistance to devitrification, the stability of the glass versus devitrification is not unambiguously correlated to composition. It rather increases as the number of component increases, in agreement with the confusion principle. This may explain why thermal stability range is smaller with NaF than with LiF and KF (figure 5): sodium from NaPO 3 enters glass composition and NaF incorporation does not increase the number of different cations.

The MnF 2 /NaPO 3 substitution increases microhardness and Young modulus. One may assume that the decrease in the concentration in monovalent cation (Na + ) and linear chain (PO 3 ) -results in larger lattice energy. The larger network connectivity reported above has the same influence.

Density increases as MnF 2 replaces NaPO 3 , which is partly explained by the smaller atomic weight and the larger size of Na by comparison to Mn. In the same way, density increases when zinc content rises from 10 to 20 mol % at the expense of NaPO 3 (figure 6). This evolution is consistent with the density values of the crystalline materials: 2.6 for NaPO 3 , 3.9 for MnF 2 and 4.9 for ZnF 2 [28]. The K/Li substitution also leads to smaller density (table 2). Although the alkali atomic mass of potassium is larger, the size effect is predominant and decreases molar volume.

A c c e p t e d m a n u s c r i p t 8

Optical transmission is restricted to the visible spectrum because the electronic transitions of Mn 2+ cations in octahedral ligand field lead to strong UV absorption [START_REF] Feuerhelm | [END_REF]13]. It is limited to 4.5 micrometers in the mid-infrared by the first overtone of the P-O vibrations, as this has been shown in fluoride glasses doped with phosphates [14,15,29].

Thus, these glasses may be used as filters. They contain large amounts of divalent manganese, and further studies could focus on possible magneto-optic or photo-induced phenomena in addition to the more classical fluorescence.

Conclusion

Bulk glasses rich in manganese ( > 30 mol % MnF 2 ) have been obtained in the MnF 2 -NaPO 3 -MF n systems, with M = Ba, Sr, Zn, Li, Na, K. Glass transition temperature is close to 250 °C and depends on composition. Main physical properties, including density, refractive index n D , microhardness, and thermal expansion, have been measured. The incorporation of manganese fluoride at the expense of NaPO 3 leads to the increase of Tg, n D, E (Young modulus) and microhardness, while chemical durability is enhanced. Optical transmission is limited in the UV spectrum by electronic transitions of Mn 2+ ions in octahedral environment and in the IR spectrum by the P-O vibrations.

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Tables Table 1 Comparison between nominal and analysed cationic and anionic compositions for a ternary glass.

Composition (%mol) Mn (%cat) Zn (%cat) Na (%cat) P (%cat) O (%an) F (%an) MnF 2 -NaPO 3 -

A c c e p t e d m a n u s c r i p t 3 measurements

 3 . Young (E), Shear (G) modulus, and Poisson ratio ( ) of samples are obtained from ultrasonic measurements using the ultrasonic pulse-echo method.

A c c e p t e d m a n u s c r i p t 4 range

 4 Tx-Tg vs composition is exemplified in figure3for two sets of glasses containing 10 and 20 % mol ZnF 2 respectively, in which manganese fluoride replaces sodium metaphosphate NaPO 3 . The general trend is that Tg decreases and Tx-Tg increases when NaPO 3 is incorporated at the expense of MnF 2 .

A c c e p t e d m a n u s c r i p t 6 a

 6 ) UV-Visible transmission

A c c e p t e d m a n u s c r i p t Figures Captions Fig. 1 .

 Captions1 Figures Captions

Fig. 2 .Fig. 3 . 2 .Fig. 4 .Fig. 5 .

 23245 Fig. 2. DSC scans of 50MnF 2 -50NaPO 3 (MNZ0) and 30MnF 2 -50NaPO 3 -20ZnF 2 (MNZ20) glasses. Heating rate: 10 K/min.

Fig. 6 . 2 Fig. 7 .

 627 Fig. 6. Evolution of density versus MnF 2 content. Glass composition : (1) x MnF 2 -(80-x) NaPO 3 -20 ZnF 2 and (2) x MnF 2 -(90-x) NaPO 3 -10 ZnF 2

Fig. 8 .Fig. 9 .Fig. 10 .Fig. 11 .Fig. 1 .

 8910111 Fig. 8. Evolution of refractive index : (a) vs MnF 2 concentration in x MnF 2 -(80-x) NaPO 3 -20 ZnF 2 system (b) vs KF concentration in 30% MnF 2 -40 NaPO 3 -(30-x) LiF-x KF glasses

Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .

 2345 Fig. 2. DSC scans of 50MnF 2 -50NaPO 3 (MNZ0) and 30MnF 2 -50NaPO 3 -20ZnF 2 (MNZ20) glasses.Heating rate: 10 K/min.

Fig. 6 . 2 Fig. 7 .

 627 Fig. 6. Evolution of density versus MnF 2 content. Glass composition : (1) xMnF 2 -(80-x) NaPO 3 -20 ZnF 2 and (2) xMnF 2 -(90-x) NaPO 3 -10 ZnF 2

Fig. 8 .Fig. 9 .

 89 Fig. 8. Evolution of refractive index : (a) vs MnF 2 concentration in x MnF 2 -(80-x) NaPO 3 -20ZnF 2 system (b) vs KF concentration in 30 MnF 2 -40 NaPO 3 -(30-x) LiF-x KF glasses

Fig. 10 .Fig. 11 .

 1011 Fig. 10. IR transmittance spectra of thin (1mm) and thick (e=2.4mm) samples of the MNZ20 glass: 30 MnF 2 -50 NaPO 3 -20 ZnF 2 .

Table 2

 2 Characteristic temperatures and densities of 30 MnF 2 -40 NaPO 3 -(30-x) LiF-x KF glasses. Tg for glass transition, Tx for onset of crystallization, Tp for exotherm maximum.

		ZnF 2	28.6	14.3	28.6	28.6	50	50	Nominal
		40 -40 -20	24.3	15.4	30.3	30.0	48.3	51.7	Analysed
	Sample	x (%)	Tg (± 2°C)	Tx (± 2°C)	Tp (± 1°C)	Tx -Tg (°C)	Density (g/cm 3 )
	MNLK0	0	200	247		258; 330	47		2.958
	MNLK5	5	203	276		289; 331	73		2.942
	MNLK10	10	212	321		328	109		2.930
	MNLK15	15	211	335		340	124		2.919
	MNLK 20	20	214	286		292	72		2.912
	MNLK 25	25	213	274		280; 339	61		2.905
	MNLK 30	30	233	287		295; 434	53		2.894

Table 3

 3 Coefficient of thermal expansion of selected glasses.

	Glass Compositions	( 10 -7 K -1 )
	05MnF 2 -20ZnF 2 -75NaPO 3	188
	15MnF 2 -20ZnF 2 -65NaPO 3	189
	25MnF 2 -20ZnF 2 -55NaPO 3	189
	30MnF 2 -20ZnF 2 -50NaPO 3	185
	35MnF 2 -20ZnF 2 -45NaPO 3	188
	40MnF 2 -10ZnF 2 -50NaPO 3	188
	20MnF 2 -10ZnF 2 -70NaPO 3	183
	40MnF 2 -20KF-40NaPO 3	210
	20MnF 2 -10SrF 2 -70NaPO 3	207
	30MnF 2 -20BaF 2 -50NaPO 3	226

Table 4

 4 Young (E) and shear (G) modulus, Poisson ratio ( ) of fluorophosphate glasses in the x MnF 2 -(80-x) NaPO 3 -20 ZnF 2 system. Longitudinal V L and transverse V T velocities are obtained from ultrasonic measurements.

	MnF2 (mol %)	Density (kg/m 3 )	VL (10 3 m/s) VT (10 3 m/s)	E (GPa)	G (GPa)
	0	2.783	4.32	2.34	39.3	15.2	0.290
	05	2.812	4.48	2.47	43.8	17.2	0.275
	15	2.929	4.92	2.57	50.2	19.3	0.301
	20	2.999	4.59	2.60	51.6	20.2	0.276
	25	3.059	4.88	2.62	53.4	21.0	0.272
	30	3.109	4.80	2.68	56.9	22.3	0.273
	35	3.205	5.10	2.63	58.4	22.2	0.317
	40	3.264	4.86	2.69	60.0	23.5	0.275