Lisa A Gottlieb

Nora Al Jefairi

Dounia El Hamrani

PhD Jérôme Naulin

MSc Jérôme Lamy

PhD Nadjia Kachenoura

PhD Marion Constantin

MSc Bruno Quesson

PhD Hubert Cochet

PhD Ruben Coronel
email: rubencoronel@gmail.com

MD Nora Al-Jefairi

PhD Dounia El Hamrani

PhD Lukas Rc Dekker

Gottlieb

Reduction in left atrial and pulmonary vein dimensions after ablation therapy is mediated by scar

Keywords: AF, Ablation scar, Pulmonary vein isolation, Cardiac magnetic resonance, Contractility Atrial fibrillation, Ablation scar, Pulmonary vein isolation, Cardiac magnetic resonance, Atrial contractility. Ablation-induced atrial downsizing

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

Introduction

Atrial fibrillation (AF) initiates a structural remodeling of the atrial myocardium involving loss of contractile fibers and an increase in interstitial collagen accumulation (fibrosis). [START_REF] Ausma | Structural changes of atrial myocardium due to sustained atrial fibrillation in the goat[END_REF][START_REF] Boldt | Fibrosis in left atrial tissue of patients with atrial fibrillation with and without underlying mitral valve disease[END_REF] Often, AF patients have enlarged atria and a reduced atrial contractile function. [START_REF] Gupta | Left atrial structure and function in atrial fibrillation: ENGAGE AF-TIMI 48[END_REF] Ablative pulmonary vein isolation (PVI) is a therapeutic option for AF when pharmacological therapy fails and is recommended in drug-resistant patients with paroxysmal AF (episodes of AF lasting less than 1 week).( 4) On longterm follow-up, PVI cures 59% of paroxysmal AF patients. [START_REF] Kis | The Short and Long-Term Efficacy of Pulmonary Vein Isolation as a Sole Treatment Strategy for Paroxysmal Atrial Fibrillation: A Systematic Review and Meta-Analysis[END_REF] Successful PVI ablation causes a decrease in PV and left atrial (LA) dimensions within months after the ablation procedure. [START_REF] Scharf | Anatomy of the pulmonary veins in patients with atrial fibrillation and effects of segmental ostial ablation analyzed by computed tomography[END_REF][START_REF] Tsao | Morphologic remodeling of pulmonary veins and left atrium after catheter ablation of atrial fibrillation: insight from long-term follow-up of three-dimensional magnetic resonance imaging[END_REF][START_REF] Jeevanantham | Meta-analysis of the effect of radiofrequency catheter ablation on left atrial size, volumes and function in patients with atrial fibrillation[END_REF] This reduction in PV and LA dimensions is attributed to a decrease in AF burden and thereby reverse structural remodeling in the atria. [START_REF] Maille | Reverse electrical and structural remodeling of the left atrium occurs early after pulmonary vein isolation for persistent atrial fibrillation[END_REF][START_REF] Jayam | Atrial volume reduction following catheter ablation of atrial fibrillation and relation to reduction in pulmonary vein size: an evaluation using magnetic resonance angiography[END_REF][START_REF] Solheim | N-terminal pro-B-type natriuretic peptide level at long-term follow-up after atrial fibrillation ablation: a marker of reverse atrial remodelling and successful ablation[END_REF][START_REF] Arana-Rueda | Reverse atrial remodeling following pulmonary vein isolation: the importance of the body mass index[END_REF] Reverse remodeling is a return towards the pre-disease state and involves the decrease of structural (hypertrophy, fibrosis, and atrial dilatation) and functional (decreased contractility) abnormalities. [START_REF] Ausma | Reverse structural and gap-junctional remodeling after prolonged atrial fibrillation in the goat[END_REF] However, there is no evidence underlying the association between the PVI-induced reduction in atrial dimensions and reverse remodeling on the one hand, and the subsequent scar formation on the other.

We hypothesized that the effect of ablative energy and subsequent scar formation explain the reduction in PV and LA dimensions. With cardiac magnetic resonance (CMR), we therefore studied PVI-induced changes in 1) PV dimensions, atrial volumes and LA contractility in AF patients with successful ablation and 2) PV dimensions in healthy sheep without AF. We anticipated that reverse structural remodeling initiated by normalization of the cardiac rhythm 1) only occurs in the setting of AF remodeling, 2) similarly takes place in the LA and right atrium (RA), 3) is characterized by an increase in contractile fibers and thereby contractility, and 4) exert an effect on LA dimensions in all cardiac phases. We tested this in patients with paroxysmal AF that were successfully treated with PVI ablation and in sheep without AF, and provide evidence that the changes in LA and PV diameter are the result of the ablative energy delivery and subsequent scar formation and not of reverse remodeling induced by decreased AF-burden.

Ablation-induced atrial downsizing Gottlieb et al. 4

Methods

Patient selection

Fifty-one paroxysmal AF patients referred for a first PVI ablation included in a previously published study [START_REF] Jefairi | Relationship between atrial scar on cardiac magnetic resonance and pulmonary vein reconnection after catheter ablation for paroxysmal atrial fibrillation[END_REF] were retrospectively analyzed. The study was approved by the Institutional Ethics Committee (Comité de Protection des Personnes Sud-Ouest et Outre Mer III, approval reference 2012-A01494-39). Informed consent was obtained from all patients. All patients were re-catheterized 3 months after the ablation procedure to test for recurrence of electrical PV-LA conduction. We included only patients with a clinically successful PVI defined as presence of sinus rhythm during the postablation CMR and re-catheterization both scheduled 3 months after the ablation procedure. Additional exclusion criteria were comorbidity of valvular disease, heart failure, administration of ablative radiofrequency (RF) outside the PVs, AF during pre-ablation CMR. Twenty-one paroxysmal AF patients were included of whom 7 were female (33%). Their age was 62(9) years and body mass index was 26.6(3.5) kg/m 2 . AF diagnosis had been made 60[60] months previously.

Patient CMR acquisition

CMR studies were conducted on a 1.5 Tesla system (MAGNETOM AVANTO, Siemens, Erlangen, Germany) equipped with a 32-channel body coil and a 18-channel cardiac coil. Cine imaging was performed by ECG-gated steady-state free precession pulse sequence during breathholding in order to acquire a stack of 4-chamber images covering the entire heart (slice thickness 6mm without interslice gap) as well as a 2-chamber slice (slice thickness 6mm; temporal resolution: 15ms).

A late gadolinium enhancement (LGE) CMR acquisition was begun 17 minutes after intravenous injection of 0.2mmol/kg gadoterate meglumine (Guerbet, Aulnay-sous-bois, France) and consisted of a trans-axial three-dimensional orientation using an ECG-gated and respiration-navigated gradient-echo pulse sequence.

The following imaging parameters were used: in-plane resolution = 1.25 x 1.25mm 2 ; flip angle = 22°; echo time = 2.4 ms; repetition time = 6.1 ms; generalized auto-calibrating partial parallel Ablation-induced atrial downsizing Gottlieb et al. 5 acquisition (GRAPPA), acceleration factor of 2 with 42 reference lines. The pre-ablation CMR was performed within 3 days before the ablation procedure, while the post-ablation CMR was performed 91±29 days after the pre-ablation CMR.

Patient ablation procedure

The ablation procedure has previously been described in depth.( 14) Shortly, patients were randomized to complete PVI ablation with either a conventional point catheter (NAVISTAR THERMOCOOL, Biosense Webster, Irvine, USA; n=11/21) or a circular catheter (nMARQ, Biosense Webster; n=10/21). Endocardial LA access was achieved by trans-septal puncture and followed by heparin administration (0.5-0.8mg/kg). RF energy was delivered for 60s per application in a unipolar mode with a temperature limitation of 45°C and simultaneous irrigation with 0.9% saline (60mL/min).

All patients had successful immediate procedural PVI confirmed by activation mapping that persisted after adenosine administration. We consider that the variations in lesion localization within catheter type differ equally when comparing both types.

Sheep CMR acquisition

The sheep study was carried out in accordance with the EU Directive 2010/63/EU for protection of animals used for scientific purposes and approved by the local ethical authorities at University of Bordeaux, France (approval number 7995).

CMR acquisition was performed in-vivo in healthy female sheep (n=12, 2-3 years old, 54 [START_REF] Calkins | HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation. Europace : European pacing, arrhythmias, and cardiac electrophysiology : journal of the working groups on cardiac pacing, arrhythmias, and cardiac cellular electrophysiology of the[END_REF] kg; sheep strain: Charmoise) before and 3-4 months after RF ablation of the right pulmonary vein (RPV). The sheep were anesthetized (pre-medication: 20 mg/kg ketamine + 0.1 mg/kg acepromazine, induction: 1 mg/kg propofol, maintenance: 2 % isoflurane) before placed on their back on the scanner table. The CMR studies were conducted on a 1.5 Tesla system (MAGNETOM Aera, Siemens, Erlangen, Germany) equipped with a 32-channel body coil and an 18-channel cardiac coil. Cine imaging was performed by ECG-gated steady-state free precession pulse sequence during forced breath-hold in order to acquire a stack of trans-axial images. Slice thickness was 4 mm (n=7) or 6 mm (n=5) and did not differ between pre-and post-ablation CMR in the same animal.

Ablation-induced atrial downsizing Gottlieb et al. [START_REF] Scharf | Anatomy of the pulmonary veins in patients with atrial fibrillation and effects of segmental ostial ablation analyzed by computed tomography[END_REF] The following acquisition parameters were used: in-plane resolution = 1.3 x 1.3 mm 2 ; flip angle = 58°; bandwidth = 992 Hz/pixel; echo time = 1.34 ms; repetition time = 21.98 ms; GRAPPA; acceleration factor of 3 with 75% partial Fourier acquisition. The pre-ablation CMR was performed within 1 week before ablation, while the post-ablation CMR was performed 120±11 days after the preablation CMR.

Sheep ablation procedure

The sheep were catheterized via the femoral veins under general anesthesia and sterile conditions. LA access was achieved by trans-septal puncture using a steerable sheath. A circular ablation catheter (PVAC Gold, Medtronic, Minneapolis, USA) was placed in the RPV ostium under fluoroscopy guidance, and RF energy was administered 2 x 60s with bipolar:unipolar 2:1 phasing and a temperature limitation of 55 °C. The ablation reference electrode was placed on the lower back of the sheep. The animals recovered under surveillance for 1 week before returning to a hosting farm.

Image analysis

Only pre-and post-ablation images containing the same anatomical landmarks (the sternum, the vertebrae, and the aorta) in similar stack slices and being without artefacts in the LA and PV regions were included in the analysis. The PV diameter was measured at the ostium at the moment of maximum LA dilatation (just before opening of the mitral valve) in the patients (figure 1A) and sheep (supplementary figure 1) if the PVs remained temporally in plane in both pre-and post-ablation images.

In the patients, we analyzed the LA volume at the moment of maximum LA dilatation, and immediately before (pre-contraction) and after (minimum LA volume) atrial contraction by tracing the endocardial LA contours on each slice of the 4-chamber stack while excluding the PVs and LA appendage (LAA) in the software Syngo.via (Siemens, Erlangen, Germany; figure 1C). The volume was calculated as the sum of each LA area multiplied by the slice thickness. The maximum RA volume was similarly measured while excluding the caval veins from the RA areas (figure 1C).

Ablation-induced atrial downsizing

Gottlieb et al. 7

We defined the total LA ejection fraction as (maximum LA volume -minimum LA volume)/maximum LA volume*100%, the LA expansion index as (maximum LA volume -minimum LA volume)/minimum LA volume*100%, the LA conduit fraction as (maximum LA volume -precontraction LA volume)/maximum LA volume*100%, and the LA active emptying fraction as (precontraction LA volume -minimum LA volume)/pre-contraction LA volume*100%.

We analyzed local LA strain and motion fraction throughout the cardiac cycle with a featuretracking algorithm previously described. [START_REF] Evin | Assessment of left atrial function by MRI myocardial feature tracking[END_REF][START_REF] Huber | Cardiac MR Strain: A Noninvasive Biomarker of Fibrofatty Remodeling of the Left Atrial Myocardium[END_REF] LA endocardial markers (>30) were positioned on a single phase of the cardiac cycle, and longitudinal strain (tangential deformation to the considered LA wall) and radial motion fraction (towards the LA center of mass) of defined regions were computed.

We identified the anterior and posterior wall of the right superior PV (RSPV) and right inferior PV (RIPV) as well as the posterior wall of the left superior PV (LSPV; figure 1B). The tracking of the slightly kinked anterior LSPV wall towards the LAA was less precise and therefore excluded from the analysis. Also, 3 LA regions outside the PVs were defined: a septal LA wall in the 4-chamber stack (in the slice with the RSPV; figure 1B) and a posterior and an inferior LA wall in the 2-chamber slice (figure 1E). The PV ostia and LAA were excluded from these regions. The temporal beginning of the strain/motion fraction curve was the moment immediately after atrial contraction, and we considered the maximum magnitude of the last 20% of the curve as an indicative of local contractility. A contractility index of the PV regions and of the LA regions was calculated in each patient as the sum of the logarithmically transformed values of local contractility (2 values per wall region: 1 value each from the strain and the motion fraction curve. The PV contractility index was calculated based on the 5 PV regions; the LA contractility index on the 3 LA regions).

Scar burden was quantified on the LGE CMR images (figure 1G) using the MUSIC software (IHU Liryc, Bordeaux, France) as previously described.( 14) Shortly, the LA wall was manually traced, and LGE was segmented using histogram analysis, applying the full width at half maximum method.

Thresholding of myocardial voxel intensity (50-70% of maximum signal intensity) was performed, and scar in the PVs and LA was quantified as the difference between pre-and post-ablation measurements and expressed in ml.

Ablation-induced atrial downsizing Gottlieb et al. 8

Histology

Three sheep hearts were explanted after post-ablation CMR acquisition in accordance with the ethically approved euthanasic methods. The RPV and left PVs (LPV) were dissected and fixated in paraformaldehyde (4%) at 5°C for 2 weeks before cut in the longitudinal direction thus including the atrial-PV junction and the distal PV. Specimen were dehydrated automatically (Leica HistoCore Pearl processor, Wetzlar, Germany) before being embedded in paraffin and sectioned with a microtome (6μm slice thickness). A Masson's trichrome staining was applied to the slices to visualize cardiomyocytes (red), nuclei (black), and collagen (green) before digitally scanned with an objective x 20.

Statistical analysis

Data are expressed as mean (standard deviation) or median [interquartile range] depending on normality tested with a Shapiro Wilk's test. Pre-and post-ablation CMR data were tested with a twotailed paired Student's t-test or a Wilcoxon signed rank test, as appropriate. P-values<0.05 were considered as statistically significant. The 95% confidence intervals (CI95%) are included for parametric testing.

A mean of ablation-induced change in PV diameters (post-ablation diameter -pre-ablation diameter / pre-ablation diameter *100%) was calculated in each patient and was tested against a theoretical mean of 0 with a two-tailed one-sample Student's t-test. The change in diameter after ablation of the ablated RPV and a mean change in diameter of the non-ablated LPVs were calculated in each sheep before similar testing and Bonferroni correction (0.05/2). A two-tailed unpaired Student's t-test was used to test for differences in ablated PVs between patients and sheep.

A linear regression model was fitted to the mean PV diameter change in patients as a function of the logarithmically transformed extent of PV ablation scar. A mixed effect linear model was applied to the PV and LA contractility indexes.

Ablation-induced atrial downsizing

Gottlieb et al. 9

Results

Not all PVs were accessible for analysis. The left inferior PV (LIPV) in the AF patients was rarely visible and comparable between the pre-and post-ablation datasets (n=4) and was discarded from the analysis. In patients, 14 RIPVs, 18 RSPVs, and 16 LSPVs were analyzed. None had left or right single ostium PV or unilateral triple PVs. In sheep, 11 RPVs, 10 LSPVs, and 11 LIPVs were measured (a RIPV is absent in sheep).

Ablation-caused decrease in PV diameter

Patients: PVI ablation caused a decrease in PV diameter in the AF patients (18.9(4.2) vs.

PV scar

Patients: Three months after PVI, ablation scar was observed in the PV regions in all patients

LA volume reduction in AF patients

The volume analysis was based on 20 AF patients because the CMR stack did not cover the whole atrium in 1 patient. The maximum and pre-contraction LA volume in patients decreased by 9(18) and 9(15) %, respectively, after ablation, whereas the minimum LA volume remained unchanged (table 1). The maximum RA volume did not change after successful PVI in patients (81 [START_REF] Ravelli | Acute atrial dilatation slows conduction and increases AF vulnerability in the human atrium[END_REF] vs. 78(27) ml, prevs. post-ablation, CI95%=[-8.2 13.2], p=0.629).

Decrease in active LA emptying fraction after successful PVI in AF patients

The active LA emptying fraction decreased by [START_REF] Tsao | Pulmonary vein dilation in patients with atrial fibrillation: detection by magnetic resonance imaging[END_REF][42] % after ablation in the patients (table 1).

The total LA ejection fraction remained stable after ablation as reflected in an unaltered passive LA conduit fraction (table 1). We observed a tendency towards a lower LA expansion index after ablation (table 1). Heart rate did not change in the patients (62 [START_REF] Ausma | Reverse structural and gap-junctional remodeling after prolonged atrial fibrillation in the goat[END_REF] vs. 66 [START_REF] Marsan | Comparison of left atrial volumes and function by real-time three-dimensional echocardiography in patients having catheter ablation for atrial fibrillation with persistence of sinus rhythm versus recurrent atrial fibrillation three months later[END_REF] bpm; prevs. post-ablation, p= 0.170) and, therefore, did not constitute a confounding factor.

Decrease in PV wall contractility in AF patients

Figure 3 shows the longitudinal strain curves before (A) and after (B) ablation of the RSPV posterior wall in patients. The maximum magnitude of the last 20% of the strain curve was considered an indicative of local contractility. The PV contractility index decreased after ablation (5.57 (2.43) vs. [START_REF] Calkins | HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation. Europace : European pacing, arrhythmias, and cardiac electrophysiology : journal of the working groups on cardiac pacing, arrhythmias, and cardiac cellular electrophysiology of the[END_REF].69 (2.38) arb. unit, prevs. post-ablation, CI95%=[0.26 1.50], p=0.004), while the LA contractility index was unaltered by PVI (5.74 (2.17) vs. 5.51 (2.18) arb. unit, prevs. post-ablation, CI95%=[-0.37 0.82], p=0.749; figure 3C).

Thus, in patients with AF, we observed that the LA volume reduction after PVI ablation was associated with a local decrease in contractility in the PV regions in which RF energy was delivered, but not in regions remote form the site of energy delivery.

Ablation-induced atrial downsizing Gottlieb et al. 11

Discussion

We investigated the changes in atrial dimensions and mechanics in paroxysmal AF patients after successful PVI and in healthy sheep after ablation in a single PV. Our results showed that the diameter of ablated PVs decreased similarly in the patients and in healthy sheep, and that a linear correlation existed between the decrease in PV dimension and extent of PV scar in patients. The maximum volume of the LA was smaller, whereas that of the RA remained unchanged, after PVI in patients. Finally, the PV walls contracted less, while the contractility of the LA walls remote from RF energy delivery did not alter after PVI in patients.

Reverse structural remodeling

AF causes structural remodeling of the atrial tissue by fibrosis, fat accumulation, myocardial hypertrophy, and loss of contractile fibers.(1, 2, 17) A consequence of structural remodeling is atrial dilatation that is common in AF patients and is an independent risk factor for AF in humans. [START_REF] Gupta | Left atrial structure and function in atrial fibrillation: ENGAGE AF-TIMI 48[END_REF][START_REF] Vaziri | Echocardiographic predictors of nonrheumatic atrial fibrillation. The Framingham Heart Study[END_REF] Observations of counteracting processes in the atria have been made upon restoration of sinus rhythm, and this phenomenon has been termed reverse remodeling.(13)

PVI-induced decrease in PV dimensions

A PVI-induced reduction in PV diameter of about 10% is observed by others.(7, 10) AF patients have enlarged PVs compared to control patients, and structural remodeling due to AF burden is considered to be the cause. [START_REF] Tsao | Pulmonary vein dilation in patients with atrial fibrillation: detection by magnetic resonance imaging[END_REF] Therefore, a reverse structural remodeling by cessation of AF has been proposed as causing the decrease in PV dimensions after successful PVI. [START_REF] Jayam | Atrial volume reduction following catheter ablation of atrial fibrillation and relation to reduction in pulmonary vein size: an evaluation using magnetic resonance angiography[END_REF] We observed that PVI ablation in healthy sheep without AF, in which structural remodeling due to arrhythmia cannot be present, caused a similar chronic decrease in PV diameter as in AF patients. It is therefore more likely that the PV size reduction is the direct causal effect of ablative energy delivery and subsequent scar formation rather than reverse remodeling processes. Indeed, we observe a statistically significant linear correlation between scar extent in the PVs and PV diameter decrease after successful PVI in AF patients.

Ablation-induced atrial downsizing Gottlieb et al. [START_REF] Arana-Rueda | Reverse atrial remodeling following pulmonary vein isolation: the importance of the body mass index[END_REF] Healthy animals served as a control to the successfully ablated AF patients instead of patients with AF recurrence because in the case of AF recurrence even a persistently isolated PV (as observed on re-do catheterization) is still subject to AF remodeling (and thereby continuous dilatation). Indeed, dilatation of RSPV is observed in patients still in AF after PVI. [START_REF] Tsao | Morphologic remodeling of pulmonary veins and left atrium after catheter ablation of atrial fibrillation: insight from long-term follow-up of three-dimensional magnetic resonance imaging[END_REF].

Ablation-induced reduction in LA volume

A meta-analysis of 8 studies supports our findings of a decrease in LA volume after successful AF ablation, [START_REF] Jeevanantham | Meta-analysis of the effect of radiofrequency catheter ablation on left atrial size, volumes and function in patients with atrial fibrillation[END_REF] and the LA volume decrease itself has been interpreted as a marker of reverse remodeling. [START_REF] Maille | Reverse electrical and structural remodeling of the left atrium occurs early after pulmonary vein isolation for persistent atrial fibrillation[END_REF][START_REF] Solheim | N-terminal pro-B-type natriuretic peptide level at long-term follow-up after atrial fibrillation ablation: a marker of reverse atrial remodelling and successful ablation[END_REF][START_REF] Arana-Rueda | Reverse atrial remodeling following pulmonary vein isolation: the importance of the body mass index[END_REF]) Ausma and colleagues report that 2 months of sinus rhythm (after 4 months of AF) decreased AF-induced atrial myolysis in goats, although a complete normalization was (still) not observed after 4 months of sinus rhythm. [START_REF] Ausma | Reverse structural and gap-junctional remodeling after prolonged atrial fibrillation in the goat[END_REF] This emphasizes that reverse remodeling processes does occur, but at a slow rate and most likely beyond the 3-month blinding period after AF ablation.

In patients with AF recurrence, in whom a reverse structural remodeling is not expected, a similar decrease in LA volume has been observed after AF ablation.(10) Thus, the chronic effects of ablative energy must directly have caused the LA volume reduction seen after PVI, rather than the restoration of sinus rhythm. Indeed, the volume of the RA, in which no RF energy was delivered, remained unchanged 3 months after successful PVI in our patients, although AF was equally absent as in the LA.

Loss of contractility at ablated sites

Our observation of a decrease in active LA emptying fraction after PVI is confirmed by others. [START_REF] Jeevanantham | Meta-analysis of the effect of radiofrequency catheter ablation on left atrial size, volumes and function in patients with atrial fibrillation[END_REF] We reason that a reverse remodeling that includes normalization of the contractile fibers, should lead to an increase and not a decrease in the active LA function. An increase in active LA emptying fraction from 22 to 33% is observed after successful PVI by Marsan et al.(20) However, 45% of these patients were medicated with angiotensin receptor blocker that is known to attenuate AFinduced atrial fibrosis. [START_REF] Kumagai | Effects of angiotensin II type 1 receptor antagonist on electrical and structural remodeling in atrial fibrillation[END_REF] We report that the contractility of 3 LA regions remote from the sites of RF delivery was unchanged after PVI. This observation questions a global reverse remodeling based on restoration of Ablation-induced atrial downsizing Gottlieb et al. [START_REF] Ausma | Reverse structural and gap-junctional remodeling after prolonged atrial fibrillation in the goat[END_REF] sinus rhythm. Instead, we observed a decrease in contractility in the PV walls, possibly due to loss of myocytes caused by the ablative energy. In alignment with our findings, others report a positive correlation between the amount of ablation scar (measured by late gadolinium enhancement) and a decrease in total LA ejection fraction. [START_REF] Wylie | Left atrial function and scar after catheter ablation of atrial fibrillation[END_REF] 4.5 Stretch-reduction as an antiarrhythmic factor A reduction in PV and LA dimensions, under the assumption of unaltered LA pressure, decreases wall stress according to Laplace's law. Because increased atrial and PV stretch is proarrhythmic, [START_REF] Ravelli | Acute atrial dilatation slows conduction and increases AF vulnerability in the human atrium[END_REF][START_REF] Chang | Mechanoelectrical feedback regulates the arrhythmogenic activity of pulmonary veins[END_REF] and relief of chronic stretch is antiarrhythmic, [START_REF] Coronel | Left atrial pressure reduction for mitral stenosis reverses left atrial direction-dependent conduction abnormalities[END_REF] the ablation-induced decrease in atrial dimensions may play a role in preventing AF by reduction in local wall stress. Indeed, the plasma concentration of B-type natriuretic peptide (BNP), a marker of atrial stretch, is decreased after successful PVI. [START_REF] Solheim | N-terminal pro-B-type natriuretic peptide level at long-term follow-up after atrial fibrillation ablation: a marker of reverse atrial remodelling and successful ablation[END_REF] The authors observed a correlation between post-ablation arrhythmia burden and BNP, and they proposed BNP as a marker of reverse structural remodeling. However, it is more likely that the ablation scar decreases atrial stretch by dimension reduction and thereby correlates with a lower arrhythmia burden.

Our observation of an unaltered minimum LA volume, despite smaller maximum LA volume, after ablation indicates that PVI prevented LA dilatation by a stiffening of the chamber. Indeed, we observed a tendency towards a lower expansion index after PVI. A future goal of research is to optimize ablation therapy such that local ablative energy delivery is sufficient to decrease LA and PV dimensions to prevent proarrhythmic dilatation but small enough to prevent loss of contractile function.

Study limitations

The reproducibility of CMR before and after PVI prevented us from analyzing all PVs. Also, the in-plane resolution was 1.25x1.25mm 2 in patients, and therefore the measurement of radial strain of the LA myocardial wall (changes in myocardial thickness) during the cardiac cycle was not feasible. We instead analyzed the motion fraction of the wall towards the LA center of mass.

Ablation-induced atrial downsizing Gottlieb et al. 14

Conclusion

We demonstrated that the reduction in LA and PV dimensions after successful PVI is attributed to the direct effect of ablative energy delivery and scar formation rather than reverse structural remodeling based on restoration of sinus rhythm. The curative potential of reverse structural AF remodeling is therefore smaller than previously estimated. An antiarrhythmic effect of PVI may lie in the downsizing of the LA and PVs.

Data statement

The data supporting the study findings can upon reasonable request be made available from the corresponding author. is the moment immediately after atrial contraction. We considered the last 20% of the curve as the atrial contractile phase (blue shade). Localized contractility was quantified as the magnitude of the strain during this phase. C: The PV contractility index decreased after ablation whereas the LA contractility index remained unchanged.

Tables

Figure legends

16. 5

 5 (3.8) mm, prevs. post-ablation, respectively; PVI-induced change: -11.0 (10.3) %, CI95%=[-15.9 -6.0], p<0.001, hypothesis test against theoretical mean equaled 0; figure 2A) Sheep: The PV diameter of the ablated RPV in sheep also decreased after ablation (13.8(2.3) vs. 12.3(1.6)mm, prevs. post-ablation, respectively; PVI-induced change: -9.2(11.0)%, CI95%=[-16.6 -1.8], p=0.020; figure 2B), whereas the control left PVs remained unchanged (15.6(2.3) vs. 15.5(2.2)mm, prevs. post-ablation, respectively; PVI-induced change: 1.5(12.0)%, CI95%=[-6.6 9.5], p=0.689; figure 2B). The percentage decrease in ablated PV diameter was similar (-11.0(10.3) and -9.2(11.0)% in patients and sheep, respectively, CI95%=[-6.4 10.0], p=0.660).

( 7 .

 7 4 (2.1)ml; figure 1H). LA scar was observed in 1 patient (1.5ml). A negative linear relation existed between ablation-induced PV scar and PV diameter change (R 2 =0.388, H0 slope equal to 0: p=0.004, slope = -44.3 CI95%=[-72.8 -15.9]; figure 2C). Sheep: Three months following PVI, acellular collagenous ablation scar was observed in the RPVs of sheep (figure 2D left panel), but not in the LPVs (figure 2D right panel). Ablation-induced atrial downsizing Gottlieb et al. 10

Figure 1 :

 1 Figure 1: CMR images in AF patients

Figure 2 :

 2 Figure 2: Ablation-induced changes in PV diameter and scar

Figure 3 :

 3 Figure 3: Regional wall contractility before and after ablation in patients

Table 1 :

 1 Left atrial dimensions and mechanics before and after ablation in patients

		Pre-ablation	Post-ablation	Pre-ablation vs. post-ablation
	Maximum LA volume, ml	103.4(37.6)	91.6(31.1)	CI95%=[3.9 19.8]; p=0.006
	Pre-contraction LA volume, ml	87.9(31.3)	78.8(26.5)	CI95%=[3.3 14.9]; p=0.004
	Minimum LA volume, ml	68.2[47.0]	57.9[33.6]	p=0.391
	Total LA ejection fraction, %	37.3(9.7)	32.5(10.3)	CI95%=[-0.1 9.6]; p=0.053
	Active LA emptying fraction, %	26.5(10.7)	21.8(10.6)	CI95%=[0.5 9.1]; p=0.031
	Passive LA conduit fraction, %	14.5(5.9)	13.7(6.4)	CI95%=[-3.7 5.3]; p=0.701
	LA expansion index, %	63.0(24.7)	51.6(23.9)	CI95%=[-0.1 22.9]; p=0.051
	Legend

table 1 :

 1 The LA volumes were manually measured on CMR images at the moment of maximum LA dilatation, and immediately before and after atrial contraction. The mechanical parameters were calculated from these volumes. Values are expressed as mean(standard deviation) or median[interquartile range] dependent on normality. The 95% confidence intervals are included if testing was parametric.

Ablation-induced atrial downsizing Gottlieb et al.

[START_REF] Tsao | Pulmonary vein dilation in patients with atrial fibrillation: detection by magnetic resonance imaging[END_REF]

Acknowledgements

We thank Medtronic for proving the ablation catheters for the animal experiments. This work was supported by Medtronic (unrestricted research grant, recipient LRCD, Catharina Hospital (research grant, recipient LRCD), and Leducq Foundation Rhythm [16CVD02] (grant recipient RC).

Medtronic, Catharina Hospital, and Leducq foundation Rhythm[16CVD02].

Discloses

The authors have no disclosures.

Author statements


Zahira El Khalidi
email: zahira@uic.edu

Ayoub Daouli

Hicham Jabraoui
email: hicham.jabraoui@cea.fr

Bouchaib Hartiti

Amal Bouich

Marí Bernabé

Elisabetta Soucase

Hashitha M M Comini

Salah Munasinghe Arachchige

Philippe Fadili

Thevenin

Zahira El Khalidi

Bernabé Marí Soucase

Elisabetta Comini

Hashitha M M Munasinghe Arachchige

Salah Fadili

Philippe Thevenin

Abderrafi Kamal

Abdellatif Hasnaoui

Michael Badawi
email: michael.badawi@univ-lorraine.fr

Impact of Sn doping on the hydrogen detection characteristics of ZnO thin films: Insights from experimental and DFT combination

Keywords: Sn-doped ZnO, ZnO thin films, Synthesis, Characterization, TEM, XPS, DFT calculations

Introduction

Because of its advantages such as combustion efficiency and low density, hydrogen (H2) could be classified as a source of clean and sustainable energy [START_REF] Mirzaei | An overview on how Pd on resistive-based nanomaterial gas sensors can enhance response toward hydrogen gas[END_REF]. However, the storage and transportation of H2 raised significant concerns, especially its leakage from pipelines [START_REF] Abdalla | Hydrogen production, storage, transportation and key challenges with applications: A review[END_REF]. The large explosive concentration range of dihydrogen [START_REF] Korotcenkov | Review of Electrochemical Hydrogen Sensors[END_REF]and the difficulty of its detection by human sensory organs [START_REF] Kabcum | Ultra-responsive hydrogen gas sensors based on PdO nanoparticle-decorated WO3 nanorods synthesized by precipitation and impregnation methods[END_REF] make it a threat for environmental safety. Therefore, an evaluation of H2 concentration in the air, with a suitable gas sensor is required. Previous studies were conducted to evaluate the efficiency of H2 sensors [START_REF] Esfandiar | Pd-WO3/reduced graphene oxide hierarchical nanostructures as efficient hydrogen gas sensors[END_REF][START_REF] Annanouch | Aerosol-Assisted CVD-Grown PdO Nanoparticle-Decorated Tungsten Oxide Nanoneedles Extremely Sensitive and Selective to Hydrogen[END_REF]. However, a sensor that detects H2 with a minimum detection/recovery time, high selectivity, and sensitivity is still needed. To ensure high sensor performance factors, semiconductor materials were suggested as the best alternatives in gas sensors technology [START_REF] Mizsei | Forty Years of Adventure with Semiconductor Gas Sensors[END_REF]. Many semiconductor oxides like SnO2 [START_REF] Hijazi | Ambient temperature selective ammonia gas sensor based on SnO2-APTES modifications[END_REF], TiO2 [START_REF] Alev | Gas Sensor Application of Hydrothermally Growth TiO2 Nanorods[END_REF], and NiO [START_REF] Nakate | Ultra thin NiO nanosheets for high performance hydrogen gas sensor device[END_REF] have been studied, besides ZnO with its promising properties desired in many fields like photovoltaic energy [START_REF] Zang | Enhanced fluorescence imaging performance of hydrophobic colloidal ZnO nanoparticles by a facile method[END_REF], Bio-imaging or LEDs [START_REF] Boyadjiev | Preparation and characterization of ALD deposited ZnO thin films studied for gas sensors[END_REF], and the detection of liquid or gaseous species [START_REF] Boyadjiev | Preparation and characterization of ALD deposited ZnO thin films studied for gas sensors[END_REF][START_REF] Chi | Control of hydrophobic surface and wetting states in ultra-flat ZnO films by GLAD method[END_REF].

ZnO can be synthesized using physical and chemical processes. In the physical deposition with the steam phase, the material's deposition is the result of condensation on a substrate by the jets of neutral or ionized molecules and atoms. Among the main physical processes used to develop thin layers, we cite: Sputtering [START_REF] Singh | Tuning the optical and electrical properties of magnetron-sputtered Cu-ZnO thin films using low energy Ar ion irradiation[END_REF], Pulser-laser Deposition [START_REF] Köseoğlu | A simple microwave-assisted combustion synthesis and structural, optical and magnetic characterization of ZnO nanoplatelets[END_REF], and other techniques that present a distinct advantage providing a high composition control, film thickness, and sharp transition. Despite all previous qualities, and in addition to the high operating system cost required by this process, other drawbacks remain, such as the need of the cooling system and skilled control of vacuum and temperature. For this purpose, researchers were attracted by chemical methods for their simplicity and low cost, besides the good adhesion of the layers and the porosity of the treated surfaces which is the basic quality needed for a better gas detection. In this category we cite: Sol-Gel [START_REF] Tiwari | The effects of Sn-In co-doping on the structural, optical, photoluminescence and electrical characteristics of the sol-gel processed ZnO thin films[END_REF] and Spray Pyrolysis [START_REF] Sree | Enhanced UV light induced photocatalytic degradation of Methyl Orange by Fe doped spray pyrolysis deposited ZnO thin films[END_REF].

In a recent research works, Kumar et al. [START_REF] Kumar | UV-light enhanced high sensitive hydrogen (H2) sensor based on spherical Au nanoparticles on ZnO nano-structured thin films[END_REF] detected H2 gas with a high percentage at 1000 ppm of the gas concentration, using ZnO thin films decorated by Au nanoparticles. Another work reported by Gaspera et al. [START_REF] Gaspera | Enhanced optical and electrical gas sensing response of sol-gel based NiO-Au and ZnO-Au nanostructured thin films[END_REF] stated that the nanostructured ZnO doped thin films showed a higher response compared to intrinsic zinc oxide. Generally, doping exhibits a significant influence on the optoelectronic properties of materials [START_REF] Papagiorgis | The Influence of Doping on the Optoelectronic Properties of PbS Colloidal Quantum Dot Solids[END_REF]. In particular, and based on previous reports, the addition of Sn dopant increases the electrical conductivity of the host ZnO material.

Free electrons are released without creating a huge lattice distortion during the substitution of Zn by Sn. Al dopant exhibited a similar influence on ZnO thin films and improved the sensing properties of the gas sensor by improving its response and reducing its response/recovery time as reported by Yoo et al. [START_REF] Yoo | Highly sensitive gas sensor based on Al-doped ZnO nanoparticles for detection of dimethyl methylphosphonate as a chemical warfare agent simulant[END_REF].

Our work aims to improve ZnO sensors using a straightforward experimental process. The basic outcome is to assess and improve hydrogen detection through the addition of Sn and Al. The experimental gas testing of Sn-doped ZnO exhibited high sensing performance compared to Al-doped ZnO. Along with the experimental efforts, we performed density functional theory (DFT) calculations to understand, at the atomic scale, experimental results such as the poor response of Al-doped ZnO and the mechanism of H2 gas detection by ZnO and explain how the Sn and Al affect the sensitivity of ZnO to reducing gas. The accuracy of DFT is sufficient to predict and understand interactions between organic molecules and solids in general ZnO-based gas detection mechanisms in particular [START_REF] Zhang | Al doped narcissus-like ZnO for enhanced NO2 sensing performance: An experimental and DFT investigation[END_REF][START_REF] Khalidi | Behavior of NiO thin films sprayed at different annealing time[END_REF][START_REF] Jabraoui | Ab initio screening of cation-exchanged zeolites for biofuel purification[END_REF][START_REF] Khalil | Selective Capture of Phenol from Biofuel Using Protonated Faujasite Zeolites with Different Si/Al Ratios[END_REF][START_REF] Khalil | Biofuel purification: Coupling experimental and theoretical investigations for efficient separation of phenol from aromatics by zeolites[END_REF]. The outcome and the novelty of our work is to assess and improve the hydrogen detection through the addition of Sn and Al to ZnO thin films.

Our goal is to reach a high sensor response and interpret theoretically the low response observed for Al doped ZnO. Herein, the experimental gas testing of Sn-doped ZnO exhibited high sensing performance reaching 200 at 500 ppm, for 400°C compared to Al-doped ZnO, ZnO, and other previous works from literature. The low Al doped ZnO response was evaluated by the density functional theory (DFT) calculations and provided an atomic understanding of H2 gas trapping by ZnO and explained how the Sn and Al affect the sensitivity of ZnO to reducing gas. The response we reached in our work, was higher at low gas target concentration, compared to some reports from literature such as Drmosh et al [START_REF] Drmosh | Hydrogen sensing properties of sputtered ZnO films decorated with Pt nanoparticles[END_REF], who reached a response between 80-95, at the operating temperature of 300˚C, under a high gas concentration of about 1200 ppm. The other research work [START_REF] Mokrushin | Chemoresistive gas-sensitive ZnO/Pt nanocomposites films applied by microplotter printing with increased sensitivity to benzene and hydrogen[END_REF], that was recently published, the authors claimed that the gas response was around 13, with lower operating temperature 250˚C, but the gas concentration was above 1500 ppm.

The paper is organized as follows: first, we detail our experimental and computational procedures, then, the obtained results with both experimental and DFT tools are presented and discussed focusing on the explanation of the high and low sensitivity of Sn and Al-doped ZnO, respectively. Finally, we outline the main conclusions of our study.

Materials and methods

Experimental section

The chemical reagents used in the experiments are zinc chloride (Cl2Zn, 98%), aluminum nitrate extra pure Al (NO3)3.9H2O and tin (II) chloride dehydrate, extra pure Ph Eur, DAB (SnCl2.2H2O) brought from SIGMA-ALDRICH. The deposition of thin films was performed using a cost-effective chemical spray pyrolysis method. The substrate preparation and the deposition process were similar to our previous work [START_REF] Khalidi | Behavior of NiO thin films sprayed at different annealing time[END_REF]. Three aqueous solutions were prepared, containing distilled water and 0.05 M of zinc oxide powder. The first solution contains pure zinc oxide, the second and the third solutions containing respectively, zinc oxide with the ratios [Sn 4+ ]/ [Zn 2+ ] = 4% and [Al 3+ ]/ [Zn 2+ ] = 4% as precursors. Each solution was agitated for 30 min at 60°C.

Characterization of the elaborated thin films

The structural characterization of the samples was established with a monochromator X-Ray Diffraction (Diffractometer system = XPERT-PRO) using Cu-Kα radiation and a wavelength of 0.15406 nm. The voltage was maintained at 20 kV, the measurements were obtained according to Bragg's law, and the detector used was point on arm. The surface morphology of the thin films was identified using scanning electron microscopy (FEI Quanta 650 FEG Environmental). Scanning Electron Microscopy (SEM) system was set at an acceleration voltage of 25 kV. We used a HORIBA monochromator iHR320 to measure the Raman spectra. The system was configured at room temperature using a grating of 1800 gmm -1 , connected to a Peltier-cooled Synapse CCD. The He-Cd laser (442 nm) was targeting the sample by a fiber coupled optical microscope at 100× magnification. We selected the wavelength of 200-800 cm -1 range. A detailed description of the equipment are described concisely in our previous work [START_REF] Khalidi | Behavior of NiO thin films sprayed at different annealing time[END_REF]. We used High Resolution Transmission Electron Microscope 100 kV (TEM 100) to perform additional structural analysis. TEM (JEO-JEM-1010) measurement conditions were 2.5 kV at various magnifications. The X-ray photoelectron spectroscopy is (XPS, SPECS GmbH) type, using AlKα X-rays with the same XRD wavelength.

Computational methodology

The detection performance of pure ZnO and Al/Sn doped ZnO surfaces to H2 gas was investigated through the periodic density functional theory (DFT) [START_REF] Hohenberg | Inhomogeneous Electron Gas[END_REF][START_REF] Sham | One-Particle Properties of an Inhomogeneous Interacting Electron Gas[END_REF]. Vienna Ab initio Simulation Package (VASP) is used to run our DFT calculations [START_REF] Kresse | Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set[END_REF][START_REF] Kresse | Ab initio molecular dynamics for liquid metals[END_REF] , with a plane-wave basis set and projector augmented wave (PAW) [START_REF] Blöchl | Improved tetrahedron method for Brillouin-zone integrations[END_REF][START_REF] Kresse | Norm-conserving and ultrasoft pseudopotentials for first-row and transition elements[END_REF] pseudopotential. The functional of Perdew, Burk, and Ernzerhof (GGA-PBE) [START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF] was combined with the Tkatchenko-Scheffler scheme with iterative Hirshfeld partitioning (TS/HI) [START_REF] Jabraoui | Ab initio screening of cation-exchanged zeolites for biofuel purification[END_REF][START_REF] Jabraoui | Adsorption of volatile organic and iodine compounds over silver-exchanged mordenites: A comparative periodic DFT study for several silver loadings[END_REF] correction to account for dispersion forces. The total energy convergence for the self-consistent [START_REF] Kohn | Self-Consistent Equations Including Exchange and Correlation Effects[END_REF] iterations was set to 10 -6 eV.

Gaussian smearing with a smearing width of 0.05 eV was considered for Brillouin-zone integrations on a 2×2×1 k-mesh. The geometry optimization was performed until the forces were smaller than 0.02 eV/Å per atom [START_REF] Kresse | Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set[END_REF]. The adsorption energies of gaseous molecules on the ZnO surface have been calculated at 0 K according to the following equation, the same trend has been used in many works [START_REF] Jabraoui | Ab initio screening of cation-exchanged zeolites for biofuel purification[END_REF][START_REF] Kohn | Self-Consistent Equations Including Exchange and Correlation Effects[END_REF][START_REF] Hessou | Ab initio screening of zeolite Y formulations for efficient adsorption of thiophene in presence of benzene[END_REF][START_REF] Jabraoui | Atomic Insights into the Events Governing the Borosilicate Glass-Water Interface[END_REF][START_REF] Hessou | Adsorption of Toluene and Water over Cationic-Exchanged Y Zeolites: A DFT Exploration[END_REF]:

= ( ) -( + ) (1)
where E(ZnO-gas) is the energy of the ZnO (0002) surface with the adsorbate, EZnO and Egas are the energies of the ZnO surface and the isolated molecule in the gaseous phase, respectively. The charge density difference (Δρ) and Bader charge [START_REF] Tang | A grid-based Bader analysis algorithm without lattice bias[END_REF][START_REF] Sanville | Improved grid-based algorithm for Bader charge allocation[END_REF][START_REF] Henkelman | A fast and robust algorithm for Bader decomposition of charge density[END_REF] difference (ΔQ) have been determined

to further improve our understanding of the interaction of molecules with pure and Al-or Sndoped ZnO. To visualize the charge density difference (Δρ) (using VESTA [START_REF] Momma | VESTA: a three-dimensional visualization system for electronic and structural analysis[END_REF]) introduced by the adsorption of H2 and O2 onto ZnO surfaces, we combine the three charge densities namely the density of the complex ρZnO-gas, the density of the clean ZnO surface ρZnO, and the density of the isolated molecule in the gaseous phase ρgas, as shown in the equation below [START_REF] Hessou | A first principle evaluation of the adsorption mechanism and stability of volatile organic compounds into NaY zeolite[END_REF]:

= - - (2)
To get the difference in Bader charge ΔQ, the following equation was used:

= - (3)
where Q ZnO-gas is the Bader charge of all atoms upon adsorption and Q ZnO&gas is the Bader charge of the separated clean ZnO surface and the separated isolated molecule in the gaseous phase.

Results and discussions

The structural characterization of the samples is described in the XRD patterns The structural identifications of the samples can be determined by calculating the lattice constants a and c [START_REF] Lin | Calculation of the lattice constant of solids with the use of valence electron structure parameters[END_REF] (Eq. 4), the crystallite size Dhkl [START_REF] Patterson | The Scherrer Formula for X-Ray Particle Size Determination[END_REF] (Eq. 5), and the dislocation density δ

[47] (Eq. 6):

= 4/3 + (4) D = 0.9 ! " # (5) δ = $ (6)
where (λ = 1.54 Å) is the incident radiation wavelength, θ represents the Bragg angle and β is the full width at half maximum (FWHM).

The results of the structural analysis are given in (Table 1) from which we observe that the dislocation is increasing while the crystallite size and lattice constants values are decreasing upon doping. This could be explained by the variation in the structure after the doping process, which leads to altering the film's crystallinity and the creation of mechanical stress caused by the substitution of Zn 2+ with lower size Al 3+ and Sn 4+ ions [START_REF] Aslan | Preparation of c-axis-oriented zinc-oxide thin films and the study of their microstructure and optical properties[END_REF]. To confirm the XRD data, Raman spectroscopy characterization was performed to identify the vibrational modes in our samples. From (Figure 2), we can observe the existence of the conventional modes related to ZnO nanoparticles, precisely at 233, 289, and 338 cm -1 in the range from 200 to 400 cm -1 . The ZnO wurtzite mode is observed at 437 and 456 cm -1 according to the E2 polar mode [START_REF] Pal | Synthesis and optical properties of ZnO nanostructures with different morphologies[END_REF]. The Optical Longitudinal (OL) E1 observed at 550 and 556 cm -1 , is attributed to the oxygen deficiency, where the multiphoton processes and resonance lead to wellresolved Raman peaks [START_REF] Mead | The temperature dependence of the raman effect in some wurtzite type crystals[END_REF].

The variation of intensities and the translation in E1 and E2 modes from pure to doped ZnO can be explained by the increase in Al and Sn concentrations, with their ions occupy and substitute those of zinc in the lattice. This mechanism can be ascribed to the active and inactive Raman phonons regarding the lattice caused by Raman shift disorder. From HRTEM image at a higher magnification, Sn doped Zn surface showed some disorder, and this might be due to oxygen vacancies. X-ray photospectroscopy (XPS) was performed to investigate the chemical state of the surface elements. From (Figure . 4, P), the intense peak located at 530.0 ± 0.1 eV is assigned to lattice oxygen bonded as O 2-ions in the ZnO matrix in our samples [START_REF] Nefedov | Electronic structures of MRhO2, MRh2O4, RhMO4 and Rh2MO6 on the basis of X-ray spectroscopy and ESCA data[END_REF]. This means that the peak around 530 eV in O1s core level spectra corresponds to oxygen vacancies and the area under this peak gives feedback on vacancies. In this regard, Sn doped ZnO is found to have the largest surface area which means higher oxygen vacancies followed by ZnO and ZnO doped Al. The electrical measurements were also carried out using 4-point probe technique. We passed a current through two outer probes and reported the voltage through the inner probes. The electrical conductivity, resistivity, and conductance were calculated and presented in Table 2.

Figure 4. EDS mapping of ZnO (a,b), and ZnO:Sn (e, f, g). HRTEM images of ZnO at 5nm (d). TEM images of ZnO (c), ZnO:Sn (h). SAED patterns of ZnO:Sn (m). ZnO:Sn at 10nm (n), 5nm (O). XPS O1s spectrum for all films (p).

The electrical resistivity was calculated using the following equation:

= %& '/( (7)
where B = π / ln2, V is the voltage across inner probes, I is the current in outer probes, and t is the thickness of the thin films. From the table we can observe the decrease in the resistivity after doping with Sn. This decrease in resistivity is probably due to the number of free electrons from the Sn donor ions incorporated in the substitutional or interstitial locations of the Zn cations. Small amounts of the dopant (Sn) introduce into the ZnO network a large number of free carriers and in this case decrease the resistivity.

Application of gas sensors

Device fabrication

The thin films were deposited on alumina substrates to form the sensor device.

Interdigitated contacts and the heaters were deposited to measure the resistance and conductance of the oxide materials. The adhesion layer was deposited by magnetron sputtering (70W argon plasma, 7 SCCM argon flow, 5.5 10 3 mbar at room temperature). The platinum contacts were placed using the same conditions to deposit the adhesion layer, and the device test was maintained in the temperature range (200˚C -500˚C).

Sensor measurements

The response of the gas sensor was performed in an isolated chamber, the humidity was set at 50% with a gas flow reaching 200 sccm at 20˚C. The temperature of each sample was controlled, each target gas was added to a synthetic air (SIAD, Italy) to fix the target concentration. The voltage between (1V-10V) was applied to measure the conductance of the devices. The sensors were heated during 10 hours before introducing the gas, then, we expose them to the desired gas and concentration.

To measure the resistance of the sensor under the analyte gas, we deposit two or more electrodes on metal oxide materials. According to the type of the adsorbed gas and the charge carrier, the resistance may increase of decrease. For our n-type devices, a high amount of oxygen is adsorbed on the sample surface and accept electrons from the material, decreasing its electron density and increasing its resistance.

The determination of the conductance variation is determined by the following expression:

S i = ∆* ∆[,] . (8)
Si is the sensitivity of the gas, R the response of the sensor (the output of the sensor, resistance, or conductance, ...), [B]i is the concentration of the gas i.

The functional ZnO sensor device is presented in Figure 6. The films were exposed to H2 flux, the conductance of the films increases due to the increase of the gas concentration. The sensor's response (Figure 7) is considered high under H2 target gas, Sn doped ZnO shows a high response compared to pure ZnO for both low and high temperatures (Figure 8), followed by Al-doped ZnO that exhibited a very low response. The optimal working temperature under 500 ppm of H2 gas is 350°C for ZnO and 400°C for Sndoped ZnO.

The variation of response/recovery time was calculated and presented in Table 3. Sn-doped ZnO showed lower values compared to literature [START_REF] Aslan | Preparation of c-axis-oriented zinc-oxide thin films and the study of their microstructure and optical properties[END_REF], where the authors observed response/recovery times fluctuating between 312 s and 349 s using other dopants such as Pd and In2O3 [START_REF] Pal | Synthesis and optical properties of ZnO nanostructures with different morphologies[END_REF]. The addition of 4 wt.% of Sn altered the sensing characteristics of the gas sensor, but the addition of Al decreased the response of the sensor. To explain this, we refer to the interpretation of the results detailed in the theoretical part.

DFT calculations

In this work, DFT calculations are employed to explain how the Sn and Al doping affect the sensitivity of ZnO (0002) towards H2. The Zn-terminated polar ZnO (0002) surface was modeled by repeated slabs containing six ZnO double layers separated by a 20 Å vacuum slab along the (0002) direction (Figure 9). To avoid any artificial charge transfer between the bottom oxygen layer and the top zinc layer (i.e., metallization effect), Hydrogen atoms with a nuclear charge of Z= 0.5|e| and an electron charge of 0.5 e were introduced to saturate the broken surface bonds on the bottom of the oxygen layer. The same procedure has been used by Kress [START_REF] Sankar Ganesh | Influence of Al doping on the structural, morphological, optical, and gas sensing properties of ZnO nanorods[END_REF]in his study of novel stabilization mechanisms on the ZnO (0001)-Zn polar surface.

The top three ZnO layers including the adsorbates have been relaxed, while the bottom four ZnO layers are kept fixed at their initial optimized bulk positions, with the equilibrium lattice constants of a = 3.281 Å and c = 5.306 Å (c/a=1.617; ideal hexagonal structure shows c/a =1.633). This is in good agreement with the experimental lattice parameters a=3.245 and c=5.210 (Table 1), and with values found by S. J. Pearton a= 3.249 Å and c = 5.206 Å [START_REF] Nguyen | High response and selectivity toward hydrogen gas detection by In2O3 doped Pd@ZnO core-shell nanoparticles[END_REF].

Figure 9. Illustrations of the supercells of ZnO (0002)-Zn

Color code: Zn is gray, and Oxygen is red.

Pure ZnO (0002) surface

The operating mechanism of gas sensors is mainly based on the change in electrical conductivity of the sensor caused by the transferred charge between the sensing materials and the gas molecules. The flow of electricity inside the sensing material is determined by the number of free electrons. It has been shown experimentally that when the sensing material is in clean air, the oxygen molecule (O2) is adsorbed on the sensor surface, and the free electrons are extracted from the conduction band. This results in the creation of O anion species, causing a change in the electrical conductance of the sensor. This means that the resistance of the sensor will increase, resulting in a decrease in the flow of electrons within the sensing material. Therefore, in the presence of reducing gases such as H2, when it reacts with oxygen, the electrons generated upon adsorption of oxygen are released back into the sensing material. As a result, sensing resistance decreases, allowing more current.

The process involved in detecting H2 gas at the ZnO (0002) surface can be described by the following reactions:

O2 (air) → O2 (adsorbed) (9)
O2 (adsorbed) + 2e (ZnO free electron)

→ 2 O - (10)
2H2 (reducing gases) + 2O -→ 2H2O + 2e (ZnO free electron) [START_REF] Zang | Enhanced fluorescence imaging performance of hydrophobic colloidal ZnO nanoparticles by a facile method[END_REF] We first investigated the interaction of O2 and H2 with the surface of ZnO (0002). The DFT calculations revealed that the H2 molecule is mainly adsorbed by dispersion forces with a 54% dispersion contribution in total interaction energy of -19 kJ/mol, it was observed that the H2 molecule interacts with the surface at 2.86 Å, leading to a charge transfer of only 0.08 |e| between the molecule (H2) and the surface (Zn). However, when the O2 molecule is adsorbed on the ZnO surface, a complex is created involving three Zn atoms from the surface, resulting in total interaction energy of -32.6 kJ/mol. We found that the interaction between O and Zn atoms presents a significant electron transfer of 0.58 |e| and an average interaction distance of 1.98 Å (Figure 10). This demonstrates that an oxygen anion layer is formed on the ZnO (0002) surface and therefore the formation of H2O molecule is occurring (Eq.9). Due to this, a small release of trapped electrons to the surfaces of ZnO (0002) of 0.14 |e| is observed, which proves the potential of this material as a gas sensor. However, the responses are expected to be low, which implies a modification of the ZnO material.

Al and Sn-doped ZnO

To account for direct contact between the dopants and the molecules, we have substituted a Zn atom of the upper layer by either Al or Sn atoms. After the relaxation of these two structures, we found that aluminum and tin are bound to oxygen atoms with an average distance of 1.81 Å (See Figure 11), 2.15 Å (See Figure 12) respectively, compared to that of Zn-O (1.92 Å).

Al-doped ZnO

Concerning the adsorption of H2 molecule, the total interaction energy is (-121 kJ/mol), this high energy is caused by the dissociative adsorption of the molecule. The difference in charge density revealed that no interaction persists between the two H2 atoms upon adsorption (Figure 10). This means that even in the absence of O2 in the air, the H2 molecule can extract electrons from the conduction band of the Al-doped ZnO, resulting in the cancellation of the electrical conductance.

In the presence of oxygen molecules, we found that the aluminum atom of the surface strongly interacts with the O2, leading to its dissociation into two oxygen ions. The electron density (Δρ)

and Bader charge (ΔQ) differences revealed that this dissociation presents a significant electron

Sn-doped ZnO

On the Sn-doped ZnO surface, the adsorption of the H2 molecule showed similar behavior to that of pure ZnO. Herein, the total interaction energy is -17 kJ/mol, with an interaction distance of 3.10 Å between hydrogen and Sn atoms. Comparing to H2, the O2

Conclusion

ZnO doped Al and Sn sensors were successfully synthesized. The experimental characterization showed the adequate surface for the adsorption phenomenon, especially for ZnO doped Sn followed by ZnO and ZnO doped Al that exhibited the lowest response. This low response was analyzed by the theoretical calculations using DFT. The influence of Sn and Al on the sensitivity of the Polar ZnO (0002) surface toward H2 gas was studied experimentally and by DFT theoretical calculations. We observed that no H2O formation has occurred on ZnO-Al surface and the charge transfer analysis showed that H2 reaction with the oxygen molecule adsorbed on Al-doped ZnO can only release a 0.08 |e| back to the surface. Thus, H2 gas cannot be detected. After Sn doping on the surface of ZnO (0002), it levels up from the surface and extends its Sn-O distance to reach 2.15 Å compared to that of pure Zn (1.92 Å). Then O2 molecules of air are adsorbed on the ZnO-Sn surface weakening the O-O bond which makes it a favorable site for H2 gas attack and H2O formation leading to a full release of the trapped electrons. thus, an important enhancement in the detection of H2 gas is observed, this agrees with the experimental results.

(

  Figure.1). We observe multiple peaks corresponding to the wurtzite structure as indicated in (JCPDS card 36-1451)[START_REF] Alim | Micro-Raman investigation of optical phonons in ZnO nanocrystals[END_REF]. The highest electrons density is translated by the highest intensity along (002) direction, which is the preferred orientation of the polycrystalline ZnO. Other peaks related to ZnO such as (220) and (222) appeared within the patterns, the other peaks proving the addition of Sn and Al are (511) and Sn (112) respectively.

Figure 1 .

 1 Figure 1. X-Ray Diffraction patterns of ZnO, Al-doped ZnO, and Sn-doped ZnO thin films.

Figure 2 .

 2 Figure 2. Raman patterns of ZnO, Al-doped ZnO, and Sn-doped ZnO thin films.

Figure 3 .

 3 Figure 3. SEM illustrations of a) ZnO, b) Al-doped ZnO, and c) Sn-doped ZnO thin films, and the corresponding grains diameter distribution (d, e, f).

Figure 5 . 8 (

 58 Figure 5. EDS spectrums of a) ZnO: Al and b) ZnO: Sn.

Figure 6 .

 6 Figure 6. The functional chemical sensor device of ZnO film.

Figure 7 .

 7 Figure 7. Responses of Sn-doped ZnO and ZnO sensors under H2 (100-250-500 ppm) at 400°C with corresponding humidity of 40% at 20°C.

Figure 8 .

 8 Figure 8. Variation of response with the temperature of ZnO, ZnO: Sn, and ZnO: Al towards hydrogen (500 ppm) with corresponding humidity of 40% at 20°C.

Figure 10 .

 10 Figure 10. Difference in electronic density (Δρ) induced by the adsorption of a) H2 b) O2 on pure ZnO (0002) surface, c) the attack of H2 on the adsorbed O2. The blue (yellow) zones correspond to the increase (decrease) in density.

transfer of 1 .

 1 31 |e| between the O and Al atoms. Therefore, the approaching H2 gases cannot extract the strongly adsorbed oxygen, and only 0.08 |e| is released back to the surface. Hence, no H2O formation will occur. This weakens the H2 detection signal and explains the extremely low response of ZnO-Al to hydrogen (Figures 8).

Figure 11 .

 11 Figure 11. Difference in electronic density (Δρ) induced by the adsorption of a) H2 b) O2 on ZnO (0002) doped aluminum surface, c) the attack of H2 on the adsorbed O2. The blue (yellow) zones correspond to the increase (decrease) in density.

  molecule is more adsorbed by around 20 kJ/mol. This adsorption led to a significant expansion of the O-O bond, from 1.20 to 1.52 Å, weakening this bond and proving the ability of Sn to facilitate the O2 ion formation on the surface. All this occurs with a charge transfer of about 0.31 |e| and -0.63 |e|, respectively for Sn and O, which makes it favorable for hydrogen attack.In the presence of the reducing gas, the H2 molecule interacts with the adsorbed O ions, leading to the formation of the water molecule. Charge transfer analysis revealed that this process led to a full release (0.30 |e|) of the trapped electrons to Sn atoms. This indicates weak adsorption of H2O on Sn-doped ZnO surface, and a significant increase of the doped material sensitivity toward H2 gas, in line with the experiment.

Figure 12 .

 12 Figure 12. Difference in electronic density (Δρ) induced by the adsorption of a) H2 b) O2 on ZnO (0002) doped tin surface, c) the attack of H2 on the adsorbed O2. The blue (yellow) zones indicate density increase (decrease).

Table 1 :

 1 Structural

	Sample	dhkl (002)	D(nm)	δ*10 5 (nm) -2	Lattice constants a(Å) c(Å) c/a
	ZnO	2.6031	22.19	203.08	3.245 5.210 1.6055
	ZnO:Al	2.6035	12.49	641.02	3.006 4.964 1.651
	ZnO:Sn	2.6025	8.98	1240	3.001 4.942 1.646

parameters of ZnO, Al, and Sn doped ZnO. JPCDS card N°: 36-1451 (a = 3.250 Å and c = 5.207Å).

Table 2 :

 2 Resistivity and conductivity of ZnO and Sn doped ZnO thin films.

	Samples	Resistivity (Ω.cm)	Conductivity (Ω.cm) -1
	ZnO	63.01x10 -4	158.7
	ZnO:Sn	62.39x10 -4	160.28

Table 3 :

 3 Response and recovery times of the sensors ZnO, Al-doped ZnO and Sn-doped ZnO.

	Sample	Response time	Recovery time
	ZnO	81	99
	Sn-doped ZnO	50	80
	Al-doped ZnO	79	101


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

Oral Session 1: Advances in risk assessment methods and tool

O1-2

Minimizing the risk of foodborne illness and analytical costs using a QMRA model for raw milk cheeses. ). Secondly, we use this QMRA model to establish efficient bio-intervention strategies or more specifically to find optimal process intervention parameters for minimizing the risk of the illness and the associated cost of intervention. This translates into a multi-objective optimization problem of a stochastic simulator.

Methodology:

The newly proposed multi-pathogens model incorporates the effects of two more pathogenic bacteria namely Salmonella and Listeria monocytogenes, which can occasionally be present in raw milk. This model accounts for, pathogen prevalence and concentration among farm animals, and the growth and decline rates of the micro-organisms in the cheese matrix. Risk for the three pathogens is assessed by combining exposure and doseresponse models. The overall risk is assessed by combining the burden of disease metrics (a.k.a Disability Adjusted Life Years) for the three pathogens. For optimization we rely on Bayesian methods, in particular, we propose a modified version of the PALS algorithm (Pareto Active Learning for Stochastic simulators) by Barracosa et al (2021), to estimate the Pareto optimal solutions for the intervention parameters.

Results:

We have considered the problem of minimizing the risk of illness and the cost of the associated interventions, in order to find the optimal parameters namely, the frequency of milk testing, the milk testing threshold, the proportion of batches tested for cheese, and the sample size for testing.

Conclusions and Relevance:

The objective of the study is to optimize the potential control measures available for minimizing the risk of foodborne illness from the consumption of raw milk cheeses and make recommendations to dairy professionals.

This work is part of the ArtiSaneFood project (grant number: ANR-18-PRIM-0015) which is part of the PRIMA program supported by the European Union. This project aims at the microbial safety of artisanal fermented food produced in the Mediterranean region.

  Minimizing the risk of foodborne illness and analytical costs using a QMRA model for raw milk cheeses Subhasish Basak, Janushan Christy, Laurent Guillier, Frédérique Audiat-Perrin, Moez Sanaa, Fanny Tenenhaus-Aziza, Julien Bect, Emmanuel Vazquez To cite this version: Subhasish Basak, Janushan Christy, Laurent Guillier, Frédérique Audiat-Perrin, Moez Sanaa, et al.. Minimizing the risk of foodborne illness and analytical costs using a QMRA model for raw milk cheeses. 12th International Conference on Predictive Modelling in Food (ICPMF12), Jun 2023, Sapporo, Japan. hal-04371085

  Subhasish Basak 1,2 , Janushan Christy 3 , Laurent Guillier 1 , Frédérique Audiat-Perrin 1 , Moez Sanaa 4 , Fanny Tenenhaus-Aziza 5 , Julien Bect 2 , Emmanuel Vazquez 2 1 Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Maisons-Alfort, France. 2 Laboratoire des Signaux et Systèmes (L2S), CNRS, CentraleSupélec, Université Paris-Saclay, Gif-sur-yvette, France. 3 ACTALIA, La Roche sur Foron, France. 4 World Health Organization (WHO), Geneva, Switzerland. 5 Centre National Interprofessionnel de l'Economie Laitière (CNIEL), Paris, France Introduction: The contribution of this work is twofold. Firstly we propose an extension to the Quantitative Microbiological Risk Assessment (QMRA) model proposed byPerrin et al (2014), which is today used by dairy professionals in France, for optimizing microbiological sampling plans regarding the risk of Haemolytic and Uremic Syndrome (HUS), caused by Shiga-toxin producing Escherichia coli (STEC


Maria Bas
email: maria.bas@univ-parisl.fr.

Pamela Bombarda
email: pamela.bombarda@cyu.fr.

Sébastien Jean

Gianluca Orefice
email: gia.nluca..orefice@da.uphine.psl.fr

Sebastien Jean

Lionel Fonta Gne

Thierry Mayer

Hillel Rapoport

Ariell Reshef

Farid Toubal

Gonzague

Firms' exports, volatility and skills: Evidence from France

Keywords: Export intensity, employment volatility, skilled labor, firm-level data. JEL classification: Fl, F16, 125, 160

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

estimate the structure of hiring and firing costs in France. While the hiring costs tend to be small and economically relevant only for CDD contracts, the firing costs are economically relevant for both types of contracts (CDI and CDD). Interestingly, the authors show that firing costs exhibit a concave shape with respect to the number of dismissed persons. Other job protection rules, such as those concerning the years of seniority of employees, have also an impact on the firing cost of firms. For example, employers with more than eleven employees have to pay severance payments in case of unfair dismissal when the employee has more than two years of service (Cahue et al., 2019, andCahue et al., 2021). 9

Kramarz and Michaud (2010) conclude that "the more skilled the labor force, as measured by the share of managers, engineers and other professionals, the higher the fixed cost for both collective and individual terminations." Their finding suggests that firing costs tend to be larger for skilled than for unskilled workers, which may contribute to explain why labor adjustments vary across skills. This conclusion is in line with the results in Blatter et al. ( 2012), who use administrative Swiss employer-employee data to show that hiring and training costs are generally higher for skilled than for unskilled workers. These findings are in line with our research question. Another reason (on top of the skilled intensive fixed export cost channel) for why firms more exposed to foreign shocks tend to adjust more easily the unskilled than the skilled labor demand is because firing and hiring costs are higher for skilled workers.

Theoretical motivation

While this paper is primarily an empirical analysis, we describe here a plausible theoretical mechanism underlying the link between export activity and employment growth volatility across skills. The mechanism we have in mind is related to the structure of firm's export costs in terms of skilled and unskilled workers. The existence of significant sunk costs associated with exporting is widely documented (Das et al., 2007), and the corresponding fixed export costs are composed in large part of logistical, coordination and distributional operations, which are usually more skill intensive than production activities (Artopoulos et al., 2011;Cavusgil and Zou, 1994). Fixed export costs reflect the adaptation to foreign standards, regulations, and taste of consumers, requiring mainly skilled workers to cover these tasks ( e.g. lawyers, engineers, marketing experts). The idea that fixed export costs are skill intensive is related to the fact that these costs involve services and tasks that require more skilled labor such as marketing, research, communication with clients or intermediaries and distribution. This idea is in line with the model developed by Matsuyama (2007) and the evidence presented 9 Other employers' obligations are related to organizing elections of staff representatives (above eleven em ployees), and to ensuring additional rights regarding hygiene, safety, employment and training (above fifties employees).

We thank the editor and three anonymous referees for very useful remarks. thank Nicolas Berman for kindly sharing the data. used to construct the instrumental variables. All errors are our own. This research has been conducted as part of the ANR project "MOQAT" 18-CE26-0011.