Carlos Diaz

Yuly Vesga

Lorenzo Echevarria

Irena G Stará

Ivo Starý

Emmanuel Anger

Chengshuo Shen

Mehdi El

Sayed Moussa

Nicolas Vanthuyne

Jeanne Crassous

Carlos Díaz

Ivo Starỳ

Antonio Rizzo

Florencio E Hernández
email: florencio.hernandez@ucf.edu

Two-photon absorption and two-photon circular dichroism of hexahelicene derivatives: a study of the effect of the nature of intramolecular charge transfer

Keywords: Two-Photon Absorption, Nonlinear Circular Dichroism, Intramolecular Charge Transfer, Helical Chirality, Circular Polarization

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INTRODUCTION

The fundamental activity of chiral molecules in biological processes 1 and the many applications optically active molecules have found in asymmetric catalysis, 2 pharmacy industry, 3 and in the world of photonics and nanotechnology, 4,5 have motivated scientists to deepen the understanding of chiral systems during the last twenty years. Efforts have been mainly driven by the development of two complementary techniques, i.e. circular dichroism (CD) and optical rotatory dispersion (ORD). [START_REF] Nakanishi | Circular Dichroism: Principles and Applications[END_REF] The former, when associated to transitions between electronic states, is called electronic circular dichroism (ECD) and is defined as the difference in onephoton absorption (OPA) between right (RCPL) and left (LCPL) circularly polarized light. [START_REF] Condon | [END_REF][START_REF] Caldwell | The Theory of Optical Activity[END_REF] During the last two decades ECD has been a standard technique for the study of the conformational and physical-chemical properties of optically active molecules. [START_REF] Nakanishi | Circular Dichroism: Principles and Applications[END_REF] However, this method presents important intrinsic limitations for the study of optically active molecules in solution and in inhomogeneous media in the UV region. [START_REF] Tinoco | [END_REF] The OPA of standard aqueous buffer solutions and common solvents in the same spectral region can mask the ECD signal of chiral molecules and, the scattering present in inhomogeneous samples can become critical at shorter wavelengths.

With the aim of surmounting the existent barriers in ECD, alternative approaches have been proposed; these include vibrational circular dichroism (VCD), [START_REF] Nafie | Vibrational Optical Activity: Principles and Applications[END_REF] and Raman optical activity (ROA), [START_REF] Barron | Molecular Light Scattering and Optical Activity[END_REF] magnetic circular dichroism (MCD), [START_REF] Mason | Practical Guide to Magnetic Circular Dichroism Spectroscopy[END_REF] and synchrotron radiation circular dichroism (SRCD). [START_REF] Miles | [END_REF] Likewise, nonlinear optics has opened a new way in this direction by the application of second harmonic generation (SHG), 14,15 sum-frequency generation (SFG), 15,16 multiphoton optical rotation, 17 nonlinear optical activity, 18 and two-photon circular dichroism (TPCD). 19 The latter is the nonlinear analogue of ECD, which is defined, in the degenerate case, as

      TPCD TPA TPA LR          , where   TPA L  and   TPA R
 are the two-photon absorption (TPA) cross-sections for LCPL and RPCL, respectively, measured at a specific wavelength  . 20- 22 TPA is particularly advantageous over OPA in many applications because the typical excitation wavelength for this nonlinear absorption process is approximately two folds longer (degenerate case) than that necessary for OPA. Therefore, the linear absorption in the twophoton excitation region is negligible and scattering can be minimized. 23,24 Furthermore, it presents a high intrinsic spatial resolution and penetration depth due to the confinement of the excitation to the focal volume, 24 and it exhibits better background discrimination and reduced photodamage. 23 Moreover, since experimental measurements of TPA in solution commonly require a relatively high concentration of the solute (typically between 10 -3 and 10 -2 M), and because the magnitude of the TPCD signal is just a small percentage (1-3%) of

 

TPA  , measurements of TPCD on one specific enantiomer are practically unaffected by the presence of small amounts of the other one. In addition to all the advantages mentioned above for TPA, TPCD is also a very sensitive spectroscopic technique to small structural distortions and with unique access into the far-UV, 25 i.e. a spectral region that is filled with important structural/conformational information but typically obscure to ECD. Therefore, the application of TPCD for the structural and conformational study of chiral molecules in this spectral region is virtually imminent. However, before reaching the full potential of this unique approach, more fundamental theoretical-experimental studies on TPCD are required.

Since the development of the double L-scan technique, 26 the study of optically active molecules in the near to far UV region using TPCD became an open possibility for the structural study and conformational understanding of chiral systems. It should be mentioned that the first step towards the experimental measurement of TPCD was made by Richardson and co-workers in 1995 27 using fluorescence-detected two-photon circular dichroism (FD-TPCD), an approach that inspired Finazzi and co-workers to develop more recently a FD-TPCD based microscopy technique. 28 Seeking for a better understanding of TPCD and its structure-property relationship we have been working on the theoretical-experimental analysis of the TPCD response of several systems. 19,25,[29][30][31][32][33] Although all these studies have contributed to the expanded knowledge of TPCD in organic molecules, more molecular systems need to be studied to better understand this nonlinear optical property and its potential applications. To help accomplish this goal, herein we present the results from our most recent work which was primarily focused on the first theoretical-experimental analysis of the TPA and TPCD spectra of two interesting aromatic molecules -hexahelicene derivatives -with helical chirality and intramolecular charge transfer (ICT) (see Figure 1.a for their chemical structures). One should remark that helicenes are very popular chiral systems employed in applications involving chirooptical photoswitches, 34 enantioselective fluorescence detectors, 35 circularly polarized luminescence for back-lighting in liquid crystals displays, 36,37 and nonlinear optical (NLO) devices. 38,39 Next, we present evidence that show that the TPA cross-section and the amplitude of the TPCD signal in helicenes are strongly affected by the strength and nature of the ICT. We demonstrate that the extension of the electronic delocalization beyond the helicene core (EXO-ICT) has a stronger effect of the nonlinear optical responses of this family of molecules than the extension of the electronic delocalization within the aromatic cluster (ENDO-ICT). This effect is directly correlated, as shown below in a series of helicene derivatives with different electron delocalization disposition (Figure 1.b), with the contributions of the magnetic dipole transition moment and the electric quadrupole transition moment to the TPCD rotatory strength of the molecules.

EXPERIMENTAL DETAILS

P-(+)-1-aza [6]helicene (hereafter A6 -Figure 1.a) was synthesized as previously reported by Stará and co-workers. 40 Briefly, A6 (Figure 1.a) was prepared by using the cobalt-catalyzed intramolecular [2+2+2] cycloaddition of a pyridine-linked triyne followed by dehydrogenation with MnO 2 . 40 [6]helicene (Figure 1.b) were obtained from racemic 2-ethynyl- [6]carbohelicene and commercially available 4bromobenzonitrile through a Sonogashira coupling reaction followed by chiral HPLC separation. 41 Although both the P and M enantiomers were obtained for this derivative, only the M enantiomer (henceforth CN6) was used for the spectroscopic measurements due to impurities that could not be removed from the solution containing the P enantiomer.

M-(-) and P-(+)-2-[4-(cyanophenyl)-1-ethynyl]carbo

OPA measurements were performed using a single-beam spectrophotometer (Agilent 8453 Diode Array Uv-Vis) from 190 to 600 nm in a 0.1 cm quartz cell in 0.210 -4 -2.010 -4 M solutions in THF. ECD spectra were completed on a J-815 CD spectropolarimeter (Jasco Corp., Tokyo, Japan) following these conditions: concentration 1.0 x 10 -5 M in THF; temperature 25 ºC; quartz cuvette length 4 mm; wavelength range 190-600 nm; 1 nm step and scan speed 50 nm/min.

TPA and TPCD measurements were carried out in solutions with concentrations ranging from 3.010 -2 M to 5.310 -2 M, employing the double-L scan technique. 26 All samples were dissolved in THF. Two-photon excitation was induced with a computer-controlled femtosecond optical parametric amplifier (OPerA Solo) pumped by an amplified laser system from COHERENT. The whole system is capable of generating 90 fs (FWHM) pulses over a wavelength range spanning from 240 nm to 2.6 μm, and with pulse energies of up to 350 J.

Experiments were performed at a repetition rate between 2 and 50 Hz to avoid any contribution from cumulative effects. The pulse width was measured using a single-shot autocorrelator from Coherent Inc. and a frequency-resolved optical gaiting (FROG) from Swamp Optics LLC.

THEORETICAL AND COMPUTATIONAL APPROACH

Although, the theoretical approach employed in this work has been extensively addressed elsewhere 22,33,[42][43][44][45] (see also Supporting Information, SI), below we briefly describe the fundamental aspects of the theoretical and computational calculations.

The molecular structures of A6 and CN6 (see Figure 1.a), as well as those of five CN6like helicenes (#CN) (see Figure 1.b), i.e. C6-CN, C6--CN, C6---CN, C7-CN and C8-CN, were optimized using Density Functional Theory (DFT), 46 employing the Becke's threeparameter exchange, Lee, Yang and Parr correlation (B3LYP) hybrid functional [47][48][49] in combination with the 6-311++G(d,p) basis set, 50,51 employing Gaussian 09. [START_REF] Frisch | Gaussian[END_REF] Solvent effects (THF) were considered as implemented in the polarizable continuum model (PCM). [START_REF] Tomasi | [END_REF] Calculations of excited states of all helicenes, in the linear (OPA and ECD) and nonlinear (TPA and TPCD) instance, were performed employing Time-Dependent DFT (TD-DFT), 46 with two different exchange correlation functionals (XCF), i.e. B3LYP and the Coulomb attenuating method-B3LYP (CAM-B3LYP) 54 . Because it is well-known that the latter is a more reliable XCF than the former in molecules with ICT, 29,44,54 in the manuscript we only present results obtained with CAM-B3LYPresults from theoretical calculations using B3LYP are available in SI.

At this point, we would like to comment on the limitations of an inherently singleexcitation based model as TD-DFT when dealing with electronic excited states dominated by double excitations. [START_REF] Marques | Fundamentals of time-dependent density functional theory[END_REF][START_REF] Ullrich | Time-dependent density-functional theory: concepts and applications[END_REF] It is well known that in some cases, for example for polyenes or polyacenes, the TPA response can be particular intense for states with this character. [START_REF] Ullrich | Time-dependent density-functional theory: concepts and applications[END_REF][START_REF] Aryanpour | [END_REF] Although, we recognize the challenges present when resorting to the computational protocol employed in this study for the study of linear and, in particular, nonlinear spectroscopies, this subject has been the theme of detailed studies within our group, cf. for instance Ref. 33. Double excitations, in the definition given usually in ab initio quantum chemistry, are somehow taken care even within standard adiabatic TDDFT approaches using approximate functional. [START_REF] Ullrich | Time-dependent density-functional theory: concepts and applications[END_REF]58 Nevertheless, it is a fact that, when double or higher excitation character of the excited state is dominant, this model is bound to fail. Although alternative approaches to adiabatic TD-DFT, able in principle to deal with double excitations, have been proposed and developed (as the use of frequency dependent or non adibatic kernels), [START_REF] Marques | Fundamentals of time-dependent density functional theory[END_REF] the debate and the current development has been mostly concerned with excitation energies and linear response properties, and we are not aware of extensions to analytical nonlinear response. On the other hand the importance of multiple excitations for two-photon spectra, has been discussed also within the ab initio quantum chemistry community, [START_REF] Aryanpour | [END_REF] and besides the well-known multiconfigurational time dependent Hartree-Fock approaches to nonlinear response 59 available in DALTON 60 now for decades -but not suitable in its current status, for the treatment of molecules of the size of those studied herethe algebraic-diagrammatic construction polarization propagator approach developed within the group of Drew and applied to octatetraene in Refs. 59, 61and 62,5 9, 61, 62app ears to be a particularly promising suitable tool to treat non-linear absorption when multiple excitation are of particular importance for sizeable molecules.

Next, we computed the oscillator strengths ( 0 f f ) and velocity rotatory strengths ( 0

ECD f R )
for the first 60 electronic excited states for all the helicene derivatives using TD-DFT at the CAM-B3LYP/6-31G(d) 63 level of theory including solvent effects (THF) via PCM, in Gaussian 09. [START_REF] Frisch | Gaussian[END_REF] The convoluted theoretical UV-vis and ECD spectra were obtained by Lorentzian broadening from 0 f f and 0 ECD f R of the calculated electronic transitions employing equations ( 1)

and (2). Note: In order to certify the reliability of the selected basis set, we performed additional TD-DFT calculations over a smaller number of excited states (first 30 electronic excited states) using a larger basis set, i.e. 6-311++G(d,p). No significant differences, compared to the linear spectra computed with 6-31G(d) were obtained. OPA spectra are reported in molar absorptivity (  ), 45, 65

    0 3 0 0 1.05495 10 , , , f OPA f f f f g            ( 1
)
where  is the circular frequency of the incident light.

ECD spectra were computed from 0 ECD f R , and they are reported as molar absorptivity difference (   ), 45, 65, 66

    1 00
2.73719 10 , ,

ECD ECD ff f gR             , (2)
OPA and ECD spectra have units of 11 mol cm l  , as long as all the elements in Equations ( 1) and ( 2) are introduced in atomic units.

TPA spectra were obtained from [START_REF] Rizzo | Computational Strategies for Spectroscopy[END_REF][START_REF] Mcclain | [END_REF]      

2 0 0 0 4 8.35150 1 2, 0 ,, TPA TPA f f f f g               (3)
where

  0 0 TPA f f



is the orientationally averaged two-photon probability for the degenerate case.

TPCD spectra were simulated according to, 20,42,[START_REF] Rizzo | Computational Strategies for Spectroscopy[END_REF]       52 0 0 0 4.87555 10 2 , , .

TPCD TPCD f f f f x g R              (4)
and spectra o tained from uations and 4 are given in ppert-Mayer units (GM), i.e., 10 -50 cm 4 •s•molecule -1 •photon -1 , as long as all the equation elements are introduced in atomic units.

RESULTS AND DISCUSSION

In order to start with the analysis, in Figure 2 we present the experimental OPA and ECD spectra of A6 (Top) and CN6 (Bottom) in THF solution, and their corresponding convoluted CAM-B3LYP stick spectra, calculated in THF using PCM and basis set 6-31G(d) (Assignment of theoretical excited states to experimental bands can be found in Tables 1.a and 1.b in SI.). To obtain a better match with the experiment, the theoretical spectra were spectrally shifted by an amount reported in the caption of the figure (this is common practice in theoretical-experimental works). 19,29,30,32,33 One should mention that the theoretical curves are consistently blue shifted with respect to the experimental. This result is not surprising since, as it has been previously reported, 19,33,45,68 in all calculations most of the depicted states are basically valence states with a significant contribution form *, and because CAM-B3LYP tends to overestimate the excitation energies. 33 The primary observation in Figure 2 is the noteworthy theoretical reproduction of the experimental OPA and ECD spectra (spectral shape, position of the bands, and the fano-type shape profile) of A6 and CN6 using CAM-B3LYP. The remarkably good performance of this particular XCF is attributed to the existent intramolecular charge transfer (ICT) in both molecules, being ICT unequivocally stronger in CN6. In this particular molecule, the cyano (CN) group, a well-known moderate electron-withdrawing group, located beyond the helicene core on one extreme of the molecule, causes not only a strong ICT effect but it increases the range of the electronic delocalization beyond the helicene core. This point can be confirmed even further by the analysis presented in Figure 3, which show the molecular orbitals involved in the transitions to excited state #3 and #2 for A6 and CN6, respectively, i.e. the most important excitations on the red side of the OPA and ECD theoretical spectra calculated with CAM-B3LYP (see Figure 2 and Tables 1.a and 1.b in SI). The observed ICT in all three excitations involved in the CN6 transition is indeed extremely important. Similar evidences have already been reported by Rizzo and co-workers in other molecular systems 29,33,45,68 and, are in accordance with previous calculations performed in CN6. 41 Next, one should highlight the fact that the amplitudes of the OPA and ECD signals in both molecules are virtually identical, though there is an obvious difference in ICT between the two. This observation shall be explained more in detail after completing the theoreticalexperimental analysis of the nonlinear spectra of A6 and CN6 presented next.

In Figures 4 we show the TPA and TPCD spectra (theoretical and experimental),

respectively, plotted at half the excitation wavelengths, i.e. in the OPA scale. The spectra of both compounds were taken in THF solution. The theoretical spectra of A6 were obtained with the CAM-B3LYP/aug-cc-pVDZ computed profiles obtained by convoluting the stick spectra (The latter simulates isolated non interacting molecules). To optimize the balance between accuracy and computational cost, calculations in CN6 were performed using the 6-311++G(d,p) basis set, which is a slightly smaller basis set than aug-cc-pVDZ. As for the one-photon spectra a shift has been applied to the computed TPA and TPCD spectra (see figure caption of Figure 4). Since the spectroscopic region of relevance is different when comparing one-and two-photon absorption profiles, the shifts applied in in SI). Also, because strong two-photon absorber states are in most cases weak one-photon absorber, the differences in spectral shift obtained for OPA and TPA are easily explained by considering that OPA and TPA are dominated (see Figure 2 and4) by different molecular states.

69, 70

First, it can be noticed that the theoretical calculations reproduce remarkably the main TPA spectral features of both helicene derivatives. However, the predicted TPA cross-sections are, in most cases, larger than the experimental. It is hard to explain this difference in amplitude considering that all experiments were performed in the femtosecond regime where excited state absorption is negligible. 71,72 Nevertheless, the chosen XCF reproduces very well the relative intensities between the main TPA bands, and it yields a suitable bandwidth and separation between the peaks.

Second, the intense peak observed at 215 nm in the TPA experimental spectrum of A6, that lead to a strong increasing band in the blue side of the TPA spectrum of this molecule is not well reproduced by the theory. This outcome could be a consequence of the limited number of electronic excited states included in the theoretical calculation of the TPA spectrum or, even more probable, due to the presence of strong TPA resonance enhancement effect found when the measurements are performed in the close vicinity of linear absorption states (see Figure 2). 73,74 In any case, this region would be better analysed with the damped approach to TPA discussed in references 67 and 68.7 5z

Next, moving onto TPCD, in Figure 4 we display the experimental (in THF solution) and theoretical (isolated molecules) spectra of A6 and CN6, obtained using CAM-B3LYP. One can notice that the theoretical TPCD spectra of A6 and CN6 reproduces notably the shape, sign, and relative intensities of the main three bands in A6 (one positive at 240 nm and two negative at 250 and 290 nm) and the three positive bands in CN6 (280, 300 and 330 nm) -see Tables 1.a and 1.b in SI.

Finally and perhaps the most interesting outcome of the nonlinear characterization of these two molecules is the obviously larger TPA cross-section (approximately ten times) and TPCD signal (ca. two-fold) of CN6 compared to that of A6 (see Figure 4). This interesting result can be explained by considering two cooperative effects: a) the strong ICT character of a wellknown moderate electron-withdrawing group (CN) on one end of CN6, and b) the extended electron delocalization over the (4-Cyanophenyl)-1-ethynyl substituent outside the helical core in this helicene derivative. In A6, however, a relatively mild ICT is effective only within the hexahelicene core due to the presence of the aza (N) group at one end of the molecule.

Although, the difference in ICT between A6 and CN6 was discussed earlier in the linear characterization of these two molecules (see Figure 4), the actual implications were not elucidated.

In order to gain more insight about the two effects mentioned above, we performed calculations of OPA, ECD, TPA and TPCD on five #CN molecules (see Figure 1.b) exhibiting different ICT conjugation length outside the [6]carbohelicene core (EXO-ICT), i.e. C6-CN, C6--CN and C6---CN, and within the aromatic cluster (ENDO-ICT), i.e. carbohelicene derivatives with six (C6-CN), seven (C7-CN) and eight (C8-CN) aromatic rings extending the helical arrangement.

In Figure 5 we show the comparative plot of the OPA and ECD, and the TPA and TPCD theoretical spectra of all five #CN. As expected from the linear analysis of A6 and CN6, the OPA and ECD signals are not strongly affected by the nature of ICT, i.e. EXO-ICT vs. ENDO-ICT. In this figure one can only observe a small increase in the amplitude of the ECD signal and a consistent red shift of the position of the fano-type bands of #CN as the conjugation length of the aromatic cluster increases from C6  C8. However, EXO-ICT seems to have no significant effect on the linear spectra of these molecules. This interesting result suggests the generation of stronger molecular circular currents as the spiral length of the ENDO-ICT increase in helical molecules. These currents can in turn generate molecular magnetic fields that couple with the magnetic dipole transition moment of #CN, 32,76,77 thus increasing the ECD response as follow:

C6-CN < C7-CN < C8-CN.

In the nonlinear optical response instance it is obvious that the effect of EXO-ICT is much stronger than ENDO-ICT. In Figure 5, one can notice that while the maximum TPA crosssections of C6--CN and C6---CN are approximately 2.4 and 4.9 times that of C6-CN, respectively, the difference in TPA between C6-CN, C7-CN and C8-CN is nearly negligiblethe small difference noticed between C6-CN, C7-CN and C8-CN, on the blue side of the spectra, is mainly attributed to the limitations of our computational resources to calculate the required number of excited states in the larger molecules (C7-CN and C8-CN) to cover the same spectral region obtained for C6-CN. In TPCD one can also remark a stronger EXO-ICT effect, i.e. the integrated area of the TPCD spectra of C6--CN and C6---CN are approximately 2.5 and 7.9 folds that of C6-CN, respectively. The difference in TPCD between C7-CN and C8-CN, and C6-CN is ca. 1.7 and 2.2, respectively, i.e. less significant than in the previous series of #CN but comparable to the small differences observed in ECD. This results strongly suggest that: (1) the electric dipole transition moment, which directly affect the TPA response of conjugated molecules, 78,79 is enhanced by increasing the electronic conjugation length beyond the helicene core, (2) the magnetic dipole transition moment could enhance the TPCD response of helical molecules via its coupling with the induced molecular magnetic field resultant from the generation of molecular circular currents within the helical core, 32,76,77 With the intention of supporting even further these results, in Figure 6 we present the integrated TPCD spectra, and the sum of the absolute values of molecular parameters B 1 +B 3 and B 2 that define the contributions of the magnetic dipole transition moment and the electric quadrupole transition moment, respectively, to the TPCD rotatory strength 20,22,42 (See SI for further detail on molecular parameters B i ). The clear tendencies observed in this plot, viz. i)

|B 1 +B 3 | (

CONCLUSIONS

In summary, we have demonstrated that the TPA cross-section and the amplitude of the TPCD signal in helicenes are strongly affected not only by the strength of the ICT but by its nature. The molecular orbital analysis of A6 and CN6, and the theoretical analysis of these effects on a series

g

  helicene derivatives were calculated employing TD-DFT in DALTON 2011,60 using CAM-B3LYP/aug-cc-pVDZ64 for A6, and employing CAM-B3LYP/6-311++G(d,p) for the rest of the helicene derivatives. For TPA and TPCD, the number of electronic excited states selected for A6(60 & 48) and CN6(60 & 40), were determined by the experimental spectral range (200 nm -450 nm) covered during the measurements. In #CN derivatives the number of electronic excited states to be considered in the calculations were established by the measured experimental spectral range for CN6 (see captions for Figures4 and 5for the accurate number of electronic excited states employed for each derivative). No solvent effects were considered in the nonlinear calculations due to the high computational costs. The convoluted theoretical TPA and TPCD spectra were obtained by Lorentzian broadening from   for the one-and two-photon cases, respectively, with half width at half maximum (FWHM) G , and centered on the computed excitation circular frequency 0 f  for a 0 f  transition. All the OPA and ECD spectra were obtained using a linewidth (  ) of 0.5 eV (FWHM). For the TPA and TPCD spectra a value of  = 0.15 eV (FWHM) was used.

Figures 4 ,

 4 chosen to improve the comparison between theory and experiment, are different from those applied in Figures 2 (assignment of theoretical CAM-B3LYP excited states to experimental bands of A6 and CN6 can be found in Tables 1.a and 1.b

  and (3) the contribution of the electric quadrupole transition moment to the TPCD signal of helical molecules with strong EXO-ICT -the extended electronic conjugation beyond the helicene core significantly enhances the nonlinear response of this type of molecular structures. While points (2) and (3) are crucial for TPCD in the series of molecules with greater EXO-ICT (C6-CN, C6--CN and C6---CN), in the other series of molecules (C6-CN, and C7-CN and C8-CN) point (2) becomes more critical.

Figure 6 .

 6 b) following the same trend observed in TPCD (Figure 6.a) within the whole series of #CN, and ii) |B 2 | (Figure 6.c) matching only the EXO-ICT series (C6-CN, C6--CN and C6---CN) confirm our initial thoughts.

  of CN6-like helicenes validated our hypothesis. Through this research we were able to show that while the extension of the electronic delocalization beyond the helicene core (EXO-ICT) enhances the contribution of the electric quadrupole and magnetic transition moments to the TPCD rotatory strength ( the electronic delocalization within the arrangement of aromatic rings (ENDO-ICT) primarily increases the contribution of the magnetic dipole

Figure Captions Figure 1 .

 Captions1 Figure Captions

Figure 2 .

 2 Figure 2. Experimental (black solid line) and theoretical (colored dotted lines) UV-vis (left

Figure 3 .

 3 Figure 3. Molecular orbitals (MOs) involved in the 3 rd and 2 nd electronic excited state of A6 and

Figure 4 .

 4 Figure 4. Experimental (black scattered squares) and theoretical TPA (left column) and TPCD

Figure 5 .

 5 Figure 5. OPA, ECD, TPA and TPCD theoretical spectra of all five #CN. OPA and ECD

Figure 6 .

 6 Figure 6. Comparative bar graph of a) integrated TPCD spectra, b) |B 1 +B 3 | and c) |B 2 |, for all five #CN.

Figure 3 29Figure 4

 34 Figure 3

ACKNOWLEDGEMENTS

Jeanne Crassous thanks the Ministère de l'Education Nationale, de la Recherche et de la Technologie and the Centre National de la Recherche Scientifique (CNRS). This work has been partially supported by the National Science Foundation through Grant Number NSF/ARRA-0840431 and NSF/DBI-1422826. The extended computing time provided by STOKES ARCC/UCF is gratefully acknowledged.


Aurélie Chagneau

Francis Claret
email: f.claret@brgm.fr

Florian Enzmann

Manfred Wolf

Benoit Madé

Thorsten Schâfer
email: thorsten.schaefer@kit.edu

Chagneau

; Claret

; Enzmann

M d ; Made, B e ; Schäfer ; Wolf

Can a

The understanding of permeability and tortuosity evolution due to porosity changes is of paramount importance for the description of various hydrogeological processes, such as fluid circulation in acid mine drainage remediation or hydrothermal systems, management of aquifer recharge, efficiency of permeable reactive barrier and the long-term geochemical evolution of host rock considered for highlevel nuclear waste repositories. The description of clogging phenomena in a reactive transport code is challenging, particularly over the long time periods (1000 to 1,000,000 years) that are of interest in most safety assessment cases. In addition to the validation of numerical scheme, experiments to validate the implemented laws used for the coupling of geochemistry (e.g. dissolution-precipitation reactions) to the transport parameters (induced porosity changes) and overall fluid flow processes (e.g. Carman-Kozeny relationship or Archie's law) are required. The so-called Archie's law [START_REF] Archie | Electrical Resistivity Log as an Aid in Determining Some Reservoir Characteristics[END_REF] is widely used in reactive transport models [START_REF] Navarre-Sitchler | Evolution of porosity and diffusivity associated with chemical weathering of a basalt clast[END_REF] and describes the relationship between the effective diffusivity (D e , in m²s -1 )of a dissolved species in a porous media and the diffusion coefficient of that same species in pure water (D w , in m²s -1 ). The D e /D w ratio is a function of the tortuosity of the material () and its constrictivity () and therefore on the accessible porosity of the material (ε a ). As both the tortuosity and the constrictivity are difficult to measure experimentally, their ratio is often described as the geometrical factor, G. The relationship can be written as follows:

𝐷 𝑒 𝐷 𝑤 = 𝛿 𝜏 = 1 𝐺 = 𝜀 𝑎 𝑛
where m is a called the cementation exponent. Though this empirical power law can accurately describe simple porous natural systems such as poorly compacted sands and sandstones, its applicability to more complex, low porosity and heterogeneous systems is questionable [START_REF] Navarre-Sitchler | Evolution of porosity and diffusivity associated with chemical weathering of a basalt clast[END_REF] In order to tackle this issue and to check the domain of validity of Archie's law, X-Ray transparent diffusion column experiments have been performed. Different porous media (clay, sand) have been used. In the present paper the focus is given on poorly compacted sand. Reduction of the pore space was induced by diffusion controlled precipitation of a simple salt, celestite (SrSO 4 ). A multi-scale and multi-technique approach has been used in order to characterize the transport properties before, during and after the reduction of the porosity as well as the amount of material that has precipitated. In this manner, (i) conservative tracer experiments (HTO) before and during the formation of a precipitation front, (ii) computed micro-tomography (µ-CT) coupled to pore-morphology modeling before and after the formation of a precipitation front, and (iii) post mortem analysis of the precipitate (dissolution, SEM-EDX) have been performed.

The conducted experiments are reproducible and after approximately 15 days the measured flux of through diffusing tracer HTO was constant and seemed to indicate that a steady state was reached, and thatthe pore space within the precipitation front was not fully clogged. Indeed, it was found that the reduced pore space in the precipitation zone remained fully connected (no isolated pores) under the resolution of CT (~6 µm), allowing for the migration of the tracer. The porosity and transport parameters prior to clogging estimated by the first 2 methods were consistent, as was the mass of precipitate estimated by the last two methods.

However, the reactive transport model built upon the experimental results underestimated the remaining connected porosity in the precipitation zone and overestimated the amount of precipitate.

The results presented demonstrate that 1D continuum models might fail in the long-term prediction of transport in geochemically disturbed systems and argue for more complex 2D pore-scale reactive transport and/or a modified diffusivity/porosity relationship.

Please tag the appropriate:

   intended to be proposed as full paper of publication in Proceedings Topic of scientific programme:  General strategy for clay based repository concepts  Geology and clay characterisation Geochemistry Mass transfer  Alteration processes  Geomechanics  Monitoring


domain: sdu chim sde

text:
Mathieu Debure

Sylvain Grangeon

Catherine Lerouge

Hélène Gailhanou

Benoît Madé

Christophe Tournassat

Car

Carbonates reactivity in temperature and interactions with Fe-minerals

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

Carbonates reactivity in temperature and interactions with

1. BRGM, French Geological Survey, D3E/SVP, 3, avenue Claude Guillemin -BP 36009, 45060 OrléansCedex 2, France (m.debure@brgm.fr, s.grangeon@brgm.fr, c.lerouge@brgm.fr, h.gailhanou@brgm.fr, c.tournassat@brgm.fr)

2. ANDRA, 1 -7 rue Jean Monnet, 92298 Châtenay-Malabry, France (benoit.made@andra.fr) *Corresponding author

The carbonate system is the one of most reactive in the mineralogical assemblages of clay-rocks. Consequently, pristine clay-rocks pore water compositions are expected to be at equilibrium with carbonate minerals. However, it is difficult to fully reconcile the concentration data obtained from seepage water sampled in equipped in situ boreholeswith the equilibrium concentrations obtained from pore water modeling (Pearson et al., 2011). It is especially true for dolomite whose solubility spans a large range of values in thermodynamic database as a function of crystallinity considerations, and for siderite/ankerite or other Fe-bearing carbonates whose compositions influence their solubility. The understanding of the carbonate system in clay-rock is necessary for a number of predictions including the effect of an increase of temperature on the pore water chemistry. In this respect, experiments were conducted to study the kinetics of equilibration of carbonate mineral assemblages as a function of temperature (25-80°C) in chemical conditions otherwise similar to those encountered in the Callovian-Oxfordian clay-rock.

Several authors reported higher Ca releases than Mg releases in solution during dolomite dissolution (e.g. Pokrowsky andSchott, 2001, Gautelier et al., 2007;Urosevic et al., 2012;Debure et al., 2013). However, others authors observed in their studies some experiments that displayed higher Ca release than Mg and some others that exhibitedonly small differences between Ca and Mg concentrations in solution (e.g. Pokrowsky et al., 2005;Zhang et al., 2007). Dolomite incongruent dissolution has been explained by the lower hydration energy of Ca 2+ compared to Mg 2+ and leading to a lower stability of Ca 2+ at the dolomite/water interface and to aCa-depleted layer at the dolomite surface (Busenberg and Plummer;1982;Pokrowsky and Schott, 2001;Zhang et al., 2007). More recently, Urosevic et al;(2012) evidence the formation of an MgCO 3 phase at the dolomite surface. However, the experimental data do not allow to quantitatively characterizing that phase. Such contradictory results underline the complexity of dolomite dissolution.In this study, dolomite dissolution in pure water wasinvestigated. Solution composition was monitored as a function of time and the mineralogy and surface chemistry was characterized before and after reaction (XRD, SEM, TEM and Raman analyses). Experiments were performed with powders (< 12 µm) and withmonocrystals. Mixed system such as dolomite/calcite and dolomite/calcite/goethite were investigated to quantify the Mg incorporation in calcite and the Fe incorporation in calcite and dolomite. Goethite was selectedfor those experiments in agreement with the presence of nanogoethite detected in COx samples [START_REF] Gailhanou | International meeting "Clays in Natural and Engineered Barriers for Radioactive Waste Confinement[END_REF].These experiments were performed in a glove box with a 99%N 2 /1%CO 2 atmosphere that is representative of the in-situ conditions [START_REF] Gailhanou | International meeting "Clays in Natural and Engineered Barriers for Radioactive Waste Confinement[END_REF] in order to preserve the systems from oxidizing perturbation and to buffer the pH.

For all the systems, high temperatures promoted high kinetic rate of dissolution and high concentrations in solution. Solution analyses did not evidence a large difference between Ca and Mg released in solution in the case of dolomite dissolution in pure water at 25°C and at 80°C. Nevertheless, TEM-EDX measurements showed the presence of incongruent Mg/Ca phases after reaction. SEM, Raman analysesdid not evidence Mg-bearing minerals precipitation but reveal the formation of hematite on the dolomite surface due to Fe impurities incorporated inside the crystals. In the calcite/dolomite systems, steady-state conditionswere quickly reached if solutions with composition closed to the expected equilibrium conditions were used as initial bulk electrolytes.

In the dolomite/calcite/goethite systems, steady state conditionswere quickly reached in solution as well (see Figure 1). TEM-EDX chemical analyses indicated a possible Fe incorporation in calcite and in dolomite. Further characterizations are in progress. Finally,solution compositions at steady-state are mostly consistent with equilibrium calculations. The initially under-saturated solutions approach equilibrium with dolomite, calcite and/or magnesite,suggesting secondary phase's formation as observed by Urosevic et al. (2012).Additional analyses (e.g.Small-angle X-ray scattering) are foreseen to investigate dolomite crystal surface and to characterize secondary carbonates minerals.

Figure 1 .

 1 Figure 1.Left : evolution of pH in the batch solution and elements concentrations in solution over the time; right: TEM micrography showing a potential Fe incorporation in calcite in the case of calcitedolomite-goethite experiment after 8 days of reaction at 80°C.


Ph.D Geert Isabelle De Groof
email: geert.degroof@uantwerp.be

Isabelle George

Sara Touj

Martin Stacho

Elisabeth Jonckers

Hugo Cousillas

Martine Hausberger

Onur Güntürkün

Annemie Van Der Linden

Ph.D George

Martin Sara Touj

Elisabeth Stacho

Ph.D Hugo Jonckers

Ph.D Martine Cousillas

Prof. Onur Hausberger

Prof Güntürkün

Prof. Annemie Van Der Linden

A three-dimensional digital atlas of the starling brain

Keywords: Songbird, European starling, High-Field MRI, brain atlas, song control system

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

INTRODUCTION

The European starling (Sturnus vulgaris) is one of the most widely used passerine species in fundamental biological research (almost 2500 starlings were used in more than 100 studies published between 2000 and 2004; [START_REF] Asher | Use and husbandry of captive European starlings (Sturnus vulgaris) in scientific research: a review of current practice[END_REF]). Starlings have been an important animal model in studies of ethology (e.g. [START_REF] Adret-Hausberger | Social Influences on the Whistled Songs of Starlings[END_REF][START_REF] Henry | The use of song repertoire changes with pairing status in male European starling[END_REF]Hausberger et al. 1995;[START_REF] Hausberger | Differential response of female starlings to shared and nonshared song types[END_REF][START_REF] Henry | Sharing Mates and Nest Boxes Is Associated W F "F d p[END_REF], behavioural ecology (e.g. [START_REF] Powell | Experimental Analysis of Social Value of Flocking by Starlings (Sturnus-Vulgaris) in Relation to Predation and Foraging[END_REF][START_REF] Tinbergen | Foraging Decisions in Starlings (Sturnus-Vulgaris L)[END_REF][START_REF] Lima | Patch Sampling Behavior of Starlings Foraging in Simple Patchy Environments[END_REF], ecophysiology (e.g. [START_REF] Grue | Use of Captive Starlings to Determine Effects of Environmental Contaminants on Passerine Reproduction -Pen Characteristics and Nestling Food-Requirements[END_REF][START_REF] Eng | Assessment of concentrations and effects of organohalogen contaminants in a terrestrial passerine, the European starling[END_REF]) and neuroethology of song production and perception [START_REF] Leppelsack | Responses of Auditory Neurons in Forebrain of a Songbird to Stimulation with Species-Specific Sounds[END_REF][START_REF] Hausberger | Categorization in birdsong: From behavioural to neuronal responses[END_REF][START_REF] Gentner | Response biases in auditory forebrain regions of female songbirds following exposure to sexually relevant variation in male song[END_REF][START_REF] Ball | Ethological concepts revisited: Immediate early gene induction in response to sexual stimuli in birds[END_REF][START_REF] George | Hemispheric specialization in the primary auditory area of awake and anesthetized starlings (*Sturnus vulgaris*)[END_REF][START_REF] Heimovics | Seasonal and Individual Variation in Singing Behavior Correlates with Alpha 2-Noradrenergic Receptor Density in Brain Regions Implicated in Song, Sexual, and Social Behavior[END_REF][START_REF] De Groof | Functional changes between seasons in the male songbird auditory forebrain[END_REF][START_REF] Ellis | Patterns of FOS protein induction in singing female starlings[END_REF].

Within the last decades, the song control system (SCS) and, to a lesser extent, the auditory systems and social behavioural network of starlings have been thoroughly examined. Up to now no atlas is available for the brain of this species, and researchers are forced to use the atlas of other songbird species like the zebra finch [START_REF] Nixdorf-Bergweiler | A Stereotaxic Atlas of the Brain of the Zebra Finch Taeniopygia guttata with Special Emphasis on Telencephalic Visual and Song System Nuclei in Transverse and Sagittal Sections[END_REF] or the canary brain [START_REF] Stokes | The telencephalon, diencephalon, and mesencephalon of the canary, Serinus canaria, in stereotaxic coordinates[END_REF]. Although zebra finch and canary brains are similar in size, the brain of a starling is almost three times the volume, making it hard to extrapolate stereotactic coordinates from either atlases to that of the starling brain. Both atlases of the songbird brain are still widely used but obviously have the limitations of all brain atlases that are based on drawings of brain sections: they not only provide a mere 2-dimensional perspective of the brain, but they are also dependent of previously specified sectioning angles.

To further neurobiological studies in starlings, we therefore decided to create the first 3D MRI atlas of the starling brain as a freely available tool for the scientific community. The atlas is based on several scanning protocols, each with their own advantages and disadvantages, to visualize a wide range of neural structures. Reflecting the traditional interest in the song production and perception processes in starling, we tried to visualize the song control and auditory systems, as complete as possible. In addition we describe the different brain subdivisions as well as the main fibre tracts. We tried to establish a combined atlas that allows not only for the identification of a larger number of brain structures, but also allows researchers to select those datasets that fit best with their scientific interests.

The starling brain atlas presented here can be easily adapted to match any surgical setup or histological protocol, and should be appealing for future birdbrain studies. We hope that this starling brain atlas will advance studies in this songbird model in an era where traditional and MRI-based studies will intermingle more and more.

MATERIALS AND METHODS

Specimen preparation

For this study, one wild adult male European starling (Sturnus vulgaris) caught in Normandy (France) in 2006 and kept in outdoor aviaries in Rennes (France) until the experiment, was deeply anesthetized with pentobarbital and transcardially perfused with a phosphatebuffered heparinized saline solution (PBS, 0.12M), followed by a mixture of paraformaldehyde (PFA, 4%) and Dotarem® (1%), a paramagnetic MR contrast agent. After decapitation, the head was post-fixed in a mixture of PFA (4%) and Dotarem (1%) for at least 5 days at 5°C. The starling (body weight of 99.4g) was perfused during the breeding season (25 th March 2009) and the colour of the beak was yellow indicating high plasma testosterone levels [START_REF] Bullough | Vulgaris L.)[END_REF][START_REF] Dawson | Plasma corticosterone in wild starlings (Sturnus vulgaris) immediately following capture and in relation to body weight during the annual cycle[END_REF][START_REF] Ball | Changes in plasma levels of sex steroids in relation to multiple broodedness and nest site density in male starlings[END_REF] at the time of perfusion.

Data acquisition

To obtain a 3-dimensional representation of the skull, the whole bird' head was imaged with a Siemens PET-CT equipped with a rotating 80 kV X-ray source (focal spot size of 50 µm) and a Siemens Inveon PET-CT Camera with a 125 mm x-ray detector. T2*-weighted 3D images were acquired on the 7T using a FISP gradient echo sequence with a 15º flip-angle, a BW of 50 kHz, an N av of 45, a TR of 14.31 ms, a TE of 6.5 ms and a scan repetition time of 7500 ms. Images had a field of view of (20.48 x 20.48 x 20.48) mm 3 with an acquisition matrix of (512 x 512 x 256) zero-filled to (512 x 512 x 512) resulting in an isotropic spatial resolution of 40 µm in all three directions. Acquisition time was 24h.

Brain area delineation and 3D reconstruction

All MRI datasets and the CT dataset were co-registered with the SPM 8 package using normalized mutual information. The MRI images were used as such to co-register to each other, however to co-register the MRI images to the CT image the brain surface was delineated from the MRI image and used as input for coregistration. The position of the ear canal and thus the most likely position of stereotactic ear-bars was established with the CTdata, and all datasets were reoriented to match a 45° angle of the ear bars and the most posterior part of the beak-opening to the horizontal axis.

All atlas delineations were performed with Amira 5.5 (Mercury Computer Systems, San Diego, CA, USA). The delineation of the skull was based on the CT dataset and was conducted automatically using a signal intensity high-pass threshold. The brain surface and the neural subdivisions of both hemispheres were manually delineated, based on the MRI signal intensity differences between brain regions (Table 1). The delineation of each brain structure and fibre tract was performed mostly in frontal plane and subsequently controlled in the two other planes.

Insert Table 1 around here

RESULTS

Scanning protocols and structural delineations

Different imaging protocols provide different and complementary possibilities to delineate neural structures. X-ray CT imaging is essential to anchor the precise position of the brain within the skull. It thus represents a crucial first step to enable the construction of a stereotactic atlas since the brain position within the skull can be co-registered with the MRbased structural positions. We first defined a reference plane from three CT imaging based reference points: both ear canals, and the most posterior end of the beak opening. The horizontal plane of the brain atlas was then defined as a plane tilted by 45 degrees to the reference plane about the axis running through both ear canals (Fig. 1). CT imaging is not useful to visualize brain areas. This is best done with the protocols outlined below (Table 1).

Insert Figure 1 around here T2-weighted imaging produces images where water-containing areas become bright and fatty/cell-dense structures become dark. This sequence produced a very good anatomical image of the birdbrain with most of the brain nuclei clearly visible and delineable (Fig. 2A andB). Also the larger fibre tracts and several of the forebrain lamina could be distinguished.

Although the strong signal deriving from the fluid-filled lateral ventricle made it easily detectable, this also caused a slight over-saturation, decreasing delineation accuracy of this structure. The images provided very nice contrast especially in the sagittal images. Due to zero-filling, we got more smoothed images for axial and to a lesser extent coronal images.

Proton-density weighted imaging provides images with an intermediate contrast between T1 and T2 weighted images, increasing delineation accuracy. Here we had less oversaturation of the liquid-filled lateral ventricle seen in the T2 weighted images (Fig. 2C). Since the images were obtained without zero-filling, we had very nice contrast and sharpness in all three orthogonal directions.

T2*-weighted imaging gives rise to the same type of contrast as normal T2-weighted imaging, but with much lower signal and contrast properties. However, because a gradientecho sequence is many times faster than a spin-echo sequence, a very high resolution can be achieved within a normal time frame. Thus at the cost of contrast, those regions that were visible could be delineated much more accurately. Also, the high resolution of these images allowed for the detailed visualization of most of the fibre tracts (Fig. 2D). The images provide very nice contrast especially in the coronal plane. Due to zero-filling we got slightly more smoothed images for axial and sagittal images.

Although T1 imaging is commonly used for anatomical inspection of human brains in clinical practices, in songbird measurements this technique offers only poor regional contrast within the brain, making it rather difficult to distinguish individual brain areas from the brain's background [START_REF] Vellema | A customizable 3dimensional digital atlas of the canary brain in multiple modalities[END_REF]. It was therefore not used here.

Insert Figure 2 around here

Data presentation and validation

The co-registered MRI and CT datasets, including skull, and 46 brain subdivisions are freely available for download from our website: https://www.uantwerpen.be/en/rg/bio-imaginglab/research/mri-atlases/starling-brain-atlas/. Data are available in Analyze format, which is supported by most 3D visualization software packages, including the free software MRIcro (http://www.cabiatl.com/mricro/mricro/mricro.html). Documentation describing how to visualize and customize the 3D datasets in MRIcro can also be found on our website.

The default data orientation is presented in a similar fashion as previously published atlases [START_REF] Stokes | The telencephalon, diencephalon, and mesencephalon of the canary, Serinus canaria, in stereotaxic coordinates[END_REF][START_REF] Nixdorf-Bergweiler | A Stereotaxic Atlas of the Brain of the Zebra Finch Taeniopygia guttata with Special Emphasis on Telencephalic Visual and Song System Nuclei in Transverse and Sagittal Sections[END_REF], with a head-angle of 45 degrees.

The 45° angle has been calculated based on the axis through the ear canal (the most likely position for fixating ear bars) and the most posterior end of the beak opening relative to the horizontal plane. When loaded into MRIcro, the junction formed by the dorsocaudal cerebral vein and two branches of the transverse venous sinus (two major blood vessels running along the brain's midline), a V-shaped stereotactic landmark often used for surgical procedures, is reset to the zero-coordinate by default. This reference-point can be manually altered however, if another zero-coordinate is preferred. In this reference frame, the X-axis ). Being a seasonal species, they are also well suited for studies of neuroendocrinology and seasonal brain plasticity (e.g. [START_REF] Bernard | Photoperiodic condition modulates the effects of testosterone on song control nuclei volumes in male European starlings[END_REF][START_REF] Ball | Steroid sensitive sites in the avian brain: does the distribution of the estrogen receptor alpha and beta types provide insight into their function?[END_REF][START_REF] Bentley | Seasonal neuroplasticity in the songbird telencephalon: a role for melatonin[END_REF][START_REF] Absil | Effect of age and testosterone on autumnal neurogenesis in male European starlings (Sturnus vulgaris)[END_REF][START_REF] Alger | Lesions to the medial preoptic nucleus differentially affect singing and nest box-directed behaviors within and outside of the breeding season in European starlings (Sturnus vulgaris)[END_REF][START_REF] De Groof | Seasonal rewiring of the songbird brain: an in vivo MRI study[END_REF][START_REF] De Groof | Structural changes between seasons in the songbird auditory forebrain[END_REF][START_REF] De Groof | Functional changes between seasons in the male songbird auditory forebrain[END_REF][START_REF] Cousillas | Seasonal female brain plasticity in processing social vs. sexual vocal signals[END_REF].

One limitation of using starling for neuroscience research is that no detailed brain maps are available. Neurobiological scientists are forced to use 2D atlases of either the canary brain [START_REF] Stokes | The telencephalon, diencephalon, and mesencephalon of the canary, Serinus canaria, in stereotaxic coordinates[END_REF] or the zebra finch brain (Nixdorf-Bergweiler and Bischof 2007) as the closest songbird brain species. However both canary and zebra finch brains are considerably smaller than that of the starling, making it sometimes hard to find the complementary structure in the starling brain (Fig 6). Also there might be a slight difference in relative positioning of the different nuclei between species. Traditional atlases are often based on drawings of histological brain sections, and are dependent on the interests of the researcher constructing the atlas both in sectioning orientation as well as delineation of brain structures. 3D imaging techniques such as MRI however, give us the opportunity to examine the brain from a 3-dimensional, whole-brain point of view [START_REF] Ma | A three-dimensional digital atlas database of the adult C57BL/6J mouse brain by magnetic resonance microscopy[END_REF][START_REF] Van Essen | A Population-Average, Landmark-and Surface-based (PALS) atlas of human cerebral cortex[END_REF][START_REF] Saleem | A Combined MRI and Histology Atlas of the Rhesus Monkey Brain in Stereotaxic Coordinates[END_REF][START_REF] Poirier | A three-dimensional MRI atlas of the zebra finch brain in stereotaxic coordinates[END_REF][START_REF] Datta | A digital atlas of the dog brain[END_REF][START_REF] Muñoz-Moreno | A magnetic resonance image based atlas of the rabbit brain for automatic parcellation[END_REF][START_REF] Güntürkün | A 3-dimensional digital atlas of the ascending sensory and the descending motor systems in the pigeon brain[END_REF][START_REF] Nie | A rat brain MRI template with digital stereotaxic atlas of fine anatomical delineations in paxinos space and its automated application in voxelwise analysis[END_REF][START_REF] Kirn | Three-dimensional reconstruction of brain structures of the rodent Octodon degus: a brain atlas constructed by combining histological and magnetic resonance images[END_REF][START_REF] Ullmann | An MRI atlas of the mouse basal ganglia[END_REF]). Here we present the first 3-dimensional MRI-based atlas of the starling brain, a model system often used for neurobiological and behavioural studies. We tried to capture as many different brain regions as possible, making this atlas suitable for many different fields of birdbrain research. There are several advantages in the brain atlas that we present here over the previously published 2D atlas of the canary brain [START_REF] Stokes | The telencephalon, diencephalon, and mesencephalon of the canary, Serinus canaria, in stereotaxic coordinates[END_REF], and similar brain atlases of other bird species (e.g. [START_REF] Karten | Livia)[END_REF][START_REF] Nixdorf-Bergweiler | A Stereotaxic Atlas of the Brain of the Zebra Finch Taeniopygia guttata with Special Emphasis on Telencephalic Visual and Song System Nuclei in Transverse and Sagittal Sections[END_REF][START_REF] Puelles | The Chick Brain in Stereotaxic Coordinates: An Atlas Featuring Neurometric Subdivisions and Mammalian Homologies[END_REF]). First and foremost, the 3-dimensional capturing methods used in this study allowed us to study the anatomy of a single brain from any possible angle, eliminating the limitations of 2D atlases that are inherently restricted to a specified orientation.

Insert Figure 6 around here

This approach is particularly useful in combination with other whole-brain imaging techniques such as functional MRI [START_REF] Van Meir | Spatiotemporal properties of the BOLD response in the songbirds' auditory circuit during a variety of listening tasks[END_REF][START_REF] Voss | Functional MRI of the zebra finch brain during song stimulation suggests a lateralized response topography[END_REF][START_REF] Poirier | Own-song recognition in the songbird auditory pathway: selectivity and lateralization[END_REF][START_REF] De Groof | Functional changes between seasons in the male songbird auditory forebrain[END_REF][START_REF] Van Ruijssevelt | Functional magnetic resonance imaging (FMRI) with auditory stimulation in songbirds[END_REF] and Diffusion Tensor Imaging [START_REF] De Groof | Structural changes between seasons in the songbird auditory forebrain[END_REF]. The atlas could be applied as a template for (future) studies of these types in starlings [START_REF] De Groof | Functional changes between seasons in the male songbird auditory forebrain[END_REF].

The possibility to reset the coordinate system of the brain-model to any preferred orientation makes it customizable to any stereotactic device that may be used for brain injections or electrophysiological recordings. Besides locating target areas based on stereotactic coordinates, the 3D brain atlas is well suited to optimize surgical protocols to avoid damage in specified regions. When injecting a tract-tracer in RA for example (e.g. [START_REF] Kirn | Three-dimensional reconstruction of brain structures of the rodent Octodon degus: a brain atlas constructed by combining histological and magnetic resonance images[END_REF][START_REF] Roberts | Telencephalic neurons monosynaptically link brainstem and forebrain premotor networks necessary for song[END_REF], the brain atlas could be used to calculate the optimal head angle to avoid any leakage from the injection tract into its afferent nucleus HVC, which is located directly above RA at certain angles. Alternatively, one might want to angle the head to the side to avoid puncturing the lateral ventricle, which could lead to an unwanted spread of a chemical through the brain.

The reorientation possibilities of the atlas are also well suited to calculate the best angle for making brain sections that contain multiple regions of interest (Fig. 3). This could be very useful for histological staining procedures or in situ hybridization protocols (e.g. [START_REF] Jarvis | Motor-driven gene expression[END_REF][START_REF] Tramontin | Seasonal plasticity and sexual dimorphism in the avian song control system: Stereological measurement of neuron density and number[END_REF][START_REF] Metzdorf | Distribution of aromatase, estrogen receptor, and androgen receptor mRNA in the forebrain of songbirds and nonsongbirds[END_REF] in which two or more brain areas are requested in a single brain section, potentially making comparative quantification studies more reliable and less time consuming.

For electrophysiological recordings in brain slices [START_REF] Mooney | The HVC microcircuit: the synaptic basis for interactions between song motor and vocal plasticity pathways[END_REF][START_REF] Gale | Physiological properties of zebra finch ventral tegmental area and substantia nigra pars compacta neurons[END_REF][START_REF] Meitzen | Plastic and stable electrophysiological properties of adult avian forebrain song-control neurons across changing breeding conditions[END_REF] it can be useful to calculate the ideal sectioning angles, not only to include two targets of interest, but also to include intact fibre tracts running between the two regions within one recording slice. The same is true for high density multi-electrode recordings [START_REF] Amin | Selective and efficient neural coding of communication signals depends on early acoustic and social environment[END_REF][START_REF] Cousillas | Seasonal female brain plasticity in processing social vs. sexual vocal signals[END_REF], where the final measurement sites are highly dependent on the insertion angle of the electrode array.

Finally, because the brain atlas has been constructed with the skull intact, without the need for dehydration or freeze-protection steps, the shape of the brain will be relatively close to the in-vivo situation. In contrast, 2D atlases that are based on histological sections have to deal with inaccuracies that are introduced during the histological procedures [START_REF] Ma | A three-dimensional digital atlas database of the adult C57BL/6J mouse brain by magnetic resonance microscopy[END_REF][START_REF] Vellema | A customizable 3dimensional digital atlas of the canary brain in multiple modalities[END_REF]. Dehydration steps that are often necessary for histological preparations cause a severe shrinkage of the brain, and additional shape-artefacts are virtually unavoidable during the cutting and mounting process.

As with 2D histological atlases, this 3D MRI brain atlas is based on one individu ' d , in this case a male starling during the breeding season. This is important because of both sexual dimorphisms and seasonal differences in brain nuclei size. One should therefore be cautious when attempting to obtain coordinates for certain nuclei of female starling studies (although starlings show less sexual dimorphism than e.g. the zebra finch; [START_REF] Bernard | Sexual dimorphism in the volume of song control nuclei in European starlings: assessment by a Nissl stain and autoradiography for muscarinic cholinergic receptors[END_REF][START_REF] Ball | Sex-Differences in the Volume of Avian Song Control Nuclei -Comparative-Studies and the Issue of Brain Nucleus Delineation[END_REF])) or non-breeding season male starlings. Regions known to be variable in size according to season are most of the song control nuclei (e.g. HVC, RA and Area X) [START_REF] Ball | Neuroendocrine mechanisms mediating the photoperiodic and social regulation of seasonal reproduction in birds[END_REF][START_REF] Tramontin | Seasonal plasticity in the adult brain[END_REF], nuclei of the social behaviour network (e.g. POM [START_REF] Riters | Seasonal changes in courtship song and the medial preoptic area in male European starlings (Sturnus vulgaris)[END_REF]) and NCM [START_REF] De Groof | Structural changes between seasons in the songbird auditory forebrain[END_REF]).

The 3D starling brain atlas presented here could be used as a framework for researchers working on the starling brain, and for avian brain research in general. Together with our previously published 3D brain atlases of the zebra finch [START_REF] Poirier | A three-dimensional MRI atlas of the zebra finch brain in stereotaxic coordinates[END_REF], of the canary [START_REF] Vellema | A customizable 3dimensional digital atlas of the canary brain in multiple modalities[END_REF]) and of a non-songbird species (e.g. the pigeon brain [START_REF] Güntürkün | A 3-dimensional digital atlas of the ascending sensory and the descending motor systems in the pigeon brain[END_REF])), we now have four detailed, easily adaptable brain atlases for four commonly studied species of birds that should appeal to scientists from different disciplines working on the function, physiology and anatomy of the avian brain. T 2 weighted. Canary data is from [START_REF] Vellema | A customizable 3dimensional digital atlas of the canary brain in multiple modalities[END_REF] and zebra finch data is from [START_REF] Poirier | A three-dimensional MRI atlas of the zebra finch brain in stereotaxic coordinates[END_REF]. The approximate volume of the brain per species is indicated below each slice.

Figure Captions

  to-right axis, the Y-axis corresponds to the posterior-anterior axis, and the Z-axis corresponds to the dorsal-ventral axis of the brain. The stereotactic coordinates of a specific brain area can be easily acquired by moving the cursor onto the desired region.The 3D datasets can also be used to determine the optimal head angle for stereotactic operations or the best cutting angle for sectioning. If it is important to have multiple brain areas of interest in one single brain section, rotation and oblique slicing tools can easily be used to estimate the best cutting angle (Fig3).Insert Figure 3 around hereDifferent datasets and delineations can be superimposed and manipulated synchronously to attain the desired brain image. The delineation sets can further be used to generate a 3dimensional representation of the brain that gives an accurate description of the relative location, shape and volume of brain areas (Fig.4 and 5). sophisticated vocal behaviour, their social nature, their high plasticity and their robustness, starlings have become an important model species that is widely used in the field of neuroscience (e.g.[START_REF] Bigalke-Kunz | Tonotopic organization and functional characterization of the auditory thalamus in a songbird, the European starling[END_REF][START_REF] Bee | Primitive auditory stream segregation: A neurophysiological study in the songbird forebrain[END_REF][START_REF] Austad | Candidate Bird Species for Use in Aging Research[END_REF][START_REF] Feinkohl | Processing of transient signals in the visual system of the European starling (Sturnus vulgaris) and humans[END_REF], and especially in studies of neuroethology of song production and perception (e.g.[START_REF] Bernard | Age-and behavior-related variation in volumes of song control nuclei in male European starlings[END_REF][START_REF] Bolhuis | Bird brains and songs: neural mechanisms of birdsong perception and memory[END_REF][START_REF] Alger | Lesions to the medial preoptic nucleus affect immediate early gene immunolabeling in brain regions involved in song control and social behavior in male European starlings[END_REF][START_REF] George | How social experience shapes song representation in the brain of starlings[END_REF][START_REF] Ellis | Patterns of FOS protein induction in singing female starlings[END_REF]

Fig. 1

 1 Fig. 1 Overlap of MRI starling brain with delineated structures within the CT head data and the

Fig. 2

 2 Fig. 2 Different imaging protocols offer different contrast properties. Each row shows 3 sagittal views

Fig. 3

 3 Fig. 3 Oblique slicing. 3D renderings of the brain surface showing the best cutting angle to include

Fig. 4

 4 Fig. 4 A: 3D rendering of the brain with subdivisions. B: Transparent overlay showing the brain (T 2

Fig. 5

 5 Fig. 5 3D rendering of the brain. A: Non-transparent rendering showing the outer brain surface with

Fig. 6

 6 Fig. 6 Para-sagittal MRI slice through HVC, RA (Robust nucleus of arcopallium) and LMAN (Lateral

Figure 1

 1 Figure 1 Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

Figure 3

 3 Figure 3 Click here to download high resolution image

Figure 4

 4 Figure 4 Click here to download high resolution image

Figure 5 Figure 6

 56 Figure 5 Click here to download high resolution image

Table 1

 1

Table 1 :

 1 Delineated Structures, their systems

	Table 1
	System	Abbreviation Structure
	Song Control System	HVC	HVC
		RA	Robust nucleus of arcopallium
		X	Area X
		MMAN	Medial magnocellular nucleus of anterior nidopallium
		LMAN	Lateral magnocellular nucleus of anterior nidopallium
		DLM	Dorsolateral nucleus of medial thalamus
	Auditory System	Field L	Field L
		NCM	Caudal medial nidopallium
		CMM	Caudomedial mesopallium
		Ov	Nucleus ovoidalis
		MLd	Dorsolateral nucleus of mesencephalon
	Visual system	E	Entopallium
		Rt	Nucleus rotundus
		TeO	Optic tectum
	Olfactory system	OB	Olfactory bulb
	Social Behaviour Network POM	Medial preoptic nucleus
		TnA	Nucleus taeniae amygdala
		GCt	Midbrain central grey
		LS	Lateral septum
		MS	Medial septum
		PVN	Paraventricular nucleus
		VMH	Ventromedial nucleus of the hypothalamus
		VTA	Ventral tegmental area
	Fibre tracts	TSM	Septopallio-mesencephalic tract
		CoA	Anterior commissure
		CoP	Posterior commissure
		N3	Oculomotor nerve
		OM	Occipito-mesencephalic tract
		MFB	Medial forebrain bundle
		LFB	Lateral forebrain bundle
		QF	Quintofrontal tract
		FA	Fronto-arcopallial tract
		Opt	Optic tract

ACKNOWLEDGEMENTS

We are grateful to Steven Staelens and Steven Deleye from MICA (Universiteit Antwerpen) for support during CT scans. This research was supported by a joint "Tournesol" grant (p j c N 037 T ) f F d F c I.G. "PICS" (project Nr 5992) from the French CNRS to I.G. and G.DG and grants from the Research Foundation -Flanders (FWO, project Nr G030213N and G044311N), the Hercules Foundation (Grant Nr AUHA0012), Concerted Research Actions (GOA funding) from the University of w p d I y c P (I P) ('PL STOCIN ': P7/ 7) .VdL. I.G., H.C. and M.H. are supported by the University of Rennes 1 and by the French CNRS. O.G. was supported by the DFG via SFB874. G.DG is a Postdoctoral Fellow of the Research Foundation -Flanders (FWO).

Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.


Mohamed Sabt
email: mohamed.sabt@orange.com

Mohammed Achemlal
email: mohammed.achemlal@orange.com

Abdelmadjid Bouabdallah
email: madjid.bouabdallah@hds.utc.fr

Over-the-Internet: Efficient Remote Content Management for Secure Elements in Mobile Devices

We propose Over-the-Internet (OTI), a novel system that manages secure element based applications. We demonstrate our solution in the context of NFC ecosystem and show that it can be effectively used for transmitting big applications to the secure element. Our system leverages the GlobalPlatform card specification as well as the GlobalPlatform user-centric ownership model. Our solution integrates the different actors of the NFC ecosystem in its architecture. We propose to leverage the concept of security domain, so that service providers can manage their applications independently from the SE issuer. We implement our solution within available platforms and show that it is secure, fast, reliable and easily deployable.

I. INTRODUCTION

The use of mobile devices such as smartphones has greatly increased in the last years. This rise has the effect of spurring service providers on to deploy their own services for mobile markets. Some of these services, such as mobile payment and e-ticketing, require user authentication and data confidentiality; hence the need to a highly secure environment in which applications should run. The most popular example of such services are NFC applications. The NFC technology is primarily used to make contactless transactions, including those for access, payment and ticketing. The secured area that is used to provide secure execution for applications is commonly referred to as Secure Element (SE). The secure element is an environment in which application code and application data can be securely stored. Secure elements come in several flavors. They could be implemented either by an embedded smart card chip, in an SD card that can be inserted in the mobile, or in the SIM/UICC which is used by phone operators to authenticate subscribers on their network.

Regardless of their form-factor, secure elements have known enormous improvement and enhancements in the last two decades. They become programmable and can support multiple applications [START_REF] Markantonakis | The Case for a Secure Multi-Application Smart Card Operating System[END_REF]. Furthermore, applications could be downloaded to secure elements that already have been issued to users. This is a great evolution in the world of secure elements, since applications were traditionally pre-installed on the card and no post-issuance application loading was possible. This inflexible model is no longer appropriate in the open world of smartphones where there exists a huge number of applications proposed by many service providers and users usually prefer to freely choose the applications that they want to use on their mobiles. For applications based on secure elements to succeed, secure elements need to be 'smart', in the sense that users could easily manage their content. For instance, a user might install an NFC payment application that she uninstalls a few days later because she finds another application that proposes better services. The service provider might need to update their application in order to offer more services or to fix some security vulnerabilities.

Post issuance management of the smart secure elements can be quite complex. Unfortunately, management systems have not followed the evolution of secure elements. Indeed, current systems are not adapted to download an application of several kilobytes to the secure element. Instead, they were designed to accomplish two main tasks. Firstly, they send a code to activate the pre-installed application after authenticating the user. Secondly, they send some personalization parameters of only a few bytes of size to customize the activated, generic application. Unlike the personalization parameters, the size of secure element applications is measured in kilobytes. Their size is often above 9 kilobytes. Being designed to send a few bytes only, the existing management systems are incapable of reliably transmitting such amount of data for thousands of users.

Nowadays, the technology of secure elements is quite mature. However, the market is still reluctant to adopt it because of the lack of remote content management systems. This lack proves problematic for both users and service providers. Users of smartphones are accustomed to choose between a wide range of applications, and therefore they are naturally uncomfortable with any system that forces them to choose from a limited list of pre-installed applications. As for service providers, they cannot easily deliver their applications to users. Even simple operations, such as updating or deleting applications become hard to accomplish. In summary, Remote content management is essential for applications based on secure elements to achieve their potential. However, the current systems are not reliable enough to perform complex content management operations, including downloading several kilobytes of code, updating code or credentials, and deleting applications. Programmable secure elements offer great opportunities due to their high security, but their future suffers from outdated remote content management systems.

In this paper, we propose an Internet-based remote content management system for secure elements in connected devices, such as smartphones. In contrast to previous work, our work does not require changes to existing secure elements or mobile devices and is compatible with the existing content management standards. We leverage the widely accepted card specifications of GlobalPlatform. We make the following contributions:

• We provide a complete and feasible solution for remote management of secure elements in connected devices. We call our solution Over-the-Internet (OTI) as it makes use of TCP/IP connection to transmit code or management commands to secure elements. We leverage the growing speed of the Internet and the proven reliability of the TCP protocol.

• We leverage the recently defined GlobalPlatform consumer-centric model. This model allows end users to control the applications that are installed on their secure elements.

• We demonstrate our solution in the context of NFC applications. There exist various contexts for the secure element applications, and designing a general content management system for a general context is difficult. We have selected the NFC ecosystem because nowadays NFC applications that are based on secure element are increasingly popular. It is worth noting that our solution is naturally extensible to manage any kind of secure element based applications.

• We present full implementation of our solution using commercially available secure elements and devices without any changes. Our prototype shows that OTI is by far faster and more scalable than the existing technologies.

The rest of the paper is structured as follows. Section II defines the problem that we address in more detail. We give a background information on secure elements and OTA architecture in section III. In section IV, the Over-the-Internet architecture is presented, including analysis of its security properties. Section V provides implementation details and performance evaluation. Section VI surveys related work, and we end the paper with a brief summary.

II. PROBLEM STATEMENT

Smart cards have evolved from a single application platform to a feature-rich multi-application platform. However, multi-application smart cards have not successfully evolved into multi-purpose cards. The card issuer, the entity that issued the card, still has full control over the applications installed on its cards. In the traditional smart card industry, cards are issued for one or a specific list of service providers. Cardholders use their smart cards as service enablers, and not as a secure computer platform which can host several applications developed by independent service providers. Traditional multi-application smart cards are not really 'smart'.

The advent of the Near Field Communication (NFC) has influenced the smart card technology. It enables a mobile device to emulate a contactless smart card. The emulated NFC card can be accessed by an external contactless reader, such as contactless-enabled point-of-sale terminals [START_REF]Information technology -Telecommunication and information exchange between systems -Near Field Communication -Interface and Protocol (nfcip-1)[END_REF]. The NFC technology allows the introduction of smart cards, called secure elements, into mobile devices. Modern mobile devices, namely smartphones, and secure elements have conflicting traditions. Secure elements are issuer-centric where users are not allowed to freely install applications, while smartphones are user-centric where an average user can easily navigate, search, install third party applications. In addition, the application developers do not have to negotiate with the mobile operators in order to be able to propose their applications to users.

Coupled with a smartphone, the secure element cannot be isolated from the so-called "iPhone effect". In 2012, Global-Platform proposed a user-centric model for secure elements in [START_REF] Globalplatform | A New Model: The Consumer-Centric Model and How It Applies to the Mobile Ecosystem[END_REF]. This new model enables secure elements to achieve the full potential of the multi-application smart card technology. Nevertheless, the absence of appropriate application management system has hindered the flourishing of the new usercentric secure elements.

At present, there is no dedicated platform to perform application management. Instead, applications are managed by the OTA platform that is used for UICC remote provisioning. The transport bearer of the current OTA platform is the SMS bearer. The SMS bearer is suitable for small amount of data. More precisely, one SMS can contain only 140 bytes of data. Therefore, it is not suitable for loading heavy applications (e.g., above 9 kilobytes). Some solutions suggest to use the Bearer Independent Protocol (BIP) [START_REF]Card Application Toolkit (CAT)[END_REF] because it is more reliable and faster than SMS. In spite of being standardized, BIP is not widely implemented in mobile devices [START_REF] Giesecke&devrient | Bearer Independent Protocol (BIP)[END_REF].

Our goal is to design a content management system for NFC enabled services that use the secure element in the mobile device. In the following, we detail the requirements for any efficient and secure solution to this problem.

We first aim to design a solution that could be deployed with ease. A solution that involves many changes to the existing architecture is unlikely to be welcomed in the production environments. Deployability is a non-functional requirement, and like most non-functional requirements, it is often overlooked. However, it is a highly regarded requirement for the NFC ecosystem, where standards are predominate and cannot be neglected. Nevertheless, our primary goals are efficiency and security, and they are not traded off against deployability. The deployability requirement implies that our solution must be compliant with the GlobalPlatform Card Specification [START_REF] Globalplatform | GlobalPlatform Card Specification[END_REF] which is considered as the de facto standards for the management of secure elements.

Second, the content management must be performed via a wireless technology. This is beneficial for both users and service providers. Wireless technology allows NFC applications to become a few clicks away from users' mobile devices. The users would enjoy the experience, and the service providers would attract more clients with less cost. The wireless technology must be fast enough to allow new applications to be installed in a reasonable time, and reliable so that no transmission errors affect the correct delivery of data.

Third, security is of paramount importance. Only authenticated applets from trusted service providers could be installed into the secure element. Otherwise, an attacker might install malicious code to exploit security flaws in the secure element software. The proposed solution must ensure a secure end-toend connection between the secure element and the service provider to ensure a secure management for the content of the secure element. Therefore, some secret keys must be shared between the secure element and the service provider.

Fourth, an NFC application running in the secure element is often accompanied by a graphical user interface running in the mobile device. This user interface is the feature that gives NFC applications advantage over contactless smart cards because it improves users interaction with the application. Applets and graphical interface are interdependent. Indeed, any update to the secure element applet might entail updating the user interface, otherwise the NFC application may not function properly. The problem is that if the user interface is a native mobile app, its life-cycle is managed independently from that of the secure element. A content management system for secure elements must include mechanisms to update the user interface when necessary.

III. BACKGROUND

A. Secure Element

A secure element (SE) is a tamper-resistant smart card chip capable of running applications (called applets or cardlets) with a high level of security. A smart card is essentially a minimal computing environment with a CPU, ROM, EEPROM, RAM, and I/O port. Recent cards also include cryptographic coprocessors that implement common algorithms such as AES and RSA [START_REF] Rankl | Smart Card Handbook[END_REF]. Modern smart cards can host multiple applications, but only one application can be executed at a time.

The two main de facto standards of multi-application cards are Java Card [START_REF] Microsystems | Java Card(tm) Specification 2.2.2 Final Release[END_REF] and GlobalPlatform card specification [START_REF] Globalplatform | GlobalPlatform Card Specification[END_REF].

1) Java Card: Java Card is a Java-based smart card. It is designed by adding a lightweight Java bytecode interpreter to a smart card's OS and downloading Java class files, which were converted to a specific format beforehand. The system architecture of the Java Card is mainly composed of three layers: the Java Card Virtual Machine (JCVM), the Java Card Runtime Environment (JCRE) which provides a wellstructured framework to access the system-level services, and finally the Java Card firewall which isolates the different applets present on the card from each other. The Java Card specification does not specify the mechanism of installing, updating, or deleting applications. The application management is defined by GlobalPlatform card specification.

2) GlobalPlatform: The Card Manager is the entity that controls the smart card environment. It contains the OPEN framework that controls the downloading and installing of applications. In addition, it contains the issuer security domain. The security domain is a generic term used to divide the card into independent regions. Each region is associated to separate cryptographic keys that allow the security domain owner to manage its applications without the card issuer's involvement. Each application is associated to a security domain. The security domain created by the card issuer, commonly called the issuer security domain, is the component that controls the security domains. However, it has no access to their content or to their cryptographic keys.

B. Over-the-Air (OTA)

Over-the-Air (OTA) platform was originally designed by mobile operators to provide remote management of their The structure of the APDU command is defined in [START_REF]Secured packet structure for UICC based applications (Release 9)[END_REF]. ETSI has introduced the TS 102 226 [START_REF]Remote APDU structure for UICC based applications (Release 9)[END_REF] standard in order to support security services for OTA communication.

IV. OVER-THE-INTERNET DESIGN

A. Overview of the Architecture

The Over-the-Internet framework (OTI) applies the usercentric model to the NFC ecosystem. It allows users to install secure element based NFC applications as effortlessly as they install any other smartphone app. OTI transmits applications to the SE via Internet (e.g., wifi or 3/4G connection). OTI leverages the concept of security domain to enable secure management of applications developed by different service providers. In addition, it leverages the secure channel protocol (SCP) to provide end-to-end secure connection between the service provider and the secure element.

Figure 1 shows an overview of the OTI platform. The architecture consists of the following components:

1) Secure Element Issuer: it is the owner of the SE and the responsible of its long-term content management. All secure elements have a mandatory representative of the issuer, known as issuer security domain (ISD).

In most cases, issuers delegate the life cycle management of SE to a trusted service manager. 2) Trusted Service Manager (TSM): the TSM acts as the connection point between service providers and the SE issuer. It creates security domains on the SE in order to provide secured blocks of space for service providers. 3) Service Provider (SP): in the user-centric model, it is the service provider that manages the life cycle of applications in their security domain created by the TSM. The TSM has no access to the content of an SP security domain. However, the TSM still could delete the SP security domain and all its contents in case of disagreement. 4) NFC-Enabled Smartphone: most NFC applications are not composed only of an applet on the SE, but they also include a graphical interface. The graphical interface displays some messages to the user. 5) Secure Element (SE): it hosts the sensitive part of the NFC application.

The OTI framework works as follows: the user looks for an applications on her favorite smartphone app store. She selects an application to install on her mobile device. The install process is started, and the corresponding service provider accomplishes three tasks. First, it installs the graphical interface on the mobile device. Second, it asks the TSM to create a security domain in the corresponding SE. The TSM sends the credentials of the recently created security domain to the SP. Third, the SP installs the applet on the SE. The process of installing is described in the following subsection.

B. Application Management

The graphical interface includes a module whereby the SP can exchange command/response messages with the SE. We call this module the "SE Manager". The management of SE applets includes installing, updating and deleting the applet. Each operation is performed by a specific set of commands. We leverage the GlobalPlatform card specification to build these commands. Sending the management commands to the SE is achieved by three steps.

First, the SP sends a push notification to the SE manager. Once notified, The SE manager opens a TCP connection with the SP server. As well, it opens a communication session with the SE. Second, the SP establishes a secure connection with the SE using the credentials of its security domain. The secure connection protects the confidentiality, integrity and freshness of the management commands. Such protection is necessary, since the commands go through untrusted parties, including the SE manager. Third, the management commands are encapsulated in the appropriate format before being transmitted to the SE. Once received, the SE performs some verification operations before executing the commands. The SP is notified of the execution status by a cryptographically protected response from the SE.

Figure 2 shows the layer model of the OTI application management.

C. Secure Channel Protocol (SCP)

Secure communication between the service provider and the secure element is of paramount importance. The integrity and the confidentiality of the management commands should be protected. The SE manager shall see nothing, but encrypted messages received from the SP and to be forwarded to the SE. We leverage the secure channel protocol 03 (SCP03) [START_REF]Secure Channel Protocol 03[END_REF] to establish a secure communication channel between the the SP and the SE. SCP03 is an improved version of the SCP02. It is a symmetric key protocol that is divided into three phases:

Secure channel initiation: this includes mutual authentication between the SP and the related security domain on the SE. It also includes the generation of session keys.

Secure channel operation: the SP and the SE exchange messages within the cryptographic protection of the secure channel session. The channel protects the integrity and the confidentiality of the exchanged messages. The cryptographic operations are done by the AES encryption algorithm.

Secure channel termination: termination occurs when either of the communicating entities determines that no further communication is required or allowed via the secure channel session.

D. Design Analysis

The OTI framework overcomes the limitations of today's commonly used technology for remote applications (e.g., SMS based OTA). Its objectives are twofold. First, it enforces the "freedom of choice" of users. This is done by creating a space in the SE for each service provider. The SP manages its applications without the SE issuer's involvement. The OTI framework supposes that the infrastructure of the service provider is secure enough to protect the credentials of the created security domains. Second, and most importantly, the OTI framework leverages various technologies to provide faster and more reliable management for SE applications. Applications are transmitted from the SP server to the SE over TCP/IP connection. Since there is no direct connection between the SP and the SE, the communication must go through a bridge that receives the TCP/IP packets from the SP and forwards them to the SE. This bridge is a module installed on the mobile device. For more usability, this module is integrated in the graphical interface of the NFC application. The security of the transmitted application is guaranteed by the well-known and proven SCP03 protocol.

We use web technology for the graphical interface. All current smartphone systems allow native apps to display web pages and execute JavaScript code. Web technology allows for seamless updates of the graphical interface and the SE manager. This is important in order to avoid execution failure due to incompatible applications.

V. IMPLEMENTATION AND EVALUATION

We implemented a proof-of-concept of the OTI platform. Our prototype is divided into five parts:

1) Secure element: we used Oberthur UICC JavaCard 2.2.2 as the secure element. The used UICC is compliant with the GlobalPlatform card specification. The secure element is the only component in the OTI platform that does not require any modification or additional module. 2) NFC-enabled smartphone: we used Samsung Galaxy SII with Android 4.0.3 (ICS). SmartCard API [START_REF]Secure Element Evaluation Kit for the Android platform[END_REF] was installed to send APDU to the UICC. 3) User interface: the user interface was implemented as webview in the Android system in order to avoid the hassle of using the browser. The graphical interface is encoded in HTML5 and CCS3. The SE module was implemented as a JavaScript library. We called this library Ajase (Asynchronous JavaScript and Secure Element). Ajase is the implementation of the SE API recently defined in unofficial draft issued by Gemalto and Deutsche Telekom [START_REF] Gemalto | Secure Element API, Unofficial Draft[END_REF]. The user interface is registered as Google Cloud Messaging (GCM) services to receive notification messages from the service provider. 4) Service provider: we integrated a full implementation of the GlobalPlatform card specification 2.2.1 written in Java 7 to the service provider. This library is used to generate the management commands and establish the secure channel. 5) TSM: we used OTA platform to create security domains for service providers.

Performance Evaluation

We conducted a comparison study between OTI (Over-the-Internet) and the SMS-based OTA (Over-the-Air). The study includes OTW (Over-the-Wire) because OTW is considered as the lower-bound of content management systems. OTW is the fastest and the most reliable content management system, since the secure element is directly connected to the TSM via a wire. However, in real context, OTW is not convenient because users prefer receiving updates via a wireless medium.

Table I shows the download time of JavaCard applet of size 9 kilobytes for different content management platforms. We notice that OTI largely outperforms OTA and comes very close to OTW. OTI is not only faster than SMS-based OTA, but it is also more reliable. More comprehensive evaluation study is kept for future work.

VI. RELATED WORK

The NFC ecosystem management is still an overlooked problem. Most work suggest to use SMS-based OTA platform to manage NFC applications, or more generally SEbased applications [START_REF] Madlmayr | Managing an NFC Ecosystem[END_REF]. An industrial proposal is to replace SMS-based OTA by LTE-based OTA [START_REF] Giesecke&devrient | The OTA Platform in the World of LTE[END_REF]. LTE is the fourth generation of mobile networks, commonly called 4G. LTE is a good substitute for SMS. However, unlike OTI, LTE-based OTA does not apply the user-centric model. The SE issuer has full control over the applications installed on the SE. GlobalPlatform proposes in [START_REF] Globalplatform | GlobalPlatform Card, Remote Application Management over HTTP, Card Specification v2.2 -Amendment B[END_REF] to send management commands to the SE using HTTPS connection. This supposes that SE is able to receive and understand HTTP messages. This is true for JavaCard 3.0. However, most of the deployed secure elements are JavaCard 2.x. In [START_REF] Badra | Toward SSL Integration in SIM SmartCards[END_REF], Badra et al. propose to combine SSL with BIP in OTA platform. As explained earlier, OTI does not rely on BIP because it is not widely supported by mobile devices. Compared to other work, OTI platform has the particularity to combine both effectiveness and deployability.

VII. CONCLUSION

We introduced OTI, an efficient management system for secure element based NFC applications in mobile devices. OTI transmits management commands over a TCP connection.

Hence, it is more reliable than SMS-based OTA. In addition, it is more secure, since OTA platforms are vulnerable to wellknown threats [START_REF] Madlmayr | Risk Analysis of Over-the-Air Transactions in an NFC Ecosystem[END_REF]. OTI solves the dilemma that previous approaches face because it does not have to trade off deployability and effectiveness. This paper also introduced the current OTI implementation and experimental evaluation. Results indicate that OTI is an effective and practical management system for real world NFC ecosystem. Our future work will focus on providing a comprehensive framework for comparative evaluation of different application management systems.

Fig. 1 .

 1 Fig. 1. An Overview of the OTI Architecture

Fig. 2 .

 2 Fig. 2. OTI Layer Model

TABLE I

 I

	.	COMPARISON OF DOWNLOAD TIME OF
		9-KILOBYTE-JAVACARD APPLET
	Content Management Platform	Average of Download Time
	OTW (Over-the-Wire)	18.2 seconds
	OTI (Over-the-Internet)	25.7 seconds
	OTA (Over-the-Air)	5.42 minutes