M Parrot

Observations by DEMETER of man-made MF waves escaping from the ionosphere by

Keywords: ionosphere, man-made MF waves, ionospheric trough

Key points :

-DEMETER observed MF waves due to man-made activities above the topside ionosphere when the ionospheric density is anomalously low -The observations are mainly related to the ionospheric trough region -These observations also occur in the South Atlantic Anomaly and during high magnetic activity Abstract : DEMETER was a low altitude satellite on a polar and circular orbit devoted to the study of ionospheric perturbations in relation with seismic activity. Its scientific payload with waves and plasma measurements also allows to study man-made waves. The aim of this paper is to check the conditions of apparition of these man-made waves in the MF range (300 kHz, 3 MHz). These waves normally propagate in the Earth-ionosphere waveguide but it is shown that they can escape above the ionosphere when there is a decrease in the electron density.

Introduction

Man-made Medium Frequency (MF) waves are in a frequency range between 300 kHz and 3 MHz. They are typically used by broadcasting, radio-navigation (maritime and aeronautical), and maritime mobile communications. They propagate in the Earth-ionosphere waveguide [START_REF] Milsom | Surface waves, and sky waves below 2 MHz in Propagation of radio waves[END_REF]. Normally they are reflected and cannot cross the ionosphere because their frequencies are lower than the plasma frequency in the F layer. But there is a

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possibility for these waves to cross the ionosphere if they follow a magnetic field line.

Individual observations of broadcast transmitters above the F layer have been already done [START_REF] Labelle | Natural and man made emissions at 1.0-5.6 MHz measured between 10 and 18 RE[END_REF]Kaiser et al., 1996;[START_REF] Kelley | LF and MF observations of the lightning electromagnetic pulse at ionospheric altitudes[END_REF][START_REF] Hashimoto | Hectometric line spectra detected by the Arase (ERG) satellite[END_REF].

Concerning the low altitude satellite DEMETER, if the recorded MF spectra are averaged over a large time interval, the transmitter locations appear on a terrestrial map of wave activity as hot spots which are a little bit shifted equatorwards due to their propagation along a magnetic field line [START_REF] Füllekrug | Transionospheric attenuation of 100 kHz radio waves inferred from satellite and ground based observations[END_REF]Parrot, 2018;[START_REF] Nagy | What are the sources of MF signatures recorded on DEMETER satellite?[END_REF].

MF man-made waves also appear in DEMETER MF spectrograms at very different locations and the purpose of this paper is to systematically study all bursts of man-made waves which emerge from time to time in these MF spectrograms. The DEMETER data are briefly described in Section 2. Various events are shown in Section 3. Discussions and conclusions are given in Sections 4 and 5, respectively.

2. The data DEMETER was in operation between 2004 and 2010 on a polar and circular sunsynchronous orbit with an altitude of 710 km (decreased to 660 km in 2005). Among the different experiments of the scientific payload there was an electric field experiment to continuously measure onboard the satellite the electric field spectrum up to 3.33 MHz with a frequency resolution of 3.25 kHz and a time resolution of 2.048 s [START_REF] Berthelier | ICE, The electric field experiment on DEMETER[END_REF].

The electron density was in situ measured by a Langmuir probe [START_REF] Lebreton | The ISL Langmuir Probe experiment and its data processing onboard DEMETER: Scientific objectives, description and first results[END_REF]. Due to technical reasons data are only recorded at invariant latitudes less than 65° except for specific campaigns in relation with HAARP or EISCAT. All data files and plots are organised by half-orbits [START_REF] Lagoutte | The DEMETER science mission centre[END_REF]. In this paper, the up-going half-orbits corresponding to night time (22:30 LT) are only used because the ionospheric electron density is too high

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during day time to observe MF waves coming from below. A total of 28710 night time halforbits have been checked.

The observations

The top panel of Figure 1 shows the MF spectrogram of an electric field component recorded on 24 September 2006 during 4 minutes. One can see a set of frequency lines over a large frequency range between 0.6 and 1.6 MHz. The intensity of some frequency lines is very important (0.3 µV 2 .m -2 .Hz -1 ) as it is of the order of the wave intensity in the VLF range (< 20 kHz). The spectrogram is recorded above Canada and the observed frequency lines correspond to emissions from various transmitters at different frequencies because the MF band has been shared between many different users. The bottom panel of Figure 1 displays the electron density which is lower at the time of the burst of the frequency lines. The density decrease fits well with the apparition of the MF waves. The shape of the electron density decrease (slow in the equatorward direction and sharp in the pole ward direction) is typical of the phenomenon called mid latitude ionospheric trough. This ionospheric trough which is a night-side event is well documented since a long time and, at both the F layer and the topside ionosphere, it corresponds to the plasmapause position [see for example, Taylor and Walsh, 1972;[START_REF] Karpachev | The dependence of the main ionospheric trough shape on longitude, altitude, season, local time, and solar and magnetic activity[END_REF]Yizengaw and Moldwin, 2005;Rothkaehl et al., 2008;[START_REF] Grebowsky | Altitude variation of the plasmapause signature in the main ionospheric trough[END_REF][START_REF] He | A study on the nighttime midlatitude ionospheric trough[END_REF][START_REF] Chen | The midlatitude trough and the plasmapause in the nighttime ionosphere simultaneously observed by DEMETER during 2006-2009[END_REF][START_REF] Karpachev | Longitudinal variations of the ionospheric trough position[END_REF].

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A third event different from the two others is shown in Figure 3. It was recorded on 8 November 2008, a day of moderate magnetic activity with Dst = -17 nT and kp = 3.3. The electron density in the bottom panel presents plasma depletions which are simultaneous with the apparition of bursts of frequency lines in the top panel.

A fourth example is shown in Figure 4 where the data are recorded on 15 December 2006 at the time of high magnetic activity (Dst = -156 nT, kp = 7.7). This corresponds to a rare half-orbit where we have at the beginning of the plot an event due to the magnetic activity with a dramatic decrease of the density as in Figure 3, and at the end of the plot an event related to an ionospheric trough as in Figure 1. One can notice that the two frequency bands are different because the satellite is not above the same countries (close to Peru around 03.51.40 UT and above USA around 04.06.00 UT).

Discussions

All in all we have observed 1066 events related to the trough (of which only 184 are in the South hemisphere), 85 events related to an anomalous low density, and 29 events related to a high magnetic activity. The locations of all these events are displayed in Figure 5 with symbols of different colors. The events related to the trough (black symbol) are observed at mid and high latitudes. They also correspond to inhabited areas where MF transmitters are located but there are some unexplained gaps close to the South Africa and the South America which will be discussed later on. It is remarkable to see that the events related to low density (green symbol) are mainly located in the South Atlantic Magnetic Anomaly (SAMA). One can also say that these events are recorded during winter time in the South hemisphere which contributes to the decrease of the electron density together with low F10.7cm solar flux in the decreasing part of the solar cycle 23. A second set of green symbols is located in the Indian

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Ocean at the magnetic equator. The high magnetic activity events (red symbol) do not have particular locations.

We cannot have all trough locations in our database because we have a limitation in latitude and we only record MF events close to inhabited areas where man made waves can be observed. But it is interesting to check the evolution of the detected troughs as function of the time. This is done in Figure 6 where the L values of the trough positions in the North hemisphere are shown as function of the years (L is the Mcllwain parameter). Several points can be underlined: i) there is a large variability of the trough positions, ii) the troughs are mainly detected during winter time, and iii) there is a trend as the L values increases with the time. This last variation is related to the decreasing part of the solar cycle 23 because it is known that the trough positions move to lower latitudes when the geomagnetic activity increases [Rothkaehl et al., 2008;[START_REF] Chen | The midlatitude trough and the plasmapause in the nighttime ionosphere simultaneously observed by DEMETER during 2006-2009[END_REF]. This is well illustrated in Figure 4 where the geographic latitude of the trough is around 36°.

As it was noticed before, we only observe a very few number of events close to the South Africa and the South America in Figure 5. This is due to the position of the trough in the South hemisphere. It has been shown (see Figure 3 of [START_REF] Chen | The midlatitude trough and the plasmapause in the nighttime ionosphere simultaneously observed by DEMETER during 2006-2009[END_REF] that the trough is at latitudes much higher than the inhabited area latitudes except for Australia and New Zealand. Then even if there are emissions of MF waves in South Africa, Chile and Argentina they cannot be observed because the locations of these emissions are too far from the trough position. However we have a few events and the MF event located at lat = -67.6° and long = 270.7° is displayed in Figure 7 as example. Data from a complete half-orbit are shown in this

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they cannot be attributed to MF transmitters located in Chile or Argentina. As the trough corresponds to the plasmapause at higher altitudes it has been shown by [START_REF] Blagoveshchensky | Main ionospheric trough as a channel for MF propagation in the magnetosphere[END_REF] that this plasmapause could be a waveguide for the MF waves. Therefore these emissions observed above the South Pacific Ocean must come from the magnetically conjugate region in the other hemisphere which is in Canada (close to lat = 44.25°, long = 280.33°). In Figure 7, the other MF emission observed at ~ 05.50.30 UT in the North hemisphere is also recorded above Canada. It can be noticed that they have a very similar frequency band, and that, due to the propagation, the MF emission in the South hemisphere have lower amplitude than the MF emission in the North hemisphere. Two other points must be underlined concerning this event in Figure 7: (i) the data are recorded during a period of moderate magnetic activity (Dst = -51 nT, kp = 6.0) which means that the positions of the trough are not usual, and (ii) to observe the MF emission coming from above the satellite a plasma depletion is additionally needed. Concerning the observations just close to the New Zealand in Figure 5 it also means that all these events are certainly not due to transmitters located in this country but a small part could be related to emissions in the North hemisphere because this area is magnetically conjugate to Alaska.

In Figures 1-4 and7, the electron density is measured at the altitude of the satellite but we do not know the density below. It is possible to obtain a rough value considering that (i) the plasma frequency in kHz is equal to 9 Ne ½ where the electron density Ne is in cm -3 , and (ii) the MF waves cannot cross the ionosphere if their frequencies are lower than the plasma frequency. For example in Figure 1 the minimum frequency observed in the spectrogram is of the order of 0.6 MHz, and this means that the maximum electron density below the satellite is ~ 4.4 10 3 cm -3 . This is higher than the minimum density observed at 660 km in the bottom panel of Figure 1 as expected because in this part of the ionosphere the density decreases when the altitude increases.

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Conclusions

For the first time an extensive study of the man-made MF waves observed by a low altitude satellite in the top of the ionosphere has been done. It is shown that these waves can escape from the ionosphere at the locations where the electron density is particularly low.

There are three possibilities:

-The largest number of events occurs when the satellite is close to the mid-latitude ionospheric trough during winter time. At higher altitudes the trough corresponds to the plasmapause which could be a waveguide for the MF emissions [START_REF] Blagoveshchensky | Main ionospheric trough as a channel for MF propagation in the magnetosphere[END_REF]. Therefore, these MF waves can propagate in the opposite hemisphere as it is the case for the event shown in Figure 7. Concerning their possible influence in the magnetosphere, interaction of man-made MF waves with natural waves at large distances has been revealed by [START_REF] Hashimoto | Hectometric line spectra detected by the Arase (ERG) satellite[END_REF].

-A second set of events is observed in the SAMA and could be related to the very low solar flux at the end of the solar cycle 23.

-The third possibility happens during high magnetic activity periods which produce large and sharp plasma depletions in the ionospheric density.

Figure 2

 2 Figure 2 is similar to Figure 1 but the data are recorded on 3 rd August 2008 in a region

Figure .

 . Figure. One can see in the top panel the two MF emissions observed in the South hemisphere

Acknowledgments: The satellite DEMETER was operated by the French Centre National d'Etudes Spatiales (CNES). This work is based on observations with the electric field experiment ICE and the Langmuir probe experiment ISL embarked on DEMETER. The author thanks J. J. Berthelier and J.P. Lebreton the PIs of these experiments for the use of the data. Many thanks are also due to J.Y. Brochot who was deeply involved in the data handling since the beginning of the mission. The DEMETER data shown in this paper can be obtained

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This article is protected by copyright. All rights reserved. Parrot, M. (2018). DEMETER observations of man-made waves that propagate in the ionosphere, Comptes Rendus Physique, 19(1-2), 26-35. Rothkaehl, H., Krakowski, A., Stanislawska, I., Błęcki, J., Parrot, M., Berthelier, J. J., and J.P. Lebreton (2008). Wave and plasma measurements and GPS diagnostics of the main ionospheric trough as a hybrid method used for Space Weather purposes. Annales Geophysicae, European Geosciences Union, 26 (2), 295-304.

Taylor, H.A. and W.J. Walsh (1972), The light ion trough, the main trough and the plasmapause, J. Geophys. Res., 77(34), 6716-6723. Yizengaw, E. and M.B. Moldwin (2005), The altitude extension of the mid-latitude trough and its correlation with plasmapause position, Geophys. Res. Lett., 32, L09105, doi:10.1029/2005GL022854.

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This article is protected by copyright. All rights reserved. Figure 1: (top panel) MF spectrogram up to 2 MHz onboard recorded on 24 September 2006 between 03:04:30 and 03:08:30 UT. The power spectral density is color-coded according to the scale on the right. (bottom panel) Electron density in situ recorded during the same period. The parameters below the panels indicate the Universal Time, the Local Time, the geographical latitude and longitude, and the McIlwain parameter L.

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This article is protected by copyright. All rights reserved. Figure 2: Same as Figure 1 but for data recorded on 3 rd August 2008 between 00:53:00 and 01:05:00 UT.

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This article is protected by copyright. All rights reserved. Figure 3: Same as Figures 2 and 3 but for data recorded on 8 November 2008 between 03:44:00 and 03:50:00 UT.

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This article is protected by copyright. All rights reserved. Figure 4: Same as Figures 1, 2 and 3 but for data recorded on 15 December 2006 between 03:49:00 and 04:07:00 UT. The two white lines in the spectrogram correspond to a lack of data.

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Emmanuelle Anceaume
email: emmanuelle.anceaume@irisa.fr

Christophe Bisière
email: christophe.bisiere@tse-fr.eu

Matthieu Bouvard
email: matthieu.bouvard@tse-fr.eu

Quentin Bramas
email: bramas@unistra.fr

Catherine Casamatta
email: catherine.casamatta@tse-fr.eu

Daniel Cremers

Marc Langheinrich

Dorothea Wagner

Preface

This volume includes the published papers of Tokenomics 2020, the second edition of the International Conference on Blockchain Economics, Security and Protocols.

Tokenomics is an international forum for theory, design, analysis, implementation and applications of blockchains and smart contracts. The goal of the conference is to bring together economists, computer science researchers and practitioners working on blockchains in a unique program featuring outstanding invited talks and academic presentations.

The conference was initially planned on May 11 th and 12 th , 2020. Due to the COVID-19 pandemic, it eventually took place on October 26 th and 27 th of the same year at Toulouse School of Economics (TSE) in an hybrid format with some of the speakers and moderators presenting in front of an audience in TSE new buidling and the other conference participants joining through videoconferencing.

For this second edition, there were 45 papers submitted: 12 papers in computer science (11 as regular papers and 1 as a short paper) and 32 papers in economics. The economics program committee selected 14 papers for presentation at the conference. The computer science program committee selected 6 regular papers for presentation at the conference and publication in this volume. Additionally, 4 submissions were accepted as short papers, for presentation at the conference and publication in this volume.

In addition to accepted papers, we had the pleasure to welcome four distinguished invited keynote speakers:

Ittai Abraham, senior researcher at vmware research. Abraham discussed the use of game theoretical tools into computer science to model blockchains and cryptocurrencies Long Chen, Secretary-General of the Luohan Academy, an open research institute initiated by Alibaba, and former Chief Strategy Officer at Ant Financial. Chen gave an overview of the ongoing changes in financial services driven by progresses in information technologies. Jean Tirole, Researcher at TSE and 2014 laureate of the Sveriges Riksbank prize in economic sciences in memory of Alfred Nobel. Tirole discussed the challenges faced by cryptocurrencies using the framework of economic theory. Timothy Zakian, Software Engineer, Novi, Facebook. Zakian presented Move, the programming language developed to implement transactions and smart contracts on the Libra blockchain.

Together with the computer science contributions gathered in these proceedings, the papers presented in the economics track tackled a wide range of issues reflecting the vitality of the research on blockchains and cryptocurrencies in economics. This growing interest reflects the current and potential impact of blockchain-based applications for consumers, businesses and governments. It also captures a fundamental feature of blockchains: implementing a distributed consensus is as much an incentive problem as it is a technological challenge.

A first subset of these papers focuses on the functioning of the blockchain itself. In keeping with an earlier stream of papers in computer sciences and economics, Ebrahimi, Routledge and Zetlin-Jones ("Getting Blockchain Incentives Right") use game theory to analyze miners' equilibrium strategies under proof of work and the possibility they may fail to ensure consensus. Amoussou-Guenou, Biais, Potop-Butucaru and Tucci-Piergiovanni ("Rational vs Byzantine Players in Consensus-based Blockchains") use a similar game-theoretic toolbox to analyze the strategies of committee members in a Byzantine Fault Tolerant blockchain. Garatt and van Oordt ("Why Fixed Costs Matter for Proof-of-Work Based Cryptocurrencies") show how miners' cost structure, notably the existence of sunk equipment costs, affect their A second set of papers adopts an industrial organization approach to understand the distinctive features of blockchain-powered businesses. Lyandres ("Product Market Competition with Crypto Tokens and Smart Contracts") shows how native tokens and smart contracts alter the nature of the competition between an incumbent firm and a potential entrant. Cong, He and Wang ("Token-Based Platform Finance") combine the industrial organization angle with corporate finance implications: they evaluate the dual role of tokens as influencing users' adoption of a platform, and as providing entrepreneurs with a source of funding. Finally, Bakos and Halaburda ("When Do Smart Contracts and IoT Improve Efficiency? Automated Execution vs. Increased Information") draw on contract theory to clarify the capabilities of smart contracts. In particular, they distinguish between two features of smart contracts, the expansion of the contracting space thanks to IoT sensors and the automatization of the contract execution.

Last, a third set of papers approaches tokens from an asset pricing side. Pratt, Danos and Marcassa ("Reversible and Composable Financial Contracts") show how the value of utility tokens can be derived from users' benefits from immediately accessing the services of a platform. Dai, Jiang, Kou and Qin ("From Hotelling to Nakamoto: The Economic Meaning of Bitcoin Mining") propose a model that relates Bitcoin prices to miners' decisions to warehouse or sell the bitcoins they earn by confirming blocks. This model delivers quantitative predictions and is calibrated to the data. Shams ("The Structure of Cryptocurrency Returns") studies the comovement of multiple cryptocurrency prices and empirically connects high correlations to common demand factors. Finally, Benigno, Schilling and Uhlig ("Cryptocurrencies, Currency Competition, and the Impossible Trinity") study the implications of the adoption of a global cryptocurrency for monetary policies and exchange rates between fiat moneys.

Overall, the breadth of the topics explored by the participants to this conference illustrates the fruitful interaction between computer science and economics for understanding the implications of blockchain-based solutions. It also suggests much more ground to cover and we hope this conference will further stimulate research in this area.

We thank the authors for submitting their work at the conference and the program committee who worked hard in reviewing papers and giving feedback to the authors.

Catherine, Christophe, Emmanuelle, Matthieu and Quentin

  2nd International Conference on Blockchain Economics, Security and Protocols (Tokenomics 2020). Editors: Emmanuelle Anceaume, ChristopheBisière, Matthieu Bouvard, Quentin Bramas, and Catherine Casamatta OpenAccess Series in Informatics Schloss Dagstuhl -Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany incentives to deploy hashpower in response to the cryptocurrency price movements. Finally, Hinzen, John and Saleh ("Bitcoin's Fatal Flaw: The Limited Adoption Problem") model how the information time lags inherent to fully distributed consensus create a hard technical constraint on the throughput of permissionless blockchains.


Edurne Luque-Michel

Laurent Lemaire

Maria J Blanco-Prieto

María J Blanco-Prieto

SPION and doxorubicin-loaded polymeric nanocarriers for glioblastoma theranostics

Keywords: Tween 80, MRI, tumor doubling time, glioma, U87-MG, PLGA, SPION Glioma cells injection NP injection MAGNET Tumor growth

Glioma is a type of cancer with a very poor prognosis with a survival of around 15 months in the case of glioblastoma multiforme (GBM). In order to advance in personalized medicine, we developed polymeric nanoparticles (PNP) loaded with both SPION (Superparamagnetic Iron Oxide Nanoparticles) and doxorubicin (DOX). The former being used for its potential to accumulate the PNP in the tumor under a strong magnetic field and the later for its therapeutic potential. The emulsion solvent and evaporation method was selected to develop monodisperse PNP with high loading efficiency in both SPION and DOX.

Once injected in mice, a significant accumulation of the PNP was observed within the tumoral tissue under static magnetic field as observed by MRI leading to a reduction of tumor growth rate.

INTRODUCTION

Glioblastoma (GBM) is the most common and the most malignant variant in the wide spectrum of intrinsic glial brain tumors [START_REF] Vleeschouwer | Glioblastoma[END_REF]. Morphologically, GBM is highly heterogeneous, diffuse and infiltrative in nature, which makes surgical removal particularly difficult [START_REF] Gaikwad | Nanotechnologybased strategies as novel therapies in gliomas[END_REF]. In parallel, the failure of chemotherapy to reach the brain, as it is protected by the dynamic Blood Brain Barrier (BBB), and the presence of stem cells lead to tumor recurrence [START_REF] Gaikwad | Nanotechnologybased strategies as novel therapies in gliomas[END_REF][START_REF] Valenciano | Importancia de Células Madre Tumorales y Cultivos de Neuroesferas en Neurooncología[END_REF]. In the area of cancer nanomedicine, drugs are encapsulated inside nanocarriers that provide controlled release of the drug once in the tumor. In this sense, nanoparticles (NP) designed to achieve BBB uptake could increase the drug concentration in glioma and then increase the effectiveness of treatment. This is the case of polymeric nanoparticles (PNP) made with surfactants like Tween 80 (T80), which have received increasing attention over the last few years. Widely accepted in the literature, this strategy takes advantage of the adsorption of apolipoproteins across blood plasma onto the T80 NP surface, promoting recognition by LDL receptors in the brain capillary endothelial cells [START_REF] Malinovskaya | Delivery of doxorubicin-loaded PLGA nanoparticles into U87 human glioblastoma cells[END_REF][START_REF] Kreuter | Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood-brain barrier[END_REF][START_REF] Neves | Braintargeted delivery of resveratrol using solid lipid nanoparticles functionalized with apolipoprotein E[END_REF]; moreover, both neurons and glial cells express LDL receptors [START_REF] Pinzón-Daza | The association of statins plus LDL receptor-targeted liposome-encapsulated doxorubicin increases in vitro drug delivery across blood-brain barrier cells[END_REF]. Then, nanomedicine could help antineoplastic drugs not able to cross the BBB naturally to reach the glioma. This is the case of DOX, an antineoplastic agent widely used in the treatment of various cancers which, although toxic against glioma cells, does not cross the BBB [START_REF] Battaglia | Solid Lipid Nanoparticles for Potential Doxorubicin Delivery in Glioblastoma Treatment: Preliminary In Vitro Studies[END_REF][START_REF] Rousselle | New advances in the transport of doxorubicin through the blood-brain barrier by a peptide vector-mediated strategy[END_REF][START_REF] Luque-Michel | Coencapsulation of superparamagnetic nanoparticles and doxorubicin in PLGA nanocarriers: development, characterization and in vitro antitumor efficacy in glioma cells[END_REF]. Above that, theragnosis, defined as any material that combines the modalities of therapy and imaging into a single package [START_REF] Luque-Michel | Clinical advances of nanocarrier-based cancer therapy and diagnostics[END_REF], is emerging as a very promise strategy to image and treat brain cancers [START_REF] Israel | Magnetic iron oxide nanoparticles for imaging, targeting and treatment of primary and metastatic tumors of the brain[END_REF]. In this work, we suggest an association of doxorubicin (DOX) and SPION (superparamagnetic iron oxide nanoparticles) within T80-coated PNP. SPION were selected since they can be monitored non-invasively by magnetic resonance imaging (MRI) and, moreover, they can be targeted to tumor with the use of magnets [START_REF] Shen | Magnetic enhancement of stem cell-targeted delivery into the brain following MR-guided focused ultrasound for opening the blood-brain barrier[END_REF][START_REF] Qiu | Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions[END_REF]. These theranostic SPION-DOX PNP thus reflect the real-time characteristics of the tumor in each patient and could allow for earlier disease detection, more accurate prognostic information and an enhanced ability to monitor the efficacy of treatment [START_REF] Weinstein | Superparamagnetic Iron Oxide Nanoparticles: Diagnostic Magnetic Resonance Imaging and Potential Therapeutic Applications in Neurooncology and Central Nervous System Inflammatory Pathologies, a Review[END_REF]. Besides, the evaluation of the NP accumulation in healthy tissues would allow us to assess the risk of patients developing off-target side effects or to screen patients who are likely to respond positively to the treatment [START_REF] Li | Cancer nanomedicine: mechanisms, obstacles and strategies[END_REF].

Specifically, the SPION-DOX-NP developed were injected intravenously in mice, and their ability to reach the GBM under the influence of an external magnetic field was evaluated.

METHODS

SPION-DOX PNP synthesis and characterization

SPION-DOX PNP were formulated by the simple emulsion and solvent evaporation method as described before [START_REF] Luque-Michel | Coencapsulation of superparamagnetic nanoparticles and doxorubicin in PLGA nanocarriers: development, characterization and in vitro antitumor efficacy in glioma cells[END_REF]. Briefly, the SPION were covered with oleic acid (10 mg of iron/mL of oleic acid) by 24 hours incubation with continuous shaking, and after that, they were washed and dissolved in dichloromethane. Separately, 0. Once the organic solvent had evaporated, the formulation was washed three times (centrifugations at 17000 g for 10 min at 4º C) and lyophilized along with 37% (w/w with respect to the amount of PLGA) of the cryoprotector trehalose. SPION-DOX PNP size and surface charge were characterized using a Zetasizer Nano ZS (Malvern Instruments, UK), and the size of the NP was measured by TEM images. The DOX loading efficiency was determined fluorimetrically in a Tecan GENios microplate reader (Tecan Group Ltd, Maennedorf, Switzerland) at an excitation and emission wavelength of 485 and 580 nm. In addition to this, the encapsulation of SPION was calculated and analyzed spectrophotometrically (λ= 300 nm) using a microplate PowerWave XS Microplate Spectrophotometer (BioTek). Fluorescence and absorbance were converted into µg/mL DOX or SPION using a calibration curve previously set.

A scheme of the in vivo studies performed is presented in the Fig 1.

All institutional and national guidelines for the care and use of laboratory animals were followed. Animal care and use were in accordance with the regulations of the French Ministry of Agriculture and approved by the Pays de la Loire Ethics in Animal Experimentation Committee under project number 01858.03.

Intracranial inoculation of GBM cells

Tumor implantation was performed via stereotaxic inoculation of the human U87 glioma cells in 8-week-old female nude mice. As described previously [START_REF] Lemaire | Perfluorocarbon-Loaded Lipid Nanocapsules to Assess the Dependence of U87-Human Glioblastoma Tumor pO2 on In Vitro Expansion Conditions[END_REF], mice were anesthetized with Xylazine/Ketamine (50/30 UI; 20 µL/g) and placed in a stereotaxic holder with a heating pad to maintain the appropriate physiological temperature. First, a hole in the skull was drilled to, secondly, inject 4 μL (at 0.5 uL/min) suspension of 8 x 10 4 glioma cells, on the fixed coordinates according to the bregma: 0.5 mm anterior, 2.5 mm right lateral, and 4 mm depth. After surgery, mice received a single 30 μg/kg subcutaneous injection of Buprecare (buprenorphin). In vivo experiments were performed 9-10 days later, after checking by MRI that the tumor had developed. The different groups of mice were formed maintaining an equitable distribution of tumor sizes.

Magnetic targeting and MRI monitoring

To assess the magnetic and contrast capability of SPION-DOX PNP, 12 tumor-bearing animals were equitably divided into two treatment groups: NP targeted using a magnet (n=6) and NP without magnetic targeting (n=6). Each group received intravenously 16 mg/kg of Fe and 5 mg/kg of DOX in SPION-DOX PNP synthetized with surfactant T80 and reconstituted in physiological serum (0.2 mL). As previously described [START_REF] Marie | Superparamagnetic Liposomes for MRI Monitoring and External Magnetic Field-Induced Selective Targeting of Malignant Brain Tumors[END_REF], an external 0.4-T (190 Tm -1 magnetic field gradient) targeting magnetic field, 8 mm diameter/4 mm high disk-shaped neodymium magnets (Supermagnete, Gottmadingen, Germany) were placed onto the top of the head of the mice for 1 hour. During the MR protocol, mice were anesthetized with 0.5% isoflurane and respiration was monitored. Furthermore, animal body temperature was maintained throughout the experiment at 36.5-37.5 ºC by using a feedback-regulated heating pad. MRI was performed using a 7T scanner (Biospec 70/20 Avance III, Bruker Wissembourg, France) equipped with BGA12S gradient system (675 mT/m). Prior to injection, animals were controlled to assess tumor sizes using a Rapid Acquisition with Relaxation Enhancement sequence (TR = 3200 ms; TE = 33 ms, RARE factor 4, matrix size = 256 x 128; FOV =2 x 1 cm, slice thickness = 1 mm). A multiple gradient echo image, using the same geometrical parameters, was also used to produce T2* maps prior to injection; susceptibility weighted images (SWI) were also collected before NP injection (TR=350 ms; TE=18 ms; Slice thickness 1 mm; matrix size=384 X 192; FOV=2 x 1 cm). Half of each group (3 mice/group) was continuously analyzed by MRI for 2.5 hours and the other half was imaged only 4 hours after the injection. SPION-DOX PNP deposition was qualitatively observed using susceptibility weighted images (SWI) and quantitated from the multiple gradient echo set of images by calculating the relaxation rate of transverse magnetization (R2*=1/T2*). Since the accumulation of PNP is often heterogeneous, R2* were averaged over the entire tumor.

Efficacy studies

To determine the efficacy of the treatment with SPION-DOX PNP, tumor-bearing mice were treated twice with a time interval of 48 h by intravenous injection of SPION-DOX PNP (at a DOX dose of 5 mg/kg). One group was exposed to 1 h external magnet targeting after each injection (n=7), while the other was not exposed to the external magnet (n=5). To monitor the efficacy of the treatment, tumor size was measured from the MRI dataset prior to the first PNP injection (day 0), day 2, day 5, and day 7. Volumes were calculated from manually drawn regions of interest (ROI). The counted pixel within the ROI were converted into volumes by timing the number of pixels by their individual volume. Afterwards, the tumor volume growth curves were fitted with an exponential function using the least squares methods; and then the time constant of the exponential was converted into a doubling time value to compare the two groups [START_REF] Lemaire | Perfluorocarbon-Loaded Lipid Nanocapsules to Assess the Dependence of U87-Human Glioblastoma Tumor pO2 on In Vitro Expansion Conditions[END_REF]. Finally, to assess survival, the body weight and mobility of each mouse were measured daily. Survival time was calculated from day 0 (tumor inoculation) to the day of sacrifice when experimental limit points were reached.

Statistical analysis

All results are expressed as mean ± standard deviation (SD). To demonstrate statistical differences, two-way ANOVA, unpaired twotailed t-test or log-Rank (mantel-Cox) survival tests were performed, using the software GraphPad Prism 5 for Windows.

RESULTS AND DISCUSSION

SPION-DOX PNP characterization

As previously described [START_REF] Luque-Michel | Coencapsulation of superparamagnetic nanoparticles and doxorubicin in PLGA nanocarriers: development, characterization and in vitro antitumor efficacy in glioma cells[END_REF], the NP developed had a uniform size distribution (Fig 2a). The mean size was 227.4 ± 18.2 nm by DLS (PDI: 0.066 ± 0.021) and 116.2 ± 37.4 nm measured on TEM images (size of minimum 150 NP measured on TEM images). A representative TEM image is presented in Fig 2b . A PDI less than 0.1 indicates that the sample consisted of a single size mode, without aggregates. Moreover, the zeta-potential value (-14 mV) ensures that the PNP will repel each other and avoid aggregation. On the other hand, the observed differences in size between DLS and TEM may arise from the drying process and the subsequent shrinkage that polymeric chains underwent during TEM sample preparation [START_REF] Luque-Michel | Coencapsulation of superparamagnetic nanoparticles and doxorubicin in PLGA nanocarriers: development, characterization and in vitro antitumor efficacy in glioma cells[END_REF][START_REF] Luque-Michel | A simple approach to obtain hybrid Au-loaded polymeric nanoparticles with a tunable metal load[END_REF], as well as, to the different size-measuring method itself [START_REF] Fischer | Pitfalls and novel applications of particle sizing by dynamic light scattering[END_REF]. In this context, the amount of Fe and DOX encapsulated in SPION-DOX PNP was 5.7 ± 0.6 µg/mg of DOX and 19.0 ± 1.6 µg/mg of Fe, in both cases corresponding to more than 80% encapsulation efficacy.

In vivo studies

Magnetic targeting and MRI monitoring

An orthotopic U87 glioma model in female nude mice was used to evaluate whether the magnetic field influenced or not the retention of the NP inside the tumor. For that, we compared by MRI the GBM deposition of non-targeted and magnetically-targeted SPION-DOX PNP (at 16 mg Fe/kg body weight).

Continuous monitoring

As can be seen in once the NP of the cerebral bloodstream receded, SPION-DOX PNP extravasation was observed only at the tumor region, with the R2* value in the healthy region decreasing rapidly whereas the R2* in the tumor area remained higher for the entire experimental time. This sustained difference between both tissues during the entire experimental period was found to be highly significant in a two-tailed ANOVA statistical study (p = 0.0017). As previously described in detail, it would be possible for a SPION-DOX PNP to cross the BBB after being trapped within its endothelial cells due to a SPION mutual dipole-dipole attraction developed in a continuous magnetic field, which would lead to NP internalization by the malignant cells [START_REF] Marie | Superparamagnetic Liposomes for MRI Monitoring and External Magnetic Field-Induced Selective Targeting of Malignant Brain Tumors[END_REF].

Monitoring at 4 hours

To evaluate the long/median term retention of the NP in the GBM after one hour external magnetic targeting, SWI images were acquired 4 hours after i.v. injection in 3 mice (Fig 4). Results appeared heterogeneous as in one mouse the susceptibility effects of NP were still observed at the tumor site (Fig 3a), in another there was a slight susceptibility effect (Fig 4b ) and in the third one, no effect was detected (Fig 4c). On the other hand, no contrast was detected in the nontargeted tumors after 4 hours (n=3). Therefore, although 4 hours seemed to be the limit of NP retention intravenous SPION-DOX PNP were directed to GBM with a limited one hour of magnet guidance, the magnet targeting could be adapted to each mouse response attaining an individualized therapy.

All the results thus demonstrate the effectiveness of magnetic targeting to enhance preferential accumulation of the SPION-DOX PNP within brain tumors in an amount properly perceptible by MRI, but not within the healthy cerebral areas. Moreover, this study establishes the effectiveness of SPION-DOX PNP as contrast agent in vivo as well as the usefulness of MRI in non-invasive in vivo monitoring. In the same way, a 30 min or 1h exposure to external magnet was shown to target starch coated nanocarriers magnetic nanocarriers to orthotopic tumors in a 9L-glioma bearing rat [START_REF] Chertok | Glioma selectivity of magnetically targeted nanoparticles: A role of abnormal tumor hydrodynamics[END_REF] as well as in mice bearing subcutaneous 9L-glioma tumors respectively. [START_REF] Zhou | Enhanced and selective delivery of enzyme therapy to 9Lglioma tumor via magnetic targeting of PEGmodified, β-glucosidase-conjugated iron oxide nanoparticles[END_REF].

Efficacy studies

The therapeutic effectiveness of SPION-DOX PNP magnetically targeted to the brain in an orthotopic U87 nude mice was evaluated. The final goal was to investigate whether magnetic targeting could further improve the access of PNP-entrapped DOX to the tumor and tumor growth inhibition as well as survival rates. For that, SPION-DOX PNP (at 5 mg DOX/kg body weight) developed with the surfactant T80 were administered twice with a time interval of 48 h by intravenous injection. In one group the NP were targeted with the use of 1 hour of magnet (n=7) and in a second group the NP were not targeted magnetically (n=5).

Tumor growth monitoring

To assess treatment efficiency, tumor progression was monitored using T2-weighted MRI (Fig 5a andb), from which the tumor doubling time was calculated (Fig 5c). SPION-DOX PNP magnetically-targeted tumors were characterized by a 4.4 ± 1.5 days doubling time, whereas non-targeted tumors doubled in size in only 2.3 ± 1.2 days, being 2.93 ± 0.77 days [START_REF] Lemaire | In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth[END_REF] and 2.0 ± 0.2 days [START_REF] Lemaire | Perfluorocarbon-Loaded Lipid Nanocapsules to Assess the Dependence of U87-Human Glioblastoma Tumor pO2 on In Vitro Expansion Conditions[END_REF] the tumor doubling time for untreated mice in previous studies. Besides, the slower growth of the tumor when treated with magnetically-targeted SPION-DOX PNP was significantly different (two-tailed unpaired t-test study (P <0.05)). These results indicated a positive therapeutic effect on the tumor as well as suggesting the enormous potential of the magnetically targeted-SPION-DOX PNP to deliver DOX to glioma cells, as free DOX displays very poor penetration into gliomas and cannot gain access to brain tumor cells [START_REF] Peiris | Treatment of invasive brain tumors using a chain-like nanoparticle[END_REF]. Therefore, like other authors [START_REF] Schleich | Comparison of active, passive and magnetic targeting to tumors of multifunctional paclitaxel/SPIOloaded nanoparticles for tumor imaging and therapy[END_REF], we demonstrate therapeutic benefits based on the superparamagnetic characteristics of SPION that allow guidance by an external magnet and simultaneously provide contrast in MRI.

Survival rate

The overall median survival time was nonstatistically significantly improved by the therapeutic scheme used even though a light tendency is observed (Fig 5d) (31 vs 28 days, p =0.0962). Despite the dosage of DOX (5mg / kg; 2 doses) is within what is usually tested in glioma therapy in mice (1-10 mg/kg), more than 2 doses are often administered [START_REF] Sun | Application of dual targeting drug delivery system for the improvement of antiglioma efficacy of doxorubicin[END_REF][START_REF] Zhang | Dual-modified liposome codelivery of doxorubicin and vincristine improve targeting and therapeutic efficacy of glioma[END_REF][START_REF] Li | Reductionresponsive PEtOz-SS-PCL micelle with tailored size to overcome blood-brain barrier and enhance doxorubicin antiglioma effect[END_REF][START_REF] Belhadj | Design of Y-shaped targeting material for liposome-based multifunctional glioblastoma-targeted drug delivery[END_REF][START_REF] Fang | Targeted glioma chemotherapy by cyclic RGD peptidefunctionalized reversibly core-crosslinked multifunctional poly(ethylene glycol)-bpoly(ε-caprolactone) micelles[END_REF][START_REF] Liu | Gene and doxorubicin co-delivery system for targeting therapy of glioma[END_REF][START_REF] Chen | c(RGDyK)-decorated Pluronic micelles for enhanced doxorubicin and paclitaxel delivery to brain glioma[END_REF]. Moreover, the therapeutically scheme implied a first injection 10 days post cell inoculation and a second one 12 days post inoculation, meaning that the survival was evaluated at about 2 weeks after the end of the therapy, definitely enough time to let the fast growing U87 cells [START_REF] Lemaire | Perfluorocarbon-Loaded Lipid Nanocapsules to Assess the Dependence of U87-Human Glioblastoma Tumor pO2 on In Vitro Expansion Conditions[END_REF] to give rise to a deadly tumor. Therefore, checking the therapy impact In a recent study, Ucakar B. et al. [START_REF] Ucakar | Magnetic targeting of paclitaxelloaded poly ( lactic-co -glycolic acid ) -based nanoparticles for the treatment of glioblastoma[END_REF] found no significant difference between the brain accumulation of T80-coated and uncoated SPION-paclitaxel PNP in an orthotopic GBM model. However, in a subsequent study by the same authors [START_REF] Ucakar | Magnetic targeting of paclitaxelloaded poly ( lactic-co -glycolic acid ) -based nanoparticles for the treatment of glioblastoma[END_REF][START_REF] Ganipineni | Paclitaxel-loaded multifunctional nanoparticles for the targeted treatment of glioblastoma[END_REF], the magnetic targeting was found to enhance the brain accumulation of these NP, their antitumor efficacy and the survival rate. A significant prolongation in the survival rates of mice was obtained using a longer targeting time (4h vs. 1h), a stronger magnet (1.4 T vs. 0.4 T) and a larger number of doses (6 vs. 2). In our study we demonstrate the theranostic efficacy of the NP developed, since they were monitored by MRI at the same time as they treated the tumors. Nevertheless, to improve the survival rates, a larger number of doses [START_REF] Sun | Application of dual targeting drug delivery system for the improvement of antiglioma efficacy of doxorubicin[END_REF][START_REF] Liu | Gene and doxorubicin co-delivery system for targeting therapy of glioma[END_REF][START_REF] Chen | c(RGDyK)-decorated Pluronic micelles for enhanced doxorubicin and paclitaxel delivery to brain glioma[END_REF] or a longer magnet exposure [START_REF] Schleich | Comparison of active, passive and magnetic targeting to tumors of multifunctional paclitaxel/SPIOloaded nanoparticles for tumor imaging and therapy[END_REF][START_REF] Shen | Magnetic Enhancement of Stem Cell-Targeted Delivery into the Brain Following MR-Guided Focused Ultrasound for Opening the Blood-Brain Barrier[END_REF] could be used to achieve a higher DOX concentration in the tumor.

CONCLUSIONS

The objective of this study was to evaluate the theranostic properties of newly developed doxorubicin-SPION loaded polymeric nanoparticles coated with T80 in a glioblastoma context. Taking advantage of the magnetic properties of the SPION, a significant enhancement of SPION-DOX PNP retention in the tumor was obtained after their magnetic targeting, with a significant decrease in the growth rate of the tumors. Further in vivo studies are needed to better the posology required to enhance survival rates, but magnetically targeted SPION-DOX PNP have proven to be a promising drug delivery system to enhance DOX delivery to brain tumors while also allowing non-invasive MRI monitoring of NP distribution.

Fig. 1 :

 1 Fig. 1: Diagram of the in vivo studies performed.

Fig. 2 :

 2 Fig. 2: a) Characterization of SPION-DOX PNP: size, polydispersity (PDI) and Z potential measured by DLS; size of minimum 150 NP measured on TEM images; and DOX and Fe loading inside the PNP. b) TEM image of a representative SPION-DOX PNP.

Fig 3 ,Fig. 3 :

 33 Fig. 3: NP brain uptake evaluation in tumor-bearing mice. a) Brain SWI images before and 80 minutes after i.v. injection of SPION-DOX PNP. b) Brain SWI images before and 90 minutes after i.v. injection of magnetically targeted SPION-DOX PNP. c) Relaxation rate (R2*) in tumor and non-tumor tissue after i.v. injection of SPION-DOX PNP (n = 3). d) Body transverse T2 images before and after i.v. injection of SPION-DOX PNP (n = 1). e) Relaxation rate (R2*) in tumor and non-tumor tissue after i.v. injection of magnetically targeted SPION-DOX PNP; significant differences detected (statistical study ANOVA 2 tails (P = 0.0017)). White brackets indicate the tumor location, plain white arrow the liver and dashed white arrow the spleen.

Fig. 4 :

 4 Fig. 4: SWI images of 3 magnet-exposed GMB before and 4 h after i.v. injection of SPION-DOX PNP. White brackets indicate tumor location.

Fig. 5 :

 5 Fig. 5: Tumor bearing mice brains intravenously treated with SPION-DOX PNP (2 times, in alternative days with (n=7) or without (n=5) magnetic targeting. a, b) T2-weighted images on days 0, 2, 5 and 7 after the first injection c) Time needed for the tumors to double their size (P <0.05). D) Survival rates.

In vivo studies

ACKNOWLEDGEMENTS

This work was supported by the Fundación Caja Navarra and Navarra Government [grant number 411001-41210-4800-322302]. The authors would like to thank Angers Hospital & University Animal Facility (SCAHU), Plateforme de Recherche en Imagerie et Spectroscopie Multimodales (PRISM-IRM-Angers) for providing access to their facilities.


Pierre Bourdier

Priscilla Duboz

Enguerran Macia

Abdou Ka

Julie-Anne Nazare

Emmanuel Cohen

Gilles Boëtsch

Stéphane Blanc

Chantal Simon

Audrey Bergouignan
email: audrey.bergouignan@iphc.cnrs.fr

Gilles Boetsch

How interdisciplinary research at the crossroad between socio-cultural anthropology, nutritional and physical activity physiology can help addressing the obesity epidemic

Keywords: Epidemiological transition, metabolism, anthropology, body weight, physical activity, pre-industrial populations Transition épidemiologique, métabolisme, anthropologie, Poids corporel, activité physique, populations pré-industrielles

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

Introduction

The pandemic of obesity places tremendous burdens on societies and health care systems [START_REF] Mitchell | Obesity: overview of an epidemic[END_REF]. Despite intensive efforts developed across the globe to reduce the prevalence of obesity, all attempted strategies have failed [START_REF] Hill | International strategies to address obesity[END_REF]. This may be explained by the fact that how excessive weight gain is affected by biological, environmental, and social factors remains one of the most debated research topics [START_REF] Blair | Commentary: Luke and Cooper are wrong: physical activity has a crucial role in weight management and determinants of obesity[END_REF][START_REF] Luke | Physical activity does not influence obesity risk: time to clarify the public health message[END_REF].

This may be due to the fact that past socio-ecological transitions that initiated excess weight gain related to an excess of fat mass, in our modern industrial societies ended decades ago, and long-term studies in relevant populations are lacking.

The diet, demography, health and physical activity transitions are often reunited in the concept of epidemiological transition, i.e. the change in the pattern of mortality and disease from high children mortality and infectious diseases to chronic diseases. How these transitions impact the social, ecological, biological, behavioral and cultural factors associated with diet and physical activity, and ultimately weight and fat mass regulation, are part of the debate on the etiology of obesity. Rather than being independent we believe the effects of those transitions are progressive and synergistic.

Understanding the respective contribution and synergistic effects of the biological, behavioral and socio-eco-anthropological factors to the global obesity epidemic requires novel approaches. Our

interdisciplinary group of research believes that studying pre-industrial populations who are currently facing major socio-ecological transitions will help addressing these challenges. In this viewpoint, we will present the limitations of past traditional research, the gaps in knowledge that need to be addressed and the arguments that support the use of pre-industrial populations for future research.

The obesity pandemic, a complex problem

According to the law of energy conservation, body weight gain can only result from a positive energy balance, i.e. an excess of calories intake over calories expended. If the theoretical principle of energy balance seems trivial [START_REF] Hill | Energy balance and obesity[END_REF], its regulation is complex. It involves intertwined genetic, epigenetic, physiological, ecological, social and cultural dimensions [START_REF] Hill | Reducing obesity will require involvement of all sectors of society[END_REF]. Because the epidemic has mainly occurred over the past 50 years, blaming genetics alone is inappropriate. The regulation of energy stores is a complex and dynamic process influenced by proximal factors, including sex, age, genes, aerobic fitness and others, that modify both intake and expenditure. It is also influenced by distal environmental factors at the societal, cultural and global levels which modify lifestyles and behaviors [START_REF] Hill | Reducing obesity will require involvement of all sectors of society[END_REF]. The influence of these combined factors triggering weight gain is recapitulated in the notion of diet, demographic and physical activity transitions, all somehow reunited in the epidemiological transition model. This transition includes changes in diet (high-fat and energy dense food), energy expenditure (low physical activity levels), lifestyle (sedentary behaviors, feeding behaviors and job occupations) and culture, which are the primary socio-ecological drivers of the obesity pandemic [START_REF] Groves | Pandemic obesity in Europe[END_REF][START_REF] Frank | Origins of the obesity pandemic can be analysed[END_REF][START_REF] Popkin | Global nutrition transition and the pandemic of obesity in developing countries[END_REF][START_REF] Swinburn | The global obesity pandemic: shaped by global drivers and local environments[END_REF][START_REF] Egger | An "ecological" approach to the obesity pandemic[END_REF].

The key role of physical activity in the regulation of body weight

The role of exercise and physical activity in the regulation of body weight has recently been the topic of animated debates in both the scientific community and the media. Diet-centric research groups argue that the diet transition is essentially responsible for the onset of the obesity epidemic. By contrast, our view is that as long as physical activity is above a certain level, diet, genes and other factors play a role, but their contribution is less preponderant.

In the 1950s, Jean Mayer, a French American nutritionist, suggested that beyond being the most variable component of total energy expenditure (TEE), physical activity is a major determinant of energy intake (see [START_REF] King | Exercise, appetite and weight management: understanding the compensatory responses in eating behaviour and how they contribute to variability in exercise-induced weight loss[END_REF]). In addition, below a certain physical activity level (PAL=TEE/RMR, where RMR is resting metabolic rate) threshold (likely around 1.7-1.8) that we have most certainly reached in our modern societies, energy intake, no longer TEE, drives weight regulation. These early findings were later supported by observational and intervention studies [START_REF] Stubbs | A decrease in physical activity affects appetite, energy, and nutrient balance in lean men feeding ad libitum[END_REF][START_REF] Shook | Low levels of physical activity are associated with dysregulation of energy intake and fat mass gain over 1 year[END_REF]. More recently, we showed that physical activity energy expenditure (PAEE), but not TEE, predicts nutrient partitioning between oxidation and storage [START_REF] Bergouignan | Activity energy expenditure is a major determinant of dietary fat oxidation and trafficking, but the deleterious effect of detraining is more marked than the beneficial effect of training at current recommendations[END_REF]. The effect of physical activity on food intake and metabolism is likely the result of its action on metabolic flexibility, i.e. ability of the body to adjust substrate use to changes in substrate availability and energy demand. The concept of metabolic flexibility implies that health is determined by the ability to adapt to conditions of temporary stress, such as overfeeding, and thus maintain or regain homeostasis. Chronic stress conditions may induce adaptation processes that go beyond the limits of normal metabolic flexibility leading to progressive inflexibility, which in turn contributes to a disease onset. Our view is that inability to maintain this flexibility in an organism as a phenotype appears with insufficient levels of physical activity; this is the metabolically inflexible phenotype.

In support, we showed that physical activity predicts metabolic flexibility [START_REF] Bergouignan | Physical inactivity as the culprit of metabolic inflexibility: evidence from bed-rest studies[END_REF][START_REF] Bergouignan | Effect of contrasted levels of habitual physical activity on metabolic flexibility[END_REF][START_REF] Rynders | Sedentary behaviour is a key determinant of metabolic inflexibility[END_REF], and that physical inactivity induces an inability to adjust fuel use to match the changing fuel availability and energy demand (i.e. metabolic inflexibility). Metabolic inflexibility precedes the development of glucose intolerance in the pathophysiology of insulin resistance [START_REF] Rudwill | Metabolic Inflexibility Is an Early Marker of Bed-Rest-Induced Glucose Intolerance Even When Fat Mass Is Stable[END_REF]. Importantly, these associations between PAEE and nutrient partitioning and metabolic flexibility were observed independent of changes in energy balance, and even in absence of an increase in TEE [START_REF] Lefai | Exercise training improves fat metabolism independent of total energy expenditure in sedentary overweight men, but does not restore lean metabolic phenotype[END_REF]. This suggests an independent role of physical activity and PAEE in the regulation of metabolism. In support of this, we recently showed that the inability to both decrease time spent sedentary and adjust fuel use (metabolic inflexibility) in response to an acute overfeeding episode is correlated with long-term weight gain [START_REF] Creasy | Free-Living Responses in Energy Balance to Short-Term Overfeeding in Adults Differing in Propensity for Obesity[END_REF][START_REF] Rynders | Associations among dietary fat oxidation responses to overfeeding and weight gain in obesity-prone and resistant adults[END_REF]. Because of the high occurrence of overfeeding events over a lifetime, these findings are highly relevant. Finally, we believe that the effects of physical activity on metabolic flexibility may contribute to the mismatch between excess body weight and adiposity and the absence of metabolic disorders that is commonly observed in metabolically healthy obese individuals. This is supported by a meta-analysis conducted by Barry et al. [START_REF] Barry | Fitness vs. fatness on all-cause mortality: a meta-analysis[END_REF] showing that regardless of BMI, normal weight unfit individuals have a risk of mortality twice higher than their normal-weight fit counterparts. Furthermore, fit individuals with overweight or obesity had similar mortality risks as normal weight fit individuals.

Taken altogether, this data suggest that physical activity plays a key role in the regulation of energy expenditure, energy intake and metabolic control, which are the main tenants of body weight

regulation. Physical activity levels below a certain threshold are associated with a metabolically inflexible phenotype that impairs appetite regulation, energy intake, and metabolic control promoting weight and fat mass gain over the long term.

We hypothesize that environmental changes lead to changes in physical activity, diet and lifestyle/culture (Figure 1).  During the first phase of the nutrition transition, improvement in food intake, i.e. increase in protein, micro-nutrients and energy intakes, decreases the prevalence of under-nutrition and malnutrition. Healthier food intake improves the ability of the body to adapt to physiological stresses and increases body weight to healthy values, mainly due to increase in fat-free mass. However, sugar, high fat content and salt start to appear in the diet.  During the physical activity transition, physical activity decreases leading to drops in PAEE (when normalized per kg of body weight) and PAL that reaches a threshold corresponding to a metabolic tipping point. This is associated with an alteration of the regulatory mechanisms of food intake leading to an uncoupling between energy intake and expenditure, and a metabolically inflexible state, which are favoring body weight gain and excessive fat mass accumulation. Metabolically inflexible phenotypes are then more vulnerable to the impact of poor diet but also to the adverse health effects associated with social and cultural changes, which increase the overall disease burden associated with obesity [START_REF] Van Ommen | Phenotypic flexibility as key factor in the human nutrition and health relationship[END_REF]. Other factors influencing body weight and composition regulation such as poor diet (high-fat, high saturated fatty acid, high fructose), increased stress, altered social structures and interactions are therefore appearing.  With more advanced technology as those present in most developed countries, additional factors such as poor sleep hygiene, endocrine disruptors, high levels of CO 2 , etc. will come at play and influence the regulation of metabolism and energy balance.

In summary, as long as sufficient levels of physical activity are maintained, the prevalence of overweight is kept low even in presence of dietary changes. However as soon as physical activity levels drop under a certain threshold and meet the metabolic tipping point, the "perfect storm" is in place to trigger and boost the obesity epidemic. We believe that to understand which factors are contributing to the epidemic of obesity, we need to investigate this overall phenomenon. The challenge resides in the available approaches to test this global hypothesis.

What have we learnt so far?

The low prevalence of obesity in most developing countries and pre-industrial populations compared to the high prevalence in Western countries supported the hypothesis that energy expenditure and diet differs between populations at different stages of epidemiological transitions [START_REF] Eaton | Physical Inactivity, Obesity, and Type 2 Diabetes: An Evolutionary Perspective[END_REF]. If the shifts in diet (diet transition) have been well documented, the changes in physical activity (physical activity transition) remains a major gap in our knowledge [4, 13]. A commonly shared view is that populations in developing countries must have higher PAEE than industrial societies [START_REF] Hamilton | The ecological and evolutionary energetics of hunter-gatherer residential mobility[END_REF]. Recent meta-analysis compiled studies during which free-living total energy expenditure (TEE) was measured with the gold standard doubly labelled water (DLW) method (see figure 2 and insert 1). Unexpectedly, the metaanalysis did not show differences in TEE adjusted for body weight or PAL between low and middle human development index countries [START_REF] Dugas | Energy expenditure in adults living in developing compared with industrialized countries: a meta-analysis of doubly labeled water studies[END_REF]. One of the meta-analysis even suggested that PAEE slightly increased in industrial countries whereas PAL remained stable [START_REF] Westerterp | Physical activity energy expenditure has not declined since the 1980s and matches energy expenditures of wild mammals[END_REF] in the 1980s-1990s, a time period during which obesity prevalence increased. While informative, these studies cannot replace longitudinal data. To our knowledge, only the elegant METs study provided longitudinal data on body weight gain and physical activity in five populations spanning different degrees of the epidemiological transition (rural Ghana, USA, urban South Africa, Seychelles, urban Jamaica) [START_REF] Luke | Protocol for the modeling the epidemiologic transition study: a longitudinal observational study of energy balance and change in body weight, diabetes and cardiovascular disease risk[END_REF]. No associations between accelerometry-derived activity at baseline and 2-year BW change were observed [START_REF] Dugas | Accelerometer-measured physical activity is not associated with two-year weight change in African-origin adults from five diverse populations[END_REF].

Altogether these findings questioned the role of reduced TEE and physical activity in the onset of body weight gain and have generated animated debates. However, the paucity of data in traditional populations have precluded clear conclusions and fed the debate.

-Please add figure 2 and insert 1 here -

Answers to promises and pitfalls of current data depends on methodological issues and interdisciplinary science

A lack of field data. Most previous reports are compilations of existing literature [START_REF] Dugas | Energy expenditure in adults living in developing compared with industrialized countries: a meta-analysis of doubly labeled water studies[END_REF][START_REF] Westerterp | Physical activity energy expenditure has not declined since the 1980s and matches energy expenditures of wild mammals[END_REF][START_REF] Black | Physical activity levels from a meta-analysis of doubly labeled water studies for validating energy intake as measured by dietary assessment[END_REF]. In the 1980s, when the DLW method was developed in humans, most industrial countries had already been subjected for multiple decades to the epidemiological transition. Changes in TEE had therefore plateaued and DLW studies were measuring TEE at a new and lower steady state. Similarly, most studies in developing countries compared populations who were already sedentary in a mix of suburban/sub-rural environments. Although these data are important, they did not allow to fully detect the changes in the determinants. To address this challenging question, the appropriate scientific approach is to conduct longitudinal studies in populations who have not transitioned from a pre-industrial lifestyle to an urban/modern lifestyle yet.

The regulation of TEE is debated in Westernized populations. Because physical activity is the most variable component of TEE, it is thought that the greater moderate-to-vigorous intensity activity (MVPA) is, the higher is TEE. Pontzer et al. [START_REF] Pontzer | Energy expenditure and activity among Hadza hunter-gatherers[END_REF][START_REF] Pontzer | Hunter-Gatherer Energetics and Human Obesity[END_REF] recently challenged this simple relationship.

Although hunter-gatherers engage in large volume of MVPA, no relationship was found between time spent in MVPA and TEE. They hypothesized that high activity levels are offset by decreases in the other components of TEE, i.e. RMR, thermic effect of food (TEF) or light intensity physical activity (any body movement of daily life including walking, gardening, taking the stairs, doing dish washing, etc.). They proposed the concept of constrained TEE [START_REF] Pontzer | Constrained Total Energy Expenditure and the Evolutionary Biology of Energy Balance[END_REF], in which TEE increases with physical activity at low levels but plateaus at higher activity levels as the body adapts to maintain TEE within a narrow range. This hypothesis challenges our current knowledge of the regulation of energy balance and warrants further studies. However, Westernized populations are not suited for such studies as they have engineered activity out of daily life. Traditional populations are ideal to test this hypothesis.

The precision of existing data on energetics is disputable. Previous comparative studies measured TEE and its components with relatively low precision and accuracy. DLW-derived TEE requires experimental and analytical precisions to reach 1-2% accuracy and 5-6% precision as obtained in our lab; however large variability existed and still exists across laboratories [START_REF] Roberts | Multiple laboratory comparison of the doubly labeled water technique[END_REF]. Because of the constraints inherent to field conditions, most past studies have estimated, rather than measured, RMR using equations known to have low precisions [START_REF] Blanc | Energy requirements in the eighth decade of life[END_REF]; and TEF has been assumed to be constant (10% of TEE). With the propagation of errors, the estimation of PAEE (PAEE=TEE-RMR-TEF) has necessarily low precision. This is critical given that a daily energy gap (difference between energy expenditure and intake) not higher than 50-100 kcal/d [START_REF] Schutz | Energy gap in the aetiology of body weight gain and obesity: a challenging concept with a complex evaluation and pitfalls[END_REF] is thought to be sufficient to induce unhealthy weight gain and excessive fat mass at the population level. Normalization of metabolic rates is another debated topic. For example, Gallagher et al. [START_REF] Gallagher | Small organs with a high metabolic rate explain lower resting energy expenditure in African American than in white adults[END_REF] reported that lower RMR among African-American adults was likely due to lower volume of high metabolically active organs contributing to RMR, which was not accounted for by statistical adjustments. Long-term studies in pre-industrial populations using state-of-the-art techniques are needed to assess TEE and its components with precision achieved in clinical trials. Metabolic rates need to be properly adjusted by including body size, as measured via body surface and volume, and a dissociation between low and high metabolically active organs contributing to RMR.

Physical activity is not a homogenous component. Most past studies assumed that PAEE is a homogeneous compartment of TEE. However, PAEE represents energy expended during the complete 24h-nycthemeral cycle, from sleep and sedentary activities to vigorous exercise. Activities differing in intensity and in bouts duration might not have the same effect on energy balance [START_REF] Bergouignan | Physical Inactivity Differentially Alters Dietary Oleate and Palmitate Trafficking[END_REF]. We and others have also questioned the impact of daily light-intensity activities [START_REF] Lefai | Exercise training improves fat metabolism independent of total energy expenditure in sedentary overweight men, but does not restore lean metabolic phenotype[END_REF] and sedentary behavior patterns on weight regulation, independent of MVPA [START_REF] Jong | Breaking up Sedentary Time in Overweight/Obese Adults on Work Days and Non-Work Days: Results from a Feasibility Study[END_REF]. We further showed that a comprehensive view of the activity/sedentary components is needed to prevent excess weight gain during adolescence, another transition situation [START_REF] Simon | Overweight Prevention in Adolescents by Increasing Physical Activity: 6-Year Results of the Randomized Icaps Study[END_REF]. Moreover, although the more recent studies used 3D-accelerometry [START_REF] Raichlen | Physical activity patterns and biomarkers of cardiovascular disease risk in hunter-gatherers[END_REF][START_REF] Tucker | Physical activity in U.S.: adults compliance with the Physical Activity Guidelines for Americans[END_REF], the commonly used algorithms overestimate MVPA and do not give precise information on sedentary behaviors, light-intensity activities and posture. Understanding activity behavior in relation with the environmental context also needs information on the location and context where the activity is performed. The phenology of the complete 24h activity cycle needs to be precisely assessed to understand the effects of the physical activity transition on weight regulation and health [START_REF] Rosenberger | The 24-Hour Activity Cycle: A New Paradigm for Physical Activity[END_REF].

Diet patterns and quality, an unavoidable determinant of energy balance and metabolic health.

Improved dietary profile has been associated with reduced BW and healthier metabolic profile [START_REF] Liese | The Dietary Patterns Methods Project: synthesis of findings across cohorts and relevance to dietary guidance[END_REF].

Moreover, beyond its energy and macronutrient composition, a better nutritional quality of the diet has been associated with a lower mortality risk [START_REF] Nazare | Changes in both global diet quality and physical activity level synergistically reduce visceral adiposity in men with features of metabolic syndrome[END_REF][START_REF] Harmon | Associations of key diet-quality indexes with mortality in the Multiethnic Cohort: the Dietary Patterns Methods Project[END_REF]. Because food is rarely consumed in isolation, food profile analysis allows to analyze how different food components aggregate together and integrate the synergistic effects of diet as a whole, compared to food items taken in isolation. For this purpose integrative methodologies that consider the whole diet by deriving dietary patterns, datadriven methods such as principal component analysis (PCA) or clustering [START_REF] Newby | Empirically derived eating patterns using factor or cluster analysis: a review[END_REF] are needed.

The socio-anthropological dimension, a parameter influencing health. During epidemiological transitions in rural populations, the modernization/urbanization processes impact living conditions and lifestyles that include eating patterns, transhumance patterns and professional/social activities, needing a sociocultural approach to better understand the impact of social background and cultural identities.

This complementary approach will allow to detect changes at smaller scales and better understand the socio-cultural dynamics involved in behavioral changes [START_REF] Fourat | Forms of Food Transition: Sociocultural Factors Limiting the Diets'Animalisationin France and India[END_REF]. For instance, the rapid evolution from a traditional rural pastoralist to an urban lifestyle, as observed in other rural populations exposed to urbanization process [START_REF] Cohen | Biocultural determinants of overweight and obesity in the context of nutrition transition in Senegal: a holistic anthropological approach[END_REF] can disrupt the social structure affecting lifestyle and quality of life, and involving higher social stress level. Precisely, the urban transition affects local dietary and physical activity habits and their associated representations as well as body weight norms [START_REF]Demographic Transitions and Modernity[END_REF][START_REF] Jodelet | Les représentations sociales [Social Representations[END_REF]. Social representations of a population correspond to a form of knowledge, socially elaborated and shared, contributing to the construction of a common reality [START_REF] Jodelet | Les représentations sociales [Social Representations[END_REF]. They consist as much of knowledge and opinions as of beliefs intended to legitimize behaviors and practices. Rapid changes in the social representations and practices of diet and physical activity are inseparables with structural changes of the society. Clearly, feeding and physical activity must be considered in their complex interactions with the environment, biology, and local socio-cultural norms as developed by Poulain et al. [START_REF] Poulain | Malaysian Food Barometer (MFB): a study of the impact of compressed modernisation on food habits[END_REF] and Drenowski & Poulain [START_REF] Drewnowski | What Lies Behind the Transition From Plant-Based to Animal Protein?[END_REF].

Both poor social relationships [START_REF] Holt-Lunstad | Social relationships and mortality risk: a metaanalytic review[END_REF] and stress [START_REF] Darviri | Determinants of self-rated health in a representative sample of a rural population: a cross-sectional study in Greece[END_REF] caused by a degraded sociocultural environment have been associated with adverse health outcomes. Stress has also been linked to unhealthy behaviors such as poor diet [START_REF] Darviri | Determinants of self-rated health in a representative sample of a rural population: a cross-sectional study in Greece[END_REF]. Given the rapid urbanization process in different rural populations, future research will therefore need to employ a bio-socio-anthropological approach to assess changes in the social representations of body weight and lifestyles related to body weight within the context of rapid sociocultural changes potentially associated with social stress levels and quality of life, and their interactions with energy balance-related determinants will need to be assessed.

Answers reside in populations who have not completed their epidemiological transition

Systemic and holistic studies of pre-industrial populations are needed. In the above reported metaanalyses, data from pre-industrial populations and populations from low to middle income countries were underrepresented. This bias masked a large inter-populations variability in the socio-ecological environments they are living in. Although food availability, social structure, and lifestyles influence diet and physical activity patterns and health, they have been overlooked in previous comparative studies [57] . Yet they differ between pastoralists, agriculturalists, horticulturalists, hunter-gatherers, etc.

For example, the "traditional" societies in South Africa (38±34 min/d) and Seychelles (38±34 min/d) had much lower levels of MVPA than the Hadza (135±91 min/d), but still greater than their counterparts from the USA (Urban Chicago: 26±32 min/d) [58] . Similarly, non-technology dependent agricultural societies have daily activity levels 3-fold higher than levels shown in hunter-gatherers [59] .

On the other side, we showed that consumption of processed energy-dense food was higher in urban Cameroonians compared to rural ones and that valorization of overweight was associated with obesity status [START_REF] Cohen | Nutrition Transition and Biocultural Determinants of Obesity among Cameroonian Migrants in Urban Cameroon and France[END_REF]. To fully assess the determinants of body weight regulation and metabolic health in relation to the epidemiological transition and the specific impact of physical activity, future research will need to simultaneously consider the anthropo-socio-ecological environment, diet and other lifestyles (e.g. sleep), as well as their interactions.

The need for longitudinal and comparative holistic and systemic studies in traditional populations.

For all the reasons mentioned above, we are convinced that only longitudinal studies of populations from pre-industrial countries spanning different phases of the epidemiological transition, including the few remaining traditional populations of agriculturalists, pastoralists and horticulturalists

around the world, will improve our understanding of the causes of the obesity epidemic. This is challenging because the populations must be selected based on specific anthropo-socio-ecological parameters that will allow for defining a near-zero time of changes that closely approximates human lifestyles prior to industrialization, as it is impossible to find nowadays a "true zero time".

Conclusions

The respective contribution of the different factors associated with the Western lifestyle and known to pose a high risk of obesity remains highly debated. However, diet, physical activity and lifestyle of pre-industrial populations provide the best model to address this challenge. Future research will need to compare these factors in industrial and pre-industrial populations, but also to track the impact of the ongoing transitions on these factors and the associated changes in body weight and metabolic health.

Exciting findings are likely to come from this emerging field of research at the crossroad between metabolism, ecology and anthropology. O dose. Total body water is enriched with stable isotope and urine samples are frequently collected to measure the enrichment of 2 H and 18 O. While 2 H is excreted from the body as 2 H 2 O, 18 O exists the body as both H 2 18 O and C 18 O 2 . The difference between 2 H and 18 O elimination rate (in blood, saliva or urine samples) corresponds to the production of CO 2 production. By using the classical equations of indirect calorimetry, we can estimate the average daily TEE over the period of measurements (usually between 7 and 14 days).

-TEF and RMR are measured by indirect calorimetry (measured of CO 2 excretion and O 2 consumption). RMR is measured in fasting state, at thermoneutrality and while the person is awake. TEF is measured for 4-5 hours following a standard meal and corresponds to the increment in energy expenditure above the RMR. TEF can also be estimated as 10% of TEE.

-PAEE is determined by calculation with the formula or . Moreover, the pattern of physical activity (i.e. time spent in sedentary activities or light, moderate or vigorous activities) can accurately be recorded with 3D-accelerometers measuring both acceleration and postures. PAEE can further be derived by using validated algorithms.

Insert 1:

 Daily Total energy expenditure (TEE) Is defined as the sum of physical activity-related energy expenditure (PAEE), thermic effect of food (TEF -i.e. energy needed to assimilate and store meal-derived energy) and resting metabolic rate (RMR -i.e. the minimal energy required to ensure vital function maintenance).  Physical activity-related energy expenditure (PAEE) corresponds to energy required to perform exercise (i.e. structured physical activity) and other daily life physical activities.  Thermic effect of food (TEF) is the amount of energy required to assimilate and store nutrients after a meal.  Resting metabolic rate (RMR) is the minimal energy needed to maintain vital functions at rest.

Figure 1 .

 1 Figure 1. Working model. Simplified illustration of the impact of epidemiological transition induced by environmental changes on cultural/lifestyles, nutrition and physical activity, and on energy intake, physical activity energy expenditure, and body weight and fat mass.

Figure 2 .

 2 Figure 2. Total energy expenditure and its components: definition and methods -TEE is calculated with the doubly labelled water method (DLW). The subject ingests a 2 H 2 18

Acknowledgements

We thank the CNRS Labex DRHIIM, l'Observatoire Homme-Milieu Téssékéré, CNRS MITI, FRM/Danone Institute and Société Française de Nutrition (SFN) to provide us with the opportunity to conduct an interdisciplinary research that will help addressing some of the questions raised in this manuscript. We are also very grateful to our local collaborators at the University of Dakar (UCAD) in Senegal.

Declaration of interest

The authors declared having no conflict of interests or competing interests.