Bruno Malet-Damour
email: bruno.malet-damour@univ-reunion.fr

Damien Ali

Hamada Fakra
email: fakra@univ-reunion.fr

Thermal and spectral impact of building integrated Mirrored Light Pipe to human circadian rhythms and thermal environment

Keywords: Building applications, building design, daylight performance, daylight, circadian rhythm, thermal conditions, spectral characterization

Light holds an important place in the way of life of humans. The first type of lighting which could be exploited was daylight. Tubular Daylight Guidance Systems (TDGS) transport and distribute this daylight inside a building. This article focuses on one of the best known: the Mirrored Light Pipe. MLPs are multiple specular reflectance transporters. This paper presents (i) state of the art on thermal and spectral studies on light pipes from 1998 to 2021 and (ii) proposes an experimental analysis on MLP in the climatic environment with a high solar resource for different spectral bands of the daylight spectrum. The results show that the thermal impact of the light pipe is limited. Moreover, it is demonstrated that this device promotes natural circadian rhythms. A comparison with artificial lighting highlights this device as an ideal solution for bioclimatic building design.

Nomenclature Introduction

Today, humans spend nearly 90% of their time in indoor environments [START_REF] Frontczak | Literature survey on how different factors influence human comfort in indoor environments[END_REF]. This environment, particularly in the thermal and luminous domains, is far from how the human species developed and set up its mechanisms of adaptation and physiological regulation. To create interior spaces adapted to human health, daylighting technologies must be exemplary: limited heat input and conservation of the properties of transmitted light. Although less and less energy-consuming, artificial lighting is often at the origin of chronobiological regulations modulated by a hormone: melatonin. The color temperature of light sources, whether natural or artificial, affects user performance. The higher the color temperature (towards white), the higher the performance [START_REF] Veitch | Office Light Source Spectrum: Effects of Individual Control on Perception, Cognition, and Comfort[END_REF]. This is related to the increase in vigilance caused by the inhibition of the nocturnal secretion of melatonin. Recent studies show that they are linked to the direct activation of brain structures involved in cognition, wakefulness and sleep regulation, and circadian rhythms [START_REF] Prayag | Light Modulation of Human Clocks, Wake, and Sleep[END_REF]. Concretely, insufficient or inappropriate exposure to light can disrupt traditional human rhythms, with adverse consequences on cognitive performance, comfort, and health [START_REF] Hatori | Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies[END_REF][START_REF] Hicks | How good is the evidence that light at night can affect human health? Graefe's[END_REF][START_REF]Agence nationale de sećurité sanitaire de l'alimentation (Anses)[END_REF].

The many existing international Tubular Daylight Guidance Systems [START_REF] Malet-Damour | Technological Review of Tubular Daylight Guide System from 1982 to 2020[END_REF] are among those devices that meet both energy and health criteria. They allow the transport and distribution of daylight into dark rooms, away from traditional openings. Previous work [START_REF] Malet-Damour | Photometrical analysis of mirrored light pipe: From stateof-the-art on experimental results (1990-2019) to the proposition of new experimental observations in high solar potential climates[END_REF][START_REF] Malet-Damour | Study of tubular daylight guide systems in buildings: Experimentation, modelling and validation[END_REF] LW Long-wavelength [-] in extreme sunlight situations (maximum global illuminance of about 200 Klux). Moreover, they seem to offer the advantage of minimizing light loss and heat gain while guaranteeing that the light retains all its healthrelated properties. However, although mirrored light pipe seems to transmit less solar infrared (heat) than visible light, few experimental studies have confirmed this.

This paper presents observations from experiments conducted over 2018-2019 on Mirrored Light-Pipe (MLP) in Reunion Island, France. The climate in which the investigation was undertaken is presented with a focus on solar irradiance. In 2021, this study is the only one to analyze the thermal impact of this type of device in a tropical climate with extreme sunlight conditions. The experimental results, differentiated by the sky typology, allow to qualitatively analyze the impact of the light pipe on (i) the infrared transmission, (ii) the energy flux effectively transmitted, and (iii) measure the profile of the electromagnetic spectrum at the device exit. This last study allowed (i) to evaluate the shape of the solar spectrum and its impact on human perception and (ii) to compare the wavelength of the light pipe to that of the main artificial lighting.

A short review on thermal and spectral light pipe investigations 1.Light spectrum transmitted by the light pipe

The luminous intensity of a source and the color of the transmitted light has an important impact on the perception of ambiance. It is generally assumed that spectral variations generated by reflective light pipes do not affect the color of the transmitted daylight. In 2014, Nilsson et al. [START_REF] Nilsson | Spectrophotometric measurements and ray tracing simulations of mirror light pipes to evaluate the color of the transmitted light[END_REF] confirmed this assumption. To do so, they use a numerical approach and spectrophotometric measurements (controlled environment) to qualify the illuminances (direct/diffuse) entering inside the pipe, neglecting the presence of the collection and diffusion device. The MLP appears to have achromatic properties. The author also states that the spectroscopic behavior may depend on the aspect ratio of the light pipe. It is also noted that the angle of incidence of the incoming beam affects the spectral transmittance of the tube. Low angles of incidence (less than 50°) allow rays of length between 400 and 800 nm (visible) to penetrate. The angles of incidence up to 50 ° extend the spectrum of rays: from 400 to 1000 nm (visible + near IR).

In 2000, Shao and Riffat conducted an experimental study in England (real environment, northern hemisphere, max illumination: 20 Klux) on a system combining light pipe and passive thermal ventilation [START_REF] Shao | Daylighting using light pipes and its integration with solar heating and natural ventilation[END_REF].

Part of their study showed that this system transmitted (slightly) less IR than visible light. Under cloudy skies, IR radiation is about three times less and more uniform than under clear skies.

In 2003, Callow [START_REF] Callow | Air-clad optical rod daylighting system[END_REF] studied mirrored light pipes and another type of light pipe (light rods) for two different real-world climatic environments: England (temperate oceanic climate, max illuminance: 80 Klux)

and Singapore (equatorial climate, max illuminance: 120 Klux). He showed that the light rods (based on fiber optics) could provide more significant amounts of light without compromising the thermal performance of the building. The transmission properties of the PMMA material that constitutes the light rods (but also the collector of the MLP) favor the transmission of visible and near-infrared wavelengths but effectively limit the mid-infrared and beyond, which was confirmed by the study of Nilson [START_REF] Nilsson | Spectrophotometric measurements and ray tracing simulations of mirror light pipes to evaluate the color of the transmitted light[END_REF].

In 2019, Omishore [START_REF] Omishore | Thermal testing of the light pipe prototype[END_REF] experimented with a light pipe prototype associated with an active collection device (parabolic collector with concentrating mirror). The device is subjected to an artificial IR light source (controlled environment). The author then records the temperature profiles and takes thermographic pictures.

The temperature measurement data show a significant increase in the temperature at the surface of the light pipe in response to the IR radiation. The author indicates that the light pipe could be supplemented with an additional vented pipe to prevent any overheating problems, which has been confirmed by other authors [START_REF] Varga | Ventilation terminals for use with light pipes in buildings: A CFD study[END_REF][START_REF] Oakley | TripleSave -The Investigation and Monitoring of a Combined Natural Daylighting and Stack Ventilation System[END_REF][START_REF] Šikula | Thermal CFD analysis of tubular light guides[END_REF][START_REF] Šikula | Thermal analysis of light pipes for insulated flat roofs[END_REF][START_REF] Ait-Taleb | Numerical simulation of coupled heat transfers by conduction, natural convection and radiation in hollow structures heated from below or above[END_REF].

Impact on indoor thermal conditions and study of solar heat gain

The thermal behavior of a light pipe in a real-world environment has been little studied, and even less in tropical climatic conditions. The literature referenced only one author, Williams [START_REF] Williams | Nvestigating the Thermal and the Lighting Performance of Light Pipes for Sunny and Cloudy Conditions in Insular Tropical Climate[END_REF] in 2014. Through an experimental study in a real environment, the author investigated the photometric and thermal behaviors of the device for two types of the day (clear and overcast) for a seminar room located in Kingston, Jamaica (northern hemisphere). The results show a significant temperature gradient during the day, increasing the energy consumption of an active cooling system to maintain a comfortable temperature of 25°C. Indeed, on a clear day, the diffuser was 6.68°C warmer than the ambient temperature, and the collector was nearly 4.45°C

warmer than the diffuser. At night, the author shows that the light pipe behaves as a thermal bridge because the temperature of the dome is always lower than the temperature of the diffuser and the room.

In another climatic context (measurement in a real Canadian environment), Harrison [START_REF] Harrison | Preliminary evaluation of the daylighting and thermal performance of cylindrical skylights[END_REF], in 1998, conducted a thermal study of a commercial light pipe measuring 0.33 m in diameter by 1.83 m in length. A winter period was chosen. His experiment reported an average effective thermal resistance of 0.279 m 2 .K.W -1 .

However, this value reflects a measurement of the outside air temperature to the inside air temperature.

Other authors [START_REF] Callow | Air-clad optical rod daylighting system[END_REF][START_REF] Callow | Daylighting Using Tubular Light Guide Systems[END_REF][START_REF] Laouadi | Transparent domed skylights: Optical model for predicting transmittance, absorptance and reflectance[END_REF][START_REF] Mccluney | Rating of Tubular Daylighting Devices for Visible Transmittance and Solar Heat Gain -Final Report[END_REF][START_REF] Bencs | Simultaneous measurement of velocity and temperature field in the downstream region of a heated cylinder[END_REF][START_REF] Perčić | A three-dimensional numerical analysis of complete crossflow heat exchangers with conjugate heat transfer[END_REF][START_REF] Hien | Daylighting Through Light Pipe for Deep Interior Space of Buildings With Consideration Heat Gain[END_REF][START_REF] Wu | Experimental investigation on light-thermal effects of solar light pipes used in USTB gymnasium under sunny conditions in Beijing[END_REF] have also attempted to understand the thermal impact of the type of light pipe and its geometric characteristics, the modes of heat transfer, and the influence of climate. Overall, although the device has a limited impact on thermal conditions, the ratio of light input to heat input is still attractive. The heat input generated by the light pipes has a lower impact than the artificial lighting.

Stack ventilation with light pipe and CFD Study

Several authors have been interested in studying the light pipe associated with a thermal draft [START_REF] Varga | Ventilation terminals for use with light pipes in buildings: A CFD study[END_REF][START_REF] Oakley | TripleSave -The Investigation and Monitoring of a Combined Natural Daylighting and Stack Ventilation System[END_REF][START_REF] Šikula | Thermal CFD analysis of tubular light guides[END_REF][START_REF] Šikula | Thermal analysis of light pipes for insulated flat roofs[END_REF][START_REF] Ait-Taleb | Numerical simulation of coupled heat transfers by conduction, natural convection and radiation in hollow structures heated from below or above[END_REF].

In single or double tubes, the light pipe is tested to quantify the impact it could have on natural ventilation. In double tubes, the temperature rise of the inner tube walls is limited by the convective phenomena occurring in the outer tube. The authors have demonstrated a definite potential to couple these two phenomena through numerical simulations in fluid dynamics or experimental measurements (temperature profiles).

The originality of the paper

The studies reviewed in the literature from 1998 to 2021 report thermal and spectrometric findings summarized and classified by theme in Table 1. These different observations were conducted in a real environment (temperate, equatorial, or tropical climates) or controlled (laboratory measurements) and exclusively in the northern hemisphere. For tropical countries, the use of daylighting technologies is a significant concern because of the heat associated with sunlight, which can increase the cost of cooling, thus reducing the energy savings resulting from the reduction of the lighting load. This paper presents the only experimental data in the southern hemisphere (different solar path) and for extreme sunlight conditions.

Under these climatic conditions, we come to refute, confirm or bring a new perspective on the impact of the Mirrored Light Pipe on thermal and spectral conditions, its impact on natural human biological rhythms.

After an annual analysis of the solar irradiation of the study site (St-Pierre, LA REUNION, southern hemisphere) to identify the extreme climatic conditions, we oriented our approach by proposing measurements on several spectral bands of daylight (see Figure 1). We sought to confirm that, despite the intense solar radiation, the light pipe brings little IR GLO (pyrgeometer measurements), that the thermal effect of the transmitted energy remains low compared to the visible part (pyranometer measurements), and that the wavelength spectrum of the penetrating radiation (i) is close to the spectrum of the sunlight, to the benefit of the suppression of UV (responsible for the aging of the materials) (ii) and of better quality than any artificial lighting. We associate our spectroscopic analysis to the quality of the transmitted light about its adequacy with the natural biological (circadian) human rhythms. All these results represent a further advance in understanding the behavior of tubular mirrored daylight guidance systems and contribute to the continuous improvement of this technology.

Table 1: synthesis of numerical and experimental studies from 1998 to 2020

Reference Findings

Light spectrum transmitted by the light pipe MLP Nilsson et al. [START_REF] Nilsson | Spectrophotometric measurements and ray tracing simulations of mirror light pipes to evaluate the color of the transmitted light[END_REF] Achromatic properties Spectroscopic behavior depends on the aspect ratio

The angle of incidence affects the spectral transmittance: (i) less than 50°: wavelengths between 400 and 800 nm (visible); more significant than 50°: from 400 to 1000 nm (visible + near IR) Shao and Riffat [START_REF] Shao | Daylighting using light pipes and its integration with solar heating and natural ventilation[END_REF] Under cloudy skies, the number of IR rays is about three times lower and more uniform than under clear skies Callow [START_REF] Callow | Air-clad optical rod daylighting system[END_REF] Nilson et al. [START_REF] Nilsson | Spectrophotometric measurements and ray tracing simulations of mirror light pipes to evaluate the color of the transmitted light[END_REF] PMMA (collector material) favors the transmission of visible and near IR wavelengths while effectively limiting the medium and far IR Omishore [START_REF] Omishore | Thermal testing of the light pipe prototype[END_REF] Significant increase in the surface temperature of the light pipe in response to IR radiation

Impact on indoor thermal conditions and study of solar heat gain

Williams [START_REF] Williams | Nvestigating the Thermal and the Lighting Performance of Light Pipes for Sunny and Cloudy Conditions in Insular Tropical Climate[END_REF] An only experimental study in tropical climatic conditions (northern hemisphere)

Large temperature gradient during the day Thermal bridge at night

Harrison [START_REF] Harrison | Preliminary evaluation of the daylighting and thermal performance of cylindrical skylights[END_REF] The average effective thermal resistance of 0.279 m 2 .K.W -1 for a light pipe with Ap = 5.55

Callow [START_REF] Callow | Air-clad optical rod daylighting system[END_REF], Callow [START_REF] Callow | Daylighting Using Tubular Light Guide Systems[END_REF], Laouadi [START_REF] Laouadi | Transparent domed skylights: Optical model for predicting transmittance, absorptance and reflectance[END_REF], McCluney [START_REF] Mccluney | Rating of Tubular Daylighting Devices for Visible Transmittance and Solar Heat Gain -Final Report[END_REF], Bencs [START_REF] Bencs | Simultaneous measurement of velocity and temperature field in the downstream region of a heated cylinder[END_REF], Perčić [START_REF] Perčić | A three-dimensional numerical analysis of complete crossflow heat exchangers with conjugate heat transfer[END_REF], Hien [START_REF] Hien | Daylighting Through Light Pipe for Deep Interior Space of Buildings With Consideration Heat Gain[END_REF], Wu [START_REF] Wu | Experimental investigation on light-thermal effects of solar light pipes used in USTB gymnasium under sunny conditions in Beijing[END_REF] The heat contribution of a light pipe is lower than artificial lighting

Stack ventilation with light pipe and CFD study

Varga [START_REF] Varga | Ventilation terminals for use with light pipes in buildings: A CFD study[END_REF], Oakley [START_REF] Oakley | TripleSave -The Investigation and Monitoring of a Combined Natural Daylighting and Stack Ventilation System[END_REF], Šikula [START_REF] Šikula | Thermal CFD analysis of tubular light guides[END_REF], Šikula [START_REF] Šikula | Thermal analysis of light pipes for insulated flat roofs[END_REF], Ait-taleb [START_REF] Ait-Taleb | Numerical simulation of coupled heat transfers by conduction, natural convection and radiation in hollow structures heated from below or above[END_REF] Interesting coupling of thermal draft + light contribution Double ducting limits the temperature rise of the reflective surface

Materials and methods

Reunion Island is very particular because of its high solar irradiation. As we have seen in a previous study [START_REF] Malet-Damour | Photometrical analysis of mirrored light pipe: From stateof-the-art on experimental results (1990-2019) to the proposition of new experimental observations in high solar potential climates[END_REF] and confirmed by the literature, it is necessary to study the environment of the light pipes to understand their behavior. For this reason, we have chosen to set up an experiment to verify and validate the literature observations. Three experimental objectives have been identified:

- Thanks to the results of the experiments set up, we will understand the impact of the light pipe on the interior ambient conditions and the comfort of the individual.

Experiment set-up

The study support is an experimental cell of 9m 2 located in Saint-Pierre (Reunion Island). This cell is oriented North/North-West (9° N) and positioned on a platform without any distant mask impacting the cell.

The dimensions of the test cell are described in

Measurement & data acquisition

To meet each of the established objectives, we wished to set up different studies.

Measurements of short wavelength from pyranometer sensor

Two CMP11 pyranometers are used to measure most solar radiation (285 to 2800 nm) and mainly short wavelengths. These measurements offer elements of response to objectives 1 and 2. To answer the objective N°1, it was necessary to obtain a complete year of measurement to put forward the seasonal profile of the place of study by using two pyranometers (global and diffuse radiation). Then, to answer partly to the objective N°2, wishing to identify the filtering effect of the light pipe, we used two pyranometers. One is placed directly under the diffuser with a black plastic cap, and the other is placed outside in the plane of the sensor (Figure 3). Since this instrument is intended for outdoor measurements, its sensitivity is relatively high for indoor measurements (7 W/m2). However, we wish to use the same instruments to have the same sensitivity wavelengths (285 to 2800 nm). In this case, failing to quantify rigorously, we adopt a qualitative approach "binary." If the transmitted energy is close to the sensitivity, the measurement will show that the light pipe transmits (1) or does not transmit (0) energy. If the measured data are low (close to the sensitivity), the experiment will demonstrate that the device transmits little energy in the spectral range of the pyranometer. The devices are connected to a CAMPBELL SCIE® brand data acquisition system and are synchronized with each other. They record in one-minute time steps, like the scanning.

The pyranometer specifications are given respectively in Table 2.

Measurements of long-wavelength from pyrgeometer sensor

To pursue objective N°2, two CGR3 pyranometers (Campbell Scientific®) were used to evaluate the global irradiation for the spectral band 4500 nm -42000 nm (thermal effect). The installation methodology is the same as with the pyranometers. The first is placed under the diffuser with a black plastic cap and polystyrene insulation to reduce the radiative effect of the cell walls, which are themselves insulated (Figure 4.a). The second is placed on the roof in the plane of the device collector (Figure 4.b).

As with the pyranometers, the pyrgeometers are devices for measuring in an outdoor environment, with a sensitivity of about 5 W/m 2 . To perform the most rigorous study possible on the effect of the light pipe, the outdoor and indoor sensors must have the same spectral bands (4500 nm -42000 nm). The sensitivity of this sensor may be close to the values recorded indoors; the measurement must be qualitative, like the study with pyranometers. In this case, failing to quantify rigorously, the qualitative binarized approach will show that the light pipe transmits (1) or does not transmit (0) energy on the spectral interval 4500 nm -42000 nm. If the measured indoor data are low, the study will show that the light pipe transmits little long wavelengths. As for the pyranometers, the pyrgeometers are connected to a CAMPBELL SCIE® data acquisition system and synchronized. The time step and the scanning are also of the order of one minute.

The pyrgeometer specifications are given respectively in Table 3.

Spectrophotometer utility in the experiment

This study aims to bring elements of the answer to the objective N°3, seeking to establish the electromagnetic spectrum profile transmitted by the light pipe. In addition, it is a question of understanding the role that the light pipe can play on the visual comfort of the occupant and its circadian efficiency. A spectrophotometer was used to measure the electromagnetic spectrum profile at the entrance and exit of the light pipe. The spectrometric measurements are performed by an Ocean Optics® spectrophotometer, model JAZ (Figure 5). This apparatus allows measuring the UV/Visible part of an electromagnetic source. The measurement was performed in 2 quasi-synchronous phases: first, the spectrophotometer cell was placed orthogonally under the diffuser. The cell device was placed outside in the plane of the collector. The recording was instantaneous. The specifications of the spectrophotometer are given in Table 4.

Summary of the experiments conducted

All experimental scenarios were synthesized in Table 5.

Results and discussion

Annually solar resource for brief climate study

We carry out a brief climatic analysis of the environment in which the light pipe is positioned. The objective is to understand the solar potential of the city of Saint-Pierre, Reunion Island (France). We seek to discern the annual evolution of the global outdoor irradiance measured with an external pyranometer (CMP11). From May to September, the southern winter is the period when the maximum irradiance is around 100 to 800 W/m 2 . The days are shorter. Mornings and ends of the days provide less than 200 W/m 2 . The trend changes as early as September when the days recover from hours of sunshine. Indeed, the length of the days also changes according to the season: in the first summer period, the light pipe can benefit from an irradiance of almost 11 hours. In the second period (southern winter), the length of the days is shortened to an average of 9 hours. In the third period (close to the summer solstice), the days are the longest, with more than 12 hours of sunshine.

Seasonality does not seem to be adequately related to the lighting needs. Indeed, a system using daylight within a building will offer significant efficiency from October to April and less for the rest of the year.

Characterization of day types

Many approaches have been made to characterize the type of sky [START_REF] Dumortier | analyse et modélisation du gisement lumineux : Application à l'évaluation des performances de l'éclairage naturel des bâtiments[END_REF], but few have been made to define typical days. We note in the literature the models of Kittler [START_REF] Kittler | Luminance models of homogeneous skies for design and energy performance predictions[END_REF], Perez [START_REF] Perez | Modeling sky luminance angular distribution for real sky conditions: Experimental evaluation of existing algorithms[END_REF], Perraudeau [START_REF] Perraudeau | Luminance models[END_REF], and a simplified model [START_REF] Pattanasethanon | All sky modeling daylight availability and illuminance/irradiance on horizontal plane for Mahasarakham, Thailand[END_REF]. The latter is based on the ratio of diffuse to global irradiance. Fakra [START_REF] Fakra | A simple evaluation of global and diffuse luminous efficacy for all sky conditions in tropical and humid climate[END_REF] evaluated the characterization given by these indices over three types of the day (clear, intermediate, and overcast skies). He concludes that the simplified model seems to be more suitable. Apart from sunrise and sunset for each day, the SR index characterizes the day type well overall. We will use this indicator for further experimentation.

The Sky Ratio can be evaluated from the following ratio :

𝑆𝑅 = 𝐼 𝑑,out,ℎ 𝐼 𝐺,out,ℎ (1)
The proposed characteristic values are listed in Table 6. This characterization will be used to analyze our results according to the different types of days.

Irradiance through the light pipe (285 to 2800 nm)

The objective of this measurement was to qualitatively assess the magnitude of energy transmitted through the light pipe inside the experimental cell for three typical days (clear, intermediate, and overcast).

Using the SR, we select three typical days: April 3, 6, and 7. The graphs (Figures 7 and8) that follow are drawn at the 10-minute time step to reduce the impact of climatic contingencies. April 4 is an overcast day (Figure 7). The global irradiance profile is almost similar to diffuse irradiance, which is typical of a cloudy sky. This classification is confirmed by the SR, which reaches an average value of 0.9. Events appear around 9 AM and 11 AM. They should probably be associated with a partially and momentarily clear sky.

April 6 is considered a clear day, with an average SR of about 0. We also plot the indoor and outdoor solar irradiance evolution on the same graph (Figure 8). The indoor irradiance level is low for each day and close to the sensor's sensitivity (±7 W/m 2 , for more information about this value, see Table 2). These values do not allow us to give a precise quantity of transmitted irradiance.

However, the data collected shows that low intensity is transmitted through the light pipe.

For the overcast day of April 4, the maximum indoor irradiance integrating the sensor's sensitivity is approximately 4.8±7 W/m 2 at 11 AM against 580 W/m 2 outside at the same time or more than 120 times less.

For April 6 (clear sky), at 12:50 PM, the nominal global outdoor irradiance is 920 W/m 2 against 7.5±7 W/m 2 indoors, or more than 123 times the transmitted energy. The trend is verified for April 7 (intermediate sky),

where the maximum indoor irradiance is close to 8±7 W/m 2 at 12:50 PM against nearly 918 W/m 2 outside.

The shape of the interior irradiance curve seems to be different from that of the exterior irradiance. The Outdoor irradiance (𝑊/𝑚 2 )

Overcast sky Clear sky Intermediate sky specular reflection inside the light pipe must significantly impact the amount of energy transmitted, just as it was noticed for the illuminance in later works [START_REF] Malet-Damour | Photometrical analysis of mirrored light pipe: From stateof-the-art on experimental results (1990-2019) to the proposition of new experimental observations in high solar potential climates[END_REF]. When we study the relationship between outdoor and indoor irradiance (Figure 9), we notice a strong positive linear correlation between the two parameters with an R 2 coefficient of 96.9% (see the red frame of Figure 9). The polynomial form of this correlation allows us to understand that the energy contribution by the light pipe is linear up to 780 W/m 2 but grows less quickly beyond. Moreover, the fact that the internal irradiance continues to expand beyond this level indicates the energy transmitted not by the light pipe but most probably by the diffuse thermal interreflection in the inner surface of the cell walls (see the green frame of Figure 9).

These findings allow us to conclude that the energy transmitted by a light pipe in the wavelength range between 285 and 2800 nm is very low, even negligible. This wavelength includes most of the electromagnetic spectrum of light: the visible range and much of the infrared (IR-near). These measurements reveal that the thermal effect, even if it is not finely expressed (related to the sensitivity of sensors), is minimal with the light pipe, compared to a window.

Estimation of the luminous efficacy of the light pipe

Considering the results obtained in previous work evaluating the nominal luminous fluxes transmitted by the same light pipe for the same climate [START_REF] Malet-Damour | Study of tubular daylight guide systems in buildings: Experimentation, modelling and validation[END_REF], we can deduce the luminous efficiency of the mirrored light pipe based on the measurements performed in the previous section.

For this, we use the most unfavorable values of the radiant flux (the largest, because of the high sensitivity of the sensor: +7W/m 2 ), we obtain : Irradiance from the LP Irradiance from cell walls and the LP L D is the downward radiation in LW received by the pyrgeometer from the measured object (i.e., in our case, two situations are presented: outdoor, for the sky and, indoor, for the light pipe diffuser). It is the fraction of infrared radiation (infrared irradiance) emitted by the target object. L net (net radiation) represents the radiative exchange between the sensor surface (𝜖. 𝜎𝑇 4 with 𝜖 = 1 if we considered black body case, given by the manufacturer) and the observed object L D (sky or light pipe).

𝜂 𝑐𝑙𝑒𝑎𝑟 𝑚𝑖𝑛 =
𝐿 𝑛𝑒𝑡 = 𝐼 𝑜𝑏𝑗𝑒𝑐𝑡 -𝐼 𝑠𝑒𝑛𝑠𝑜𝑟 (4)
𝐿 𝑛𝑒𝑡 = 𝐿 𝐷 -𝜎. 𝑇 4

(5)

The sensor interprets L net as the ratio between the voltage delivered by the pyrgeometer and its sensitivity.

L D being always positive (the flow being always descending), the sign of L net allows giving the direction of the radiative flow, and thus the direction of the exchange. Thus, in theory: when the diffuser is warmer than the collector (scenario of an overcast sky, or a beginning/end of the day), the radiative flux is rising, so the radiation L net is negative (the sensor measuring positive downward). In the clear sky (or at the peak of solar radiation), the diffuser is colder than the collector, the radiative flux is downward, so the radiation L net is positive. In this approach, L net is zero when there is no exchange between the collector and the diffuser. of the winter solstice in Reunion Island). This observation seems coherent: at the beginning and at the end of the day (when the Sun is not yet visible), there is no radiative exchange between the ground and the sky (i.e., 𝐿 𝐷 = 𝜎. 𝑇 4 ). When the Sun rises and sets, the radiative flux is rising (towards the sky) because the ground temperature increases. The net radiation L net,out is then negative and increases when the ground temperature rises more than that of the sky. Inside the cell and under the light pipe diffuser, the LW L D,in is very high, with a nominal value of more than 520 W/m 2 in the early afternoon (2:30 PM). This can be justified by the fact that the sensor measures radiation from the light pipe elements rather than the sky. As these elements rise in temperature, the infra-reds recorded are those coming from the diffuser and collector. We also note that at the beginning and end of the day, when the diffuser is warmer than the collector, L net,in is negative because the radiative exchange is from the cell to the outside and reverses during the day (Figure 10.c). The values vary between 3 W/m 2 , which is very low, marking the low infrared exchange between the diffuser and the collector. Finally, on June 26 (Figure 10.a), the profile of the global irradiance is almost similar to that of the diffuse irradiance, which is typical of the evolution of the irradiance during an overcast sky. This classification is confirmed by the SR moy, which reaches an average value of 0.9. L D,out is close to 475 W/m 2 , which is consistent with the type of sky. Instead of measuring the radiative exchanges with the sky (colder), the latter measures the radiation of water droplets at air temperature (Figure 10.b). The net radiation L net,out is thus close to zero due to the overcast condition (Figure 10.c). For the net fraction, the difference for long wavelengths is negative and increases during the day. Inside the cell and under the light pipe diffuser, the downward radiation in LW L D,in is low, with a nominal value of more than 474 W/m 2 in the early afternoon (1:20 PM). This can be explained by the fact that the angle of incidence is higher. Thus the radiation arrives directly on the sensor, with fewer inter-reflections within the tube. This finding had been noted in the literature for a controlled environment [START_REF] Nilsson | Spectrophotometric measurements and ray tracing simulations of mirror light pipes to evaluate the color of the transmitted light[END_REF].

We also note that when the diffuser is warmer than the collector at the beginning and end of the day, L net is negative because the radiative exchange is from the cell to the outside. However, the value reached by L net is wider under cloudy skies than under clear skies, which can be explained by the low-temperature rise of the light pipe components. Moreover, the measured radiation probably corresponds to radiative exchanges with the water droplets trapped in the tube. However, this observation must be modulated because the values reached for L net,out have a reduction factor of nearly ten compared to the outside, with a maximum value of L net,in an overcast sky of about 12 W/m 2 , which confirms the weak contribution of IR by the light pipe.

Moreover, L D,in presents higher values in the clear sky than in the overcast sky, which translates to an inverse behavior than the one observed in an outdoor environment when the presence of clouds strongly modulates the infrared radiation (emission in the infrared of water drops or ice crystals).

Spectral irradiance transmitted by a light pipe (200 to 1100 nm)

The objective is to establish the profile of the electromagnetic wave passing through the light pipe for the only two more extreme types of the day (clear sky and overcast sky). With this, we can answer the objective N°2 (to establish the profile of the wavelength transmitted). The measurements with the spectrophotometer were carried out on these two days at solar noon (i)

indoors by placing the sensor in the center of the light pipe diffuser and (ii) outdoors, in the plane of the collector, by pointing at the Sun.

The graphs below represent the solar irradiance spectrum at sea level (called AM1.5). We find the atmosphere's absorption bands in the ultraviolet (notably by ozone) and the infrared (notably by water vapor and carbon dioxide). In the clear sky (Figure 12) and outside, the profile of the daylight spectrum shows us a relatively continuous spectral irradiance in the visible range. At the exit of the light pipe, no UV is detected. This is probably due to the polymethylmethacrylate (PMMA) properties, which compose the dome of the light pipe collector. This thermoplastic has the advantage of maintaining its transparency, lightness, resistance to UV (responsible for the aging of materials), and corrosion. These properties make it ideal for a collector for a dome subjected to intense mechanical or solar constraints. This same material also seems to act on the near IR, as indicated by Nilson [START_REF] Nilsson | Spectrophotometric measurements and ray tracing simulations of mirror light pipes to evaluate the color of the transmitted light[END_REF] and Callow [START_REF] Callow | Air-clad optical rod daylighting system[END_REF] in their studies.

Regarding the heat input, represented on the graph by the amount of A-IR, we note that the light pipe transmits only a relatively small part of near-infrared (on average, three times less than the external resource) with a substantial decrease beyond 950 nm. This analysis allows us to confirm the results of the literature [START_REF] Shao | Daylighting using light pipes and its integration with solar heating and natural ventilation[END_REF] and the measurements made with the pyrgeometer: the light pipe is only responsible for a small part of the daily heat input. The internal spectral curve also shows that the device smoothes the spectrum of the external electromagnetic wave. The spectral irradiance at the outlet of the device is balanced on each of the visible wavelengths. Our experiment in real weather conditions (i.e., in situ) confirms the results of Nilsson et al. [START_REF] Nilsson | Spectrophotometric measurements and ray tracing simulations of mirror light pipes to evaluate the color of the transmitted light[END_REF] in a controlled environment that the MLP has achromatic properties. In overcast conditions (Figure 13), the spectral irradiance values drop by 2 to 4 times compared to the clear sky condition. Outdoors, for UV and IR-A, the electromagnetic waveform remains unchanged. The changes occur mainly for the spectral range 460-630 nm, where the sky absorbs a significant part of the visible spectrum. The profile of the spectrum transmitted by the light pipe remains unchanged compared to the clear sky condition, with a reduction of the amplitude of the spectral irradiance by a factor of 4. This value in a tropical climate is higher but close to the factor of 3 found in temperate climate by Shao and Riffat [START_REF] Shao | Daylighting using light pipes and its integration with solar heating and natural ventilation[END_REF] in their work in a real environment (northern hemisphere, max illumination: 20 Klux). Based on these findings, it is interesting to compare the spectrum of the electromagnetic wave transmitted by the light pipe with that of artificial lighting devices.

The efficiency of light pipe on human circadian rhythm compared by artificial lighting

The objective is to compare the spectrum of radiation from the light pipe for two types of the day (clear sky and overcast sky) and from two artificial lighting devices the most used and which we can easily find on the market: LED (T8) and fluorescent lamp (T8). Moreover, this artificial lighting is the most efficient, conforms to Table 7. With this comparative analysis, we can answer objective N°3, namely, to evaluate the concordance of the transmitted light with natural biological rhythms. The visual system is composed of different biological sensors, photopic (retinal cells: cones and rods), and melanopic (ipRGCs), which ensure, respectively, biological synchronism (chronobiology) and the perception of the environment. The excitation peak of ipRGCs is not the same as that of retinal cells for image formation. It is located between 446 and 480 nm [START_REF] Brainard | Action spectrum for melatonin regulation in humans: Evidence for a novel circadian photoreceptor[END_REF] [36] (towards the blue), whereas the nominal efficiency of photopic vision is found at yellow-green wavelengths, close to 555 nm [START_REF] Van Bommel | Visual, biological and emotional aspects of lightings: Recent new findings and their meaning for lighting practice[END_REF]. Thus, the quality of illumination, and more precisely, the wavelength spectrum transmitted, will influence circadian rhythms and cognitive performance. The higher the color temperature (towards white), the higher the performance [START_REF] Veitch | Office Light Source Spectrum: Effects of Individual Control on Perception, Cognition, and Comfort[END_REF].

Moreover, Cajochen [START_REF] Cajochen | High sensitivity of human melatonin, alertness, thermoregulation, and heart rate to short wavelength light[END_REF] showed that prolonged exposure in the late evening to monochromatic light of 460 nm (blue color) essentially reduced the secretion of melatonin compared to the exact duration of exposure to monochromatic light of 550 nm (green color). The literature [START_REF] Vandewalle | Light as a modulator of cognitive brain function[END_REF][START_REF] Boyce | The Benefits of Daylight through Windows Sponsored by : Capturing the Daylight Dividend Program The Benefits of Daylight through Windows[END_REF][START_REF] Phipps-Nelson | Daytime exposure to bright light, as compared to dim light, decreases sleepiness and improves psychomotor vigilance performance[END_REF][START_REF] Rüger | Article in AJP Regulatory Integrative and Comparative Physiology[END_REF][START_REF] Viola | Blue-enriched white light in the workplace improves self-reported alertness, performance and sleep quality[END_REF][START_REF] Vandewalle | Brain responses to violet, blue, and green monochromatic light exposures in humans: prominent role of blue light and the brainstem[END_REF][START_REF] Zeitzer | Sensitivity of the human circadian pacemaker to nocturnal light: Melatonin phase resetting and suppression[END_REF] T8 LED 5000 CCT T8 Fluorescent 4100 CCT Light-pipe under overcast sky (%)

Light-pipe under clear sky (%)

Intensity (normalized)

Visible biological day, whereas during the biological night, they perform poorly due to temporary attention losses.

Therefore, daylight, the most effective stimulus of the master clock, influences the cognitive abilities of human beings.

Our study compares daylight (from the light pipe) and artificial lighting (Figure 14). Whatever the type of sky, the spectrum of the wave coming from the light pipe dominates the blue-green colors while remaining relatively balanced. As we have seen previously, the light from the device is achromatic. The LED has two peaks of intensity: a maximum at 450 nm (blue color) and one at 580 nm (yellow color). This technology has a direct effect on the production of melatonin. Used during the day allows inhibiting the hormone and accentuates vigilance. However, the light issued is monochromatic with bluish dominance, creating discomfort on the color rendering index inside. The spectrum is not complete, so the health benefit is limited. The LED studied should be used in dark spaces, not used at night (scotopic vision). On the other hand, the compact fluorescent lamp emits an irregular and rich spectrum in the green and yellow colors (540 nm and 610 nm), with a value of its spectral intensity low in the blue wavelengths (440 nm). This type of lighting is thus to be proscribed for photopic lighting (daily).

Overall, the experimental results show that the light pipe emits a full spectrum of light in clear and overcast skies. This light favors chronobiological balance and promotes cognitive performance. The intermittence of its luminous flux also allows the occupant to be connected to the climatic variation outside, an important point in the study of visual comfort in the building.

Conclusion and perspective

A literature review allowed us to understand that the mirrored light pipe allowed the transmission of achromatic light. Some works have also noted that the transmitted wavelength depended on the angle of incidence of the rays and the aspect ratio. Another finding is that the proportion of IR radiation transmitted is influenced by the type of sky and the kind of material used for the collector. Other studies have revealed that the light pipe, during the night, could work on the thermal environment conditions by acting as a thermal bridge. During the day, the literature notes that this device has only a small heat input, lower than artificial lighting. All the studies reviewed were conducted in temperate, continental climatic conditions and only one in tropical conditions. They are all located in the northern hemisphere.

This paper presents the only experimental data in the southern hemisphere (different solar trajectories) and extreme sunlight conditions. We have aimed to address three distinct objectives: [START_REF] Frontczak | Literature survey on how different factors influence human comfort in indoor environments[END_REF] to analyze the annual solar irradiation profile of the experimental location, (2) to measure the spectral profile of the light wavelength effectively transmitted by the light pipe for different spectral bands, and (3) to evaluate the potential of the device on the respect of the human circadian cycle compared to conventional artificial lighting sources.

Several measurements were conducted on different spectral bands to meet these initial objectives, with quantitative and qualitative approaches. The studies are called "qualitative" because of the low values measured indoors, very close to the sensitivity of the sensors. The instruments are thus used to detect flux without targeting the exact value of the transmitted flux. This approach was applied to measurements in the spectral bands 285 to 2800 nm and 4500 nm to 42000 nm. We have still quantified, with sensitivity, the value of the energy transmitted by the light pipe. For example, in the clear sky, we noted a radiant flux (285 to 2800 nm) of nearly 7.5±3.5 W/m 2 inside, or less than 123 times the outdoor solar irradiance. This value allowed us to estimate the minimum luminous efficacy of the light pipe (taking into account the highest radiant flux) in a clear sky (591 lm.W -1 ) and overcast sky (85.5 lm.W -1 ), and then to compare them to the luminous efficacy of various sources. The latter shows a result nine times higher in the clear sky than a compact fluorescent lamp.

The results for the 4500 nm to 42000 nm range (LW, IR) also show that the thermal impact of the mirrored light pipe is limited. The "quantitative" studies concern the measurements associated with the spectrophotometer covering the electromagnetic wave spectrum from 200 to 1100 nm. At the exit of the light pipe, no UV is detected, and the spectral profile of the transmitted light shows us a relatively continuous spectral irradiance in the visible range. This confirms the literature findings: the light pipe, in the clear sky as overcast, diffuses an achromatic light, unlike the artificial lighting source studied.

We show that this system is well suited to promote visual comfort thanks to its non-visual effects (intermittence of the luminous flux, chronobiology, and cognitive performance). It can provide full-spectrum light in spaces that have few or no openings in the front. These rooms typically use artificial lighting that does not regulate circadian rhythms. Our experimental work has shown that the light pipe brings little heat and does not modify the visible light spectrum, thus providing beneficial light for humans.

Our experimental study was conducted on a single type of mirrored light pipe (curve, with an aspect ratio of 4.6). These results allow us to understand the thermal and spectral behavior of the light pipe. The evolution of geometric parameters (different aspect ratios, straight rather than a curved light pipe) or photometric parameters (different reflection coefficient) will modify the number of light rays transmitted. The level of interior illuminance will also change. However, this should not affect the spectroscopic profile of the light passing through it. As found in the literature, the dominant parameter to act on the spectrum profile is the angle of incidence of the incoming rays. Further studies may confirm these hypotheses.

The light pipe is the most successful daylighting device to meet the growing need for bioclimatic design in a device focused on human well-being. The study of multimodal comfort would allow orienting technical innovations so that the building can adapt to the human being, and not the opposite.

Acknowledgment

The authors thank Mr. J. Vigneron for his help in taking the measurements. Thanks are also due to Mr.

E. Jouen for his assistance in setting up the experiment and the loan of specific measuring equipment.

Figure 1 :

 1 Figure 1: Measurements made in the course of this work

Objective 1 :-Objective 2 :

 12 Analyze the annual solar irradiation profile of the experimental site to understand the profile of the available solar resource, Measure the profile of the wavelength of the light effectively transmitted by the light pipe by highlighting the filtering effect of the device for two types of day, -Objective 3: Evaluate the device's potential for the human circadian cycle compared to traditional artificial lighting sources.

Figure 2 .

 2 The vertical walls are made of composite panels (fibrocement/polyurethane/ fibrocement), and the horizontal ceiling is made of wood. Each interior face is covered with 5 cm of white polystyrene to reduce the radiative effect. The sloping roof (20°) is made of midnight blue corrugated sheet metal. A mirrored light pipe ( LP = 99.7%; Ap = 4.6) of the brand SOLATUBE® was installed in the roof plane. It is curved to ensure the horizontal of the diffuser.

Figure 2 :

 2 Figure 2: a) Cross-section view of the LGI cell b) Bottom view of the LGI c) Dimensions of the light pipe

  Polystyr ene (30) -Fi br ocement + Polyurethane (80)

Figure 3 :

 3 Figure 3: Outdoor pyranometer CMP11 for global irradiance in the plan of the light pipe

Figure 4 :

 4 Figure 4: a) outdoor pyrgeometer CGR3 for global irradiance in the plan of the light pipe b) indoor pyrgeometer CGR3 with black protection and insulation provided by polystyrene c) sensor positioned on the light pipe diffuser

Figure 5 :

 5 Figure 5: a) Ocean Optics® spectrophotometer, model JAZ for spectrometric measurements in the plan of the light pipe b) Photosensor cell of the spectrophotometer

Figure 6 :Figure 6

 66 Figure 6: Hourly distribution of𝐼 𝐺,𝑜𝑢𝑡,ℎ for Saint-Pierre (La Reunion, France) during the year 2019 Figure 6 highlights a significant seasonality in global irradiance corresponding substantially to the seasons present in Reunion Island. The southern summer extends from October to April. This is the period when the solar resource is most abundant. The days reach a maximum irradiance frequently between 700 W/m 2 and 900 W/m 2 , and, sometimes, up to 1000 W/m 2 (in a clear sky), and the beginning and end of the day close to 200 to 500 W/m 2 .

Figure 7 :Time

 7 Figure7: Global (𝐼 𝐺,𝑜𝑢𝑡,ℎ ) and diffuse (𝐼 𝑑,𝑜𝑢𝑡,ℎ ) outdoor irradiance for three types of days[START_REF] Hatori | Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies[END_REF][START_REF]Agence nationale de sećurité sanitaire de l'alimentation (Anses)[END_REF] and 7 April)

3 .

 3 The morning is clear of any clouds (the diffuse irradiance is very low), and scattered cloudy episodes may appear from 3 PM. Like those of April 6, clear sky days are relatively frequent at this time of the year in Reunion Island, where the thermal breeze regime pushes the clouds in the island's heights, which allows clearing the sky for the coastal areas where the tests take place. April 7 is an intermediate day, confirmed by the average SR reaching 0.4. The global irradiance can reach 900 W/m 2 with drops in irradiation caused by the presence of clouds.

Figure 8 :

 8 Figure 8: Indoor (𝐼 𝑑,𝑖𝑛,ℎ ) and outdoor (𝐼 𝐺,𝑜𝑢𝑡,ℎ ) irradiance for three types of days (4, 6, and 7 April), and the high sensibility of the indoor sensor

Time

  Indoor irradiance (under the diffuser)Hight sensibility for the indoor sensor Outdoor global irradiance (plan of the collector)Indoor irradiance (𝑊/𝑚 2 )

Figure 9 :

 9 Figure 9: Correlation between outdoor (𝐼 𝐺,𝑜𝑢𝑡,ℎ ) and indoor (𝐼 𝑑,𝑖𝑛,ℎ ) irradiance

Figure 10 :

 10 Figure 10: a) Global and diffuse outdoor irradiance for three types of days (21, 23, and 26 June) b) Outdoor and indoor downward longwave radiation, LD c) Outdoor and indoor net radiation Lnet June 21 (Figure 10.a) is equated with a clear sky day (SR moy < 0.3). The high values at the beginning and end of the day are related to this index [33]. The nominal global irradiance measured by an outdoor pyranometer reaches 715 W/m 2 at 12:30 PM. We can see that the downward L D,out radiation at 5:30 AM reaches nearly 450 W/m 2 and then decreases to 407 W/m 2 at 8 AM (Figure 10.b). A parabolic shape is then observed between 8 AM and 5 PM, with a maximum value of 425 W/m 2 reached noon (solar noon, because

  (plan of the collector) Ld,indoor (under the diffuser) Sensitivity range of the indoor sensor Outdoor Ld (𝑊/𝑚 2 ) (plan of the collector) Lnet,indoor (under the diffuser) Sensitivity range of the indoor sensor Outdoor Lnet (𝑊/𝑚 2 )

June 23 is

 23 an intermediate day (Figure 10.a), confirmed by the SR moy reaching 0.4. The global irradiance can reach 900 W/m 2 with drops in radiation up to 170 W/m 2 at 12:30 PM caused by the presence of clouds. L D,out increases in the presence of cloud masses (Figure 10.b).

Figure 11 :

 11 Figure 11: Global and diffuse outdoor irradiance for two types of days (2 and 4 October)To verify the sky type, we use daily global and diffuse outdoor irradiance (measurements with a CMP11 pyranometer), shown in Figure11. The first day was October 2, 2019. The sky was overcast (SR > 0.8). At solar noon, irradiance peaked at 300 W/m 2 . The second (October 4) was a clear sky with an SR below 0.3. Its irradiance was around 1000 W/m 2 .

Figure 12 :

 12 Figure 12: Amount of energy transmitted by wavelength for a day of clear sky (October 4)

Figure 13 :

 13 Figure 13: Amount of energy transmitted by wavelength for an overcast day (October 2, 2019)

Figure 14

 14 Figure 14 is based on the two days explained in the previous section: October 2, 2019 (overcast) and October 4, 2019 (clear). To provide a viable comparison, we normalize the spectral irradiance and smooth the wavelength spectra at scanning of 10 nm.

Figure 14 :

 14 Figure 14: Normalized spectral intensity of the wave: at the exit of the light pipe for two types of the sky; under a LED luminaire (T8 LED 5000 CCT); under a Fluocompact luminaire (T8 Fluorescent 4100 CCT)

  has shown the photometric performance of the light pipe TDGS

		Transmission coefficient of the light pipe	[-]
	LP	Internal reflectivity coefficient	[-]
	Ap	Aspect ratio 𝐴𝑝 = 𝐿𝑒𝑛𝑔𝑡ℎ 𝐿𝑃 𝐷𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝐿𝑃 ⁄	[-]
	𝐼 𝐺,𝑜𝑢𝑡,ℎ	Outdoor global irradiance	[W/m 2 ]
	𝐼 𝑑,𝑜𝑢𝑡,ℎ	Outdoor diffuse irradiance	[W/m 2 ]
	𝐼 𝐺,𝑖𝑛,ℎ	Indoor diffuse irradiance	[W/m 2 ]
	𝐿 𝐷,𝑜𝑢𝑡	Outdoor downward longwave radiation	[W/m 2 ]
	𝐿 𝐷,𝑖𝑛	Indoor downward longwave radiation	[W/m 2 ]
	𝐿 𝑛𝑒𝑡,𝑜𝑢𝑡	Outdoor net radiation	[W/m 2 ]
	𝐿 𝑛𝑒𝑡,𝑖𝑛	Indoor net radiation	[W/m 2 ]
	SR	Sky Ratio	[-]
	𝜆	Wavelength	[nm]
	𝜙 𝑣	Luminous flux	[lm]
	𝜙 𝑒	Radiant flux	[W]
	𝜂	Luminous efficacy	[lm.W -1 ]
	LP	Light pipe	[-]

Table 2 :

 2 Pyranometer specifications

	Accuracy

Table 3 :

 3 Pyrgeometer specifications

	Accuracy

Table 4 :

 4 Spectrophotometer specifications

	Description	Jaz UV/Visible Spectrophotometer with Remote Probe
	Grating options	14 different gratings, UV through short-wave NIR
	Detector	2048-element linear silicon CCD array; L2 collection lens
	Spectral range 𝜆	200 to 1100 nm
	Signal-to-noise ratio	250:1
	Integration time	870 𝜇𝑆 to 65 seconds

Table 5

 5

		: Experimental scenarios
	N°	Period used	Measurement	Sensors	Objectif
					Quantitative approach :
	1	1 year (2019) 3 days (April 2018)	𝐼 𝐺,𝑜𝑢𝑡,ℎ 𝐼 𝑑,𝑜𝑢𝑡,ℎ 𝐼 𝐺,𝑖𝑛,ℎ	3 x CMP11 pyranometers	Annually solar resource for brief climate study Qualitative approach : Evaluate the ability of the light pipe to transmit radiation in the spectral band (285 to 2800 nm) for 3 types of the sky
					(clear, overcast, and intermediate)
	2	3 days (June 2019)	𝐿 𝑛𝑒𝑡,𝑜𝑢𝑡 𝐿 𝑛𝑒𝑡,𝑖𝑛𝑡 𝐿 𝐷,𝑜𝑢𝑡	2 x CGR3 pyrgeometers	Qualitative approach : Evaluate the ability of the light pipe to transmit IR (4500 nm to 42000 nm) for 3 types of the sky (clear, overcast, and
			𝐿 𝐷,𝑖𝑛𝑡		intermediate)
					Quantitative approach :
	3	2 days (October 2019)	Outdoor and indoor spectral irradiance (W/m 2 /nm)	1 x JazRad spectrophotometer	Quantified the outdoor light spectrum in the plan of the collector and the indoor light spectrum under the diffuser for two
					types of the sky (clear and overcast) for
					the spectral band (200 to 1100 nm)

Table 6 :

 6 Characteristic values of SR

	Type of sky	SR
	Clear	𝑆𝑅 ≤ 0.3
	Intermediate	0.3 < 𝑆𝑅 < 0.8
	Overcast	𝑆𝑅 ≥ 0.8

Compared with the types of artificial lighting and their characteristics presented in [START_REF] Malet-Damour | Study of tubular daylight guide systems in buildings: Experimentation, modelling and validation[END_REF], we obtain Table 7. We note that the luminous efficacy of the light pipe in a clear sky is much better than the other current artificial lighting devices and equivalent to the luminous efficacy of a traditional fluorescent tube in an overcast sky. In the clear sky, despite an estimated radiant flux at the highest (15 W), the light pipe offers a luminous efficiency of about 453 lm.W -1 , which is next to 7 times more than the compact fluorescent lamp. Compared with the outdoor luminous efficacy, according to [START_REF] Fakra | A simple evaluation of global and diffuse luminous efficacy for all sky conditions in tropical and humid climate[END_REF], we can see that the low share of thermal energy transmitted in favor of a highly luminous flux allows the light pipe to improve the luminous efficacy of the transmitted daylight.

Infrared through the light pipe (4500 nm to 42000 nm)

The measurement of this radiation is carried out with a Kipp & Zonen CG4 pyrgeometer sensitive to a spectral range between 4500 nm and 42 000 nm (99% of the energy of the total infrared spectrum).

The pyrgeometer allows measuring the long wavelengths (LW). Its silicon optical window is designed to allow infrared radiation to pass through. An internal thin-film coating prevents short-wave solar radiation from reaching the LW thermopile (cutting off the shortest wavelengths of the solar spectrum). The infrared radiation received at the Earth's surface is emitted by the entire atmospheric column (CO 2 , water vapor, clouds, and other particles).

The measurement method is possible (compare outside and inside) because the light pipe has highly reflective walls ( LP = 99.7%). Measurement at the diffuser level is thus sustainable because the IR-rays coming from the light pipe will be preponderant compared to the IR-rays coming from the walls of the experimental cell. The latter contribute little to the overall measurement of IR. Indeed, the IR-rays passing through the tube are mainly reflected and therefore reach the sensor cell. In addition, the absorbed and transmitted parts of the radiation are minimized primarily to the thinness of the tube.


Keywords: virus genome equivalent, vRNA, viral RNA, WHO, world health organization Hepatitis B virus, Polo-like-kinase 1, small-interfering RNA, lipid nanoparticle, antiviral, hosttargeting agent, hepatocellular carcinoma

A link between HBV and PLK1 was clearly evidenced in HBV-driven carcinogenesis, and we have also recently shown that PLK1 is a proviral factor in the early phases of HBV infection. Moreover, we have shown that BI-2536, a small molecule PLK1 inhibitor, was very efficient at inhibiting HBV DNA neosynthesis, notably by affecting nucleocapsid assembly as a result of the modulation of HBc phosphorylation. Yet, as small molecule kinase inhibitors often feature poor selectivity, a more specific and safer strategy to target PLK1 would be needed for a potential development against chronic HBV infections. Here, we analysed using both freshly isolated primary human hepatocytes and differentiated HepaRG, the anti-HBV properties of an LNP-encapsulated PLK1targeting siRNA. Standard assays were used to monitor the effect of LNP siPLK1, or controls (LNP siHBV and LNP siNon-Targeting), on HBV replication and cell viability. A dose as low as 100ng/mL of LNP-siPLK1 resulted in a >75% decrease in secreted HBV DNA (viral particles), which was comparable to that obtained with LNP siHBV or 10 µM of Tenofovir (TFV), without affecting cell viability. Interestingly, and in contrast to that obtained with TFV, a strong inhibition of viral RNA and HBe/HBsAg secretions was also observed under LNP siPLK1 treatment. This correlated with a significant intracellular decrease of vRNA accumulation, which was independent of any change in cccDNA levels, thus suggesting a transcriptional or post-transcriptional modulation. Such an effect was not obtained with a biochemical approach of PLK1 inhibition, suggesting an enzymaticindependent role of PLK1.

INTRODUCTION

Hepatitis B virus (HBV) infection represents the main cause of hepatocellular carcinoma (HCC), which is the 2 nd leading cause of cancer deaths worldwide (WHO data). Out of the hundred million people exposed to HBV, 257 million remain currently chronically infected (CHB) (WHO data; global hepatitis report, 2017). If left untreated, these patients have a 100 fold increased risk of developing HCC compared to the general population [START_REF] Chan | Infection and Cancer: The Case of Hepatitis B[END_REF]. When patients are successfully and continiously treated with safe nucleos(t)ide analogs (NUCs), their risk of developing HCC is 2 to 5 times higher compared to normal individual [START_REF] Yoo | Update Treatment for HBV Infection and Persistent Risk for Hepatocellular Carcinoma: Prospect for an HBV Cure[END_REF][START_REF] Durantel | New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus[END_REF]. This reminiscent risk of HCC under treatment is likely due to the fact that NUCs do not allow the elimination of the HBV replicative intermediate called cccDNA (i.e., covalently closed circular DNA), which is responsible for viral persistence and rebound when treatment is stopped [START_REF] Bloom | Gene Therapy for Chronic HBV-Can We Eliminate cccDNA?[END_REF][START_REF] Lucifora | Attacking hepatitis B virus cccDNA--The holy grail to hepatitis B cure[END_REF]. In addition to the development of drugs that may target directly cccDNA [START_REF] Bloom | Gene Therapy for Chronic HBV-Can We Eliminate cccDNA?[END_REF][START_REF] Lucifora | Attacking hepatitis B virus cccDNA--The holy grail to hepatitis B cure[END_REF], the discovery of host-targeting agents (HTA) acting both on HBV replication and HCC initiation and/or progression could increase the efficacy of current standards of care (SoC) for curing patients from the infection and associated liver diseases.

We previously identified the serine/threonine polo-like-kinase 1 (PLK1) as such a host target [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF].

PLK1 is known to be overexpressed in many human cancers [START_REF] Strebhardt | Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy[END_REF], and was demonstrated an important driver of HBV-induced HCC [START_REF] Studach | Subset of Suz12/PRC2 target genes is activated during hepatitis B virus replication and liver carcinogenesis associated with HBV X protein[END_REF][START_REF] Wang | Proteins ZNF198 and SUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication[END_REF][START_REF] Andrisani | Gene signatures in hepatocellular carcinoma (HCC)[END_REF][START_REF] Studach | Polo-like kinase 1 inhibition suppresses hepatitis B virus X protein-induced transformation in an in vitro model of liver cancer progression[END_REF]. Moreover, we found that PLK1 is a proviral factor of HBV and that small inhibitory molecules of PLK1 (here after abbreviated SM-PLK1i) (e.g., BI-2536) had a negative impact on HBV replication [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF]. The inhibition of PLK1 could therefore be of interest for the treatment of CHB patients with potential effects on both virologic and oncologic aspects of the disease.

Due to their poor specificity (i.e., they target several kinases, not a single one) and overall low safety profile, as demonstrated in the oncology field, SM-PLK1i cannot be reasonably clinically trialed in CHB patients, who require long-term and very safe treatments. Interestingly, small interfering RNAs directed against PLK1 were also shown to be efficient at targeting PLK1 oncogenic activity [START_REF] Spankuch-Schmitt | Effect of RNA silencing of pololike kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells[END_REF]. Chemically modified and injectable versions of PLK1 siRNA were also validated in preclinical cancer animal models [START_REF] Yao | Targeted delivery of PLK1-siRNA by ScFv suppresses Her2+ breast cancer growth and metastasis[END_REF][START_REF] Judge | Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice[END_REF], and one PLK1 siRNA encapsulated in proprietary lipid nanoparticles (LNPs) was clinically trialed in patients with advanced HCC (NCT02191878).

In this study, we found that the use of LNP siPLK1 in HBV-infected primary human hepatocytes (PHHs) lead to a decrease in the amount of intracellular HBV RNAs, that subsequently affect the amount of secreted viral particles and HBe/HBs antigens. Knowing that a loss of HBsAg in patients represents a clinical end-point goal [START_REF] Durantel | New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus[END_REF], our finding could have relevant therapeutic implications.

Moreover, these data point-out a novel PLK1 proviral function, which seems independent of its kinase activity. Indeed besides being involved in HBV reverse transcription through the regulation of capsid assembly [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF], our data suggest that PLK1 could also be involved in the regulation of HBV RNA accumulation.

METHODS

Chemicals, antibodies, and others reagents

PLK1 inhibitor BI-2536 was purchased from Selleckchem. All other chemicals were purchased from Sigma-Aldrich. Rabbit polyclonal anti-HBc antibodies were purchased from Dako (B0586) or Abcam (ab115992) and monoclonal anti-HBc antibodies from Abcam (ab8637; Clone C1). Rabbit polyclonal anti-PLK1 antibodies from Abcam (ab21738) were used for detection of phospho-PLK1-S 137 ; rabbit monoclonal anti-PLK1 antibodies from Abcam (ab115095) were used for detection of phospho-PLK1-T 210 and rabbit polyclonal anti-PLK1 antibodies from Abcam (ab109777) for detection of total PLK1.

HepaRG and primary human hepatocyte cultures

Human liver progenitor HepaRG cells [START_REF] Marion | The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies[END_REF][START_REF] Gripon | Infection of a human hepatoma cell line by hepatitis B virus[END_REF] were cultured and differentiated (dHepaRG) as previously described [START_REF] Hantz | Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells[END_REF]. Primary human hepatocytes (PHH) were freshly prepared from human liver resection obtained from the Centre Léon Bérard (Lyon) with French ministerial authorizations (AC 2013-1871, DC 2013 -1870, AFNOR NF 96 900 sept 2011) as previously described [START_REF] Lecluyse | Isolation and culture of primary hepatocytes from resected human liver tissue[END_REF].

Production of siRNA-containing liponanoparticles and use in cell culture

The forward and reverse sequences of siRNA against PLK1 are respectively, 5'-AGAUCACCCUCCUUAAAUAUU-3' and UAUUUAAGGAGGGUGAUCUUU-3', and feature 2'OMe modification at indicated position (bold underlined) as previously described [START_REF] Judge | Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice[END_REF]. This dupleix siRNA was formulated in liponanoparticles as previously descrived [START_REF] Jeffs | A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA[END_REF]. The lipid formulation contains 1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dilinoleyloxy-N,N-dimethylpropylamine, 3-N-[(ω-methoxypoly(ethyleneglycol) 2000 )carbamoyl]-1,2-dimyristyloxy-propylamine and cholesterol.

The equivalence between molarity of siPLK1 and amount of LNP siPLK1 in ng/mL is: 1 nM of siPLK1 is equivalent to 13.3 ng/mL of LNP siPLK1. Controls LNP-siRNAs were manufactured the same way.

The sequences of siRNA against HBV are Fw: 5'-CGACCUUGAGGCAUACUUCUU-3' and Rv: 5'-GAAGUAGCCUCAAGGUCGUU-3' ON-TARGETplus duplex. LNP siRNAs were administrated to cells by direct dilution into the culture medium. Cells were exposed to LNPs for 24 hours, then washed with culture medium to remove excess of LNPs. Treatment were repeated as indicated in figure legends.

HBV infection and analysis of viral replication

Differentiated HepaRG cells (dHepaRG) and PHHs were infected with HBV genotype D (concentrated from HepAD38 cells supernatants) as described previously [START_REF] Lahlali | Novel potent capsid assembly modulators regulate multiple steps of the Hepatitis B virus life-cycle[END_REF]. After treatment procedures, levels of intracellular and/or extracellular HBV RNAs and DNAs, HBc, secretion of HBeAg and HBsAg were monitored by quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), immunoblot and enzyme-linked immunosorbent assay (ELISA) respectively, as previously described [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF][START_REF] Lahlali | Novel potent capsid assembly modulators regulate multiple steps of the Hepatitis B virus life-cycle[END_REF]. Briefly, HBeAg and HBsAg were quantified in culture medium using a chemiluminescence immunoassay kit (Autobio Diagnostics) according to manufacturer's instructions. Total intracellular DNAs and RNAs as well as secreted viral DNAs/RNAs were purified from infected cells using NucleoSpin® 96 Tissue kit, NucleoSpin® 96 RNA kit, NucleoSpin® 96 virus kit (Macherey-Nagel) respectively, according to manufacturer's instructions. Reverse-transcription of RNA into cDNA was performed with the SuperScript® III First-Strand Synthesis kit (Invitrogen) according to manufacturer's instructions. All qPCR analyses were performed with LightCycler™ 96 and 480 systems (Roche), except for cccDNA and pgRNA specific PCRs that were processed under a QuantStudio™ 7 (Applied), using homemade TaqMan assays [START_REF] Lahlali | Novel potent capsid assembly modulators regulate multiple steps of the Hepatitis B virus life-cycle[END_REF].

Primary human hepatocytes preparation from chimeric FRG mice and use for antiviral

This study was approved by the WuXi IACUC (Institutional Animal Care and Use Committee). FRG™ (Fah -/-/Rag2 -/-/Il2rg -/-) mice were purchased from Yecuris (Tualatin, OR, USA), engraphted with PHH and infected with 1.10 9 virus genome equivalent (vge) of HBV genotype D. When HBV viremia had reached a maximum (i.e., around 10e9 vge/mL), the liver was digested by in situ collagenase perfusion and recovered hepatocytes seeded in DMEM medium into 24-well plates (2.7.10 5 cells/well). LNP treatments were conducted every 4 days starting at day 0 and BI-2536 treatment was started on day 0 and replenished every tow days. DNA in the culture supernatants was isolated using QIAamp 96 DNA Blood Kit (Qiagen) and quantified by qPCR. HBsAg was quantified using specific CLIA kit (Autobio).

Separation of cytoplasm and nucleopasm

Extractions of nuclear and cytosolic proteins were performed using NE-PER Nuclear and Cytoplasmic kit (Thermo Scientific), according to the manufacturer's instructions. Sodium orthovanadate, Complete™ EDTA-free protease inhibitor cocktail (Roche) and sodium fluoride were added freshly to all lysates. Nuclear and cytosolic RNAs were isolated as described in Weil et al., [START_REF] Weil | Mature mRNAs accumulated in the nucleus are neither the molecules in transit to the cytoplasm nor constitute a stockpile for gene expression[END_REF], followed by a purification with TRI reagent (Merck) and precipitation with isopropanol.

Purity of nuclear vs cytoplasmic extracts, was assessed by RT-qPCR using primers to amplify the nuclear U6 snRNA (Fw: 5'-CTCGCTTCGGCAGCACATATAC-3' / Rv: 5'-GGAACGCTTCACGAATTTGCGTG-3') and the cytoplasmic Human Tyr-tRNA (Fw: 5'-CCTTCGATAGCTCAGCTGGTAGAGCGGAGG-3' / Rv: 5'-CGGAATTGAACCAGCGACCTAAGGATGTCC-

3').

Capsid migration assay

The intracellular HBV nucleocapsids were analyzed by agarose electrophoresis in native condition followed by transfer onto an enhanced chemiluminescence membrane (Amersham) and western blot analysis, as previously described [START_REF] Lahlali | Novel potent capsid assembly modulators regulate multiple steps of the Hepatitis B virus life-cycle[END_REF].

Southern Blotting

Total DNAs extracted with NucleoSpin® 96 Tissue kit (Macherey-Nagel) were pooled, precipitated using 5M NaAc and ethanol and analyzed by Southern blot as previously described [START_REF] Lucifora | Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector[END_REF]. DNAs from HepG2-NTCP cells extracted using a modified Hirt procedure were used as positive controls [START_REF] Cai | A southern blot assay for detection of hepatitis B virus covalently closed circular DNA from cell cultures[END_REF].

Statistical analysis

Statistical analysis was performed using two-way analysis of variance, t tests, or non-parametric Mann-Whitney tests using GraphPad Prism software, version 7.04. For all tests, P ≤ 0.05*, P ≤ 0.01**, and P ≤ 0.001*** were considered statistically significant.

RESULTS

Efficacy and safety of LNP siPLK1 in hepatocytes

Compared to control LNP siRNAs, LNP siPLK1 were efficient at inhibiting PLK1 expression, with a 70% loss of PLK1-mRNA at 13.3ng/mL without toxicty in proliferating HepG2 cells. As expected, LNP siPLK1 induced a 50% mortality in proliferating HepG2 cells at 133ng/mL due to active cell division and crucial role of PLK1 in G2/M transition (Fig. Sup. 1). Considering these data, as well as the concentration correspondence (i.e., 1 nM of naked siPLK1 is equivalent to 13.3 ng/mL of LNP siPLK1) and our previous work done using naked siRNA targeting PLK1 [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF], we tested the effect of LNP siPLK1 and controls in HBV-infected primary human hepatocytes, and related dHepaRG cells, with a range of concentration going from 100 to 300 ng/mL. We observed a strong reduction in the levels of PLK1 mRNA in both dHepaRG and PHH (Fig. Sup. 2A and Fig. 1A ) and in the level of PLK1 phosphoprotein in PHH (Fig. 1B). Interestingly, as previously reported, LNP siHBV led also to a reduction of PLK1 mRNA and PLK1 phosphorylation to levels comparable to those observed in uninfected cells (compare lanes LNP-siHBV and NI; Fig. 1A and1B), thus confirming that HBV strongly increases PLK1 activity in PHH. Confirming also our previously published data [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF], the inhibition of PLK1 activity was not cytotoxic in PHH (3 different batches tested) even when LNP siPLK1 was administrated 3 times at 300ng/mL (Fig. 1C). Moreover PHH remained functional, as measured by the apolipoprotein B (ApoB) secretion (see apoB dosing in Fig. 2A), a component of VLDL (very low density lipoprotein) that are made by healthy hepatocytes.

LNP siPLK1 strongly, and specifically, inhibits viral particle and antigen secretion in HBV-infected primary human hepatocytes

To evaluate the antiviral activity of LNP siPLK1, freshly isolated PHH from three different donors were infected with HBV at a high multiplicity of infection, and at day-4 post-infection (p.i.), when the average HBsAg production reached around 1000 IU/mL (which is a level signing a high replication of HBV), cells were treated three times every three days with increasing LNP siPLK1 doses or control molecules. Secretion of viral DNA (i.e., infectious virion) and RNA (i.e., RNAcontaining particles; [START_REF] Wang | Serum hepatitis B virus RNA is encapsidated pregenome RNA that may be associated with persistence of viral infection and rebound[END_REF]) were significantly inhibited by LNP siPLK1 (Fig. 2A). These inhibitons were comparable to those observed with LNP siHBV in the model. The lack of dose-dependency of the phenotype suggests that even a weak loss of PLK1/phospho-PLK1 (Fig. 1B) was associated with a strong inhibition of virus replication. Interestingly, we also observed a 70% inhibition of viral antigen HBeAg and HBsAg secretion (Fig. 2A), without any effect on ApoB secretion, highlighting a specificity of LNP siPLK1 action on those secreted viral proteins. In the mean time, tenofovir (TFV)

did not affect HBV antigens secretion and inhibition of HBV antigen secretion was stronger with LNP siHBV, as expected due to the direct targeting of HBV RNAs encoding those antigens.

This strong inhibition of HBeAg and HBsAg secretion was also confirmed in the HBV-infected dHepaRG cells with the same experimental setting (Fig. Sup. 2B). Moreover, the inhibitory phenotype on virion and HBsAg secretion was also observed by ex vivo treatment with LNP siPLK1 of freshly isolated hepatocytes from HBV-infected liver-humanized FRG mice [START_REF] Bissig | Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment[END_REF], which replicate HBV at high level (i.e., around 1.10 9 copies/mL at day-26 post-infection), mimicking the highly viremic « immune tolerant » phase in human. In this model, 300ng/mL of LNP siPLK1 led to a decrease of 60% in HBV DNA and 65% in HBsAg, whereas treatment with LNP siHBV was very efficient (Fig. Sup. 3).

LNP siPLK1 induces a reduction of intracellular viral RNA accumulation without significant modification of cccDNA amount

To further describe the anti-HBV potential of LNP siPLK1 and get insigths on the observed phenotype on secreted HBV antigens, intracellular viral parameters were also analyzed. We (analyzed by qPCR or Southern Blot) (Fig. 3). These data suggest that the decrease of HBV RNA observed with LNP siPLK1 is a consequence of a transcriptional and/or post-trancriptional event.

LNP siPLK1 does not inhibit better HBV replication in pre-infection treatment setting

To determine if PLK1 affects HBV infection establishment, we treated PHH twice with LNP siPLK1

or control molecules, prior to HBV infection and a third treatment was done at the time of HBV inoculation in order to maintain PLK1 protein level low from HBV entry to cccDNA formation.

Analyses were done at 5d.p.i. PLK1 mRNA knockdown was still >75% with 100 and 300ng/mL of LNP siPLK1 (Fig. 4A). As seen before LNP siHBV allowed a PLK1 mRNA decrease, in accordance to the level found in uninfected cells. Interestingly a similar phenotype was observed with an entry inhibitor (i.e., PreS1 peptide), which very efficiently prevented HBV infection in this setting (as shown in other panels of Fig. 4). Regarding antiviral phenotypes obtained with LNP siPLK1, they were very similar to that obtained in post-infection treatment setting (compare results of Fig. 2 and4). Importantly, cccDNA establishment was not fully blocked by either LNP siPLK1 or LNP siHBV, as compared to that observed with PreS1 peptide, which acts a genuine entry inhibitor. The slight decrease amount of cccDNA observed with LNP siPLK1 and LNP siHBV was poorly significant compared to controls (LNP si Ctrl and TFV), and could therefore not explain per se other observed inhibitory phenotypes. It is worth mentioning here that PreS1 peptide did not lead to an absence of detection of intracellular HBV DNA in this setting, as compared to other papameters (cccDNA, intracellular RNA, HBeAg, HBsAg), thus confirming that residual HBV DNA from inoculation perturbs analyses, as mentioned earlier. This is the reason why results obtained from supernatant analyses are more reliable, and without ambiguity demonstrate the overall strong effect of LNP siPLK1 on HBV replication.

PLK1 knockdown results in a partial nuclear vRNA sequestration in HBV-infected PHH

In HBV-infected PHH, activated pPLK1-S 137 was mainly localized in the nucleus (Fig. Sup. 4 and refer also to [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF]). As we observed a decrease in HBV RNAs in absence of change in the level of cccDNA under LNP siPLK1 treatment, we wondered if PLK1 could be involved in the modulation of HBV RNA levels. To address this, we infected PHHs, treated them as above and harvested cells three days after the last treatment. We performed a cytosolic/nuclear fractionation in order to quantify HBV RNAs in both compartiments. RNA purity of our extracts was checked using nuclearspecific U6 snRNA and cytosolic-specific Tyr-tRNA. LNP siHBV resulted in a decrease of HBV RNAs levels in both compartments, whereas LNP siPLK1 led to a dual phenotype, depending on subcellular compartments. Indeed, in the cytosol, a 50% decrease of HBV RNAs levels was observed, similar to that observed with whole cell extracts (Fig. 5). By contrast, in the nuclear compartment, a dose-dependent accumulation of HBV RNAs was observed under LNP siPLK1 treatment (Fig. 5C). As the amount of cytosolic HBV RNAs was overall 10 times higher than that observed in the nucleus, this accumulation of HBV RNAs in the nucleus induced by LNP siPLK1 did not modify the overall observed phenotype (i.e., reduction of total RNA accumulation), but certainly accounted for the decrease in viral protein synthesis.

DISCUSSION

Hepatocellular carcinoma ranks second in term of cancer-induced death worldwide, with an estimated 600,000 death/year burden. HCC development is the consequence of the interplay between genetic predispositions, environmental factors (e.g., exposure to aflatoxins, alcohol, etc…), as well as oncogenic viruses; the latter are involved in 80% of cases, and HBV for itself accounts for 50% of HCC worlwide. As 257 million people are chronically infected with HBV and only a minority of them (<1%) are effectively diagnosed and treated, the HBV-mediated HCC burden is far from being eradicated. If a better diagnostic of infected patients and a more universal use of antiviral therapies are needed, it would be also intresting to identify novel molecules that could target both viral replication and hepatocellular cancer cell growth.

Our previous study demonstrated that PLK1 is activated upon HBV infection and acts as a proviral factor. Consequently, we showed that its inhibition by BI-2536, a SM-PLK1i, leads to an inhibition of HBV replication. In addition, the group of Dr O. Andrisani have shown in several studies that PLK1, which is induced by HBx, is a driver of murine hepatocyte cancerous transformation in vitro and that its inhibition by BI-2536 could prevent it [START_REF] Studach | Polo-like kinase 1 inhibition suppresses hepatitis B virus X protein-induced transformation in an in vitro model of liver cancer progression[END_REF][START_REF] Studach | Polo-like kinase 1 activated by the hepatitis B virus X protein attenuates both the DNA damage checkpoint and DNA repair resulting in partial polyploidy[END_REF]. Moreover PLK1 being a validated target in oncology, important research efforts to identify potent SM-PLK1i were made, as reviewed recently [START_REF] Liu | PLK1, A Potential Target for Cancer Therapy[END_REF]. However most of SM-PLK1i are not strictly specific to PLK1 itself and cross-inhibit of other kinase functions; this may account for the poor toxicologic profiles and side effects of these chemical inhibitors. The possibility to use siRNA to more specifically target kinases involved in cancer has opened novel perspectives. In this line, the clinical trial NCT02191878 performed with TKM-080301 in subjects with advanced HCC was instrumental and led us to consider LNP siPLK1 as a good candidate for early inhibition of HBV replication, in the dual perspective of use described above.

Here we confirmed that PLK1 is crucial for HBV replication in non-dividing hepatocytes by using PLK1-targeting-siRNAs encapsulated in lipid nanoparticles (Arbutus Bioparma proprietary composition; the LNP-siPLK1 used in this study was closely related, but different from TKM-080301). In particular, we demonstrated that LNP siPLK1 inhibits more efficiently HBV replication than naked siRNA or SM-PLK1i (reported in [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF]) in the absence of cell toxicity. Indeed 100 to 300ng/mL of LNP siPLK1 (corresponding to 7.5 to 22.5nM siPLK1) led to a >75% decrease in HBV viremia in vitro. Moreover, in contrast to BI-2536, the SM-PLK1i that was previously profiled in depth in vitro and in liver-humanized FRG mice [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF], we found that LNP siPLK1 was also capable to potently inhibit the secretion of HBV antigens in the supernatant of treated cells. This indirectly indicates that a kinase-independent function of PLK1 could account for production and secretion of HBV antigens. PLK1 could interact with a viral or host factor independently of its kinase activity and through this interaction contribute to an optimal production of HBV antigens. This proteinprotein interaction model to explain activitywas precedently exemplified for PLK1 by Vitour and colleagues [START_REF] Vitour | Polo-like kinase 1 (PLK1) regulates interferon (IFN) induction by MAVS[END_REF]. In their study, it was shown that the polo-box domain of PLK1 could bind to the mitochondria-bound innate immunity adapter MAVS and inhibit its ability to activate the IRF3 and NF-kappaB pathways, without any requirement of the kinase activity.

Such an unexpected phetotype of LNP siPLK1 on HBV antigens production is particularly relevant, as the next step for the improvement of current HBV therapy in clinic is precisely to be capable to reduced antigenemia in patients, in particular HBsAg, to potentially restore immune responses and control infection [START_REF] Durantel | New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus[END_REF]. The LNP siPLK1-induced inhibition of HBeAg and HBsAg in supernatant correlated with a reduced accumulation of intracellular HBV RNAs in LNP siPLK1 treated cells, in the absence of any change in cccDNA amount. This observation points out toward a role of PLK1 in transcriptional and/or post-transcriptionnal regulation. In this study, we limited our work to the analysis of the impact of LNP siPLK1 on the accumulation of viral RNA in nucleoplasm and cytoplasm after fractionation, and found that HBV RNAs tended to accumulate in the nucleus under LNP siPLK1 treatment, while the total amount of RNA was decreased. Although the precise underlying mechanistic of this transcriptional and/or post-transcriptional regulation (i.e., cccDNA activity, vRNA stability, vRNA trafficking…) is yet to be defined, it is tempting to speculate that the previously described interplay between PLK1 and HBc [START_REF] Diab | Polo-like-kinase 1 is a proviral hostfactor for hepatitis B virus replication[END_REF] could also be at work. Indeed, HBV core protein is known to interact with cccDNA [START_REF] Bock | Structural organization of the hepatitis B virus minichromosome[END_REF] and potentially in the regulation of transcription [START_REF] Chong | Role of hepatitis B core protein in HBV transcription and recruitment of histone acetyltransferases to cccDNA minichromosome[END_REF]. Moreover, it has been suggested that the HBc CTD could be determinant for HBc-cccDNA interaction [START_REF] Chong | Role of hepatitis B core protein in HBV transcription and recruitment of histone acetyltransferases to cccDNA minichromosome[END_REF] and in our precedent study, we have shown that PLK1 targets HBc CTD. Finally, HBc is also known to be an RNA-binding protein physically interacting with vRNAs. Of course, a role of PLK1 independently of HBc should also be considered. In this case PLK1 could interact with other known or unknown host-regulators of both/either cccDNA transcription and/or posttranscriptional events, and regulate their functions in a kinase-independent manner. Whether this is yet to be demonstrated regarding the pro-HBV role of PLK1, there are in literature many exemples of kinases, which feature kinase-independent regulatory functions [START_REF] Audetat | A Kinase-Independent Role for Cyclin-Dependent Kinase 19 in p53 Response[END_REF][START_REF] Jang | Kinase-independent role of nuclear RIPK1 in regulating parthanatos through physical interaction with PARP1 upon oxidative stress[END_REF][START_REF] Li | Hepatitis C virus infection activates an innate pathway involving IKK-alpha in lipogenesis and viral assembly[END_REF][START_REF] Sulzmaier | FAK in cancer: mechanistic findings and clinical applications[END_REF][START_REF] Vivanco | A kinase-independent function of AKT promotes cancer cell survival[END_REF][START_REF] Wang | p21-activated kinase 1 (PAK1) can promote ERK activation in a kinase-independent manner[END_REF]. Taken together, this provided a nice hypothetical landscape for further mechanistic studies to document LNP siPLK1 mode of action (MoA).

Of course, it would also be important to evaluate the anti-HBV efficacy of LNP siPLK1 in a liver humanized mouse model to confirm its potential therapeutic interest on the virology side, and to compare it or combine it to siRNA targeting HBV itself. Whether siRNA targeting HBV are currently in clinical trials in CHB patients (e.g., phase-2 for ALN-HBV, NCT02826018; phase-2 for JNJ-3989 ,NCT03982186 and NCT04129554) and remains the most straightforward and relevant RNAi option against HBV replication, the co-administration of siRNAs targeting both HBV and PLK1 could be an original way to further improve such a therapeutic approach.

Regarding the possibility to test the relevance of using LNP siPLK1 as a double bullet compound, which would be able to both contribute to HBV replication inhibition in "add on therapy" with SoC and prevention of HBV-driven carcinogenesis, we would need a preclinical model able to recapitulate the entire pathogenesis process. A mouse model based on immune-competent mice transduced with AAV-HBV vectors could be of interest, as it features a persistent viral replication [START_REF] Dion | Adeno-associated virus-mediated gene transfer leads to persistent hepatitis B virus replication in mice expressing HLA-A2 and HLA-DR1 molecules[END_REF], in the context of an HBV immune tolerance [START_REF] Martin | an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBVpersistent mice[END_REF], and was also reported to support HCC development [START_REF] Huang | A murine model of hepatitis Bassociated hepatocellular carcinoma generated by adeno-associated virus-mediated gene delivery[END_REF]. Alternatively other transgenic mouse models featuring the overexpression of HBV proteins, HCC development and a demonstrated implication of PLK1 in carcinogenesis could be used to this end. Recently, it was shown that genomic integrity of hepatocytes was disrupted by viral LHBS and that PLK1 acted as a major modulator in LHBS-mediated G2/M checkpoint override. In this model, SM-PLK1i resulted in a G2/M checkpoint restauration and suppression of tumorigenesis [START_REF] Li | Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis[END_REF].

To summarize, in this study we have confirmed the proHBV role of PLK1 and reported a potent and specific antiviral activity of LNP siPLK1 in HBV-infected hepatocytes, which includes a strong loss of intracellular vRNA accumulation, leading to reduction of HBV antigens secretion. As it is now rather well accepted that PLK1 is an important HCC driver, its inhibition by RNA interference in patients featuring an HCC from HBV etiology, in which HBV replication would be still active, could result in a combined effect on the virus and cancer cell growth. These results warrant additional investigations in preclinical animal model to further determine the usefulness of such a therapeutic strategy.

  observed a dose dependent decrease of intracellular HBV DNAs and RNAs in HBV-infected PHH or dHepaRG cells treated with LNP siPLK1 (Fig. 2B and Fig. Sup. 2C). The highest dose of LNP siPLK1 resulted in a decrease of 55% (70% in dHepaRG) and 50% (also 50% in dHepaRG) of HBV DNA and RNA respectively (Fig. 2B and Fig. Sup. 2C). LNP siHBV treatment resulted in a stronger effect, with 80% and 90% drops, whereas only viral DNA amount decreased with TFV (65% in PHH and 90% in dHepaRG) as expected. The lower inhibition of intracellular DNA amount for all molecules tested, as compared to what observed with secreted DNA is likely due to the detection of remaining input HBV DNA from HBV inoculum as discussed below. Interestingly and contrary to what we described with SM-PLK1i [6], we observed a decrease in the levels of HBV RNAs upon LNP siPLK1 treatment of HBV-infected hepatocytes without observing any significant changes in cccDNA amount

Figure 1 .

 1 Figure 1. Evaluation of the efficacy and toxicity of LNP siPLK1 in primary human hepatocytes.

Figure 2 .

 2 Figure 2. Antiviral activity of LNP siPLK1 in non-transformed and non-dividing HBV-infected PHH.

Figure 3 .Fig. 2 ,

 32 Figure 3. LNP siPLK1 does not affect cccDNA amount. (A) A representative Southern blot,

Figure 4 .

 4 Figure 4. Pre-infection treatment with LNP siPLK1 in primary human hepatocytes. Experiments

Figure 5 .

 5 Figure 5. Localization of HBV RNAs under LNP siPLK1 treatment. Differentiated HepaRG cells

1 L 1 L

 11 Figure 1

ACKNOWLEDGEMENTS

The authors thank Maud Michelet, Océane Floriot, Jenifer Molle, Laura Dimier, Anaëlle Dubois and Judith Fresquet for PHHs isolation as well as the staff from Michel Rivoire's surgery room for providing us with fresh liver resections. This work was supported by a grant from ANRS (French national agency for research on AIDS and viral hepatitis; CSS4) and by INSERM core grant to DD. This work was also supported by the DEVweCAN LABEX (ANR-10-LABX-0061) of the "Université de Lyon", within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR) to DD. The work was also supported by a research contract with Arbutus Biopharma.

research grant from Arbutus Biopharma for this work; AD and AL are Arbutus Biopharma's employees; AS, JL and MR: no conflict of interest

SUPPORTING FIGURE LEGENDS

Supporting Figure 1. Efficacy of LNP siPLK1 and toxicity profile in HepG2 cells. (A) HepG2 cells were treated with increasing doses of LNP siPLK1, ranging from 1.3ng/mL to 133ng/mL. RNA were extracted, reverse transcribed and PLK1 mRNA quantified using specific qPCR. Normalized to