Christis Z Chrysostomou

Nicholas Kyriakides

Panagiotis Kotronis

Martin Poljansek

Fabio Taucer

Panayiotis Roussis

Antonis Kosmopoulos

M Poljanšek

Seismic retrofitting of RC frames with RC infilling

Keywords: RC infill Walls, Retrofitting Existing Structures, Full Scale Psd Testing

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

INTRODUCTION

The construction of new walls is the most effective and economic method for retrofitting multi-storey reinforced concrete buildings, especially those with pilotis (soft-storey). Their structural and economic effectiveness increases when they result from infilling full bays of the existing RC frames. The experimental research performed to the present day covers sufficiently the other frequently used types of retrofitting -in particular the use of fibre reinforced polymers (FRP) and the concrete jackets -but not the RC infill walls. For them, experimental research did not target what is necessary, but what is feasible: due to the practical difficulties of testing large specimens with high force resistance, only one-or two-storey specimens have been tested. Regarding code provisions, Eurocode 8 -Part 3 covers fully and adequately retrofitting with FRP or concrete jackets, while it does not say anything about new walls created by infilling frame bays. Other guidelines, like KANEPE in Greece, refer to the design of these walls only in terms of forces, without providing tools for calculation of their characteristic deformations (at yield and failure) and stiffness, unless the infill wall can be considered integral with the bounding frame. The inadequacy of design codes in this respect is due to our poor knowledge of the behaviour of walls created by infilling with RC a bay of an existing frame.

To the present day, experimental research on reinforced concrete frames converted into walls by infilling with RC has been carried out almost exclusively in Japan and Turkey. The experiments in Japan (Hayashi et al. 1980, Higashibata et al. 1978[START_REF] Kato | Construction and Performance Test of Precast Tana ilitation Which Used Pull-Out Preventive Teym ixed shotctrete walls to retrofit low ductile RC frames, 14 Turk einforced Concrete Frames with Reinforced A of Structural Engineering[END_REF], Shiohara et al. 1984, Tanaka et al. 1984, Masuo et al. 1998, Takeyama et al. 1998, Sugimoto et al. 1999) were performed on 27 in total 1:3 to 1:4 scale single-storey one-bay RC-infilled frames with RC infill thickness 25% to 50% that of the frame members, which were compared in most cases with monolithically cast specimens of the same geometric characteristics (in which the frame and the infill wall were cast at the same time and integrally connected). The thickness of the RC infill varied from 26% to 60% (on the average 43%) of the width of the members of the frame. The connection of the RC infill to the bounding frame was done with epoxy-grouted dowels (17 specimens), or mechanically (shear keys, dowels without epoxy etc., 6 specimens). In four other cases the thickness of a pre-existing thin wall was increased by 100% to 150% without any direct connection of the new wall with the bounding frame. The failure mode of all the specimens was in shear (including sliding at the interface). It is interesting to note that for epoxy-grouted dowels the force resistance of the infilled frame was on average 87% of that of the integral one, while for mechanical connection it was 80% on average. For the increased thickness of an existing thin infill wall, the force resistance was on average 92% of that of the monolithic specimen, while the displacement at failure was on the average 13% smaller than in the integral specimen. For the epoxy grouted dowels the ultimate deformation was on average 55% larger than in the integral specimen and for the mechanical connection by 115% larger on the average. Therefore, although the deformable connection gives a somewhat reduced strength, it increases considerably the ultimate deformation of the retrofitted structure.

Of the specimens tested in Turkey, those of Teymur et al. (2008), Anil and[START_REF] Altin | An experimental study[END_REF] were single storey one-bay 1:2 and 1:3 scale, with RC infill thickness 25% or 33% that of the frame members. Those of Altin et al. (1992), Turk et al. (2003), Cambay et al. (2003), Sonuvar et al. (2004), Kara and Altin (2006) were two-storey one-bay scaled at 1:3, with infill wall thickness 1/3 and 40% of the width of the members of the bounding frame. The RC infill was in most cases fully connected on the perimeter with dowels, in some cases though (Teymur et al. 2008) there was a gap between the infill and the columns, while in some other cases there was no connection other than simple bearing. Altin et al. (1992) have also tried to weld the rebars of the infill to those of the members of the frame, instead of using dowels. Only Altin et al. (1992) included some monolithic specimens, however not exactly similar to the infilled ones. Finally, the specimen of Erdem et al. (2004 and[START_REF] Kara | Behavior of reinforced concrete frames with reinforced concrete partial infills[END_REF] was twostorey three-bay scaled at 1:3, with the middle bay infilled with a wall with thickness 63% of the width of the frame members. The connection was with epoxy grouted dowels and the failure mode predominantly flexural. In all other cases the single storey walls failed in shear, while the two storey ones failed by a combination of flexure and shear sliding at the base.

The low-aspect ratio test specimens used in the experiments described above are non-representative of the behaviour of a multi-storey and slender wall since their behaviour and failure mode is dominated by shear. By contrast, a multi-storey and slender wall to be used in real life is controlled by bending and can be easily dimensioned so that a plastic hinge forms at its base. In such a case shear will not have a detrimental effect on its behaviour and on its energy dissipation capacity. In addition, the higher modes of vibration of the structure are not taken into account although it has been shown numerically (Keinzel 1988, Eibl andKeinzel 1990) that they may increase considerably the shears at the upper floors of a wall after the formation of a plastic hinge at the base. This aspect has never been studied experimentally even in integral walls, because their height and number of storeys was not large enough to allow higher mode inelastic response. Another common element of past tests is the small thickness of the RC infill wall relative to the width of the frame members. As a result, the weak link of the structural system is either the infill wall in diagonal compression, or its connection with the surrounding frame.

In order to start filling the gap of knowledge regarding infilling of existing RC frames with RC walls, the effectiveness of seismic retrofitting of multi-storey multi-bay RC-frame buildings, by converting selected bays into new walls through infilling with RC, was studied experimentally at the ELSA facility at JRC, Ispra, and the results are reported here. This research is under the project "Seismic Engineering Research Infrastructures for European Synergies" (SERIES), financed by the Seventh Framework Programme of the European Commission. The consortium consisted of the Cyprus University of Technology (co-ordinator), the Ecole Central de Nantes, DENCO, the ELSA laboratory at JRC Ispra and the University of Cyprus. In the first part of the paper the design of the bare-frame specimen is presented and in the second the details of the design of the RC infills are given. Then, the results of the testing campaign are presented and conclusions are drawn.

DESCRIPTION OF THE SPECIMEN

The specimen consisted of three bays with the central bay infilled with RC wall and it had four storeys. The scale of the model was 1:1. The centre-line length dimension of the specimen was 8.5 m (central bay 2.5 m and the two exterior bays 3.0 m), the storey height 3.0 m and the total height of the specimen (excluding the foundation) about 12.0 m (Fig. 2.1a). Two parallel retrofitted frames (spaced at 6.0 m) were used, one at each side of the test structure, linked through RC slabs 0.15 m thick (Fig. The proposed structure represents typical construction of the late 70's and beginning of the 80's in Cyprus. The structures at that time were designed for gravity loads only, since there were no provisions for earthquake loading. There was no specific design standard and the authorities were accepting any standard that was acceptable to other countries such as CP110 and BS8110, DIN, Greek Code, US code etc.

For the design of the mock-up it was decided to use the provisions of BS8110 which are very close to those of CP110 with very minor differences. In Cyprus the transition that was made was from CP114, which was an allowable stress design, to BS8110, without going through the CP110 phase. It was made sure that all the reinforcement details used for the design were according to CP110:1972 and BS8110:1983.

The material properties used in the mock-up were constrained by the availability of materials in the Italian and European market. It was finally decided to use concrete C20/25 for both the frame and the walls, of unit weight 25 kN/m 3 and modulus of elasticity, E=30.000 MPa. The yield strength of the ribbed bar reinforcing steel was f yk = 400 MPa for both bending and shear reinforcement of the frame members and the slab, while for the RC infill and the dowels to be used for connecting the wall to the bounding frame members the yield strength was specified to f yk = 450 MPa.

The frame was designed for gravity loads only, since this was the practice in the period of time that is being examined. The self-weight was calculated using the unit weight of concrete specified above. The imposed dead load was 3 kN/m 2 , including the load of masonry infill walls, and the live load was 1.5 kN/m 2 . The above loads were combined using partial factors of safety of 1.4 for self-weight and imposed dead-load, and 1.6 for live load. The material partial factors used were 1.5 for concrete and 1.15 for steel.

The reinforcement details for the beams are shown in Fig. 2.2a and the ones for the columns in Fig 2 .2b. For the beams of the frame 4Φ12 bars were used for top and bottom reinforcement, which was running the total length of the beams. The shear links were Φ8 at 200 mm intervals starting at 50mm from the face of the column. For the transverse beams 2Φ20 bars were used at the top and 5Y20 at the bottom of each of the four transverse beams. The links were Φ8 spaced at 100 mm so as to make sure that no failure will take place in the transverse beams which were used to transfer the forces from the actuators to the frames. The columns were reinforced with 4Φ20 bars and were lapped for a length of 0.55 m measured from the top face of the slab (Fig. 2.2b). This represents a compression lap, since the structure was designed for gravity loads only, and it is expected fail when subjected to tension. The shear links were Φ8 spaced at 200 mm starting at a distance of 50 mm from the top face of the slab. Similar to the transverse beams, the slab was considered as an element that will facilitate the transfer of forces from the actuators to the two parallel frames, therefore the reinforcement was increased considerably. For the slabs, nominal reinforcement of Φ10/200 was required, and Φ10/100 was specified in order to facilitate the transfer of forces.

Reinforcement details of the RC infill walls

The specimen, as it was explained above, consisted of two parallel frames connected through a slab and it had the central bay infilled. In order to facilitate the study of as many parameters as possible, the walls in the two frames were reinforced with different amounts of reinforcement, with the north one being the stronger of the two (Fig. 2.3.). 2.1. Regarding the connection detail, two distinct connection details were used. In the first detail, the web bars are connected to the surrounding frame through lap-splicing with the same diameter starter bars epoxy grouted into the frame members. Short dowels are then used in order to transfer the shear at the interface between the wall and the frame member. This detail was used to connect the wall at the bottom beam and right column at the 1 st and 2 nd floors of the specimen (Fig.

2.4a).

In the second detail, longer dowels were used to double as dowels as well as for anchorage of the web panel to the surrounding frame; to this end, the dowels are considered as lap-spliced with the nearestsmaller diameter -web bars. However, in this case, the clear distance between the dowel and the nearest web bar, violates the maximum clear distance of 50 mm or 4Φ between lapped bars, specified in Eurocode 2. This detail was used to connect the wall at the top beam and left column at the 1 st and 2 nd floors of the specimen (Fig. 2.4a). In the 3 rd floor of both the north and south frames only the second detail was used, while for the 4 th floor only two dowels per wall interface were used to provide safety against falling of the wall out of plane. The reinforcement details for the dowels and the starter bars are shown in Table 2.1. The completed wall reinforcement (including web, starter bars and dowels) for the 1 st floor of the south wall is shown in Fig. 2.4b.

Table 2.1. Reinforcement details for the RC infill walls

Since the lapping of the column reinforcement could take only compression, then it was obvious that there would be lap splice failure, which could be detrimental to the whole experiment. Therefore, in order to safeguard against this type of failure and allow the experiment to be performed without any premature failure, it was decided to reinforce the edges of the wall at the 1 st floor with three-sided CFRP for a height of 0.60m from the base of the column (Fig. 2.5).

TESTING CAMPAIGN

The testing campaign consisted of two pseudo-dynamic tests and a "funeral" cyclic test. Two 1000 kN actuators were used in the two top floors of the specimen and two 500 kN ones at the lower two floors.

The gravity load on the structure was the one corresponding to the dead and reduced live loads, as specified in section 2.1, and it was equal to 192 kN per floor. This was achieved through 15 plastic barrels per floor that were filled with water and were distributed on the slabs of the test specimen (Fig.

2.3.).

The prototype structure consisted of four frames spaced at 6 meters, and the test specimen represented the two end frames of that structure. Therefore, in the pseudo-dynamic test, the total mass of the prototype was used in the equation of motion, which was equal to 156 tons per floor. In this way, the two tested frames were subjected to the true dynamic forces that the prototype would have taken. The Heregnovi-D1-Transverse component was used as input, which was 15 seconds long and it was digitized at 0.005 seconds. For the first pseudo-dynamic test the accelerogram was scaled to an acceleration of 0.10g and for the second test to an acceleration of 0.25g. For the low level acceleration, the structure behaved very well. There were no visible cracks either on the columns or the walls. Some hairline cracks that appeared on the surface of the wall closed down when the experiment was finished. The maximum top storey displacement was 24 mm and the displacement in the opposite direction was 26 mm (Fig. 3.1.a). These displacements were the same for both the north and south frames, since the forces in the south frame were imposed in such a way so as to keep the displacements of the two frames equal, and hence avoid any torsional effects on the specimen. It should be noted that the variation of the displacements at the first and second stories are shown only up to about 11 seconds, since a problem was encountered with the data acquisition system. The variation of the base shear with the top displacement in each of the frames is shown is Fig. 3.1.b. As it can be observed from the figure, there is very little difference between the two frames. The maximum positive shears were 645 kN and 574 kN for the south and north frames, respectively, and the maximum negative shears were -634 kN and -625 kN for the respective frames. It was considered that both walls have reached their cracking moment, and this was the purpose of this experiment. The 0.25g test was performed the day after the first test, and it was designed to bring the specimen at its ultimate capacity. The maximum top storey displacement was 109 mm and the displacement in the opposite direction was -93 mm (Fig. 3.2.a). Some difference was observed in the base shear between the two frames. As it can be observed from Fib. 3.2.b, the maximum base shear in the positive direction was 1074 kN for the south frame and 1036 kN for the north, which are about the same, while a negative base shear of -843 kN was recorded for the south frame and -1011 kN for the north one. This was an indication that the south frame has suffered some damage and it could not take further load. The only visual indication of this was a crack that opened in the ground beam at the base of the wall and the lap-splice failure of the outer column on the east side of the south frame. It should be noted that the presence of the CFRP on the bounding columns of the wall, have prevented a similar failure and it allowed the completion of the experiment.

In examining Figs. 3.3. and 3.4. it can be observed that there is a steady decrease of the storey shear from about 1000 kN at the 1 st storey to about 400 kN at the top storey. What it can be also observed is that while the interstorey drift at the 1 st storey is about 20 mm, the ones for the upper three floors are about 30 mm, which shows the influence of the RC infill on the dual system that was created by connecting it to the bounding frame. It can be also observed that there are stable hysteresis loops which allow the absorption of energy.

Interstorey Regarding general behaviour of the specimen it can be said that it withstood the loading imposed on it very well. There were no visible diagonal cracks on the walls, which behaved flexurally. In nearly all the columns a horizontal crack appeared at a height of 0.55 m where the lap-splice stopped, and in some cases there was spalling of the concrete cover. Some vertical cracks appeared at the connection between the beams and the columns, but there was no severe damage, despite the fact there were no ductile connections in the structure. In general, the stronger north frame had an overall better behaviour compared to the south one, but the differences were minor.

Finally, in the "funeral" cyclic test a displacement history was imposed at the top storey (92, -92, 89, -125, 37, 0 mm) and a triangular distribution of forces was imposed. The objective of the test was to obtain a 15% reduction of the peak strength of the infill, so as to establish the strength envelope of the specimen. The base shear versus the top storey displacement is shown in Figure 3.5. As it can be observed in the first cycle the structure could reach 92 mm in both directions. In the second cycle the attempt was to reach 125 mm, but in the positive direction only 89 mm was possible to be reached, while in the negative direction 125 mm was reached. This though had as a result a sudden drop of the strength of the south frame from 838 kN at -110 mm to 553 kN at a displacement of -125 mm. This amounts to a drop in strength of 34%. After that, the displacement was reduced to 37 mm in the positive direction and from there to zero. . CONCLUSION he effectiveness of seismic retrofitting of multi-storey multi-bay RC-frame buildings, by converting was demonstrated that this is a viable method for retrofitting and it can be used to strengthen KCNOWLEDGEMENT e results has received funding from the European Community's 7th Framework e 4 T selected bays into new walls through infilling with RC, was studied experimentally at the ELSA facility at JRC, Ispra, on a full scale specimen. The main parameters of the mock-up were the connection between the RC infill and the surrounding RC frame and the percentage of the reinforcement in the RC infill. The effect of these parameters was studied during the experiment, by using different connection details and reinforcement percentages for the two infilled frames. Some preliminary findings regarding the behaviour of the structure are: a) the structure managed to sustain an earthquake of 0.25g without significant damage, b) some column lap-splices failed with concrete spalling, but the structure continued to carry load, c) the 3-sided CFRPs protected the wall bounding columns at the 1st floor and prevented lap-splice failure, d) The "weak" south frame behaved equally well as the "strong" north frame, e) there has not been visible movement at the interface between the wall and the bounding frame, f) the behaviour of the wall was mainly flexural, although on the southframe wall some diagonal cracks appeared, g) higher mode effects appeared in the response of the structure h) some vertical cracks appeared at the connection of the beams to both the exterior column and the wall columns, and i) a horizontal crack appeared at the ground-beam of the walls, and it was the main reason for loss of strength of the south frame.

It existing, ductility and strength deficient structures. The local behaviour of the structure is now under study and numerical models are being developed, so as to be able to propose design guidelines for such a retrofitting method.

A

The research leading to thes Program [FP7/2007-2012] for access to the EUROPEAN LABORATORY FOR STRUCTURAL ASSESSMENT of the EUROPEAN COMMISSION -JOINT RESEARCH CENTRE under grant agreement no.

  2.1b). The dimensions of the columns were 0.25 m by 0.40 m with the long dimension along the plane of loading, while those of the beams 0.25 m by 0.50 m (for both along the plane of the frame and perpendicular to it).

Figure 2 . 1 .

 21 Figure 2.1. Dimensions and layout of the full-scale specimen without the RC infill

Figure 2

 2 Figure 2.2. (a) Reinforcement details for beams, (b) Reinforcement details for columns

Figure 2 . 3 .

 23 Figure 2.3. Elevation of the specimen in the lab. The wall shown on the right of the picture is the south wall and the one on the background is the north wall

Figure 2

 2 Figure 2.4. (a) Dowels and starter bars, (b) Dowels, starter bars and web reinforcement

Figure 2 . 5 .

 25 Figure 2.5. Reinforcing of the column with CFRP to safeguard against column lap-splice failure

Figure 3 . 1 .

 31 Figure 3.1. Experiment at 0.10 g acceleration (a) Variation of the storey displacements with time, (b) Base shear versus top storey displacement for the north and south frames

Figure 3 . 2 .

 32 Figure 3.2. Experiment at 0.25 g acceleration (a) Variation of the storey displacements with time, (b) Base shear versus top storey displacement for the north and south frames

Figure 3 . 3 .Figure 3 . 4 .

 3334 Figure 3.3. Experiment at 0.25 g acceleration (a) 1 st storey interstorey drift, (b) 2 nd storey interstorey drift

Figure 3 . 5 .

 35 Figure 3.5. Base shear versus top storey displacem nt for the south and north wall for the cyclic test.

227887. The authors would also like to acknowledge Prof. Michael Fardis for his invaluable suggestions for the setting-up of the mock-up and testing campaign, as well as Artur Pinto, Georges Magonette, Francisco Javier Molina and all the personnel of the ELSA laboratory for their contribution in building and testing the structure.


Joanatavares Ab

David Mendescosta Ab

Ana Rafaelateixeira

Anabelacordeiro-Da-Silva Abc

In vivo imaging of pathogen homing to the host tissues

Keywords: Live imaging, Arrest, Tropism, Plasmodium, Leishmania, Trypanosoma

Highlights

•A novel method to visualize and quantify the homing of pathogens to tissues in the first minutes following intravascular inoculation.

•Once in the blood circulation, Plasmodium berghei sporozoites specifically home to the liver.

•Hepatotropism of Leishmania infantum amastigotes does not determine increased arrest in the liver shortly after intravascular inoculation.

INTRODUCTION

The use of the host circulatory system to disseminate and infect specific tissues is a recurrent characteristic observed in the lifecycle of innumerous viruses, bacteria and parasites. This tissue tropism is frequently associated with the mode of transmission of these microorganisms, as observed for pathogens that invade salivary glands of arthropod vectors and are transmitted into the host skin with the vector saliva during an infectious bite [START_REF] Ross | The role of the mosquito in the evolution of the malaria parasite[END_REF], [START_REF] Conway | Fikrig Role of the vector in arbovirus transmission[END_REF]. Tissue tropism is also associated with the pathology caused by these microorganisms as known, for example, for the hepatitis or encephalitis-causing viruses [START_REF] Schneider | Schaulies Cellular receptors for viruses: links to tropism and pathogenesis[END_REF]. In some cases this targeted infection can also be asymptomatic as observed for Plasmodium sporozoites, the infective malaria parasite stage inoculated in the skin during a mosquito bite. Following deposition in the skin tissue, sporozoites invade blood vessels [START_REF] Vanderberg | Frevert Intravital microscopy demonstrating antibody-mediated immobilisation of sporozoites injected into skin by mosquitoes[END_REF], [START_REF] Amino | Quantitative imaging of Plasmodium transmission from mosquito to mammal[END_REF] and enter into the blood circulation to specifically and transiently infect the liver [START_REF] Shortt | Pre-erythrocytic stage in mammalian malaria parasites[END_REF], [START_REF]Vanderberg Plasmodium berghei exoerythrocytic forms develop only in the liver Trans[END_REF], [START_REF] Gueirard | Amino Development of the malaria parasite in the skin of the mammalian host[END_REF].

Since mammals have a closed circulatory system, the use of the blood circulation to reach specific tissues requires the arrest of the pathogen in the target organ and the ensuing crossing of the endothelial barrier, which physically separates the blood compartment from the organ parenchyma. Some organs, such as the liver, spleen and adrenal glands, are directly exposed to blood-borne pathogens due to the presence of fenestrated vessels, allowing a direct extravasation of small pathogens (e.g., fenestra diameter of hepatic sinusoids:∼100 nm [START_REF] Wisse | The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer[END_REF]), or a local open circulation. Consequently, the arrest of pathogens can be expected to occur through their binding to molecules on the surface of either tissue-specific endothelial cells or resident immune cells, or to molecules of extravascular cells exposed to the blood circulation in the case of fenestrated sinusoids and in the open splenic circulation.

The dissection of the mechanisms used by pathogens to specifically infect a tissue is hampered by the lack of an easy and quantitative method to assess this first step of arrest in the host organs following blood dissemination. The localization and quantification of pathogens in tissues usually requires the dissection of organs and the measurement of pathogen load in the excised samples by PCR, radioactive labeling, subinoculation or in vitro culture. On the other hand, bioluminescence imaging can reveal the distribution of living luciferase-expressing cells in a non-invasive and quantitative manner in the whole body of small hosts, such as mice. This technique, which is based on an enzymatic reaction that generates photons following the oxidation of a substrate, has been frequently applied to study Plasmodium behaviour [START_REF] De Niz | An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle[END_REF] in the blood [START_REF] Franke-Fayard | Waters Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration[END_REF], [START_REF] Franke-Fayard | Real-time in vivo imaging of transgenic bioluminescent blood stages of rodent malaria parasites in mice[END_REF], liver [START_REF] Mwakingwe | Noninvasive realtime monitoring of liver-stage development of bioluminescent Plasmodium parasites[END_REF], [START_REF] Ploemen | Fayard Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging[END_REF], [START_REF] Yang | Boddey Cell traversal activity is important for Plasmodium falciparum liver infection in humanized mice[END_REF] and skin of rodents [START_REF] Gueirard | Amino Development of the malaria parasite in the skin of the mammalian host[END_REF], [START_REF] Matsuoka | Visualization of Malaria parasites in the skin using the luciferase transgenic parasite, Plasmodium berghei[END_REF]. The firefly luciferase has been preferentially used for in vivo studies when compared to the NanoLuc and Gaussia luciferase, which despite emitting more light in vitro, seem to be less efficient in vivo [START_REF] Stacer | NanoLuc reporter for dual luciferase imaging in living animals[END_REF]. This is in part due to the higher absorption and scatter by the host tissues of the blue light emitted by their substrates [START_REF] Jacques | Optical properties of biological tissues: a review[END_REF]. Accordingly, increased in vivo sensitivity has been successfully achieved by the red-shifted variant of firefly luciferase [START_REF] Foucault | Courvalin In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice[END_REF], [START_REF] Mclatchie | Highly sensitive in vivo imaging of Trypanosoma brucei expressing "red-shifted" luciferase[END_REF]. Here, using bioluminescence imaging we demonstrate the specific homing of firefly luciferase-expressing Plasmodium berghei sporozoites to the liver following the first minutes of their hematogenous dissemination.

MATERIAL AND METHODS

Parasites.

Throughout the experiments parasites used include Plasmodium berghei ANKA strain clone 676cl1 expressing a GFP-Luciferase fusion gene via the pbef1 α promoter [START_REF] Franke-Fayard | Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites[END_REF], Trypanosoma brucei brucei Lister 427 expressing the redshifted luciferase gene (PpyRE9h) flanked by 5′VSG/3′tubulin [START_REF] Mclatchie | Highly sensitive in vivo imaging of Trypanosoma brucei expressing "red-shifted" luciferase[END_REF], [START_REF] Graça | Cordeiro-da-Silva Activity of bisnaphthalimidopropyl derivatives against Trypanosoma brucei[END_REF], a cloned line of Leishmania infantum (MHOM/MA/67/ITMAP-263) expressing luciferase under the control of the intergenic region of α-tubulin [START_REF] Sereno | Ouellette DNA transformation of Leishmania infantum axenic amastigotes and their use in drug screening[END_REF].

To obtain P. berghei (Pb) sporozoites, female Anopheles stephensi mosquitoes (SDA 500 strain) reared in the Center for the Production and Infection of Anopheles at the Pasteur Institute were infected 3-4 days after emergence and kept as previously described [START_REF] Amino | Imaging malaria sporozoites in the dermis of the mammalian host[END_REF]. Sporozoites were collected from salivary glands 21-28 days after the infectious blood meal and kept on ice in PBS until inoculated into mice. Merozoites were obtained by culturing infected mouse blood in RPMI 1640 (Lonza) supplemented with 20% heat inactivated fetal bovine serum (Biowest) and 50 μg/ml neomycin (Sigma) and flushed 90 s at 1.5-2 bar pressure with a gas mixture of 5% CO2, 5% O2 and 90% N2 for 16 h at 37 °C and under shaking. Mature schizonts were separated using a Nycodenz (Axis Shield) gradient and merozoites were isolated by filtration of schizonts through a 1.2 μm filter [START_REF] Janse | Waters High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei[END_REF].

The bloodstream forms of T. b. brucei (Tb) Lister 427 were grown in HMI-9 medium (Sigma) supplemented with 10% heat inactivated fetal bovine serum (Biowest) and 100 IU/ml of penicillin/streptomycin (Lonza) at 37 °C, 5% CO2 in a humidified atmosphere [START_REF] Loureiro | Cordeiro-da-Silva Knockdown of asparagine synthetase A renders Trypanosoma brucei auxotrophic to asparagine[END_REF].

L. infantum (Li) axenic amastigotes were cultured at 37 °C, and 5% CO2 in a cell-free medium called MAA/20 (medium for axenic amastigotes growth) as described previously [START_REF] Tavares | Cordeiro-da-Silva Bisnaphthalimidopropyl derivatives as inhibitors of Leishmania SIR2 related protein 1[END_REF]. Briefly, MAA/20 consisted of modified medium 199 (Invitrogen) with Hank's Balanced Salt Solution supplemented with 0.5% trypto-casein (Oxoid), 15 mM d-glucose (Sigma), 5 mM glutamine (Lonza), 4 mM NaHCO3, 0.023 mM bovine hemin (Fluka) and 25 mM HEPES (Lonza) to a final pH of 6.5 and supplemented with 20% heat inactivated fetal bovine serum (Biowest).

Bioluminescence imaging. Parasite loads of mice infected with luciferase-expressing parasites were assessed by bioluminescence imaging using the IVIS Lumina LT system (Perkin Elmer). Prior to infection, animals had their ventral fur shaved with an appropriate clipper. In all the models, mice were infected following the intravenous injection into the tail vein of 100 μl of parasite suspension.

For the experiments with Pb, C57BL/6 mice were typically infected with either ∼1.5 × 10 5 salivary gland sporozoites or ∼2 to 6 × 10 7 blood merozoites. Infections with Tb bloodstream forms or with Li axenic amastigotes were performed in BALB/c mice following the inoculation of respectively 1 × 10 6 or 1 to 2 × 10 8 parasites. Immediately after parasite inoculation, mice were anesthetized with 2.5% isoflurane and four minutes after the infection, injected subcutaneously with d-luciferin (2.4 mg, Perkin Elmer). Mice were then transferred to the stage of an intensified charge-coupled device photon-counting video camera box where anaesthesia was maintained with 2.5% isoflurane. After a three minute incubation allowing the distribution of the substrate in the body of the anesthetized animals, a five minute signal acquisition controlled by the Living Image software (Perkin Elmer) was initiated. At the end of this period animals returned to their cage and recovered from the anaesthesia. Measurement of infection by bioluminescence one day later was performed as described above.

The detection of the bioluminescence signals by the system resulted in the generation of signal maps automatically superimposed to the grey-scale photograph of the mice. The quantifications were performed using the Living Image software (Perkin Elmer). Bioluminescent signals on the regions of interest (ROI) encompassing most of the ventral view of the animal body, thorax, liver, spleen and lower abdomen were manually defined as described below and applied to all animals. The total flux (photons/s) and average radiance (photons/s/cm 2 /steradian) within these ROIs was automatically calculated. ROI background signal was obtained from imaging non-infected mice that received d-luciferin as described above. The total flux or the average radiance background signal of the respective ROIs were subtracted to the respective total flux or average radiance measured on infected animals. The percentage of bioluminescence in the defined ROIs (thorax, liver, spleen and lower abdomen) was calculated by dividing the total flux of the respective ROI by the total flux of the ventral animal body ROI.

Statistical analysis.

All the statistical analysis and graphical representation of the data were performed using GraphPad Prism 6 software. Students t-test or a two-way analysis of variance (ANOVA) with Sidak's multiple comparisons test was performed and statistical significance was asserted whenever the value of p was lower than 0.05 by * (p < 0.05), * * (p < 0.01), * * * (p < 0.001) and * * * * (p < 0.0001).

RESULTS AND DISCUSSION

In this study we applied 2D bioluminescence imaging to investigate the accumulation of bloodcirculating Pb bioluminescent sporozoites in the liver of living mice during the first minutes following their intravascular inoculation, herein defined as homing. The pattern of parasite homing was also further compared to the successful establishment of infection as measured by the distribution of the parasite bioluminescence signal one day later.

To assess the unambiguous accumulation of sporozoites in the liver, we use different firefly luciferase-expressing blood-borne parasites with relatively similar sizes and with different specificities and capacities of invasion.

As shown in the Fig. 1A, once in the blood circulation, Pb sporozoites and merozoites invade and develop, respectively, inside hepatocytes and erythrocytes [START_REF] Ménard | Amino Looking under the skin: the first steps in malarial infection and immunity Nat[END_REF]; Li amastigotes are known to survive and multiply inside phagocytic cells, such as macrophages and dendritic cells of the liver, spleen and bone marrow [START_REF] Kaye | Scott Leishmaniasis: complexity at the host-pathogen interface Nat[END_REF], [START_REF] Mccall | Matlashewski Determinants of the development of Visceral Leishmaniasis disease[END_REF], and extracellular replicative Tb bloodstream forms live in the blood, lymphatic system and interstitial space of organs [START_REF] Kennedy | Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness)[END_REF]. Therefore, the parasites used in this study include pathogens that infect the liver (Pb sporozoites and Li amastigotes), the spleen (Li amastigotes) and the blood of the host (Pb merozoites and Tb bloodstream forms).

Taking advantage of the Living Image software that can superimpose the bioluminescence signal of parasites and the grey-scale photograph of mice, the ventrally exposed body of the host, placed in the dorsal position, was equally divided from the nostril to the perineal body in four rectangular areas, delineating the head, thorax, upper abdomen and lower abdomen (Fig. 1B). Importantly, the middle line of this zone, laying between the thorax and the upper abdominal area, coincided with the sternum, and therefore served as a robust fiducial mark. The total area of these four rectangular regions of interest (ROIs) was used to determine the total bioluminescence signal emitted by the parasites in the ventral view of the animal body (ventral ROI). Inside these rectangular ROIs and for quantification purposes, elliptical ROIs encompassing organs well known to be infected by some of the parasites listed in the Fig. 1A were defined using the Living Image software and used to quantify the relative distribution of parasite load by bioluminescence at early (homing) and late (infection) time points.

In the upper abdomen, the ROI for quantifying parasite load in the liver was defined by infecting mice with luciferase-expressing Pb sporozoites. Two days after sporozoite inoculation in the host, when parasites reach the maximum of their intracellular development inside hepatocytes, we averaged the bioluminescence signal of three infected mice and determined the corresponding ROI of the infected liver (Fig. 1B, hepatic infection, left panel). As expected, the liver was the only visceral organ to emit photons and the hepatic ROI was placed just bellow the sternum matching the model illustrated in the IVIS mouse atlas model (Fig. 1B, right panel). The position of the liver using bioluminescence was confirmed using luciferase-expressing Li amastigotes. Accordingly, the hepatic ROI obtained using amastigote infection corresponded to the bioluminescence signal of sporozoite infection (Fig. 1B, left and middle panel). In addition, amastigotes also infected a region adjacent to and distinct from the hepatic ROI, which according to its anatomical position and the tissue tropism of amastigotes should correspond to the ventral extremity of the spleen (Fig. 1B, splenic infection, middle panel). To quantify the relative load of parasites in the thorax, which contains the lungs, thymus and heart, and in the lower abdomen, which contains the intestine and bladder among other organs, the elliptical area of the hepatic ROI defined using the Pb sporozoite infection was transposed to the thorax and lower abdomen rectangular areas, and was defined as thoracic and lower abdominal ROIs, respectively (Fig. 1B, right panel).

The homing and infectivity of the different parasites were determined by measuring the intensity of the parasite bioluminescence throughout the anatomical regions defined above (thoracic, hepatic, splenic and lower abdominal ROIs) relative to that of the ventral view of the animal body (ventral ROI). For that, the total flux (photons/s) of the respective ROI was divided by the total flux of the ventral ROI, previously subtracted by the background values calculated from non-infected animals. In addition, the parasite development in the tissues was Once in the blood circulation, Plasmodium sporozoites are known to transform and multiply into merozoites, the red blood cells infective forms, exclusively inside hepatocytes [START_REF] Shortt | Pre-erythrocytic stage in mammalian malaria parasites[END_REF], [START_REF]Vanderberg Plasmodium berghei exoerythrocytic forms develop only in the liver Trans[END_REF], [START_REF] Gueirard | Amino Development of the malaria parasite in the skin of the mammalian host[END_REF].

Sporozoites can be detected in the liver a few minutes after intravascular inoculation [START_REF] Matsuoka | Visualization of Malaria parasites in the skin using the luciferase transgenic parasite, Plasmodium berghei[END_REF], [START_REF] Shin | Terzakis Direct infection of hepatocytes by sporozoites of Plasmodium berghei[END_REF].

After two hours, most of injected sporozoites accumulate in the liver [START_REF] Xu | Plasmodium yoelii: influence of immune modulators on the development of the liver stage Exp[END_REF]. Therefore, prior to invading hepatocytes, sporozoites passively transported in the blood must arrest in the liver sinusoids. This arrest has been proposed to be due to both a slower blood circulation speed in the sinusoids and the interaction of the most abundant sporozoite surface protein, the circumsporozoite protein (CSP) with the highly sulphated proteoglycans that protrude through the fenestra of endothelial cells [START_REF] Ménard | Amino Looking under the skin: the first steps in malarial infection and immunity Nat[END_REF], [START_REF] De Niz | Progress in imaging methods: insights gained into Plasmodium biology[END_REF]. Noteworthy, the current model explaining this crucial step is based on indirect experiments interpreting the binding of recombinant proteins to hepatocytes or the outcome of a liver infection by sporozoites. When and how sporozoites arrest in the liver is still an unsettled issue.

To evaluate whether Plasmodium sporozoites specifically home to the liver in the first minutes following their intravascular inoculation in C57BL6 mice, the distribution and relative intensity of the bioluminescence signal emitted by luciferase-expressing sporozoites was calculated in the previously defined ROIs and compared to that obtained after injection of luciferaseexpressing blood merozoites. Using this method, ∼70% of the total bioluminescence signal detected 7 min after the intravenous injection of sporozoites was found in the liver ROI, contrasting with only ∼20% after injection of merozoites (Fig. 2A andB). While sporozoite bioluminescence is clearly concentrated in the liver, the bioluminescence of merozoites is seen all over the body suggesting parasites are in circulation. Indeed, free merozoites recognize, attach, and invade red blood cells and this process occurs rapidly, in the order of minutes, as the antigens present on the parasite surface are susceptible to immune attack [START_REF] Cowman | Invasion of red blood cells by malaria parasites[END_REF]. Inside red blood cells, they develop through the ring, trophozoite, and schizont stages, replicating to produce between 16 and 32 daughter merozoites. After egressing from the infected cell and invading another red blood cell, these merozoites initiate a new cycle of multiplication. This cyclic process is responsible for the clinical manifestations of malaria and lasts nearly 24 h in rodents [START_REF] Cowman | Invasion of red blood cells by malaria parasites[END_REF]. Therefore, one day after merozoite injection, the bioluminescence signal remained distributed all over the animal body while concentrated in the liver of mice infected with sporozoites. Indeed, in the latter ∼90% of the total bioluminescent signal was detected in the liver versus 20% in mice inoculated with merozoites (Fig. 2C andD). Notably, the pattern of parasite distribution in the ROIs, concentrated in the liver after sporozoite injection and disseminated through the body after merozoite injection, did not change substantially from the homing and infection time points. In the first case, this indicates that sporozoites rapidly accumulate in the liver to establish the infection of hepatocytes. In the second, this similarity suggests that merozoites and infected red blood cells are principally circulating in the vasculature. The exponential multiplication of parasites in the liver (Fig. 2E) or in the blood (Fig. 2F) was assessed by quantifying the average radiance of the bioluminescence signal respectively, in the hepatic and ventral ROIs.

In the range of 75,000-300,000 injected sporozoites, a linear relationship is observed between the number of injected parasites and the average bioluminescence quantified in the hepatic ROI in the first minutes post-inoculation (Fig. 2G, R 2 = 0.99). Therefore, this method can be used to quantify not only changes in the parasite relative distribution (homing pattern) but also in the sporozoite load, early during the hepatic infection. To confirm the specificity of sporozoite homing to the liver, another blood circulating bioluminescent parasite, the extracellular replicative monomorphic Tb was intravenously injected into BALB/c mice. The relative distribution of the bioluminescence signal of Tb bloodstream forms was quantified using the pre-defined ROIs (Fig. 3) and compared to that obtained using Plasmodium parasites (Fig. 2). Immediately after intravascular inoculation, the bioluminescence signal was detected all over the body as observed following Pb merozoite injection. Similar percentages of bioluminescence in the hepatic (∼20%) and in the other ROIs strongly indicate that both Pb merozoites and Tb bloodstream forms are circulating with the same pattern in the blood, presumably without any detectable specific arrest in the organs (Fig. 3A andB). Consequently, the values obtained in the ROIs should correspond approximately to the percentual blood volume of these regions. Indeed, the value of 20% of total bioluminescence in the liver is in the range (14-30%) of the total blood percentage found in the liver [START_REF] Greenway | Capacitance effects and blood reservoir function in the splanchnic vascular bed during non-hypotensive haemorrhage and blood volume expansion in anaesthetized cats[END_REF], [START_REF] Lautt | Hepatic Circulation: Physiology and Pathophysiology, San Rafael (CA)[END_REF]. The distribution of Tb bloodstream forms in the ROIs did not also change substantially at the homing and infection time points, except for the splenic ROI. Owing to parasite multiplication the average radiance in the thorax, liver and lower abdomen increased ∼15-fold from the first minutes after infection to day 1, contrasting with the 90-fold observed in the splenic ROI (Fig. 3C).

To determine if the capacity to infect the liver is associated with the homing of parasites to this organ as seen for sporozoites, we assess the distribution pattern of bioluminescent Li amastigotes known to infect macrophages including those of the liver, as depicted in the Fig. 1B. Shortly after intravenous injection of amastigotes into BALB/c mice, bioluminescence is mostly observed in the thorax, presumably in the right and left lungs given the anatomical position and shape of the signal, as well as, in the spleen and in the liver (Fig. 4A). The spleen and the liver are known to harbour resident macrophages, which can contact the blood and clear particulate material from the circulation [START_REF] Biozzi | Quantitative study of the granulopectic activity of the reticulo-endothelial system: II: A study of the kinetics of the granulopectic activity of the R.E.S. in Relation to the Dose of Carbon Injected. Relationship between the weight of the organs and their activity Br[END_REF]. Interestingly, the bioluminescence signal of amastigotes in the liver, ∼20% of the total signal, did not statistically differ from the percentage observed using injection of blood-circulating Tb bloodstream forms and Pb merozoites (Fig. 5A), in clear contrast with the 70% of Pb sporozoites that accumulated in the liver. These results show that despite infecting the liver, Li amastigotes do not significantly accumulate in this organ shortly after intravascular inoculation. On the other hand, the accumulation of amastigotes in the spleen was significantly higher than those of Pb merozoites and Tb bloodstream forms, indicating a preference of Li amastigotes to accumulate in this organ. Regarding the distribution of amastigotes in the thoracic region, despite the distinctive pulmonary pattern, the percentage of bioluminescence was not statistically different from Tb injection but differed from Pb merozoites, complicating the interpretation of the data (Fig. 5A). Importantly, the percentage of the total bioluminescence detected in the thorax decreased from ∼40 to 5% when comparing the first minutes after Li amastigotes inoculation to day 1 (Fig. 4B), while it increased in the liver and spleen from ∼20 to >40% and from ∼20 to 35%, respectively (Fig. 5). Analysis of the average radiance shows a significant reduction in the signal detected in the thorax at day 1 comparing to 7 min post-inoculation indicating that the parasites initially observed in the thorax were cleared in the lungs in the case of a possible arrest in this region or they were simply removed from the circulation by phagocytes of the liver, spleen, bone marrow or blood (Fig. 4C).

The liver can rapidly clear microorganisms from the blood circulation [START_REF] Benacerraf | A quantitative study of the kinetics of blood clearance of P32-labelled Escherichia coli and Staphylococci by the reticuloendothelial system[END_REF] through the scavenging activity of Kupffer [START_REF] Brunner | Benacerraf Blood clearance of P32-labeled vesicular stomatitis and Newcastle disease viruses by the reticuloendothelial system in mice[END_REF], [START_REF] Zeng | Kubes CRIg functions as a macrophage pattern recognition receptor to directly bind and capture blood-borne gram-positive bacteria[END_REF] and endothelial cells [START_REF] Ganesan | Rapid and efficient clearance of blood-borne virus by liver sinusoidal endothelium[END_REF]. This clearance can be extremely fast, mediated by the direct recognition of pathogens [START_REF] Zeng | Kubes CRIg functions as a macrophage pattern recognition receptor to directly bind and capture blood-borne gram-positive bacteria[END_REF], or relatively slower, mediated by the recognition of host molecules bound to the opsonized pathogens [START_REF] Broadley | Dual-track clearance of circulating bacteria balances rapid restoration of blood sterility with induction of adaptive immunity[END_REF]. However, some pathogens, such as Li amastigotes, can survive to this clearing activity and exploit this scavenging function to arrest and specifically infect the liver. Plasmodium sporozoites are known to be captured by KCs in the lumen of liver sinusoids [START_REF] Tavares | Amino Role of host cell traversal by the malaria sporozoite during liver infection[END_REF], presumably by the interaction of CD68 with the parasite GAPDH [START_REF] Cha | Jacobs-Lorena Identification of GAPDH on the surface of Plasmodium sporozoites as a new candidate for targeting malaria liver invasion[END_REF], [START_REF] Cha | Jacobs-Lorena CD68 acts as a major gateway for malaria sporozoite liver infection[END_REF]. After a transitory interaction with KCs, most sporozoites escape from a phagocytic death by traversing these cells and either return to the blood circulation or invade the hepatic parenchyma [START_REF] Tavares | Amino Role of host cell traversal by the malaria sporozoite during liver infection[END_REF]. However, KCs do not seem to be directly involved in the homing of sporozoites to the liver, since their depletion by liposome-encapsulated clodronate, rather increases the hepatic infection [START_REF] Vreden | Broek Kupffer cell elimination enhances development of liver schizonts of Plasmodium berghei in rats[END_REF], [START_REF] Baer | Frevert Schnieder Kupffer cells are obligatory for Plasmodium yoelii sporozoite infection of the Liver Cell[END_REF]. In vivo imaging indeed indicates that sporozoites bind to endothelial cells prior to the crossing of the sinusoidal barrier after interacting, or not, with KCs [START_REF] Tavares | Amino Role of host cell traversal by the malaria sporozoite during liver infection[END_REF], [START_REF] Frevert | Intravital observation of Plasmodium berghei sporozoite infection of the liver[END_REF]. The lack of the intercellular adhesion molecule (ICAM) 1 and 2, complement factors C3 and C4 [START_REF] Sultan | Nussenzweig Sporozoites of Plasmodium yoelii infect mice with targeted deletions in ICAM-1 and ICAM-2 or complement components C3 and C4[END_REF], LDL receptor related protein [START_REF] Marshall | Plasmodium sporozoites invade cells with targeted deletions in the LDL receptor related protein[END_REF], CD36 [START_REF] Sinnis | Febbraio Plasmodium yoelii sporozoites infect CD36-deficient mice[END_REF] or macrophage scavenger receptors (SR) AI and AII [START_REF] Cunha-Rodrigues | Infection by and protective immune responses against Plasmodium berghei ANKA are not affected in macrophage scavenger receptors A deficient mice[END_REF] does not change sporozoite infectivity in mice, indicating that these molecules are also unlike to play a role in the sporozoite homing. On the other hand, the sulfated polysaccharides, dextran sulfate and fucoidan, can inhibit sporozoite infectivity in vivo [START_REF] Pancake | Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates[END_REF], while the role of SR-BI still remains controversial [START_REF] Yalaoui | Froissard Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection[END_REF], [START_REF] Rodrigues | Mota Host scavenger receptor SR-BI plays a dual role in the establishment of malaria parasite liver infection[END_REF], [START_REF] Foquet | Anti-CD81 but not anti-SR-BI blocks Plasmodium falciparum liver infection in a humanized mouse model[END_REF]. In all these cases, sporozoite infectivity is assessed by the measurement of parasite load in the liver two days post-infection, rendering impossible the discrimination of an effect of these molecules on sporozoite homing from an inhibitory effect on the invasion and/or development inside hepatocytes. This novel homing method can therefore be used to reassess the impact of these molecules and cells in the specific arrest of sporozoites in the liver. For example, combining this method with the use of mice lacking CD68 [START_REF] Song | Schindler Deletion of the murine scavenger receptor CD68[END_REF], stellate cells [START_REF] Puche | A novel murine model to deplete hepatic stellate cells uncovers their role in amplifying liver damage in mice[END_REF] or KCs [START_REF] Baer | Frevert Schnieder Kupffer cells are obligatory for Plasmodium yoelii sporozoite infection of the Liver Cell[END_REF], [START_REF] Frevert | Intravital observation of Plasmodium berghei sporozoite infection of the liver[END_REF], or alternatively, with the targeting of putative arrest/invasion receptors, such as CD81 [START_REF] Silvie | Mazier Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity[END_REF], SR-BI [START_REF] Yalaoui | Froissard Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection[END_REF], [START_REF] Rodrigues | Mota Host scavenger receptor SR-BI plays a dual role in the establishment of malaria parasite liver infection[END_REF], [START_REF] Foquet | Anti-CD81 but not anti-SR-BI blocks Plasmodium falciparum liver infection in a humanized mouse model[END_REF] and EphA2 [START_REF] Kaushansky | Malaria parasites target the hepatocyte receptor EphA2 for successful host infection[END_REF], will allow the elucidation of the role played by these factors in the sporozoite homing. Complementary to this approach, mutant sporozoites lacking adhesion molecules or neutralizing antibodies can be used to determine the impact of parasite proteins on the specific accumulation of sporozoites in the liver. Finally, this method can be adapted for the quantification of the homing of bioluminescent humaninfecting sporozoites to the liver of humanized mice harboring human hepatocytes [START_REF] Yang | Boddey Cell traversal activity is important for Plasmodium falciparum liver infection in humanized mice[END_REF], [START_REF] Foquet | Anti-CD81 but not anti-SR-BI blocks Plasmodium falciparum liver infection in a humanized mouse model[END_REF].

CONCLUSION

A complete picture of increased arrest and subsequent development of parasites in organs, defined here respectively, as homing and infection is shown in the Fig. 5. The increased arrest leading to parasite accumulation in organs was defined by statistical comparison with the distribution of blood-circulating parasites, Pb merozoites and Tb bloodstream forms. Based on this parameter, we conclude that Pb sporozoites specifically home to the liver in contrast to Pb merozoites, Tb bloodstream forms and even liver-infecting Li amastigotes. Interestingly, using this methodology, we observe the splenic and hepatic infection of amastigotes, but only the parasite accumulation in the spleen statistically differs from the distribution of both bloodcirculating control parasites. These results allow us to clearly discriminate at an early time point of analysis the increased arrest (homing step) from the regular arrest of parasite in organs, both leading to a specific tissue infection (tissue tropism).

The combination of this non-invasive and quantitative imaging method with genetically modified pathogens and transgenic mouse strains lacking surface molecules on putative target cells will be key to dissect the molecular mechanisms involved in the homing of microorganisms to tissues following hematogenous dissemination. Finally the use of hosts immunized against pathogen molecules involved in this arrest step certainly will assist in the design of better strategies to prevent infection.

TABLES & FIGURES

  Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 evaluated by comparing the average radiance (photons/s/cm 2 /steradian) of ROIs at early (homing) and late (infection) time points.

  Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00

Figure 1 .

 1 Figure 1. Defining pathogens and regions of interest in the host. (A) Schematic representation of parasite species and stages used in this work to evaluate by ventral body in vivo bioluminescence imaging the parasite homing and infectivity to the host organs. (B) Defining the Regions of Interest (ROIs) used for quantitative analysis. The ventral view of the animal body was divided into four main rectangular regions to delineate the head, thorax, upper abdomen and lower abdomen. Inside these regions and for quantification purposes elliptical ROIs were drawn with Living Image software. The liver and spleen ROIs in the upper abdomen were defined based on the bioluminescence signal of mice infected with P. berghei sporozoites (day 2; left) and L. infantum amastigotes (day 2; middle). The schematic mice model (right) was obtained from the IVIS digital atlas.

Figure 2 . 00 Figure 3 .

 2003 Figure 2. P. berghei salivary gland sporozoites home to the liver immediately after intravascular inoculation. C57BL/6 mice were injected intravenously with luciferase expressing salivary gland sporozoites (PbSPZ) or blood merozoites (PbMZ), and the distribution of parasites was evaluated by bioluminescence imaging at 7 min (A-B) and day 1 (C-D). (B;D) The percentage of the bioluminescence signal in the thorax, liver, spleen and lower-abdomen was calculated dividing the total flux (photons/s) of the respective ROIs by that of the ventral ROI previously subtracted by the background values calculated from non-infected mice. The graphs show the mean + standard deviation (n = 3) and are representative of at least 2 independent experiments. Statistical significance between sporozoites and merozoites was determined using 2-way ANOVA with Sidak's multiple comparisons test. (E) Quantification at 7 min and day 1 of liver infection in average radiance (photons/s/cm 2 /steradian) following sporozoite injection. (F) Quantification at 7 min and day 1 of whole body infection in average radiance (photons/s/cm 2 /steradian) following merozoite injection. (E-F) Statistical significance calculated by Students t-test. ** p < 0.01; *** p < 0.001; **** p < 0.0001. (G) Quantification of the liver average radiance (photons/s/cm 2 /steradian) at 7 min following intravenous injection of different numbers of sporozoites. The graph shows the linear regression of the bioluminescent signal with the 95% confidence bands (R 2 = 0.99) Symbols represent mean ± standard deviation (n = 3).

Figure 4 . 00 Figure 5 .

 4005 Figure 4. Homing and infection of L. infantum amastigotes. (A-C) BALB/c mice were injected intravenously with luciferase expressing axenic Li amastigotes and the distribution of parasites evaluated by bioluminescence imaging at 7 min and at day 1. (B) The percentage of the bioluminescence signal in the thorax, liver, spleen and lowerabdomen was calculated dividing the total flux (photons/s) of the respective ROIs by that of the ventral ROI previously subtracted by the background values calculated from non-infected mice. (C) Quantification of the bioluminescence signal in average radiance (photons/s/cm 2 /steradian) in the thorax, liver, spleen and lower abdomen. (B-C) The graphs show the mean + standard deviation (n = 3) and are representative of 2 independent experiments. Statistical significance between 7 min and day 1 for the several regions of interest was determined using 2-way ANOVA with Sidak's multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001.

  All experiments were carried out in accordance with the IBMC.INEB Animal Ethics Committees and the Portuguese National Authorities for Animal Health guidelines or in accordance with the Animal Care and Use Committee of Institut Pasteur both according to the statements on the directive 2010/63/EU of the European Parliament and Council. Four to six weeks old C57BL/6, Swiss, NMRI or BALB/c mice were purchased from Elevage Janvier or Charles River.

Mice.

ACKNOWLEDGEMENTS

We would like to acknowledge the team of the Center of Production and Infection of Anopheles (CEPIA), Institut Pasteur, in particular Marek Szatanik, Catherine Thouvenot and Sylvain Golba for providing the Anopheles stephensi female mosquitos; the team of the Platform of Dynamic Imaging, Institut Pasteur, in particular Spencer Shorte and Marie-Anne Nicola for the help with bioluminescence imaging; Martin Taylor from the London School of Hygiene and Tropical Medicine for kindly providing the construct used to generate the T. b. brucei Lister 427 strain expressing the red-shifted luciferase.

FUNDING

This work was supported by funds from the Fundação para a Ciência e Tecnologia (FCT)/Ministério da Educação e Ciência (MEC) co-funded by FEDER (EXPL/JTAVARES-IF/00881/2012/CP0158/CT0005, EXPL/IMI-MIC/1331/2013) under the Partnership agreement PT2020, through the Research Unit No.4293, the Institut Paris -Paris, the French National Research Agency (Grant No. ANR-10-JCJC-1302-PlasmoPEP), the French Government's Investissement d'Avenir program, Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (Grant No. ANR-10-LABX-62-IBEID). JT is an Investigator FCT funded by National funds through FCT and co-funded through European Social Fund within the Human Potential Operating Programme. DC is funded by FCT (SFRH/BD/123734/2016).


Joseph F Messina

Wave-particle duality and the unitary nature of electric charge

Keywords: 04, 20, Cv -Fundamental problems and general formalism 03, 65, Ta -Foundations of quantum mechanics 12, 38, Qk -Experimental tests

Wave-particle duality and the unitary nature of electric charge

Joseph F.

Introduction. -The fact that quantum mechanics was developed in the absence of a fundamental understanding of the unitary nature of electric charge must be regarded as one of the most remarkable achievements in the annals of theoretical physics. Nevertheless, the question of why the unit of electric charge e, which is considered to be a structureless entity, is observed to be identical on all charged particles, despite wide variations in their other characteristics, remains one of the great unanswered questions of particle physics. Indeed, aside from the fact that electric charge is the source of the electromagnetic interaction, and its conservation is understood, it stubbornly continues to elude our understanding at a fundamental level, which suggests that there is no good reason to maintain the prevailing view that electric charges are structureless entities. The present paper is a natural outgrowth of the algebraic relationship between the Stoney system of absolute units and quantum theory, whose interpretation of sub-microscopic phenomena has shown itself to be so wonderfully adept at rendering transparent such fundamental problems.

Passage to a sub-Planckian length scale. -What quantities one chooses to regard as fundamental depends on the domain one seeks to investigate. For example, if one seeks to set the scale for atomic and molecular sizes, then the Bohr radius 10 -9 cm appears as the appropriate fundamental unit of length. However, if one's objective is to set the scale for determining the nature of the elementary charge then one finds that the Stoney scale [1] is the (a) E-mail: jfmessina77@yahoo.com appropriate scale since in addition to giving prominence to the electromagnetic interaction it has the advantage of being an order of magnitude smaller than the Planck length, which affords the possibility of investigating sub-Planckian spacetime. Its base units for mass, length, and time consist of the electric charge (e), the Newtonian gravitational constant (G), and the velocity of light (c). By taking powers and ratios, Stoney was able to construct a system of absolute units, which has the form

M 0 = e 2 G 1/2 = 1.86 × 10 -6 g (1)
L 0 = e 2 G c 4 1/2 = 1.38 × 10 -34 cm (2) T 0 = e 2 G c 6 1/2 = 4.60 × 10 -45 s (3)
where we have employed the modern values (NIST), e = 4.803 × 10 -10 g 1/2 cm 3/2 s -1 G = 6.674 × 10 -8 cm 3 g -1 s -2 c = 2.998 × 10 10 cm s -1 .

As we shall see below, these absolute units are especially revealing when applied to quantities such as charge and mass.

Conceptualization. -We shall take eq. ( 1) as the proper starting point by expressing the gravitational mass equivalent of the elementary charge's electrostatic potential energy in terms of Einstein's mass-energy relation

E 0 = M 0 c 2 (4) = 1.67 × 10 15 erg
which is advantageous, since it allows us to substitute the elementary charge's electrostatic potential energy with its gravitational mass equivalent, M 0 c 2 , in the context of the Stoney system of absolute units whose mathematical simplicity and unity provides a road map to a quantumtheoretical formulation of the elementary charge that enters its tenets, in a natural manner, as the product of the energy M 0 c 2 and the absolute unit of time T 0 , which has the dimensions of an "action," and is quantitatively equal to the ratio of two universal constants, namely e 2 /c, which is given added significance by the fact that it is inseparably linked to M 0 c 2 . We are thus as good as forced to consider its implications for the corresponding momentum, M 0 c, since energy and momentum are fundamentally linked in the closest possible way. Hence, it is essential that both be considered on a completely equal footing, which implies that it should be possible to express the "action" constant e 2 /c in terms of the product of the momentum M 0 c and the sub-Planckian absolute unit of length L 0 , in the form

(M 0 c) L 0 = 7.70 × 10 -30 momentum • length = e 2 c (5)
where [(M 0 c) L 0 ] is the emergent sub-Planckian length gravitational mass equivalent of the electrostatic quantum of "action" e 2 /c. The significant aspect of eq. ( 5) is the quantization of the absolute unit of length from the more exact expression

L 0 = e 2 /c (M 0 c) (6)
which is physically meaningful since it sets the scale at which something peculiar happens to the structure of space. More succinctly, it provides a basis for formulating a consistent quantum-theoretical conceptualization of the elementary charge in a natural and unambiguous manner that corresponds to reality. It is tacitly understood from eq. ( 5) that the elementary charge's experimentally determined value can be expressed as a squared quantity, in the form

e 2 = M 0 c 2 L 0 . (7)
In order to give physical meaning to this expression it is necessary to consider the nature of electric charge in an entirely new way that is consistent with the general principles of quantum theory, which tend to be simpler the greater their generality. It is therefore quite reasonable to assert, upon close inspection, that eq. ( 6) clearly suggests that the elementary charge can be characterized as a quantum-theoretical wave-particle entity at the sub-Planckian length L 0 . Since in quantum theory the simplest assumption often turns out to be the most natural, we shall assume, on the basis of eq. ( 6), that at the sub-Planckian length, L 0 , the fabric of spacetime vibrates with an intrinsic energy equivalent to the gravitational mass equivalent of the elementary charge's electrostatic potential energy, M 0 c 2 . We are then able to ascribe to the fabric of spacetime a vibrational frequency, v 0 , that is inversely proportional to the absolute unit of time, T 0 , and directly proportional to M 0 c 2 . The vibrational frequency and energy can then be linked through the equation

jν 0 = M 0 c 2 (8)
where, for simplicity of presentation, j denotes the sub-Planckian scale quantum of "action" [(M 0 c) L 0 ], deriving from eq. ( 5), and jν 0 is the quantized intrinsic vibrational energy of the fabric of spacetime. This is the decisive quantum-theoretical condition that equates the intrinsic vibrational energy of the fabric of spacetime with the elementary charge's electrostatic potential energy. Now, since we know from eq. ( 8) that a quantum of vibrational energy jν 0 has a momentum jν 0 /c denoted by p 0 , and a wavelength that can be expressed in terms of momentum as

λ 0 = j p 0 (9)
we can then directly identify eq. ( 7) with eqs. ( 8) and (9) as a wave-particle phenomenon by demonstrating that the vibrational energy of the fabric of spacetime, per cycle, corresponds to the universal constant e as a squared value, in the form

e 2 = (jν 0 )λ 0 (10)
= 4.80 × 10 -10 esu in quantitative agreement with the experimental value from which it draws its justification.

We have thus achieved an easily interpreted, observationally consistent, wave-particle conceptualization of electric charge as a manifestation of the quantization of the intrinsic vibrational energy of the fabric of spacetime at a sub-Planckian length scale.

Application. -A simple illustration of the application of this conceptualization of electric charge is afforded by the vexing question of the origin of the electron's rest mass. If we subscribe to the view that the electron's rest mass, m e , is entirely electromagneic in origin, then it must have a mass equivalent to

m e = (m e c 2 ) c 2 (12)
that is associated with the momentum

m e c = j r e (13)
which is required for the electron's localization through

m e c 2 = j r e c (14)
where r e is the so-called "classical" electron radius. It then follows, in a natural manner, from eq. ( 14), that the electron has a quantized intrinsic vibrational frequency, ν e , whose value is given by

ν e = m e c 2 j (15) = 1.06 × 10 23 s -1
from which it follows that

jν e = m e c 2 (16)
which links the electron's intrinsic vibrational frequency ν e to its mass-energy m e c 2 . Hence,

m e = jν e c 2 (17)
= 9.11 × 10 -28 g which is easily seen to be equivalent to our basic equation ( 12). This result furnishes strong support for both the electromagnetic origin of the electron's rest mass, and the wave-particle conceptualization of electric charge.

Conclusion.

-It is possible, in the context of the Stoney system of absolute units, to extend the quantum mechanical concept of wave-particle duality to the elementary charge. The elementary processes imposed by the Stoney system of units on the elementary charge leads, in the simplest possible manner, to a conceptualization of the elementary charge as a manifestation of the quantization of the intrinsic vibrational energy of the fabric of spacetime at a sub-Planckian length scale; which is particularly satisfying since it provides a basis for an exact evaluation of the electron's intrinsic vibrational frequency, whose validity can be subjected to experimental verification.

To conclude, the foregoing considerations demonstrate, in an instructive manner, that no theory of particles that deals only with particles will ever explain particles.

= 2 .

 2 30 × 10 -19 erg • cm and e = (jν 0 )λ 0