P Maurice

S Lemonnier

N Kohili

L Cavagnac

G Mornieux

Biomechanical effects of using a passive upper-limb exoskeleton to assist firefighters during vehicle extrication maneuver

Keywords: Exoskeleton, Ergonomics, Physiological Measures

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Firefighters' work involves numerous strenuous tasks, which often lead to the development of musculoskeletal disorders (MSDs) (Kodom-Wiredu 2019; [START_REF] Nazari | Prevalence of musculoskeletal symptoms among Canadian firefighters[END_REF][START_REF] Frost | Firefighter injuries are not just a fireground problem[END_REF]. Among these tasks, vehicle extrication is particularly demanding because it requires the manipulation of heavy cutting tools (20 to 30 kg) in constrained and non-ergonomic postures. While adequate physical training is of utmost importance and can help reduce MSDs risk, it is not sufficient alone, especially with the current aging of the workforce. In industry, occupational exoskeletons have been proposed as a potential solution to alleviate the physical load on workers, and thereby reduce MSDs risk [START_REF] De Looze | Exoskeletons for industrial application and their potential effects on physical work load[END_REF][START_REF] Theurel | Occupational exoskeletons: overview of their benefits and limitations in preventing work-related musculoskeletal disorders[END_REF]. While exoskeletons are envisioned as a promising solution for firefighters as well [START_REF] Taborri | Preventing and Monitoring Work-Related Diseases in Firefighters: A Literature Review on Sensor-Based Systems and Future Perspectives in Robotic Devices[END_REF], their benefit is strongly task-dependent. So far, exoskeletons for firefighters have mainly been developed as whole-body powered devices for strength enhancement in load carrying tasks [START_REF] Osipov | Fire exoskeleton to facilitate the work of the fireman[END_REF]. However, the use of lighter and less cumbersome occupational exoskeletons with MSDs reduction in mind has, to the best of our knowledge, not been studied. This work therefore investigates the biomechanical effects of using a passive upper-limb exoskeleton during the vehicle extrication maneuver.

Methods

Participants and Protocol

Twelve male firefighters (7 professionals and 5 volunteers; age: 31±9 yrs; experience: 11±9 yrs; height: 175±5 cm; mass 73±6 kg) performed a vehicle extrication maneuver (Fig. 1), both with (WE) and without (NE) the exoskeleton. The maneuver consisted in a timed sequence of three subtasks performed at different heights on the vehicle, with two different cutting tools (about 22 kg each), for a total duration of 3 min. The two conditions were performed in random order, with a 15 min break in between. The study was approved by INRIA's ethical committee COERLE, and was conducted in accordance to the Declaration of Helsinki.

Instrumentation

The participants were equipped with the Xsens MVN Link inertial motion capture system (capture rate 240 Hz) to record whole-body kinematics. Fourteen Delsys Trigno Snap-Lead EMG sensors were placed according to the Seniam recommendations bilaterally on the erector spinae (ES), latissimus dorsi (LD), anterior deltoid (AD), upper trapezius (TR), biceps brachii (BB), triceps brachii (TB) and brachioradialis (BR) to record back, shoulder and arm muscle activity (capture rate 1926 Hz). Finally, a Garmin Forerunner 345 sensor was placed on the participants' chest to monitor heart rate (capture rate 1 Hz).

At the end of each condition, participants answered a Borg CR10 questionnaire to evaluate the effort perceived in five body areas: shoulders, upper-arms, forearms, abdominal and lumbar [START_REF] Borg | Borg's perceived exertion and pain scales[END_REF].

Exoskeleton

The exoskeleton used in this study is the MATE-XT (Comau, Torino, Italy). It is a lightweight (3 kg) passive upper-limb exoskeleton designed to provide shoulder support in tasks involving arm elevation [START_REF] Pacifico | An Experimental Evaluation of the Proto-MATE[END_REF]. The MATE was selected as a test candidate following a preliminary study of the non-assisted extrication maneuver, in which the shoulder was identified as the primary joint to assist. Among commercially available exoskeletons for shoulder support, the MATE was deemed a good compromise between the level of assistance provided and the device compactness imposed by the task.

In the present study, the firefighters wore the exoskeleton over their work clothes, and the highest level of assistance was always used (6 kg per arm). Before starting the task, participants could move around and manipulate the cutting tools during a few minutes to familiarize with the exoskeleton.

Data analysis

The Due to the non-normality of the data, Wilcoxon signed-rank tests were conducted for all dependent variables to evaluate the effect of the exoskeleton. A significance level of 5% was used.

Results

Kinematics: When using the exoskeleton, participants spent more time with their left arm in the 45-90 deg elevation zone and less in the 0-45 deg zone, compared to without it (+7% in average). There was no significant difference for the right arm or back.

Muscle activity: The use of the exoskeleton caused a significant increase in the activity of the right BB (by 10% in average). All other muscles were unaffected.

Even the activity of the left shoulder muscles remained similar, despite more time spent with the arm elevated when using the exoskeleton.

Heart rate: The cardiac cost was not significantly affected by the use of the exoskeleton. Perceived effort: A significant reduction was observed for the shoulder (by 22% in average), upperarm (by 22% in average), and forearm (by 26% in average) areas when using the exoskeleton (Fig. 2).

For the abdominal and lumbar areas, the exoskeleton also reduced the perceived effort, but not significantly.

Figure 2: Distribution across participants of the Borg CR10 scores of the 5 body areas, with and without the exoskeleton.

Discussion and Conclusion

This study suggests that there is no physiological benefit in using the MATE exoskeleton for the extrication maneuver, except possibly a mild assistive effect on the left shoulder (similar muscle activity despite higher arm elevation). This may be due to the low arm elevation angle adopted by most participants (<45 deg during most of the task), where the MATE provides only limited assistance, especially compared to the weight of the cutting tools. Interestingly though, participants reported a reduction of perceived effort with the exoskeleton, which suggests either a placebo effect of the device, or biomechanical changes that are too small to be detected with the proposed methodology, given the large task's degree of freedom.

Figure 1 :

 1 Figure 1: Firefighters equipped with the MATE exoskeleton performing a vehicle extrication maneuver.

Acknowledgements

This work has received funding from the Université de Lorraine Interdisciplinary Program (Pompexo project).


Jean-Paul Thibaud

Philip Tidwelll

Ed ) Helsinki

Tapio Wirkkala- Rut

Bryk Foundation

Installing an Atmosphere

Just like the night, an ambiance has no outline, no clearly defined shape, no exact limits. It is more of an indistinct sensory backdrop, before which the world presents itself and is

Introduction

Luce Irigaray published L'Oubli de l'Air -The forgetting of Airin 1983. 'Always there, the air is neither absent nor present,' she wrote in the book's final pages (p. 148). This is certainly one of the reasons why western philosophy disregarded air for so long. Too constant to be worthy of note, too evanescent to be grasped, too insignificant to merit attention, there was no reason to investigate it. So it was quite simply overlooked.

But the situation has changed in the past 30 years. With the growing volume of work on the sensory -what some call the sensorial turn -the next step is to make room for the fragile, transient entities we refer to as atmosphere, ambiance or climate. It is time to open our senses to the atmospheric existence of the world, to give consistency to the sensory environments in which we are constantly immersed. Or in other terms, the current urban ecology is increasingly underpinned by explicit strategies to sensitize lived and inhabited spaces. Air, sound, light, fragrance, warmth, smell and other sensory streams are thus becoming the focus of creative and experimental endeavour, debate and theory-building. An aesthetic of the immaterial, the ephemeral and shapeless is gradually supplanting the ontology of things and substance. Humans are in the process of becoming 'designers of atmospheres' [START_REF] Desroches | L'homme comme designer d'atmosphère. Sloterdijk et la critique des milieux métaphysiques[END_REF].

So what can be said about the sensory fabric of contemporary territories? How are we to account for the staging of atmospheres in urban spaces? What happens when the goal is not only to design a space but also to install an atmosphere?

Learning to unfocus

To start with, we need to learn to unfocus and recognise the fundamentally pervasive, vague and evanescent nature of an atmosphere [START_REF] Thibaud | De la qualité diffuse aux ambiances urbaines[END_REF]. The following description of the night space by Maurice [START_REF] Merleau-Ponty | Phenomenology of perception[END_REF] is instructive in this respect: "When, for example, the world of clear and articulate objects is abolished, our perceptual being, cut off from its world, evolves a spatiality without things. This is what happen in the night. Night is not an object before me; it enwraps me and infiltrates through all my senses, stifling my recollections and almost destroying my identity. I am no longer withdrawn into my perceptual look-out from which I watch the outlines of objects moving by at a distance. Night has no outlines." (p. 283) experienced, a tonality which colours the whole situation. We may learn a great deal too, from a brief detour via the 'viewing chambers' of artists such as James Turrell. With his work on space constructions and arrangements of elementary materials, the artist has shown time after time that light is not just a matter of illuminating objects, but also the power to instil tactile qualities which mobilize the whole body. In his experimental works, light gradually becomes denser, palpable, saturated and homogeneous. Combined with a suitable spatial treatment -no objects in the room, an enclosed space, attention to borders and edges -Turrell reveals the atmospheric power of light [START_REF] Didi-Huberman | homme qui marchait dans la couleur[END_REF]. As the artist puts it himself, we move from 'looking at' to 'looking into'. We are no longer in front of discernable objects but part of a compact luminous field with no sense of depth.

These borderline experiences make manifest the sensory atmosphere, in which we are immersed in daily life, by intensifying it. They reveal that the ambient space cannot in any way be reduced to an objective space, geometrical and divisible -in the manner of partes extra partes. On the contrary it is more an enveloping milieu of the intensive and dynamic order. In the terms of Merleau-Ponty, ambiance involves a 'spatiality of situation', rather than a 'spatiality of position'. Turrell's viewing chambers also teach us that an ambiance only has a temporary, situated existence, and that it necessarily brings into play a built environment, some material device which contributes to its installation.

With ambiance, it is a matter of working on a milieu-orientated architecture, which is concerned about what happens between things and edifices, taking an interest in intervals, streams and interstices, rather than objects themselves. Various propositions by the new phenomenology have confirmed the pervasive, unassignable character of an ambiance. Gernot [START_REF] Böhme | Atmosphere as the Fundamental Concept of a New Aesthetics[END_REF] quite rightly emphasized the ecstatic nature of things and people, their capacity for overflowing, radiating around themselves and thus affecting their immediate environment. Hermann Schmitz [START_REF] Schmitz | Emotions outside the box -the new phenomenology of feeling and corporeality[END_REF] has developed the idea of surfaceless space and Tonino Griffero has looked at quasi-things which 'appear and disappear, without the possibility of asking oneself where and how they exist when they are not perceived' [START_REF] Griffero | Ambiances. Sensory Environment, Architecture and Urban Space[END_REF]). Think, for example, of wind, silence, fragrance or the weather. All these ambiant phenomena defy strict delimitation and localization, necessarily unfolding in time. So to learn to unfocus we need to develop new ways of relating to space, without immediately trying to take control of it or projecting any prior intention on it. Two main conditions play a part in this attitude of letting go which favours the installation of an atmosphere.

On the one hand, we must make ourselves available to our surroundings, and really recognise the existence of margins, horizons and fields of perception. Juhani Palasmaa (2014) has perfectly demonstrated the importance of peripheral perception in our experience and conception of a sensory space. As he so aptly puts it: "Peripheral perception is the perceptive mode through which we grasp atmospheres. The importance of the senses of hearing, smell, and touch (temperature, moisture, air movement) for the atmospheric perception arises from their essence as nondirectional and embracing character. " (p. 244) The pervasive attention one practices in psychoanalysis, the floating observation developed by ethnography and the distracted perception of the flâneur are all techniques which contribute to opening our attention. Nor should we overlook the importance of bodily sensations, tactile, kinaesthetic, proprioceptive, and vestibular impressions, which help fully experience an ambiance, even if they are subconscious most of the time. In this respect Richard [START_REF] Shusterman | Soma-esthétique et architecture : une alternative critique[END_REF] advocates developing somaesthetics to take full account of the qualities which make up an atmosphere.

Furthermore we need to let ourselves go with the flow of events rather than trying to halt them. In so doing we may take an interest in transient phenomena, in transitions and progressions, rather than scrutinizing one or other component, or concentrating on a particular stimulus. Unfocusing therefore requires us to be available to what is going on, to become part of time unfolding, to agree to take a moment and open ourselves to what may occur. This means quite simply that an atmosphere has only a temporary existence, that it can only give rise to an experience according to a dynamic, a rhythm, a quality of duration. We can only experience viewing chambers if we leave them enough time to upset our senses.

When it is time to install …

I have chosen Installing an atmosphere as the title of this article. The verb, 'install', is of prime importance when we start looking at how an ambiance or atmosphere emerges. It enables us to pinpoint the nature of the processes which play a part in the existence of an ambiance. We should start by noting that installing means to 'establish (a person, etc.) in a place'. It is an action which necessarily involves a place, drawing on its resources and testing it in some sense. The place is not an epiphenomenon, simply a framework for installation; it is a constituent part of it. In other words, installing an atmosphere involves both taking place and giving place, becoming part of a place and transforming it, making do with it while also animating it. Any ambiance is at one and the same time spatialized and spatializing.

We should also note that installing is not the same as creating or imposing. In drawing a distinction between installing and creating, I wish to question the idea of ex nihilo production. An ambiance does not emerge out of nothing but always supposes a previous ambiance, an ongoing motion, an already-there on which to build. This entails admitting that there cannot not be an ambiance, that it is omnipresent and that it constitutes the basso continuo of our existence. As Gabor Csepregi (2006) explains very clearly: "Wherever we are, in a small room or in the middle of the ocean, we are constantly exposed to a particular atmosphere. Even though we do not always notice it, the contact with an atmosphere is just as much a fundamental feature of our existence as are consciousness and language." (p. 42) Installing an atmosphere therefore always means coming to terms with an existing atmosphere, and finding ways of inflecting and transforming it.

In drawing a distinction between installing and imposing, our aim is to take the opposite course to any action that might be too peremptory or limited in time. As we shall see later in greater length, installing an atmosphere supposes a relation to time which is not necessarily of the order of an event, but involves a process characterized by discretion and nuance.

Focusing on the term 'install' thus enables us to underline that staging an ambiance involves basically a situated set of actions, always based on a process already underway and characterized by discreet effectiveness. To make this general framework more specific, we may emphasize three main features. Installing an atmosphere implies both an art of accompaniment, an art of impregnation and an art of tonalization.

An art of accompaniment

The art of accompaniment draws attention to the fact that the world is always changing and that installing an atmosphere is achieved firstly and above all in a gradual, indirect way. As I said before, it is not so much a matter of creating an atmosphere from scratch as of acting on the conditions of its possibility, feeding the processes which make it possible, and sustaining the factors which favour its emergence. In this respect the built environment plays a part in the conditioning process, creating holds for actions and perceptions which in return affect it. We might say that any piece of architecture sensitizes a lived space. In this way we might develop a sensory ecology of affordances which would show how material devices play a part in staging the ambiance of a place and lend themselves to the emergence of a sensory atmosphere.

The philosopher François [START_REF] Jullien | Françoise Jullien Les transformations silencieuses[END_REF] analysed this rationale of ambiant effectiveness, which involves accompanying the world as it unfolds. Instead of a conception based on direct, finalized action, we encounter the idea of the "potential of a situation", which allows us to activate available resources, play with circumstances, exploit propensities and prime processes. There is no decreeing an atmosphere, it cannot be forced upon a situation; it gradually establishes itself extending the motion and dynamics already at work. Jullien cites the example of agriculture, which could equally well apply to atmosphere: "We cannot force a plant to grow, nor can we leave it to get on by itself; but, by freeing it from whatever might hold back its development, it must be left to grow." (p. 112) Under such circumstances, the concept of the architect as a demiurge or a self-sufficient designer no longer stands up; on the contrary it is a matter of seconding the world. 'Seconding the world'. This fine expression, which comes from Kafka's journal, recently gave rise to an instructive commentary by Pierre [START_REF] Zaoui | Ou l'art de disparaître[END_REF], a specialist on Spinoza and Gilles Deleuze. He writes: "What matters is to second the world, to accept in other words not being the first, not being at the centre nor at the origin, yet supporting what exists, serving not oneself -a humbug, chimera or lie -nor the Other -a tyrant or fantasy -but each thing, each being and each instant. " (p. 24) This is exactly what is going on with the installation of an atmosphere. We are seconding the world at an everyday level, for the duration, while acknowledging that an ambiance is a common good and a collective work essentially anonymous in character. So we must pay attention to particulars, from one day to the next, discreetly and permanently, by recurrent gestures, little nothings which make all the difference. The aim is not to go down in history, to mark the town with a memorable event, but to take care of the ordinary permanences of a territory. In short, we shall not focus exclusively on the scene, the most obvious, spectacular components of a space, but just as much, if not more, on a territory's background and what makes up its texture and depth.

The art of accompaniment to which I refer requires a certain permanence in the sensory transformations underway. As Hannah Arendt (1958) says, this supposes that we should be "surrounded by things more permanent than the activity by which they were produced, and potentially even more permanent than the lives of their authors" (p. 96). On this depends our collective ability to look after places and take care of them.

To sum up, insisting on an art of accompaniment enables us to highlight the importance of constant, collective care in the installation of an atmosphere. What matters here is not so much the rationale of a one-off event but rather a slow, continuous relation to time, prolonging the world as it is making itself. Obviously our insistence on gestures of installation and upkeep should not be taken as a strategy to keep a territory just as it is. There is no question of freezing the city or preserving it in a particular state. Rather, I wish to convey that the urban world is in constant flux and that such changes are continuously at work, deep down. Rather than trying to create novelty out of nothing, such processes should be accompanied. This is how an ambiance is installed in time, by seizing on opportunities and acting on the conditions of the possibility of development action itself. One way of summing up this operation would be to cite Sun [START_REF] Tzu | 'art de la guerre[END_REF], who refers to 'controlling the factors behind changing circumstances'.

Art of impregnation

I shall now turn to the second point, the art of impregnation. What we need to understand here is our porosity to the ambiant milieu, emphasizing our dual capacity to inhabit a world and be inhabited by it, to incorporate and inflect tiny modulations in the sensory space. This involves a joint movement of impression and expression, incorporation and incarnation, of embodiment. From this point of view, any work of impregnation consists in developing complicity with the things surrounding us, adjusting to our environment and adjusting it in return through our movements. Merleau-Ponty refers to 'knowledge of familiarity' in this respect. What we see, hear, touch, feel, at such and such time and place, does not immediately leave us; on the contrary it plays a part in what we are and become. In the words of Jean-François Lyotard (2000): "We inhabit a place, a milieu, when our way of being is shaped by frequenting it" (p. 200). Witness the bodily habits we acquire when we are immersed for a long time in the same milieu. Gestures are fashioned, styles of conduct formed, which respond immediately to ambiant situations. Or indeed, think of the phenomena of involuntary memory described by Marcel Proust, when a present sensation meets another from the past, and transports the narrator to Combray, Balbec or Venice. In short, the installation of an atmosphere brings into play a climatic force which, though pervasive, is nevertheless operative. A memory of the body is activated, adding to the sediment of past experience, favouring the acquisition of new skills and shaping our practical, sensory relation to the world around us. I am emphasizing here our capacity to be inhabited by the milieu in which we live, but I should also take time to show how our ways of being, our everyday actions and gestures also play a part in this art of impregnation.

It is important to understand that this phenomenon of impregnation is the result of a discreet influence on beings and milieus. It is all a matter of immersion and infusion, and of tiny connections [START_REF] Laplantine | Jean-François Lyotard Misère de la philosophie[END_REF]. Much as a novel transports us with a stream of words to another world, or music imperceptibly carries us away with its movement; we are living in the novel, we belong to the music. We could build a whole lexicon to try to put words on the slight, but vital relation to the ambiant milieu: from a fragrance to a stroke, from a breath to floating, from exhaling to inclination, from the unobtrusive to the evasive, from je ne sais quoi to almost nothing. Proceeding most of the time by light brushstrokes, small inflections and subconscious phenomena, impregnation is by nature pervasive, underground, infra, unsaid. Think of the continuous sound of ventilation which we end up erasing from our field of consciousness or the smell of pollution we no longer notice, it being so omnipresent in the town where we live. One day the ventilation stops, or we return from a trip and notice something that was already there. The habituation to the milieu in which we live, also plays a part in the relative invisibility or imperceptibility of impregnation.

But here again, if impregnation remains at a subconscious level, at the limit of what is perceptible, it is also because we are dealing with low-intensity phenomena. If impregnation generally goes unnoticed, it is because it works through infinitesimal, interstitial vibrations which combine with one another to produce a continuous experience. Leibniz referred to 'little perceptions', referring to perceptions so microscopic and so mixed with each other that we have no distinct awareness of them. He liked to take as an example the roar of the sea, made up of the sound of an infinite number of barely audible ripples, which we would have difficulty picking out, but which nevertheless affect us. In a quite different context, William James, later, drew attention to the 'fringes of experience' which establish a continuity between experiences. This is one of the main argument of radical empiricism advocated by James (1912), for whom, "life is in the transitions as much as in the terms connected" (p. 87). So it is not just the infinitesimal nature of phenomena which needs to be emphasized here, but also their power of connection and transition. These micro-phenomena build imperceptible bridges from one state to the next and, to do so, blend with one another.

To the microscopic, transient nature of impregnation, we must add its fundamentally diffuse character. By this we mean that if ambiance has an underlying force, it works in an overall, pervasive, disseminated way. Just think of the effect of rain or mist in installing an ambiance. Fernando Pessoa's Book of Disquiet contains superb evocations of such barely noticeable variations in atmosphere, when the mist envelops the Tagus or drizzle conceals the Baixa neighbourhood of Lisbon. It is impossible to position an object precisely, or identify a single signal, thought to be the cause of this atmospheric effect. The whole situation is requalified, the entire perceived space reconfigured. François [START_REF] Jullien | Françoise Jullien Les transformations silencieuses[END_REF] develops the idea of 'silent transformations' to designate the continuous, overall changes which go unnoticed most of the time.

Summing up, this art of impregnation contributes to developing the world of nuances Roland Barthes craved. It is a matter of small inflections and tiny graduations which colour experience. It is also important to emphasize the significance of continuous streams rather than discreet signs, discontinuous signs, as an ambiance establishes itself in a space. This enables us to make a distinction between context and atmosphere. As Fernando [START_REF] Gil | Les petites perceptions[END_REF] points out: "This [the context] is visible and can be reduced to a set of relations or signs. It can be semiotized, whereas the atmosphere is infra-semiotic and unfolds in a continuum. " (p. 16) We may posit, that this constitutes a shift from an aesthetic of forms to one of flux.

Art of tonalization

I now come to the last point, the art of tonalization. It should by now be obvious that installing an atmosphere is not just a question of controlling the physical parameters of a particular setting, but also involves endowing a place with an overall quality which lends it a certain physionomy. For this reason, we can sum up an ambiance with a single word, such as joyous, morose, electric, friendly, peaceful or worrying. In this respect an ambiance pulls together diverse strands giving the same character to everything which appears. Anne Cauquelin (2006) explains that every atmosphere is the result of a complex conjunction of heterogeneous data: 'If I try to remember a moment in my life, a fragment of experience, this recollection is mixed with places, people, the weather and time of day, the words exchanged: a fragile fabric, which tends to unravel if you look at it too closely, its consistency depending largely on its fluidity. What emerges from this exploration is an atmosphere, an air, an envelope of odours, flavours and, here and there, a few distinct, more sharply defined items. " (p. 6-7) On this depends the capacity of an ambiance to give a situation a particular consistence and to weave together a large variety of components.

Any lived space is therefore marked by a dominant tonality, an emotional charge and by the pathic relation we entertain with the world. We should keep in mind that the term ambiance originally had a protective connotation, connecting to the idea of a 'warm embrace'. What matters here is to acknowledge our capacity for being affected by the ambiant world and for responding to its solicitation. Gaston Bachelard analysed this very well in his rhythmanalysis project and his poetics of dreaming. By proposing the idea of tonalization, using the image of the Eolian harp, he shows how our bodies enter into resonance with beings and objects, demonstrating real motor-imagination. We should therefore accept that we are not outside of, or indifferent to our surroundings, rather that we resonate with, in and through them. Adding an affective thread to the sensory no doubt helps to highlight the difference between environment and ambiance. One might say that ambiance is what breathes life into a setting, giving it an emotional value and a force of attachment.

But more specifically, what can be said about the affective experience of contemporary cities, and the way it plays a part in installing an atmosphere? The idea proposed by Jean [START_REF] Ladrière | La ville comme inducteur existentiel[END_REF] of an 'existential inductor' could well be useful here. For him: "The city is an affective essence, in so far as it is an affective inductor, or an existential shifter, in other words in so far as we can recognize in it, through resonance, a certain affective tonality which qualifies our existential movement." (p. 155)

The notion of affective tonality is of prime importance here. It enables us to query the classical dichotomy between object and subject, material and immaterial, real and imaginary. This means that an ambiance is both objective and subjective, indistinctly a feeling of me and of the world. As Pierre Sansot (1984) says in his Poétique de la Ville -Poetry of the City -in a city you can never tell "who in the night has a fever, whether it is the city lights or the busy passers-by " (p. 19).

Following this track we may consider the city as a remarkable resonator of affective tonalities, regardless of whether they take the form of warm conviviality, measured urbaneness or feelings of insecurity. So installing an atmosphere involves coming to terms with various affective tonalities and activating the tonalizing potential of inhabited spaces. If we consider the conditioned environments of shopping malls or the planted areas of eco-neighbourhoods, the heritage-oriented historic town centres or the privatized gated communities, the new scenes of the creative city or the functional atmosphere of transport hubs, every effort seems to be made to channel our feelings and make us experience a specific Stimmung. Many authors have tried to qualify the dominant tonalities of the contemporary urban space. Whereas modern urban life were marked by hyperstimulation [START_REF] Simmel | The Metropolis and Mental Life[END_REF] and an aesthetics of shock [START_REF] Benjamin | The Writer of Modern Life: Essays on Charles Baudelaire[END_REF], contemporary western megacities tend to be described in terms of detachment from reality, leading to an uncanny feeling [START_REF] Vidler | The Architectural Uncanny: Essays in the Modern Unhomely[END_REF], an almost hallucinatory experience [START_REF] Bégout | L'expérience de Las Vegas[END_REF], anaesthetized perceptions [START_REF] Ingersoll | Looking for the City on its Edges[END_REF] or a preponderance of feeble sensations [START_REF] Koolhaas | [END_REF]. It very much looks as if the urban environment is animated by a twin movement, programming festivity and enforcing security, covering a broad spectrum reaching from the ecology of fear [START_REF] Davis | Ecology of Fear: Los Angeles and the Imagination of Disaster[END_REF] to that of enchantment [START_REF] Ritzer | Enchanting a disenchanted world : revolutionizing the means of consumption[END_REF]. What needs to be developed is a proper political ecology of affects, which would show how stage-managing the ambiance of urban territories requalifies the urban experience and its contemporary evolution.

Conclusion

I shall now conclude with just few words. First of all, atmospheres belong to a precarious domain, a fragile form of existence which needs to be treated with the greatest care. In these times of rampant globalization and cross-the-board standardization, it is vital to develop our sensibility to the nuances of experience and the small marks of attention which sustain our social life. Or, to put it differently, the notions of ambiance and atmosphere lend themselves to a socio-aesthetic of lived spaces.

Secondly, installing an atmosphere prompts us to develop a new design posture, made up of modesty, caring and interdisciplinarity. This new way of designing a space, requires the separations between disciplines to be removed, making room for cross-breeding between specialities. With ambiance we are at the crossroads between architectural and urban design, human and social sciences and art. To borrow something Deleuze said, it is a matter of taking things by the middle, par le milieu, of looking for horizontal connections between built forms, sensory forms and social forms; at the meeting point between the work of designers and everyday life of inhabitants. It is also important to acknowledge the degree of indeterminacy and incompleteness in any built space, because an atmosphere is not a finished object but much more fundamentally a process of becoming.

Lastly, is there any need to say how much ambiance as we understand it differs from some cosmetic notion? Far from being a simple additive procuring comfort or luxury, no more than a marketing ploy, it helps us conceptualize the existential side of human experience. Atmosphere anchors the world of the senses right in the middle of dwelling, constituting a condition for its possibility.


Comparison of Stabilization Methods for Finite Element Method in the Context of Space Charges.

Raphaël Pile and Guillaume Parent

Univ. Artois, UR 4025, Laboratoire Systemes Electrotechniques et Environnement (LSEE), F-62400 Bethune, France.

Partial discharges have recently become an issue for electrical machines longevity. Thus, there is an interest in studying the phenomenon through numerical simulation. This task is rather complex since it requires to couple Finite Element Method (FEM) for the electrostatic field with a numerical simulation of the charge density flow using hydrodynamics model, which is known to be unstable with FEM. Thus, this communication proposes to compare the main stabilization methods with an analytical case dedicated to space charge context. It suggests that a logarithmic formulation of the problem can accurately ensure the positivity of the density as opposed to the common stabilizations methods. However, the conditions of convergence remain to be established and it comes with a significant additional computational cost.

Index Terms-Partial discharges, Finite element analysis, Numerical stability, Electrohydrodynamics.

I. INTRODUCTION

T ECHNOLOGICAL developments in electric motors and actuators over the last decade have led to increase voltage amplitude and switching frequency. These new constraints lead to the appearance, in the inter-coil insulation of the windings, of partial discharges that are particularly harmful to the longevity of the insulating layer [START_REF] Fabiani | Aging acceleration of insulating materials for electrical machine windings supplied by pwm in the presence and in the absence of partial discharges[END_REF]. In electrical engineering, the Paschen empirical law is commonly used to determine the Partial Discharges Inception Voltage (PDIV). The local evolution and properties of partial discharges remain misunderstood in the electrical engineering literature. Thus, the numerical simulation of these phenomena has recently gain interest.

Most recent simulation models of partial discharges in the literature follow the hydrodynamic approximation, where a set of transport equations for the charged particles in the gas are coupled with Poisson's equation for the electric field [START_REF] Georghiou | Numerical modelling of atmospheric pressure gas discharges leading to plasma production[END_REF]. The Finite Element Method (FEM) is more adapted to the Poisson's equation, thus it is widespread in electrical engineering field. Moreover, it enables to easily use high order space function with unstructured meshes. However, the natural choice for the transport problem would be the Finite Volume Method (FVM) or Finite Difference Method (FDM). Indeed, these methods are more adapted to the convective part of the transport equation.

In such a case, the FEM methods is often privileged because using the same method for both physics considerably ease the coupling. Nevertheless, the FEM for the convective part of the transport equations has a major stability drawback. Thus it requires stabilization methods such as Streamline Upwinding Petrov-Galerkin (SUPG), Taylor-Galerkin (TG) [START_REF] Codina | Comparison of some finite element methods for solving the diffusion-convection-reaction equation[END_REF]. Nevertheless, it is unclear why these classical stabilization methods are not used in the context of space charges. This communication proposes to clarify this point. For this purpose, a 1D validation case is proposed, derived from the Davies test case [START_REF] Davies | Solution Continuity Equations in Ioniz. Plasma Growth[END_REF]. The results are discussed in term of stability, accuracy and computation time.

II. CONVECTION-DIFFUSION SIMULATION A. Galerkin Variational Formulation

In general form, transport equation is expressed as [START_REF] Liu | An Efficient Semi-Lagrangian Algorithm for Simulation of Corona Discharges: The Position-State Separation Method[END_REF]

∂u ∂t (x, t) + ∇ • (u(x, t) • v(x, t) -D∇u(x, t)) = s(x, t) (1)
with the position x in the domain, time t ∈ [0, T], velocity field v, diffusion tensor D, and source/sink function s. In the following, x and t are omitted for ease of reading. The convective term ∇

• (u(x, t) • v(x, t)) is the critical part of this study. The compact form ∂u ∂t + L(u) = s , (2)
where L is a linear differential operator. FEM consists in solving the physics equations by using a variationnal formulation of the problem. Let us consider H s 0 , a Sobolev space with null value on the boundary. Then, a variational formulation problem of (1) can be written:

Find u ∈ H, with w ∈ H such that a(u, w) -⟨s , w⟩ = 0 (3)
where

a(u, w) = ⟨ ∂u ∂t , w⟩ -⟨u • v , ∇w⟩ + ⟨D∇u , ∇w⟩ . (4)
However, FEM encounters difficulties in dealing with the instabilities of convective problems. It can be shown that the FEM discretization introduces a purely numerical (unstable) diffusion [START_REF] Kuzmin | A guide to numerical methods for transport equations[END_REF]. Several options are discussed in the following.

B. Main Galerkin Stabilization Methods

1) Streamline Upwinding Petrov-Galerkin

An idea is to stabilize this numerical diffusion by adding an artificial diffusion term in the formulation. The SUPG method consists in adding this term in the direction of flow [START_REF] Kuzmin | A guide to numerical methods for transport equations[END_REF]. It can be performed by modifying the test function. The problem (3) becomes:

a(u, w + τ v • ∇w) -⟨s , w + τ v • ∇w⟩ = 0 ( 5
)
where τ is an arbitrary stabilization parameter, often evaluated as τ = h/(2|v|) with h the element size [START_REF] Kuzmin | A guide to numerical methods for transport equations[END_REF].

2) Taylor-Galerkin In the TG stabilization, the time discretization plays a pivotal role. Instead of manipulating the Galerkin space discretization, the time-stepping method is chosen so as to stabilize it in natural way, perhaps, under a certain time step restriction. TG algorithms do not contain free parameters and are directly applicable to multidimensional transport problems [START_REF] Kuzmin | A guide to numerical methods for transport equations[END_REF].

The idea is to use a Taylor expansion to create the time discretization scheme. For example, the second order approximation gives

u(t + ∆t) ≈ u(t) + ∆t ∂u ∂t + ∆t 2 2 ∂ 2 u ∂t 2 . ( 6
)
The time derivatives can be expressed as

∂u ∂t = -L(u) + s (7) ∂ 2 u ∂t 2 = -L( ∂u ∂t ) + ∂s ∂t = L(L(u) -s) + ∂s ∂t (8)
such that ( 6) becomes a time discretization scheme [START_REF] Codina | Comparison of some finite element methods for solving the diffusion-convection-reaction equation[END_REF], [6]

u n+1 -u n ∆t + L(u n ) - ∆t 2|v| L(L(u n+θ ) -s) = s + ∂s ∂t . (9)
It is assumed that the derivative of the source/sink term can be computed independantly (numerically or analytically). The parameter θ corresponds to the time discretization scheme.

Then the variational formulation of this problem is [START_REF] Codina | Comparison of some finite element methods for solving the diffusion-convection-reaction equation[END_REF], [START_REF] Kuzmin | A guide to numerical methods for transport equations[END_REF] a(u, w) -∆t 2|v| ⟨L(u) -s , L * (w)⟩ -⟨s + ∂s ∂t , w⟩ = 0 , (10) where L * is the adjoint operator of L.

C. Logarithmic Variational Formulation

Another method consists in a change of the unknown in (1) by using u = exp(U ) instead of u such that [START_REF] Singh | Computational framework for studying charge transport in highvoltage gas-insulated systems[END_REF]

exp(U ) ∂U ∂t + ∇ (exp(U ) • v -D exp(U )∇U ) = s . (11)
Then, the corresponding logarithmic variational formulation (LOG) is build the same way. The main issue is that the variational problem is not linear anymore. Thus, it requires to iteratively evaluate exp(U ). By construction, the positivity of the solution is ensured.

III. NUMERICAL APPLICATION

The unstabilities comes from the convection terms in (3). Hence, the diffusion tensor and the source term are neglected in this validation case.

The Davies test case [START_REF] Davies | Solution Continuity Equations in Ioniz. Plasma Growth[END_REF] is a very restrictive case to evaluate the accuracy of stabilizations methods: the density can effectively form a plateau, however local discontinuities in the derivative do not occur in areas of low density. Thus, it is proposed to replace the window shape by a clipped gaussian. The results after one period are presented in Fig. 1. The analytical solution is also the initial condition for all methods. The relative total error (using L2 norm) gives 39% for the standard Galerkin method, 37% for the SUPG stabilization, 36% for the TG, and 31% for the logarithmic method. The computation time gives about 3 s/CPU for the standard Galerkin method, 12 s/CPU for the SUPG stabilization, 20 s/CPU for the TG, and 34 s/CPU for the logarithmic method. SUPG and TG have similar accuracy. TG has the significant advantage of avoiding to evaluate stabilization parameter. The main issue is that the numerical oscillations lead to unphysical negative values for the charge density for both SUPG and TG. Even, with the proposed smoothed window case, the positivity of the solution could not be ensured. On the other hand, the logarithmic method seems to be a good candidate for the spatial charges simulation. However, it is significantly slower due to the non-linear formulation and its stability/convergence still has to be proven to the authors knowledge. For example, the method did not converge on the original Davies test case.

The obtained results validate that the FEM with logarithmic stabilization could be a viable method for partial discharge simulations. However, it comes with an added computational cost. Future research will therefore focus on the coupling with the electric field, in order to determine the computational cost associated with a change of method (FVM, Lagrangian, etc.) for the hydrodynamics.

Fig. 1 .

 1 Fig. 1. Davies test: Comparison between analytical solution and numerical schemes using 1000 elements and 200 time steps.


Kaoutar Hazim

Guillaume Parent

Stéphane Duchesne

Christophe Geuzaine

Frequency-Dependent Behavior of the Lumped Parameter Model of the Windings of Electrical Machines in Transient Simulations

Keywords: Finite elements method (FEM), frequency-dependent parameters, transient simulations, vector-fitting

This paper presents a method to take into account the frequency-dependent behavior of the elements of lumped parameter models of electrical machine windings. First, frequency-dependent inductances, resistances and capacitances are determined thanks to time harmonic finite element (FE) computations in the frequency range of interest. Then, equivalent electrical networks are deduced using the vector-fitting (VF) algorithm, which allows for their inclusion in transient circuit solvers. The resulting time-domain voltage distributions can therefore be extracted and the whereabouts of partial discharges (PD) predicted using Paschen's law. This will enable the usage of this method as a diagnostic tool before/during the design phase of the winding of electrical machines.

I. INTRODUCTION

The high dv dt imposed by the new generation of semiconductor devices generates intense inter-turn electric fields in the winding of electric machines. These high electric fields may exceed the partial discharge inception voltage resulting in partial discharges. Occurrence of partial discharges may result in an inter-turn local fault, increasing the thermal stress electrical machines have to withstand. Correspondingly, an expansion of the fault can take place followed by an eventual premature breakdown of the machines. Hence the need of a well parameterized model of the winding of machines serving as a predictive diagnostic tool. Previous research works [START_REF] Mihaila | A simulation method to predict the turn-to-turn voltage spikes in a PWM fed motor winding[END_REF]- [START_REF] Bauer | FEM-based computation of circuit parameters for testing fast transients for EMC problems[END_REF] have presented a phenomenological lumped parameter circuit (LPC) of the winding of electrical machines. However, the values of the elements of the LPC were computed at a single frequency regardless of their frequency-dependent behavior. This paper presents a method to take into account the frequencydependency of these elements in transient simulations.

II. LUMPED PARAMETER MODEL

The phenomenological approach proposed in [START_REF] Mihaila | A simulation method to predict the turn-to-turn voltage spikes in a PWM fed motor winding[END_REF][START_REF] Toudji | Determination of Winding Lumped Parameter Equivalent Circuit by Means of Finite Element Method[END_REF] enables to model each of the occurring physical phenomena within the winding of the machine using an electrically passive component. Iron losses, Joule losses (including both skin and proximity effects) and dielectric losses (both turn-to-turn and turn-to-ground) are modeled using resistances R p , R s , R t , and R m , respectively. Across each turn, the appearing electromotive force is modeled using an inductance L p . Finally, the capacitive coupling (turn-to-turn and turn-to-ground) is modeled using capacitances C t and C m , respectively. The elementary cell modelling each turn is represented in Fig. 1.

III. FREQUENCY-DEPENDENT PARAMETERS

Since the input voltage is rich in harmonics, it is important to account for the frequency-dependent behavior of the elements of the elementary cell. Accordingly, the values of the resistances R s and the inductances L p are computed over the Fig. 1. The elementary cell of one turn of the winding of the machine [START_REF] Toudji | Determination of Winding Lumped Parameter Equivalent Circuit by Means of Finite Element Method[END_REF]. The three blocks Yt, Ytt and Ytg correspond to the admittance of the turn, the coupling admittance of the turn to the remaining turns, and the coupling admittance of the turn to ground, respectively. frequency range of interest in a magnetodynamic ("magnetoquasistatic", where displacement currents are neglected) finite element approximation to consider skin and proximity effects. In the magnetodynamic approximation, the magnetic vector potential a and the scalar electric potential v satisfy:

curl µ -1 curl a + σ grad v + ∂a ∂t = 0, (1)
where µ and σ are the magnetic permeability and conductivity, respectively.

The frequency-dependent behavior of the capacitive coupling and the dielectric losses, i.e. C t , C m , R t and R m , is taken into account using a electrostatic approximation for each frequency of interest f :

div (-ε (f ) grad (v)) = 0, ( 2
)
where v is the scalar potential, ε (f ) is the dielectric permittivity of the used insulation, characterized experimentally in terms of the frequency. It is worth mentioning that the frequency-dependent behavior of the capacitive coupling is only considered for adjacent turns. For non-adjacent turns, only the capacitance computed at f =0 Hz is used.

IV. TIME-DOMAIN SIMULATION

At this stage, the elements of the LPC seen in Fig. 1 are vectors whose coordinates correspond to their values at different frequencies within the range of interest. To account for this frequency-dependent behavior in transient circuit solvers, the three admittances Y t , Y tt and Y tg presented in Fig. 1 are expressed as

                 Y t (f ) = 1 R s (f ) + 1 1 jLp(f )ω + 1 Rp(f ) , Y tt (f ) = 1 R tt (f ) + jC tt (f )ω, Y tg (f ) = 1 R tg (f ) + jC tg (f )ω, (3)
with ω the angular frequency, and are fitted using vector-fitting (VF) [START_REF] Gustavsen | Computer code for rational approximation of frequency dependent admittance matrices[END_REF]. This allows for them to be described analytically in the frequency domain and included in time domain simulations using the equivalent passive network depicted in Fig. 2. The elements of the latter are of constant values. Taking Y tg (f ) as an example, its VF expression is

Y tg fit (s) = d + se + Nr i=1 c ri s -a ri + Nc j=1 c cj s -a cj , (4)
where s = jω, d and e are real numbers, and c ri , c cj , a ri and a cj are the real and complex residues with their corresponding real and complex poles, respectively. The order of approximation is N = N r + N c , with N r being the number of the used real poles and N c the number of the used complex poles for the fitting. The fitting Y tg fit could also be represented in the form of the electrical network depicted in Fig. 2, so its expression also equals the equivalent admittance of the network:

Y tg fit (s) = 1 R 0 + sC 0 + Nr i=1 1 sL 1i + R 1i + Nc j=1 1 sL j + R j + 1 Gj +sCj . (5)
The constant values of the parameters R 0 , L 0 , R 1i , L 1i , R j , L j , C j and G j according to the computed residues and poles of the fitting can be deduced from ( 4) and (5) by identification as described in [START_REF] Gustavsen | Computer code for rational approximation of frequency dependent admittance matrices[END_REF].

Finally, to guarantee the stability of the transient simulation, the passivity of the equivalent network is enforced using the positive-definite criterion. The performed correction is based on constrained minimization by quadratic programming as proposed in [START_REF] Gustavsen | Enforcing passivity for admittance matrices approximated by rational functions[END_REF].

V. PRELIMINARY RESULTS

The proposed method was tested on a case study where the considered winding is of 34 turns with no magnetic core. The result of the frequency analysis is seen in Fig. 3, where the high frequency resonances are noticed. This result and the time domain response of the model will be compared with the results of previous research work [START_REF] Toudji | Determination of Winding Lumped Parameter Equivalent Circuit by Means of Finite Element Method[END_REF] in the extended version of the paper.

Fig. 2 .

 2 Fig.2. The equivalent network used to take into account the frequencydependent behavior of either Yt, Ytt or Ytg[START_REF] Gustavsen | Computer code for rational approximation of frequency dependent admittance matrices[END_REF].

Fig. 3 .

 3 Fig. 3. The simulated frequency response, amplitude and phase, of the studied 34 turns winding.


Guilherme C A Tolentino

Guillaume Parent

Olivier Ninet

Mathieu Rossi

Jean V Leite

Jonathan Blaszkowski

INFLUENCE OF THE GEOMETRIC PARAMETERS OF A VERTICAL ROTATIONAL SINGLE SHEET TESTER ON SAMPLE FIELD HOMOGENEITY

The horizontal Rotational Single Sheet Tester (RSST) suffers from weaknesses such as the reduced size of test samples, measurement disturbances due to magnetic flux leakage and non-homogeneity of field in the measurement area. Although the vertical RSST allows to overcome the first two aforementioned drawbacks, the heterogeneity of the field in the test sample remains an issue. Additionally, there is still a lack of device standardization to ensure test repeatability, as already is well established with the Epstein frame. In this paper, a finite element model of this device is developed in order to investigate the influence of several parameters on the field homogeneity in the test sample, such as the geometrical dimensions of the yokes and the presence of holes drilled for B-coil placement. It is expected that this study will contribute to the optimization and standardization these devices.

I. INTRODUCTION

The vertical RSST has been introduced in the early 90s [START_REF] Sievert | The measurement of rotational power loss in electrical sheet steel using a vertical yoke system[END_REF] but is still under consideration in recent publications [START_REF] Fonteyn | Numerical and experimental results from a vertical yoke system for measuring magnetic properties of Fe-Si steel sheets[END_REF]- [START_REF] Yue | Comprehensive investigation of magnetic properties for Fe-Si steel under alternating and rotational magnetizations up to kilohertz range[END_REF]. According to the literature, the advantages of this setup compared to the horizontal RSSTintroduced in the 80s are numerous [START_REF] Brix | Measurements of the rotational power loss in 3% silicon-iron at various frequencies using a torque magnetometer[END_REF]. Firstly, its ease of implementation due to similarities to the standardized Single Sheet Tester (SST). Secondly, a better homogeneity of both magnetic field H and magnetic flux density B at the center of the test sample is expected [START_REF] De La Barriere | 1-D and 2-D loss-measuring methods: optimized setup design, advanced testing and results[END_REF]. Thirdly, higher flux density levels can be reached in the test sample than with the horizontal SST [START_REF] Fonteyn | Numerical and experimental results from a vertical yoke system for measuring magnetic properties of Fe-Si steel sheets[END_REF]. It has to be noted that each publication related to that device brings its own setup in terms of numbers and dimensions of the yokes as well as on dimensions of the sensors [START_REF] Nencib | 3D analysis of a rotational loss tester with vertical yokes[END_REF], [START_REF]Performance evaluation of a large rotational single sheet tester[END_REF], denoting the lack of standardization of structure and measurement system. Nevertheless, the trend emerging from all those publications is that the larger the width of the yokes the better. Indeed, in [START_REF] De Wulf | Design and calibration aspects of small size single sheet testers[END_REF] the authors evaluate the accuracy of miniature SSTs dedicated to the characterization of small size test samples and highlight that the difference with the results obtained using an Epstein frame can be up to 5 %. The same trend is highlighted on vertical RSSTs in [START_REF] Nencib | 3D analysis of a rotational loss tester with vertical yokes[END_REF], [START_REF]Performance evaluation of a large rotational single sheet tester[END_REF]. Moreover, large yokes allow to "eliminate the influence of the demagnetizing field" [START_REF] Tumanski | Handbook of Magnetic Measurements[END_REF], leading to a better characterization of large test samples [START_REF] Sievert | The measurement of rotational power loss in electrical sheet steel using a vertical yoke system[END_REF]. However, the standard relative to the regular SST allows the use of yokes of any size "as long as the relevance of the results is ensured" [START_REF]magnetic properties of magnetic sheet and strip by means of a single sheet tester[END_REF]. To the authors' knowledge, almost no quantitative study of the influence of those parameters on the field homogeneity in the measurement area has been published except in [START_REF] Nencib | 3D analysis of a rotational loss tester with vertical yokes[END_REF], [START_REF]Performance evaluation of a large rotational single sheet tester[END_REF] in which the focus is made on the deflection of the mean value at the center of the test sample only. Above all, in all of the aforementioned studies, no link is made between the field non-homogeneity in the test sample and the actual field values that would be measured with actual sensors, which is after all the key point when characterizing a material. In particular, none of those works takes into account that the sensors employed to evaluate H and B are not of the same dimensions nor do they account for the fact that the more often B is evaluated by means of a coil requiring to drill the test sample. Yet, there is no doubt those holes have a great influence on field homogeneity.

II. TOPOLOGY OF THE STUDIED VERTICAL RSST AND FINITE ELEMENT MODEL

An overview of the topology of the studied vertical RSST is presented in Fig. 1a. It is composed of two magnetic yokes, i.e. one per phase. Each yoke consists of two C-shape cores (see Fig. 1b) arranged along each side of the test sample. Due to geometrical constraints and for the sake of magnetic balance between the two yokes, the two cores share every dimensions W yl , W y and T y but the height H y . The test sample is square with side and thickness denoted W s and T s respectively. All dimensions are summarized in table I.

The finite element simulations are conducted using the softwares GMSH and GetDP. The model is fully 3D and the problem is solved by means of the magnetostatic scalar potential formulation. Moreover, the nonlinear behavior of the test sample is taken into account by means of its first magnetization curve.

III. SOME RESULTS

In the following, the test sample is made of M260 electrical steel whose first magnetization curve is presented in Fig. 2. Moreover, for the sake of relevance of the analysis, only the worst case in terms of field homogeneity in the test sample, that is the case in which the latter is magnetically excited along its diagonal, is considered and presented.

A. Field homogeneity inside the test sample It can immediately be noted on this figure that both H and B inside the whole test sample, shown in blue, are widely heterogeneous due its large size. One might consider that this is not so much an issue since an actual sensor would only cover a small area of the test sample. Indeed, the red marks in Fig. 2 show the values of H and B occurring in a square area with a size of 50 mm located at the center of the latter. It clearly highlights that even inside a limited are the heterogeneity is still noticeable. Moreover, the mean values of H and B, which correspond to the values that would be given by an actual sensor, do not match the ones at the center of the test sample.

B. Influence of the core length T y

The values of H and B inside a square area with a size of 50 mm located at the center of the test sample with respect to T y are represented as box and whisker plots in Fig. 3. T y varies from 75 mm to 200 mm in increments of 25 mm and the magnetization level is such that ∥B∥ = 0.75 T at the center of the test sample. The first thing that can be notice in this figure is that outliers are always present whatever the value of T y . There is no doubt those outliers have an influence on the value given by actual sensors. The second thing is that the homogeneity of both H and B increases with T y . Nevertheless, Fig. 3 also highlights that as soon as T y is superior to half the width of the test sample the results are much the same.

IV. CONCLUSION AND OUTLOOKS

The accuracy of the results given by a vertical RSST largely depends on its dimensions as well as on the dimensions of the sensors used for the measurements. In the final paper, the study of the field homogeneity inside limited areas presented in Section III-A will be extended to different size and shape for the sake of comprehensiveness and relevance with respect to actual sensors. As an example, an H-coil is usually square but a B-coil is not. Moreover, the study of the influence of T y will also be extended to different magnetization levels. It will be shown that the higher T y the better the accuracy of the measurement has to be weighted to the magnetization level as well as the size of the sensors. Finally, the influence of drilled wholes, which are required to place a B-coil in the test sample, will also be addressed.

Fig. 1 .

 1 Fig. 1. Studied vertical RSST

Fig. 2 .

 2 Fig. 2. Distribution of H and B inside different areas of the test sample.

Fig. 2

 2 Fig. 2 shows the values of both H and B occurring in different areas of the test sample for different levels of magnetization and with T y = 200 mm. The first magnetization curve of M260 steel is also shown for information purpose.It can immediately be noted on this figure that both H and B inside the whole test sample, shown in blue, are widely heterogeneous due its large size. One might consider that this is not so much an issue since an actual sensor would only cover a small area of the test sample. Indeed, the red marks in Fig.2show the values of H and B occurring in a square area with a size of 50 mm located at the center of the latter. It clearly highlights that even inside a limited are the heterogeneity is still noticeable. Moreover, the mean values of H and B, which correspond to the values that would be given by an actual sensor, do not match the ones at the center of the test sample.

Fig. 3 .

 3 Fig. 3. Evolution of the values of H and B inside a square area of size of 50 mm located at the center of the test sample with respect to Ty. ∥B∥ = 0.75 T at the center of the test sample.


Smellscape Design and Monitoring

Jean-Paul Thibaud Is there such a thing as a "smellscape", and if so, is it possible to design and monitor it? By asking this question, we are required to specify what we understand by "smellscape" and to reconsider the very idea of "design". The four chapters presented in this part offer diverse responses and describe numerous case studies related to smellscape design.

One could imagine that an olfactory environment is simple to decipher and monitor: on the one hand an environment that "smells good" and need to be promoted, and on the other hand, one that "smells bad" and should be prohibited. After reading the articles, the question is more complex and certainly more interesting and subtle.

To start with, it is important to notice that odor alone is never sufficient to evaluate and characterize a smellscape. At least three additional aspects should be fully considered. First, we have to acknowledge the fact that the sensibility to odors depends very much on individuals and groups. Not only the olfactory sense is more or less developed depending on people, but the appreciation of an odor can be very diverse, even opposite sometimes. Hence, a smellscape involves not only odors but also the experience of odors and the relationship we have with them. Secondly, one need also consider the role of context: an odor can be pleasant or unpleasant depending on its concentration and its combination with other odors, depending also on the place, the time and the activity during which it is experienced. Thirdly, smellscape cannot be dissociated from the other senses such as vision, sound, taste and touch. Hence, a smellscape involves an experienced situation as a whole, it has to be considered from an intersensorial perspective.

Moreover, designing a smellscape brings stimulating issues and involves new questions related to the field of design. Let me mention and emphasize five of them.

First: integrating cultural know-how. At first glance, smellscape design seems to be a new kind of activity, a contemporary project which stems from the growing interest towards the senses. After reading these chapters, it becomes obvious that such an idea is incorrect. Not only do we learn that odorisation has been more or less appreciated depending on cultures, but also certain ancient traditions have developed a whole set of practices regarding the world of smells. Korea, China or Japan immediately come to mind, but other traditions could also be mentioned. What is interesting here is that such cultures invented material devices and promoted specific know-how, be it for example burning incense or using fragrances in gardening. It is also worth noting that the use of smell is often closely related to rituals and religious ceremonies. Hence, smellscape is part of a cultural heritage that crosses the boundaries between materiality and spirituality.

Second: paying attention to the background. Composing with odors leads to integrate very carefully the question of attention as well as that of background levels.

Often taken for granted or supplanted by other sensory phenomena, the world of smell tends to be forgotten in everyday life. It is mostly noticed when it becomes a public problem and involves a form of nuisance or disturbance. But what happens when such is not the case? Smelling is not only passive -a mere reception of an olfactory environment -but also active. It relies on the act of breathing, inhaling and exhaling, and this activity can be enhanced, become intentional and be traced back to consciousness. One of the challenges of designers is to produce a smellscape that will be fully experienced. A whole vocabulary is used to account for the threshold of smell and the sensitivity to odors. The smell environment is described and acted upon in terms of detection, concentration, dilution, and dispersion. Hence, smellscape design opens new categories within the field of design and highlights the importance of sensory background in the experience of an environment.

Third: producing all sort of effects. When designing a smellscape, one does not intend to only create a pleasant and comfortable environment but may also aim at producing more specific effects. Such effects can be very diverse, tending to reduce stress or calm people, to produce a sense of sacredness, to purify the air and the surroundings, to mark the space and foster urban identity, to attract visitors or passers-by, to trigger memories and past experiences (one could add to encourage shopping and consumption). Well-being is of course at the core of this activity, but odors may also concern the fields of health and therapy, architecture and urban planning, sociability and religion, business and marketing. Thus, the aesthetic dimension of smellscape has to be articulated with its pragmatic and praxiological dimensions. The reason why there is a need to design a smellscape and the various consequences of such a project, can raise political issues, as well as ethical ones, since odors can be effective without a fully conscious perceiver.

Fourth: composing with the elements. Natural elements seem to be the primary means by which designers create and monitor smellscapes. Flowers, trees and all sorts of greenscapes diffuse specific odors and contribute to composing an olfactory environment. For instance, orange trees and other fragrant plants have the ability to pervade a place with odors. Also, water plays an important role in the diffusion and transmission of aromas. Waterscapes produce several effects and involve diverse modes of action that are important in order to monitor a smellscape: running water may accelerate ventilation, increase air humidity, and bring a sense of freshness. Last but not least, air is the medium par excellence of the propagation of odors. This is maybe one of the most difficult problems encountered by designers: how to master air movements and airflows that are very labile, unstable and transient by nature? We have just seen that water can be very useful, but the built environment is also to be considered. Urban forms rely on different types of boundaries or semi-closed spaces that can confine odors and limit their dispersion. They also involve corridors and streetscapes that can orient and channel air movements. Thus, the monitoring of odors need to understand the complex interaction between the materiality of a built space and the atmospheric dimension of a smellscape.

Fifth: designing in accordance with the weather. Because smellscape relies basically on air and elements, weather is a key feature of this of kind of design. Humidity, temperature and wind, rain or dryness, affect the nature and the experience of an olfactory environment. One of the most stimulating lessons to learn from smellscape design is that it cannot be identical anywhere and anytime. One will not design the same way in a place subject to a tropical climate, a continental, mild or polar one. The weather conditions are not the same, neither is the flora or the sensitivity to seasonal cycles. To be sure, geographical situations and seasonal change determine largely what will be possible to do in terms of design and planning. This statement has three important implications. First, designing a smellscape means designing space-time. These two dimensions -space and time -are inseparable since the olfactory resources of a place depend very much on the weather and the season. Second, I am referring to a temporality that is cyclical and not linear. Smellscape is not only subject to variation and transformation, but also to a periodic return according to the same process (one season after the other). Third, entering the era of the Anthropocene, smellscape design has certainly a role to play in order to sensitize inhabitants to climate change. Because it is so fundamentally embedded in natural conditions, smellscape design could help raise awareness by creating new olfactory experiences significant of our time period.

Finally, smellscape design is at the crossroads of numerous issues related to the contemporary environment. In between quality of life and sensory marketing, health concerns and ecological challenges, it is particularly effective even though it is discreet, or may be because of its discreetness. Moreover, dealing fundamentally with ambient and atmospheric phenomena, diffuse and invisible components, smellscape design leads to a fresh questioning of what design is all about: a way of immersing in life, pervading bodies and places, and enhancing everyday sensory experience.


Fabienne Rayne

Solène Debaisieux

Anne Bonhoure

Bruno Beaumelle
email: bruno.beaumelle@irim.cnrs.fr

HIV-1 Tat is unconventionally secreted through the plasma membrane

Keywords: HIV-1, Tat, toxin, unconventional secretion

The Tat protein is required for efficient HIV-1 transcription. Moreover, Tat is secreted by infected cells and circulating Tat can affect several cell types, thereby contributing to HIV-1 pathogenesis. We monitored Tat secretion by transfected CD4+ T-cells. A Tat chimera carrying an N-glycosylation site did not become glycosylated when expressed into cells, while the chimera was glycosylated when mechanically introduced into purified microsomes. These data indicate that secreted Tat does not transit through the endoplasmic reticulum. The use of pharmacological inhibitors indicated that Tat secretion pathway is unusual compared to previously identified unconventional secretion routes and does not involve intracellular organelles. Moreover, cell incubation at 16°C inhibited Tat secretion and caused its accumulation at the plasma membrane, suggesting that secretion takes place at this level.

Introduction

The human immunodeficiency virus type 1 (HIV-1) transactivating protein Tat is a small (86-102 residues) and basic protein that enables efficient transcription of viral genes and has thus a key role in virus replication [START_REF] Jeang | Multifaceted activities of the HIV-1 transactivator of transcription, Tat[END_REF]. Although devoid of signal sequence that allows targeting to the ER/Golgi secretion apparatus, Tat can be secreted by infected cells [START_REF] Ensoli | Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients[END_REF]. Such unconventional secretion is also observed for some cell proteins. They include homeodomain transcription factors, HMGB, a non-histone nuclear factor as well as numerous cytokines, including FGF-1, FGF-2 and IL-1 [START_REF] Nickel | Mechanisms of regulated unconventional protein secretion[END_REF]. Their secretion pathways are collectively designed as nonclassical or unconventional export routes [START_REF] Dupont | Identification of a signal peptide for unconventional secretion[END_REF][START_REF] Nickel | Mechanisms of regulated unconventional protein secretion[END_REF]. These processes are intriguingly diverse and, so far, there seems to be as many pathways as proteins. Some, such as FGF-1, FGF-2 and homeodomains, are directly released through the plasma membrane, although by different mechanisms [START_REF] Dupont | Identification of a signal peptide for unconventional secretion[END_REF][START_REF] Prudovsky | The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export[END_REF][START_REF] Temmerman | A direct role for phosphatidylinositol-4,5bisphosphate in unconventional secretion of fibroblast growth factor 2[END_REF]. In other cases, secretion relies on intracellular organelles. This intermediate is a secretory lysosome for IL-1  [START_REF] Andrei | The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles[END_REF][START_REF] Gardella | CD8(+) T lymphocytes induce polarized exocytosis of secretory lysosomes by dendritic cells with release of interleukin-1beta and cathepsin D[END_REF], and the ER or the Golgi apparatus for the Tax protein of the human T-cell leukemia virus type 1, another human retrovirus whose transactivating protein is secreted by infected cells [START_REF] Alefantis | Secretion of the human T cell leukemia virus type I transactivator protein tax[END_REF][START_REF] Jain | Identification of human T cell leukemia virus type 1 tax amino Acid signals and cellular factors involved in secretion of the viral oncoprotein[END_REF]. The overall secretion route has been identified for most proteins that are secreted in an unconventional manner but, despite the fact that accumulating evidence suggests that circulating Tat is involved in evolution to AIDS [START_REF] Gallo | Tat as one key to HIV-induced immune pathogenesis and Tat toxoid as an important component of a vaccine[END_REF][START_REF] Huigen | Multiple effects of HIV-1 trans-activator protein on the pathogenesis of HIV-1 infection[END_REF][START_REF] Rubartelli | HIV-I Tat: a polypeptide for all seasons[END_REF], this is not the case for HIV-1 Tat.

Here we show that Tat secretion does not rely on intracellular organelles but takes place at the plasma membrane.

Materials and Methods

Plasmids and transfection

Tat (BH10 isolate, 86 residues) was cloned into pBi Tet expression vector (Clontech) that was transfected together with pUHD 15.1 Neo (Clontech) at a ratio of 10 pBi /1 pUHD to ensure expression into mammalian cells. The pET11d vector (Novagen) was used for Tat expression in E. coli. Tat-PNVSG (termed Tat-Glyc) was obtained by PCR using appropriate primers. Its coding sequence entirely sequenced (Genome Express, Meylan). The Jurkat CD4 + clone E6-1 was obtained from the ATCC, grown following their recommendations and transfected using electroporation as described [START_REF] Vendeville | HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF].

Reagents

The anti-Tat monoclonal antibodies for immunofluorescence and ELISA were from santa cruz biotechnology (sc-65912) and the Programme EVA Centre for AIDS Reagents (#ARP352), respectively. Rabbit anti-Tat (HXB2) antiserum was kindly provided by Erwann Loret (Marseille, France). Secondary antibodies were from Jackson ImmunoResearch. Chemicals and phalloidin conjugated to tetramethylrhodamine were from Sigma-Aldrich. Recombinant Tat and Tat-Glyc were produced in E.coli and purified as detailed earlier [START_REF] Vendeville | HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF]. Microsomes were obtained by centrifugation of postnuclear supernatant from homogenized Jurkat cells [START_REF] Vendeville | HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF].

Secretion assays

Jurkat cells were transfected with pBi-Tat (20 µg), pBi-firefly luciferase (2 µg) and pUHD (2 µg). Two days after transfection, cells were washed and transferred (7.5 x 10 5 cells / well, in triplicate) to an ELISA plate that had been coated overnight at 4°C with monoclonal anti-Tat antibody (1 µg/ml) before saturation with 8% (w/v) skimmed milk in phosphate-buffered saline (PBS). Dilutions of recombinant Tat (20 pg -1 ng/well) were also added to the plate for calibration purposes. After 6 h at 37°C (except when otherwise stated), cells were transferred to microtubes, centrifuged and the supernatant was frozen for later luciferase assay. Cells were then lysed and part of the lysate was used to assay intracellular Tat by ELISA, while the rest was frozen for the luciferase assay that was performed using a Promega kit. Secretion was calculated using the Tat (or luciferase) amounts present in the medium and intracellularly using the medium / (intracellular + medium) formula.

Immunofluorescence

Tat-or Tat-EGFP-transfected Jurkat cells were allowed to adhere to alcian-blue coated coverslips for 3 min at room temperature and were fixed in 3.7% paraformaldehyde before permeabilization with 0.1% saponin. Tat and cortical actin were then stained by immunofluorescence [START_REF] Vendeville | HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF]) and phalloidin-tetramethylrhodamine, respectively. Tat-EGFP was localized using EGFP fluorescence. Cells were mounted for observation using a Zeiss LSM 510 Meta confocal microscope and a 63 x objective. Median optical sections are shown.

Transactivation assays

Jurkat cells were cotransfected with 3 µg of a Tat or Tat-Glyc expressing vector, together with 7 µg of pGL3-LTR, which expresses firefly luciferase under control of the Tat-activated HIV-1-LTR promoter, and 1 µg of pRL-TK (Promega), which codes for renilla luciferase under the control of the herpes simplex virus thymidine kinase promoter. After 48 h, cells were lysed for dual luciferase assays (Promega) and transactivation activity was calculated using the firefly/renilla activity ratio [START_REF] Vendeville | HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF].

N-glycosylation analysis

To assay cell-free N-glycosylation, Tat or Tat-Glyc (10 µl at 2 mg/ml in 150 mM NaCl, 50 mM citrate, pH 7.1) were mixed with purified Jurkat microsomes (170 µl, 15 mg protein/ml in 150 mM KCl, 2 mM MgCl2, 2 mM DTT, 10 mM Pipes pH 7.1 supplemented with an antiprotease cocktail (Complete, Roche)) before sonication for 2 min and incubation at 37°C for up to 3 h. Where indicated, tunicamycin was used at 1 µg/ml. Samples were then cooled on ice, treated with 0.1 M Na2CO3 to remove unimported proteins, before centrifugation for 10 min at 160.000 g and pellet resuspension in SDS/PAGE/tricine sample buffer. After Western blotting, Tat was detected using rabbit anti-Tat antiserum and the ECL + reagent (GE Healthcare). To examine whether Tat travels through the ER during export, Jurkat cells were transfected with Tat or Tat-Glyc. After 48 h, cells were lyzed for Tat immunoprecipitation using ARP352 monoclonal antibody, followed by swine antimouse antibodies (Nordic Immunochemicals) and protein A sepharose (GE Healthcare). Immunoprecipitated proteins were resuspended in SDS/PAGE/tricine sample buffer. After Western blotting, Tat was detected using rabbit anti-Tat antiserum and the ECL + reagent.

Results and Discussion

In this study we studied Tat secretion pathway using Jurkat cells. These CD4 + T-cells were cotransfected with a Tat-encoding plasmid and a luciferase vector that was used as a control for transfected cell lysis. To prevent Tat oxidation by air oxygen that causes aggregation [START_REF] Chang | HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region[END_REF] and makes it unreactive to antibodies, we directly plated transfected cells onto ELISA plates that have been coated with anti-Tat monoclonal antibodies before cell incubation in degassed medium. Antibodies were thus allowed to capture secreted Tat before it gets oxidized in the medium, and the plate was revealed using standard ELISA procedures. Cells were recovered, washed and lysed for the assay of intracellular Tat by ELISA, using the same antibodies. Using this experimental system, we found that Tat secretion was linear for up to 6 h at 37°C (Fig. 1A). After this time, 25-30% of Tat was found in the medium, indicating that transfected cells secrete ~5% of cellular Tat per hour. Hence, Tat secretion is an efficient process that takes place in the absence of cell lysis since no release of cotransfected luciferase was observed with time, whatever the temperature (Fig. 1B). As reported earlier for COS-1 cells [START_REF] Chang | HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region[END_REF], Tat secretion was temperature-dependent and was essentially blocked at 16°C (Fig. 1A), a temperature that severely slows down membrane traffic and allows to accumulate transport intermediates in the exocytic [START_REF] Presley | ER-to-Golgi transport visualized in living cells[END_REF] or endocytic [START_REF] Magadan | Rab22a regulates the sorting of transferrin to recycling endosomes[END_REF] pathways. In the search of such structures that might be key intermediates in Tat secretion, we examined by confocal microscopy Tat intracellular localization in cells kept at 16°C for 6 h. Whatever the temperature, most Tat concentrates in the nucleus of Jurkat cells (Fig. 2). Nevertheless, while no plasma membrane labeling was detectable at 37°C, it became much more evident upon incubation at 16°C (Fig. 2, arrows). This was observed using both immunofluorescence or a Tat-EGFP chimera in either fixed (Fig. 2) or living cells (data not shown). These results indicated that Tat secretion takes place directly through the plasma membrane, and does not involve intracellular intermediates.

To further confirm this finding and characterize Tat secretion, we tried to interfere pharmacologically with this process using agents known to affect previously characterized unconventional secretion pathways. Drugs were used at concentrations that were found effective on T-cell lines [START_REF] Alami | Involvement of ATP-dependent Pseudomonas exotoxin translocation from a late recycling compartment in lymphocyte intoxication procedure[END_REF][START_REF] Vendeville | HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF]. These concentrations only induced a minute luciferase release (< 0.8%; Fig. 3), indicating that cell integrity was preserved throughout the experiment. Because Tat binds to microtubules [START_REF] Chen | HIV-1 Tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic Bcl-2 relative Bim[END_REF] and FGF-1 secretion depends on the actin cytoskeleton [START_REF] Prudovsky | The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export[END_REF], we first examined the effect of cytoskeleton poisons. Neither nocodazole, nor taxol that target microtubules significantly affected Tat secretion, while cytochalasin D that perturbs the actin cytoskeleton moderately inhibited secretion (by ~30%; Fig. 3). FGF-2 secretion requires Na + ,K + -ATPase activity that is blocked by ouabain [START_REF] Florkiewicz | The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na + ,K + -ATPase[END_REF], but this inhibitor did not affect Tat secretion. Endosome neutralizing drugs such as bafilomycin A1, monensin and nigericin prevent IL-1 import into late endocytic vesicles thereby inhibiting IL-1 secretion [START_REF] Andrei | The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles[END_REF]. None of these agents interfered with Tat secretion (Fig. 3), indicating that Tat follows an original secretion pathway.

We then examined in more details whether, as described for HTLV-1 Tax [START_REF] Alefantis | Secretion of the human T cell leukemia virus type I transactivator protein tax[END_REF][START_REF] Jain | Identification of human T cell leukemia virus type 1 tax amino Acid signals and cellular factors involved in secretion of the viral oncoprotein[END_REF], Tat transited by the ER and the Golgi apparatus on its way out. To this end we first used brefeldin A (BFA) that causes Golgi collapse and prevents exocytosis by the ER/Golgi pathway [START_REF] Chardin | Brefeldin A: the advantage of being uncompetitive[END_REF], as well as Tax secretion [START_REF] Alefantis | Secretion of the human T cell leukemia virus type I transactivator protein tax[END_REF]. As reported earlier for COS-1 cells [START_REF] Chang | HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region[END_REF], BFA did not affect Tat export by Jurkat T-cells (Fig. 3), indicating that Tat secretion does not involve the ER or the Golgi. To confirm this result using a different and drug-free approach, we prepared a Tat chimera bearing a N-glycosylation site (PNVSG) at its carboxyl terminus (Tat-Glyc). This tag should enable to trace the passage of a protein by the ER where the motif gets glycosylated [START_REF] Johannes | Retrograde transport of KDEL-bearing B-fragment of Shiga toxin[END_REF].

We first checked whether the glycosylation tag affected Tat biological activity. To this end, we used a transactivation assay. Indeed, this activity requires Tat interaction with several partners, i.e. proteins and RNA [START_REF] Jeang | Multifaceted activities of the HIV-1 transactivator of transcription, Tat[END_REF] and therefore provides information regarding the preservation of Tat structure and reactivity. Jurkat cells were cotransfected with a Tat vector, a plasmid expressing Firefly luciferase under the control of HIV-1 LTR and another vector expressing Renilla luciferase behind a CMV promoter. Transactivation was quantified as the increase in the Firefly / Renilla luciferase activity ratio [START_REF] Vendeville | HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF]. Since the Glyc tag is only 5-residues long, it was not surprising to find that Tat-Glyc retained entire Tat transactivation activity (Fig. 4A). We concluded that Tat conformation and reactivity was not affected by the presence of the Glyc tag that should not modify Tat secretion route.

We then examined whether the tag was correctly exposed so that Tat-Glyc was indeed glycosylated when reaching the ER lumen. We first tried to express Tat-Glyc behind a signal sequence to trigger Tat-Glyc import into the ER but this chimera was very poorly expressed into cells and was quickly degraded probably because, due to its basic nature, Tat could not be efficiently imported into the ER (data not shown). We thus prepared recombinant Tat-Glyc from E.coli, and microsomes from Jurkat cells [START_REF] Vendeville | HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF]. We then mechanically introduced Tat-Glyc into microsomes by sonication. Following incubation at 37°C, Tat-Glyc glycosylation was monitored by anti-Tat Western blot. An additional band (arrow) corresponding to a gain of ~5 kDa for Tat-Glyc appeared upon incubation (Fig. 4B). This band was not observed in the presence of tunicamycin, a well established glycosylation inhibitor [START_REF] Leavitt | Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses[END_REF] or when Tat was used instead of Tat-Glyc. Hence, the glycosylation motif was functional in Tat-Glyc. Nevertheless, when Tat-Glyc was immunoprecipitated from transfected cells before Western blot no glycosylation was detectable (Fig. 4C). Altogether with the absence of BFA effect on Tat secretion (Fig. 3) and the accumulation of Tat at the plasma membrane at non-permissive temperature (Fig. 2), these data indicate that Tat does not transit by the ER on its way out but is instead directly transported through the plasma membrane.

Conclusions

It should be noted that, although most cellular Tat is nuclear in cell lines such as Jurkat cells (Fig. 2) and that Tat does not follow the conventional ER/Golgi secretion pathway, Tat secretion is an efficient process. Nevertheless, it is difficult to visualize it at export sites, i.e. on the plasma membrane. This is probably due to efficiency of the export process that prevents Tat accumulation at this level. The identification of Tat partner(s) enabling its recruitment at the plasma membrane should help to understand this unconventional secretion pathway. It would also be interesting to examine whether cellular or viral proteins regulate Tat secretion. For instance, Tat owns seven Cys residues and one of them could be palmitoylated, as is the case with many proteins that associate with the plasma membrane [START_REF] Linder | Palmitoylation: policing protein stability and traffic[END_REF].

Figure Legends

Fig. 1 Time and temperature dependence of Tat secretion. Jurkat cells were transfected with Tat and luciferase expression vectors. After 48 h, they were plated onto ELISA plates precoated with anti-Tat monoclonal antibody. Secretion was allowed for the indicated time and temperature before Tat ELISA and luciferase assay. Intracellular Tat and luciferase were also assayed and secretion was calculated as medium / (cell + medium) (%) for both Tat (A) and luciferase (B). Data are mean + SEM of three independent experiments performed in triplicate.

Fig.2.

Tat is detected on the plasma membrane upon incubation at reduced temperature. Tator Tat-EGFP-transfected Jurkat cells were incubated at the indicated temperature before fixation, permeabilization and labelling with fluorescent phalloidin, which stains cortical actin just beneath the plasma membrane. Tat was detected using a monoclonal antibody and conventional immunofluorescence, while Tat-EGFP was localized using EGFP fluorescence. Representative medial confocal sections are shown. Bar, 10 µm. Fig. 3. Pharmacological characterization of Tat secretion process. Secretion was allowed for 6 h in the presence of the indicated molecule. Drugs were used at the following concentrations. Taxol, 0.1 µM; nocodazole (Noco), 10 µM; brefeldin A (BFA), 5 µg/ml; cytochalasin D (CytoD) 5 µM; ouabain, 100 µM; nigericin 0.1 µM; monensin, 1 µM; bafilomycin A1 (Bafilo), 100 nM. Higher drug concentrations were not more effective in inhibiting Tat secretion (data not shown). Data are mean + SEM of three independent experiments performed in triplicate.

Fig.4.

A Tat chimera bearing a glycosylation signal does not get glycosylated in living cells. A, Cells were cotransfected with a Tat-or Tat-PNVSG (termed Tat-Glyc) expressing vector and a plasmid expressing Firefly luciferase behind HIV-1 LTR. A plasmid encoding Renilla luciferase behind a Tat-independent promoter was used for normalization purposes, and transactivation was calculated from the Firefly / Renilla activity ratio. Data are mean + SEM of two independent experiments performed in quadruplicate. B, Jurkat microsomes were sonicated with recombinant Tat-Glyc or Tat before incubation at 37°C in the absence or presence of tunicamycin (TM) before SDS/PAGE and anti-Tat Western blot. An additional band (arrow) of molecular weight corresponding to glycosylated Tat-Glyc appeared upon incubation at 37°C in the absence of tunicamycin. C, Jurkat cells were transfected with a Tator Tat-Glyc-expression vector. One day later, Tat was immunoprecipitated before anti-Tat Western blotting. * indicates the position of Tat dimers. Data are representative results of three independent experiments.

Fig. 4 ,

 4 Fig.1, Rayne et al

Acknowledgements

We are grateful to Erwan Loret for rabbit anti-Tat antibodies. We are indebted to the AIDS Research and Reference Reagent Program, NIH, USA and the Programme EVA Centre for AIDS Reagents, NIBSC, UK for reagents.

Funding

This work was supported by grants from the ANRS and Sidaction.

Author contribution

FR and SD performed secretions assays and some immunofluorescence experiments. AB prepared Tat-Glyc and performed glycosylation experiments. BB designed the study, performed some immunofluorescence experiments and wrote the paper.