Ph Dondon

Th Samson Enseirb

Introducing embedded system concept through a multi thematic funny hexapod robot design project

Keywords: Educational reform, "teaching efficiency ", Embedded system, Multi thematic robot project, Student's motivation

In this paper, we first describe the pedagogical reform process in our school. Then, we point out the major human difficulties in reforming. The complexity of the relation between the new student generation and the teachers is briefly described. And some major changes in student's behaviour are given as example. The consequences on the teaching strategy are then indicated. At least, we show that theses evolutions require adaptation, efforts and changes in the way of teaching. And we finally present here, in our field of competence, a concrete and pedagogical robot design project (with technical details) we did, as an answer to the reform necessity.

Introduction 1.1 ENSEIRB learning overview 1.1.1 ENSEIRB Teaching reform

Since a few years, we observe in our electronic and informatics engineer school, a kind of increasing gap between the student's needs and what we gave to them. A global disaffection for all theoretical lessons appeared and our traditional pedagogical reached its limits. These tendencies we noticed in our school are confirmed by French national statistics: there is a global demotivation for the scientific curriculum. Economical, commercial studies seem to be now more attractive for this new generation of students.

In front of this situation, a Quality program has then been engaged in our school inducing a deep pedagogical reform as well on the bottom as on the form. All the scientific, electronic, and other engineering fields of the ENSEIRB program will have to be reformatted. Each one of us had to suggest improvement, modification in each own field of competence.

Reformatting fields of teaching in our electronic department

Comparing the situation in 1990 with the one of nowadays, some major evolutions appears in our department: In 1990, up to 90% of the courses were based on physics, mathematics and electronics. While 10% was dedicated to human and social sciences. The annual working time of students was around 900h. In 2005, the working time of the students decreases till 770h with a complete different repartition of the scientific and non scientific thematic : Indeed, the evolution of industrial needs, European criterion, and engineer profiles required, obliged us to redefine the relative proportion of each field as follow :

-Scientific teaching around 70% of the whole program including the main following topics: Physics and mathematics Analogue Electronic Digital Electronic (circuit and system) Informatic languages and tools, RF and Power Electronic Integrated circuit technology and design… -Non scientific field: 30% of the whole program including Foreign language, Human science training, Management and business training… In the scientific field of teaching, we observe that interest for analogue electronic is, year after year, reduced, while digital electronic (and embedded systems in particular) is more and more present.

The student's evolution

In parallel, we have attended for a few years, a change of behaviour students. The teachers are in front of a new kind of public and do not know how to manage behaviours which are not familiar. Among the major evolutions, we can extract the most important one's:

-A global disinterest for theoretical sciences is observed [START_REF]Web site[END_REF], [START_REF]Le monde de l'éducation[END_REF]: This phenomenon is confirmed by national [START_REF]Direction de l'évaluation de la prospective et de la performance note d[END_REF] and local statistics. There is a global loss of motivation for scientific matters. The total number of French students carrying on studies at a university level is decreasing with an annual rate of 1,3%. And the national rate of students choosing sciences studies is less than 2%.

-The international origin [START_REF] Ph | DONDON Plenary lecture -" An overview of psychological aspects and human factors in Scientific Engineering Education[END_REF], [START_REF] Perivoliotis | Education for Students of Different Cultural Backgrounds[END_REF] and the diversity of social origin of our students increase the difficulties of teaching: level dispersion and cultural diversity is not anymore compatible with traditional lessons in full classroom.

Figure 1a: number of foreign students in France [START_REF]Web site[END_REF] Figure 1b: international student's origin at Enseirb -Reduction of the long cycle of attention (inherent in human being and normally about 45 min with 1 hour): on a traditional course of 1 hour, we observe a progressive unhooking of the audience faster than before.

-Multiplication of the short cycles of attention: These one duration cycles from 6 to 7 minutes, are also inherent in the human being. They have appeared however more openly since a few years. They are concretised by successive unhooking and accosting during the course, phenomenon improperly qualified of "zapping" by some. These typical human behaviour are well known by neuro-linguistic programming ecialists [START_REF] Robert | Neuro-linguistic programming, the study of subjective experience[END_REF] but they are now more frequent.

Classical resistances in reforming

We often practice what we call "mismatching" in Neuro Linguistic Programming words [START_REF] Robert | Neuro-linguistic programming, the study of subjective experience[END_REF], in front of a new situation. We know all the small "killing" sentences: "Yes but…" "That will never go"... "It is a waste of time!" …. Or "it is not for us because we are not like the others!"… "Why change something since all is all right till now? This is often a manner to reject something without thinking about before. This is also the best way to lock a situat fo

Chinee se Mexico Marroco Tunisia

Black Africa The main classical locks which can obviously be nsposed in any human life evolution processes are: -Judgments : tra when judging a person or a beliefs forbid us to be n is the "not kn g and r example clothes, objects or concepts…) prevent from progressing ts like is one we suggest in this paper. This experience can st be seen as a first teaching evolution step… through atic funny project f a processor, a set of sensors with eir analogue and digital conditioning circuits and ctuators [START_REF] Kadionik | L'enseignement des systèmes numériques complexes[END_REF] Anyway, in this paper and as an example of this reform process, we describe here what some of us did to progressively move from analogue domain to embedded system field. Even if analogue electronic specialist can not become immediately a high level digital specialist, he can acquire enough new competences to manage some student's projec th ju

. Learning embedded systems 2 a multi them

Definition

An Embedded system can be defined as an electronic autonomous system dedicated to a precise task. It generally consists o th a

Teaching strategy

As a practical complementary approach of embedded system theoretical courses given b colleagues, we suggest here the design and the programming of an hexapod robot. If this concept of such project is obviously not new, its introduction in our scientific school is quite recent. The aim of this approach is to impr and to develop the curiosity of the students by a complementary practical approach. Such approach allows a soft approach to difficult rejected by the students. It also makes the student more co results. An other collateral effect of a long term project is the opportunity for the student to deve sp

Practical application

The funny robot (figure 3) (from easyrobotics company) [7] has been chosen to experiment this learning concept. As this robot's use was initially suggested for teenager's hobbyists, some modifications have been done to make this robot appropriate to a engineer level teaching: the initial processing board has been replaced by a microcontroller PIC 16F873 board in order to program in assembler or C langu la

. Description of the robot

With the use of this robot, the old full classroom course concept is now replaced by a global and practical approach : It allows a cross connection between different fields of science in one project: the "sensor world" of course, the embedded software for sensor management, the actuator control, the feed back theory, and power management. This approach covers exactly the same main fields than the old course but the human approach a completely different. In that way, an hexapod "competition" could be organized at the end of the project, t m

The set of sensors

Some sensors can be used to help the robot moving. In our case, a set of quite simple or sophisticated sensors is availa

Ultrasonic sensor

The ultrasonic module (figure 4a) [START_REF]Ultrasonic distance sensor module application sheet #28015[END_REF] can be mounted on the mobile turret (figur

Motion motors

We use here classical 200° angle servo motor for robot walking (Figure 7a) with two servomotors per leg. The rotation velocity of the servo motor is not an important characteristic to move the hexapod. So, we did not give it here.

Even Servo Odd Servo

Power management aspects

The quiescent current per servomotor is around 10mA.

When "powering on" the robot, the minimum consumption is 140mA (10mA*14). When walking, 12 among the 14 servomotors are working simultaneously, (figure 10) so that the total average supply current is close to 1.8 A. So, well sized NimH battery cells of 6,8V, 2400mAh has been chosen to power the robot. Instantaneous robot consumption is given on figure 10.

Walking strategy

Walking with an hexapod is not so easy than it could appears! We have to program and to synchronize the movements of the 6 legs and feet. For example, the figure 11a shows the 6 elementary movements sequenced to move forward. (Clear point: leg "up"; dark point: leg "down"). In a same way, we can define the other basic movements: backward, turning left and right. These movements are the four indispensable programmed movements.

Processing board [5]

Depending of the time schedule, we can use the Basic Stamp BS2 Parallax board (with a BASIC interpreter on board), (figure 12) or design our own microcontroller board.

In this case, a micro chip PIC 16F873 is often used. And a sensor C language management program can be written. Effects and advantages of "Real time programming" can be then compared to interpreted language programming.

Programming example

A motion algorithm is given as example of possible software implementation. This program contains around 400 lines of PBASIC code. The Hexapod robot moves first forward. If a front obstacle is detected, the robot stops, and looks right and left with turret ultrasonic sensor. The robot will turns in direction of free space. If there is no free space around the robot, it will execute a full half turn and move backward. In case of detection of a « suspect » movement above it with the PIR sensor, it will stops and stay in dead position during a short time.

Pedagogical flowchart

The project proceeds by group of 2 or 4 students (in second year from GEII University Technological Institute for students doing their final training period at ENSEIRB). It is distributed over a locked period of two months. Because of the multi competence aspects of our approach, the project is managed by two or three teachers. This allows sharing the knowledge and a better cross connection between the different fields.

The miles stones of the project are:

-Student group definition: Initiation with the team work. This allows a first student sensitizing to team management, From complementary courses on human resources management, the team profile is then identified (open, close synchronous, random) (cf figure 13). The main human characteristics of each team type are explained by the teachers. Then, the methodology, and the human organisation, (task management, leadership…) are described as consequences of the team profile, for a first student sensitizing to team management According to the individual profiles of each students, the student teams are then made up. The technical specifications are defined all together in order to give some freedom to the students: choice among the set of available sensors, power supply constraint, definition of the motion strategy and reaction in case of obstacle detection… -Project management initiation: Once the specifications are defined, the job of each member of the team must be defined. By a short seminar on project management, we help the students to make their task repartition, project manager designation and role attribution.

RANDOM

-Thematic bibliography: Showing the necessity of collecting information before starting working, we encourage the student to find documents, books, and articles in our technical library rather than on internet. This process is often more efficient.

-Sensors implementation: At this step of the teaching process, the students became conscious by their own, that they have to understand the physical principles and detailed description of the sensors before going further: In short lessons given by the "sensor" teacher, the main sensor physical effects are explained. Some training about conditioning circuits, calibration, is done before going back to the project. Each sensor used by the students, is individually characterized. So our students become not only "simple users" but furthermore "intelligent users"…

The following example shows the characterisation of the PIR sensor. The figure 11 shows a sensor view angle of 20° and figure 12 shows the time response of the sensor when submitted to an Infrared pulse.

-Motion: a few lessons given by the electro technical teacher are dedicated to DC motors, electronic drivers sizing, feed back theory, position and speed control. Each servomotor used by the students, is characterized (i.e, PWM signal, speed vs control signal, response time …) (cf. example in figure 13). -Semi global and global test: once the whole system is correctly manufactured, the behaviour of the sensors is individually tested. The motor behaviour is also checked. Finally, a "under true conditions test" is performed by verifying the global motion strategy. A video clip can be optionally done to illustrate the oral report.

-Final report: In this report, we request the students to reformulate what they understood during the project. This enables us to be sure that the sensors, actuators and embedded system bases were assimilated.

-Oral report: the group must orally expose the covered subject at the end of the project with slides and video tools during 20 minutes. Some technical questions are asked to the students to check the knowledge appropriation. -The funny aspects of the project are a source of interest. It is thus an opportunity to teach some unpleasing fields of electronic as simply as possible.

Advantages of "learning by multi thematic project"

-Some freedom into the design gives the impression to the students to be actor and creator. It acts like a motivation amplifier.

-While in a classical approach, the students often complain about the "bulk-heading" or the absence of connection between the courses, here, our « system » approach allows connecting different fields of electronic (Analogue, digital, sensors, embedded systems, micro programming, motor driving). It ensures a better comprehension, and causes a global interest for the lessons.

-Embedded systems concept is well received with such concrete and funny application.

-The physical and mathematical complexity of the sensors world is better received by the students through a practical approach.

-At least, this multi thematic project is the opportunity of a self encouragement to the student to deepen himself his knowledge by necessity and not obligation.

It is also a time for a human experience, a pleasant team work, and management: Each student can discover his own preferences, and personal interest in his work.

Results

Internal Statistics

Even if it is always difficult to "measure" the impact of a teaching strategy, this one seems to be more attractive than before. Each year, an opinion poll is performed (through anonymous WEB formulary) in our electronic department to get feed back from the students on the whole fields of studies. For our subject, 70% of the students answered to the poll. The last result shows that the global satisfaction rate raised from 45% up to 65%. (The satisfaction index includes technical, pedagogical and human's aspects). Of course, we are far from the perfection but what is important is that the satisfaction rate increases.

Feed back from the students

In this report, the students point out the funny aspect of the project, and also the system approach which allows mixing their different acquired technical knowledge. Some of them (the most motivated for embedded electronic) found the project too easy. However, if the technical level of this project is not very high, the most important for us was to improve the motivation, the interest, physical and mental presence of the students.

Impact on teacher's evolution

Through this multi thematic approach, it allows to the teachers to enlarge their knowledge and to move softly from their previous teaching field to newest one's like this very important "embedded systems" domain. Of course, this experience is not enough for the teachers to become immediately specialists in embedded systems practice, but we are able to help and to support the main teachers in this field of electronic.

Comparison with other experiences

Comparing to other experiences [START_REF] Avila | La pédagogie par projets[END_REF], [START_REF] Vincent | Radar de poursuite[END_REF], [START_REF] Ph | An alternating practical pedagogical approach for sensors and actuators teaching: Learning through a multi thematic mini rolling robot design project[END_REF], [START_REF] Grenier | Apprentissage par projet autour d'un robot mobile[END_REF], [START_REF] Anthierens | Julio, une plateforme educative pour la mecatronique CETSIS EEA Toulouse[END_REF], [START_REF] Vloebergh | Intégration d'une compétition de robotique au sein du cursus de formation en mécatronique[END_REF] done in different European Engineer schools, we see first that many colleagues are now testing this kind of approach in different fields of electronics. The same evolution in term of motivation is observed, even if it will never possible to obtained 100% of satisfaction rate. However, we must be careful, because "learning by project approach" has also its own limits in term efficiency and quantity of information given top the student by unit of time.

Conclusion

We first discussed in this paper the necessity of reforming our teaching approach: we had to adapt ourselves to the new emergent fields of electronic. We showed, as an example, a possible conversion from analogue to embedded field of teaching through the management of a multi thematic robot design project.

We showed also that it was possible for non specialist teacher to carry a complementary, integrated pedagogical offer to the students. Moreover, replacing some classical unpleasing lectures by "Learning by project" approach seems to be a good way to improve the efficiency of our teaching and also the behaviour, the motivation and the curiosity of the students. We are obviously conscious that pedagogy requires permanent adjustments to fit as well as possible to the student's needs and scientific evolution.

Figure 2 :

 2 Figure 2 : human cycles of attention

sp-

  Most of the students have a lot of extra scholar activities such as sport, internet electronic games, and other leisure's. They are interesting from a personal development point of view, but they also generate a too big mental energy dispersion which is fa1.1.4 Adaptation to the new dealsThe phenomenons we spoke about in the previous paragraph obliged the teachers to adapt their competencies and knowledge to this new situation. It is obviously not easy for many human and re

  theoretical courses and trainings which are nowadays nfident and responsible of his work and lop his team work irit and his management ability. age instead of the BASIC nguage from Parallax. situation, it is like putting a barrier or a limit, -Individual or collective beliefs: often the consequence of different internal mental construction, ours creative. One of the best illustratio famous sentence : owing that it was impossible, it did it" -Initial education: Family conditionin social rules format the human behaviour and reduce the power of creativity. -Conscious or unconscious links with the past (it can be fo and innovating.

Figure 3 :

 3 Figure 3 : assembled robot

3 WSEAS

 3 TRANSACTIONS on Advance in Engineering Education DONDON,SAMSON ENSEIRB,A. Schweitzer ISSN: 1790-1979 605 Issue 9, Volume 7, September 2008 3.1 General description This mini robot consists mainly of: board -A battery cells 6,8V 2400mAh is 15 cm high and 30cm length. ble. The most popular are shortly listed below. e 4b) in order to look for obstacle close to the robot. -Mechanical parts -A set of sensors, -12 servo motors and 6 legs -A rotating turret with 2 servo motors -A Basic stamp processing -A servo controller board It

Figure

  Figure 4a : ultrasonic module

Figure

  Figure 4b : ultrasonic sensors

Figure 5 : 3 . 2 . 2

 5322 Figure 5 : wall obstacle avoidance

Figure 6 :

 6 Figure 6 : Front whiskers and Top Passive Infra Red sensor (PIR) (white ball on the top)

Figure7a : servomotor

 servomotor Figure7a : servomotor

Figure 7b :

 7b Figure 7b: Typical PWM servo command signal

Figure 8b :

 8b Figure 8b : servomotors and reference angles

Figure 9 :

 9 Figure 9 : Parallax Servo controller board (16 channels)

Figure 10 :

 10 Figure 10 : instantaneous current consumption

Figure

  Figure 11a: walking forward sequence

Figure

  Figure 11b : Defence position

Figure 12 :

 12 Figure 12 : Basic stamp 2 processing board (from Parallax inc)

Figure 13 :

 13 Figure 13 : the four types of team and characteristics

Figure 11a :Figure 12 :

 11a12 Figure 11a: PIR sensor angle response

Figure 13 :

 13 Figure 13 : PWM servomotor control signal Vertical scale 5V/div, Horizontal scale 1ms/div

Figure 14 :

 14 Figure 14 : hexapod partial mechanical assembly

Acknowledgement

To Easy robotics company 77, Rue d'assevent 59131 Roussies, France, for technical support and robot free supply. WSEAS TRANSACTIONS on Advance in Engineering Education DONDON,SAMSON ENSEIRB,A. Schweitzer ISSN: 1790-1979 611 Issue 9, Volume 7, September 2008


Antoine Lonjon

Sophie Barrau

Eric Dantras

Philippe Demont
email: demont@cict.fr

Colette Lacabanne

Propriétés physiques de nanocomposites NTC-polyepoxy: influence du traitement de surface des NTC

Keywords: thermal properties, mechanical properties, conductivity, nanocomposites, CNT. MOTS-CLÉS : propriétés thermiques, propriétés mécaniques, conductivité électrique, nanocomposites, NTC

ABSTRACT. Polymeric matrix nanocomposites provide great opportunities to overcome macroscopic properties of polymers. The shape factor of nanofillers is a key parameter, so carbon nanotubes (CNTs) have attracted much interest. To avoid aggregation, mechanical stirring has been used. In those conditions, the percolation threshold of conductivity is reached at 0.15 wt % for CNTs. To improve the shape factor, amphiphilic molecules have been successfully used: in this case, the percolation threshold is at 0.04 wt % for CNTs. After the percolation threshold, such nanocomposites allow the elimination of electric charges, while keeping the same mechanical properties than the polymeric matrix. RÉSUMÉ. Les nanocomposites à matrice polymère offrent de nouvelles opportunités pour améliorer les propriétés macroscopiques des polymères. Le facteur de forme des nanocharges est un paramètre clé, ce qui constitue le grand intérêt des nanotubes de carbone (NTC). Afin d'éviter les phénomènes d'agrégation un mélange mécanique a été utilisé. Dans ces conditions, le seuil de percolation électrique est de 0.15% en masse. Pour augmenter le facteur de forme, des molécules amphiphiles ont été utilisées : dans ce cas le seuil de percolation est de 0.04% en masse. Au dessus du seuil de percolation, ces nanocomposites permettent l'écoulement des charges électrostatiques tout en conservant les propriétés mécaniques de la matrice.

Introduction

The dispersion of nanoparticles in a polymeric matrix is one of the ways to solve the electrostatic charges accumulation in insulating polymers [START_REF] Curran | A composite from poly(mphenylenevinylene-co-2,5-dioctoxy-p-phenylenevinylene) and carbon nanotubes: A novel material for molecular optoelectronics[END_REF][START_REF] Sandler | Development of a dispersion process for carbon nanotubes in a epoxy matrix and the resulting electrical properties[END_REF][START_REF] Wan | Synthesis, characterizations, and physical properties of carbon nanotubes coated by conducting polypyrrole[END_REF][START_REF] Kilbride | Experimental observation of scaling laws for alternating current and direct current conductivity in polymer-carbon nanotube composite thin film[END_REF][START_REF] Benoit | Transport properties of PMMA-carbon nanotubes composites[END_REF][START_REF] Barrau | Effect of palmitic acid on the electrical conductivity on carbon nanotubes-polyepoxy composite[END_REF]. Carbon nanotubes (CNTs) as a conductive filler will be considered to improve the electrical properties of insulating thermoset matrix. The experimental procedure was set up in order to prepare composites containing low weight content of nanofillers. Scanning electron microscopy studies were carried out in order to check the homogeneity of the dispersion.

As most polymers, epoxy resins are electrical insulators. Charges accumulation can be responsible for damages of composites. The preparation of composites with conductive fillers implies a high amount of fillers into the matrix. The use of carbon nanotubes (CNTs) as a conductive filler offers an attractive way to improve the electrical properties of polymers. In order to obtain CNTs dispersion, we added an amphiphilic molecule, palmitic acid. The hydrophobic part is adsorbed onto the nanotubes surface while the hydrophilic polar head induces electrostatic repulsions between nanotubes then preventing their aggregation.

Materials and methods

CNTs were synthesized by a catalytic chemical vapour deposition CCVD [START_REF] Flahaut | Synthesis of single walled carbon nanotube-Co-MgO composite powders and extraction of a nanotube[END_REF]. More than 85 % of CNTs were single-walled and doublewalled and 90 % had diameter d 3 nm. After extraction in HCl, CNTs were washed with distilled water and then kept in water to avoid their packing. CNTs-epoxy resin composites were prepared by dispersing weight amount of CNTs ranging from 0.04 to 0.8 wt % assisted by palmitic acid, with CNTs to palmitic acid weight ratio of unity [START_REF] Barrau | Effect of palmitic acid on the electrical conductivity on carbon nanotubes-polyepoxy composite[END_REF]. Functionalized CNTs-epoxy resin composites were elaborated by acid attack of CNT. CNTs were functionalized in nitric acid HNO 3 68% and HNO 3 16% at 120°C. Standard differential scanning calorimetry (DSC) measurements were performed using a DSC/TMDSC 2920 setup manufactured by TA Instruments. The sample temperature was calibrated using the onset of melting of tin (T m = 231.8 r 0.1 °C), indium (T m = 156.6 r 0.1 °C) and cyclohexane (T m = 6°C) with a heating rate of q h = 5°C.min -1 . The heat flow was calibrated with the heat of fusion of indium ('H = 28.5 r 0.1 J.g -1 ); its baseline was corrected using sapphire. DSC experiments were systematically carried out over a temperature range from the equilibrium state (in order to remove the effect of previous thermal history) T eq = T g + 30°C down to the glassy state T 0 = T g -50°C, and immediately followed by an heating rate q h = 5°C.min -1 . For each sample, the glass transition temperature but also the melting temperatures were measured. The dynamical mechanical analysis DMA was performed using an ARES strain controlled rheometer in the torsion rectangular mode. The maximum strain amplitude used (0.01%) was well within the linear viscoelasticity range. Dynamic storage modulus G' and tan G were recorded for temperatures ranging from -150 to 170°C at 3°C/min at the frequency of 1 Hz. AC conductivity measurements were carried out at room temperature using a Solartron -Schlumberger frequency response analyser with a Novocontrol interface in the frequency range from 10 -2 to 10 6 Hz. The real part V'(Z), of the complex conductivity V*(Z) was investigated. The DC conductivity V dc was taken as the value of the real part V'(Z) of the complex conductivity in the region of the low frequency plateau, i.e. circa 10 -2 Hz.

Results and discussion

Influence of CNTs on thermal properties of nanocomposites

Figure 1 shows the CNT content dependence of the glass transition temperature T g of the CNTs-epoxy resin composites. First, T g increases slightly at very low CNTs contents, but undergoes a weak depression ('T g = 5°C) at 0.07 wt.-%, i.e., very close to the electrical percolation threshold pc. Above p c , T g increases again by 4°C up to 0.1 wt.-% and then decreases very slowly with increasing CNTs content. This unexpected behaviour has been observed also in DMA for the temperature of the maximum of the tan G peaks (T D ) and the mechanical storage modulus G' undergo a minimum at the percolation threshold.

As expected to reinforce epoxy matrix by addition of CNTs filler, the vitreous mechanical modulus of nanocomposites is higher than the pure epoxy one, excepted for the CNTs content close to the percolation threshold.

Influence of CNTs with amphiphilic molecules on electrical properties of nanocomposites

When the dispersion of CNTs is assisted by amphiphilic molecules to decrease the percolation threshold, a T g depression is still observed at the new threshold [START_REF] Barrau | Effect of palmitic acid on the electrical conductivity on carbon nanotubes-polyepoxy composite[END_REF]. The origin of this depression is not the CNTs dispersion state in epoxy matrix. A weak decrease of molecular mobility of the epoxy matrix chain segments is observed in CNTs-resin epoxy composites excepted for the vicinity of the electrical percolation where the free volume accessible to the molecular motions of polymer chains is maximal. This behaviour has been observed too in polypyrroleepoxy resin composites [START_REF] Cassignol | Microstructure-conductivity relationship in conducting polypyrrole/epoxy composites[END_REF][START_REF] Barrau | Glass transition temperature depression at the percolation threshold in carbon nanotube-epoxy resin and polypyrroleepoxy resin composite[END_REF]. As shown in Figure 2, V DC displays a sharp increase by 8 decades between 0.05 and 0.1 wt %. This behaviour is the signature of a percolation threshold occurring at a very low mass fraction of CNT (0.04 %). Such low value is explained by the very high aspect ratio of CNTs and their good dispersion in epoxy matrix assisted by palmitic acid. The comparison with nanocomposites without palmitic acid shows clearly the improvement of CNTs dispersion by lowering the percolation threshold by a factor 4 in presence of palmitic acid. These results are consistent with SEM images (Figure 3a) which show that such nanocomposites contains a great number of individuals nanotubes while without palmitic acid, only CNTs bundles with diameter about 30-40 nm are observed (Figure 3b). As observed in CNTs -polymer composites [START_REF] Kilbride | Experimental observation of scaling laws for alternating current and direct current conductivity in polymer-carbon nanotube composite thin film[END_REF], the higher DC conductivity values appear lower than the intrinsic value for nanotubes in compressed pellets (50 S.cm -1 ) [START_REF] Benoit | Transport properties of PMMA-carbon nanotubes composites[END_REF] because a wrapping of nanotubes by epoxy matrix and preventing a direct electrical contact. However, a value of about 10 -4 S.cm -1 at 0.4 wt % is acceptable for insuring antistatic properties.

Influence of chemical functionalization of CNTs on electrical properties of nanocomposites

Figure 4 shows the influence of heating time and nitric acid solution concentration on electrical conductivity (AC) from 10 -2 Hz to 10 6 Hz. The composites were elaborated with 0.4 wt% of CNTs. The curve obtained without oxidization was represented. The value of ı'(Ȧ) at 10 -2 Hz was taken as dc conductivity (ı dc ). All the values obtained with HNO 3 at 68% shows a low electrical conductivity level around 10 -12 S.cm -1 . The oxidization with high concentration HNO 3 dropped the electrical conductivity of final nanocomposite. With low a b concentration HNO 3 solution a slight decreasing of conductivity is obtained. A decrease of electrical conductivity with oxidization is observed for all nanocomposites. The percolated network was highly disturbed by the functionalization and led to degradation of intrinsic properties of CNTs. 3), oxidized 1h in HNO 3 68% ( ), at 3h () and with HNO 3 16% during 1h (@), 3h () and 9h (&).

Influence of functionalized CNTs and amphiphilic molecules on mechanical properties of nanocomposites

Dynamic mechanical analysis permits to study the influence of CNTs on mechanic properties of nanocomposites. Samples were obtained by mechanically stirring with low concentration of CNTs (< 1 wt%). The mechanical experiments on the nanocomposites and the pristine polymer do not show variation of the vitreous modulus G'. Due to epoxy resin degradation near 160°C we cannot observe the influence of the CNTs on the rubber plateau.

Figure 5 shows the influence of palmitic acid on nanocomposites mechanical properties. The D relaxation mode associated with the viscolelastic relaxation decreases up to 6°C when the dispersion of CNTs is assisted by palmitic acid. This amphiphilic molecule has probably the same effect as a plasticizer on primary relaxation mode. The effect of chemical functionalization on mechanic properties of CNTs-epoxy resin nanocomposites was studied and reported on Figure 6. G' (T) was measured for 0.4 wt% of CNTs. CNTs were previously oxidized during 3h at 16% of HNO 3 . In the case of the dynamic stored modulus G', we do not observed the modification of mechanical properties by functionalization of CNTs.

Conclusion

The introduction of nano fillers in polymeric materials sounds a promising way to improve their macroscopic properties. Due to their very high aspect ratio, a specific interest has been devoted to CNTs. The presence of CNTs in a polymeric matrix induces only mild modifications of the physical structure of the disordered phase in the region of the glass transition. Contrarily, the evolution of electrical properties is spectacular since the electrical conductivity increases by 8 decades upon the introduction of only 0.15 wt % CNTs. By improving dispersion using amphiphilic molecules, the percolation threshold is decreased till 0.04 wt % CNTs. Since mechanical properties are not modified for such low loading, the introduction of CNTs in polymeric materials sounds an interesting route for eliminating static electric charges.

Figure 1 .

 1 Figure 1. Variation of DSC T g () and DMA T D (i) of the CNTs-epoxy resin composites with CNT weight content. Solid lines are guides for the eyes.

Figure 2 .

 2 Figure 2. DC conductivity of CNTs-epoxy resin nanocomposites at room temperature as a function of the CNTs weight content: ({) without palmitic acid and () with palmitic acid. Dashed lines are guides for the eyes.

Figure 3 .

 3 Figure 3. Scanning Electron Microscopy images of CNTs-epoxy resin nanocomposites for 0.8 wt % CNTs: (a) with palmitic acid and (b) without palmitic acid.

Figure 4 .

 4 Figure 4. AC conductivity at room temperature of CNTs-epoxy resin nanocomposites as function of HNO 3 attack time for 0.4 wt% CNTs: without functionalization (3), oxidized 1h in HNO 3 68% ( ), at 3h () and with HNO 3 16% during 1h (@), 3h () and 9h (&).

Figure 5 .

 5 Figure 5. Dynamic stored modulus G' of nanocomposites CNTs -palmitic acidepoxy resin for 0.02 ( ), 0.06 () and 0.8 () wt% CNTs

Figure 6 .

 6 Figure 6. Dynamic stored modulus G' of nanocomposites with 0,4 wt % of CNTs: without functionalization ( ) or oxidized with HNO 3 16% during 3h ().

Acknowledgements

The authors should like to thank Pr. Christophe. Laurent, Pr. Alain. Peigney and Dr. Emile Perez for many helpful and stimulating discussions.


Benoit Desnues

Amanda B Macedo

Diana Ordoñez-Rueda

Annie Roussel-Queval

Bernard Malissen

Pierre Bruhns

Marie Malissen

Lena Alexopoulou
email: alexopoulou@ciml.univ-mrs.fr

Diana Ordoñez

The transcriptional repressor Gfi1 prevents lupus autoimmunity by restraining TLR7 signaling

Keywords: Growth factor independence 1 (Gfi1), systemic lupus erythematosus (SLE), Toll-like receptor 7 (TLR7), animal model, innate immunity, dendritic cells ABC, age-associated B cell, BMDC, bone marrow-derived DC, BMF, bone marrow-derived macrophage, cDC, conventional DC, DC, dendritic cell, GC, germinal center, Gfi1, growth factor independence 1, IFN, interferon, IRF, IFNregulatory factor, MFI, mean fluorescence intensity, pDC, plasmacytoid DC, SLE, systemic lupus erythematosus, TLR, Toll-like receptor

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

Introduction

Growth factor independence 1 (Gfi1) is a transcriptional repressor that is required for multilineage blood cell development, since it controls both the differentiation of stem and progenitor cells into lymphoid and myeloid cells, and their functions [START_REF] Moroy | Growth factor independence 1 (Gfi1) as a regulator of lymphocyte development and activation[END_REF][START_REF] Phelan | Gfi1cells and circuits: unraveling transcriptional networks of development and disease[END_REF]. Gfi1 forms a complex together with other cofactors to control histone modifications that lead to silencing of the target gene promoters [START_REF] Moroy | Growth factor independence 1 (Gfi1) as a regulator of lymphocyte development and activation[END_REF][START_REF] Phelan | Gfi1cells and circuits: unraveling transcriptional networks of development and disease[END_REF]. In humans, Gfi1 mutations cause autosomal dominant severe congenital neutropenia, and dominant non-immune chronic idiopathic neutropenia of adults, which are heterogeneous hematopoietic disorders that cause predispositions to leukemias and infections [START_REF] Phelan | Gfi1cells and circuits: unraveling transcriptional networks of development and disease[END_REF][START_REF] Moroy | From cytopenia to leukemia: the role of Gfi1 and Gfi1b in blood formation[END_REF].

The Gfi1 protein consists of a six C-terminal zinc-finger domains and an N-terminal SNAG-domain linked by an intermediary region [START_REF] Chiang | Snail/Gfi-1 (SNAG) family zinc finger proteins in transcription regulation, chromatin dynamics, cell signaling, development, and disease[END_REF]. The transcriptional repression activity of Gfi1 depends on the zing finger motifs required for DNA binding, as well as the SNAG-domain responsible for nuclear localization [START_REF] Grimes | The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal[END_REF][START_REF] Zweidler-Mckay | Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a transcriptional repressor[END_REF]. Mouse studies have shown that Gfi1 is implicated in the development and function of hematopoietic stem cells, granulocytes, macrophages, dendritic cells (DCs), B and T cells and the control of endotoxin-mediated innate immune responses [START_REF] Karsunky | Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1[END_REF]. Indeed, Gfi1-deficient (Gfi1 -/-) mice are highly susceptible to LPS-induced septic sock due to exaggerated production of proinflammatory cytokines by macrophages, suggesting a negative regulatory role for Gfi1 in the TLR4-initiated signalling pathway [START_REF] Karsunky | Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1[END_REF]8]. Upon TLR4 stimulation, Gfi1 directly interacts with the p65 subunit of NF-κB through its intermediary region to control its DNA binding activity, thereby regulating the expression of a large number of NF-κB target genes [9]. Nevertheless, the implication of Gfi1 in the signaling pathway downstream of other TLRs has not been investigated.

Toll-like receptors (TLRs) sense conserved molecules from invading microorganisms, as well as endogenous molecules released from damaged tissue or dead cells and play crucial role in orchestrating both innate and adaptive immunity [10,11]. Thus, TLRs must be tightly regulated to avoid excessive activation by endogenous or exogenous ligands that can disrupt immune homeostasis and result in the development of inflammatory and autoimmune diseases, including systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disease that affects multiple organs and is characterized by production of autoantibodies against self-nucleic acids and associated proteins [12]. Accumulating evidence shows that TLRs and especially those detecting nucleic acids, are implicated in the pathogenesis of autoimmune diseases by recognition of self-molecules. The nucleic acid-sensing TLRs that are common between human and mouse include TLR3, which recognizes doublestranded RNA [13], TLR7 and TLR8 (human), which detect single-stranded RNA [14][15][16] and TLR9, which senses DNA containing CpG motifs [17]. Upon activation, TLR7, TLR8 and TLR9 recruit the adaptor molecules MyD88, while TLR3 recruits TRIF, and trigger the release of pro-inflammatory cytokines and type I interferons (IFN) through activation of NF-κB, MAP kinases and IFN-regulatory factors (IRFs) [10].

In the current study we evaluated the involvement of Gfi1 in the development of lupus and the signaling pathways that are controlled by Gfi1 in DCs. We found that both Genista mice, which carry a hypomorphic mutation in the gfi1 gene [18], and Gfi1 -/-mice [19] develop signs of spontaneous lupus autoimmunity. Our analysis revealed a previously unknown role for Gfi1 as a negative regulator of the TLR7 signaling pathway, since Genista mice deprived of TLR7 (Genista/TLR7 -/-) were protected from lupus autoimmunity. At the cellular levels, Genista DCs upon TLR7 or TLR4 stimulation produced increased amounts of TNF, IL-6 and IFN-β, which was accompanied by faster and stronger NF-κB phosphorylation and IRF7 nuclear translocation, suggesting that Gfi1 controls these signaling pathways downstream of TLR7 and TLR4. Overall, our results show that Gfi1 plays a critical role in the prevention of spontaneous lupus autoimmunity by negatively regulating TLR7 signaling.

Results

Genista and Gfi1 -/-mice develop spontaneous lupus autoimmunity

Previous studied have shown that Genista mice that carry a hypomorphic mutation in the Gfi1 gene and Gfi1 -/-mice are neutropenic and have increased numbers of monocytes, with Genista having a milder phenotype than Gfi1 -/-mice [START_REF] Karsunky | Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1[END_REF]18]. Indeed, side-by-side flow cytometry analysis revealed the Gfi1 -/-mice were severely neutropenic, while Genista mice had 4 times less neutrophils than WT mice (Fig. 1A).

Moreover, the increased percentage of blood monocytes was more profound in Gfi1 -/- than in Genista mice (Fig. 1A).

Assessment of serum levels of IgM and IgG2a isotypes revealed that Genista and Gfi1 -/-mice possess significantly increased levels compared to WT mice, whereas Gfi1 -/-mice also had significantly elevated IgG2a levels compared to Genista mice (Fig. 1B). In order to address if Gfi1 is implicated in autoimmunity the levels of IgG autoantibodies against RNA and DNA were also evaluated. Genista and Gfi1 -/-mice had significantly increased titers compared to WT sera, with Gfi1 -/-mice showing the highest increase (Fig. 1C). Immunohistological staining of kidney sections revealed stronger glomerular deposition of IgM and IgG in Genista and Gfi1 -/-mice than in WT controls, with Gfi1 -/-mice showing also increased size of glomeruli compared to Genista mice (Fig. 1D). Thus, these data suggest that Gfi1 deficiency or dysfunction can lead to lupus-like autoimmunity.

Lupus autoimmunity in Genista mice depends on TLR7 signaling

In contrast to Gfi1 -/-mice that are susceptible to infections and die at early age, Genista mice have normal viability up to 12 months, they manifest no weight loss and there is no need to be kept under long-term antibiotic treatment [START_REF] Karsunky | Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1[END_REF]18]. Therefore, we focused our further in vivo studies on Genista mice. Moreover, since there is a strong association between SLE and increased TLR7 signaling [20], we also generated Genista mice deprived of TLR7 (Genista/TLR7 -/-) and studied them in parallel with the Genista mice. Genista/TLR7 -/-mice had normal appearance, growth, fertility and normal survival rates up to 8 months of age that we followed up the mice. Assessment of IgM and IgG2a isotype levels and IgG autoantibodies against RNA, DNA and Smith ribonucleoproteins (smRNP) in sera revealed that Genista mice had significantly increased levels compared to WT controls, whereas Genista/TLR7 -/- mice had normal levels (Fig. 2A andB). Assessment of the frequencies of plasmablasts (B220 lo CD138 + ) and GCs B cells (B220 + GL-7 + CD38 -) revealed that both populations were significantly increased in Genista mice compared to WT controls, whereas in Genista/TLR7 -/-mice these populations appeared normal (Fig. 2C andD). In agreement with the FACS results, immunohistological analysis further confirmed the presence of increased splenic GCs in Genista mice versus WT or Genista/TLR7 -/-samples (data not shown). Furthermore, immunostaining of kidneys revealed that glomerular deposition of IgM and IgG was substantially greater in Genista mice than in WT controls, whereas Genista/TLR7 -/-mice showed a slightly diminished deposition versus WT mice (Fig. 2E). Collectively, these data indicate that Genista mice develop spontaneous lupus autoimmunity that it is TLR7-dependent.

In order to further validate the contribution of TLR7 in the lupus phenotype of Genista mice, we crossed the Genista mice with TLR8 -/-mice that develop spontaneous lupus due to increased TLR7 expression and signaling on DCs [21,22].

Although Genista, TLR8 -/-, and Genista/TLR8 -/-mice showed similar increased titers of IgM, IgG2a, and IgG autoantibodies against RNA, DNA and smRNP compared to WT mice (Supporting Information Fig. 1A-B), the Genista/TLR8 -/-mice displayed increased percentages of splenic plasmablasts and GC B cells, as well as more profound glomerular deposits of IgM and IgG compared to Genista or TLR8 -/-mice (Supporting Information Fig. 1C-E), suggesting that increased expression of TLR7 (due to TLR8 deficiency [21,22] in Genista mice) worsens the lupus phenotype observed in Genista.

Altered B cell compartment in Genista mice

Given that SLE is an autoimmune disease with B cell hyperactivity, and Gfi1 is implicated in B cell development, we next assessed the status of B cells. The frequencies of splenic B cells were significantly reduced in both Genista and Genista/TLR7 -/-mice compared to WT mice (Fig. 3A and Table 1). Staining for CD69 and MHC class II revealed that Genista splenic B cells were activated compared to WT or Genista/TLR7 -/-B cells (Fig. 3B), and this was accompanied by increased TLR7 expression in Genista versus WT B cells (Supporting Information Fig. 2). Moreover, since in the setting of lupus autoimmunity the populations of marginal zone (MZ), B1 and transitional T1 and T2 B cells can be affected we also evaluated these populations. Compared to WT mice, both Genista and Genista/TLR7 - /-mice had similar increased frequencies of MZ B cells (WT 9.6 ± 1.6,Genista 17.5 ± 1.8, Genista/TLR7 -/-22.4 ± 6.4) (Fig. 3C) and dramatically reduced frequencies of peritoneal B1a (WT 15.5 ± 4.9, Genista 2.3 ± 0.7, Genista/TLR7 -/-2.9 ± 1.2) and B1b B cells (WT 18.4 ± 3.4, Genista 7 ± 3.1, Genista/TLR7 -/-8.9 ± 1.1) (Fig. 3D). The frequencies of T1 B cells were similar between the three genotypes, however there was a tendency for reduced frequency of these cells in Genista mice compared to WT or Genista/TLR7 -/-mice (Supporting Information Fig. 3A). In addition, the frequencies of T2 and T3 B cells were similar in all three genotypes (Supporting Information Fig 3A). Furthermore, we also assessed the frequency of age-associated B cells (ABCs) that secrete autoantibodies and appear in autoimmune-prone mice by the time of onset of autoimmunity, through a TLR7-dependnt manner [23,24].

Genista mice had a significant increase in the frequency of ABCs in their spleens compared with age matched WT or Genista/TLR7 -/-mice (Supporting Information Fig. 3B), and the expression levels of T-bet was higher in Genista than in WT or Genista/TLR7 -/-ABCs (Supporting Information Fig. 3C).

Next, we performed ex vivo studies using mouse splenocytes. Genista B cells showed spontaneous increased expression of CD86 compared to WT B cells, and upon stimulation with R848, LPS or CpG, Genista B cells exhibited a stronger response than WT B cells (Fig. 3E and Supporting Information Fig. 4A). Moreover, Genista splenocytes produced significantly increased levels of IL-6 compared to WT splenocytes both in unstimulated conditions, as well as upon stimulation with R848, LPS, or CpG (Fig. 3F and Supporting Information Fig. 4B). As expected, Genista/TLR7 -/-splenocytes were unresponsive to R848, while upon LPS stimulation produced similar level of IL-6 as Genista cells (Fig. 3F).

B cells can proliferate and may differentiate into antibody producing cells in response to TLR triggering [25]. Therefore, we tested these parameters and their dependency on TLR7 in Genista mice. We found that WT and Genista B cells proliferate similarly in response to R848, LPS or CpG, while as expected Genista/TLR7 -/-B cells did not respond to R848 (Supporting Information Fig. 4C). However, in the course of proliferation we noticed that Genista cells showed a higher ability to differentiate into plasmablasts upon R848, LPS or CpG stimulation compared to WT cells, while Genista/TLR7 -/-B cells did not respond to R848, but upon LPS or CpG stimulation differentiated as efficiently as Genista cells (Supporting Information Fig. 4D).

Altogether, these results show that in Genista mice the B cells are more activated and respond stronger to TLR ligands.

Spontaneous activation of DCs and T cells in Genista mice

DCs and T cells are also implicated in the development and progression of lupus.

Therefore, we next assessed the frequencies and activation status of these cell types.

The frequency of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) were significantly increased in Genista mice compared to WT mice, whereas Genista/TLR7 -/-mice had normal frequencies of these cell types (Fig. 4A and Table 1). Evaluation of activation markers, such as CD86 and CD40, on the splenic DC subsets (CD11b-like, CD8α-like and pDCs), revealed that Genista pDCs (Fig. 4B and Supporting Information Table 1) and CD11b-like DCs (Supporting Information Fig. 5C and Supporting Information Table 1) were spontaneously activated compared with WT or Genista/TLR7 -/-cells.

Concerning the T cell compartment, the frequencies of splenic CD4 or CD8 T cells were similar in WT and Genista mice, while Genista/TLR7 -/-mice showed reduced frequencies of CD4 T cells and increased frequencies of CD8 T cells compared to WT or Genista mice (Table 1). Moreover, both Genista and Genista/TLR7 -/-mice had significantly reduced frequencies of naïve CD4 and CD8 T cells, and increased frequencies of effector memory (CD44 + CD62L -) CD4 and CD8 T cells compared to WT mice (Fig. 4C). The frequencies of central memory (CD44 + CD62L + ) CD4 T cells were similar in WT, Genista and Genista/TLR7 -/-mice, while central memory CD8 T cells were significantly increased in Genista and Genista/TLR7 -/-mice compared to WT mice (Fig. 4C). Thus, the increased frequencies and activation status of DCs in Genista mice are TLR7-dependent, while the increased T cell activation status in Genista mice is TLR7-independent.

Increased response of Genista DCs upon TLR stimulation

In macrophages Gfi1 expression can be induced by the TLR4 ligand LPS in a TNFindependent manner [9], but its modulation by other TLRs or its expression by DCs has not been addressed. Thus, we examined the effects of TLR ligands on Gfi1 expression in WT macrophages and DCs. Stimulation with TLR2/6, TLR3, TLR4, TLR7 or TLR9 ligands upregulated Gfi1 mRNA expression both in bone marrowderived macrophages (BMMFs) (Supporting Information Fig. 5A) and bone marrowderived DCs (BMDCs) (Supporting Information Fig. 5B), suggesting that in macrophages and DCs the expression of Gfi1 is altered upon TLR stimulation.

It has been reported previously that Gfi1 -/-BMMFs respond to LPS with increased TNF secretion [START_REF] Karsunky | Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1[END_REF]9]. Thus, we tested whether the Genista mutation also affects the responses of macrophages and DCs to TLR stimulation. Upon R848, LPS or CpG challenge, Genista BMMFs produced normal amounts of IL-6 and TNF (Supporting Information Fig. 5D), while Genista BMDCs produced significant increased amounts of IL-6 and TNF versus WT BMDCs (Fig. 5A). Moreover, upon R848 or LPS stimulation Genista BMDCs showed increased expression of IFN-β, and the type I IFN stimulated genes, Isg15 and IRF7 versus WT cells (Fig. 5B). Thus, upon TLR stimulation the Genista mutation causes an overproduction of cytokines by DCs, but not by macrophages.

We next assessed the implication of Genista mutation on the NF-κB and type I IFN signaling pathways on DCs. Upon R848 or LPS stimulation, Genista BMDCs showed stronger and faster NF-κB p65 phosphorylation than WT cells (Fig. 5C and Supporting Information Fig. 5E). In addition, we monitored the activation of the transcription factors IRF7, which resides in the cytosol of resting cells and upon cytosolic TLR stimulation undergoes serine phosphorylation, allowing it to translocate into the nucleus and induce the expression of type I IFN genes [26]. Under resting/unstimulated conditions IRF7 was already present in the nucleus of Genista, but not of WT, BMDCs (Fig. 5C and Supporting Information Fig. 5F). In addition, upon R848 stimulation, nuclear expression of IRF7 was stronger in Genista than in WT cells at 15 and 120 min, while upon LPS stimulation WT cells showed a delayed response and nuclear expression of IRF7 was stronger in Genista than in WT cells at 15 min (Fig. 5C and Supporting Information Fig. 5F). Overall, these results show that Genista BMDCs are more responsive than WT cells and suggest that Gfi1 is a negative regulator of the TLR7 and TLR4 signaling pathways in DCs.

Discussion

Engagement of TLRs activates multiple signaling pathways that have to be tightly regulated since excessive TLR activation can disrupt immune homeostasis and may lead to the development of autoimmune and inflammatory diseases [27,28]. In this study, we uncovered a critical role for the transcriptional repressor Gfi1 in the development of lupus autoimmunity by restraining TLR7 signaling. We demonstrated for the first time that neutropenic Gfi1 -/-or Genista mice develop spontaneous lupus autoimmunity. In addition, we provide evidence that the lupus phenotype in Genista mice is TLR7-dependent, since Genista mice deprived of TLR7 were protected from lupus development, while increase of TLR7 signaling (in Genista/TLR8 -/-mice) led to the exacerbation of the lupus phenotype. Our finding fits with the current notion that increased TLR7 signaling contributes to the initiation and/or exacerbation of SLE in mice [22,[29][30][31]. Interestingly, lately it has become apparent that also in humans SLE can be attributed to increased TLR7 function. Indeed, various studies revealed that the SNP rs3853839 (C-G) at the 3' untranslated region of TLR7 is associated with elevated TLR7 transcript expression and increased risk for SLE both in Asian [32][33][34] and Danish [35] populations.

Many different immune cell types are implicated in the development of SLE, of which DCs and B cells play a central role [36,37]. cDCs and B cells are major antigen presenting cells that regulate T-cell mediated autoimmunity, while pDCs drive disease by type I IFN production. DCs from lupus-prone mice display a range of alterations in their frequency and function [36,38]. Accordingly, we showed that Genista mice had increased numbers of splenic cDCs and pDCs, along with increased activation, while in Genista/TLR7 -/-mice these DC populations appeared normal, indicating that in Genista mice increased TLR7 function is responsible for the observed DC alterations. Analysis of TLR7 expression in the splenic DC subsets revealed that cDCs from Genista mice overexpressed TLR7, but this was not observed in pDCs (Supporting Information Fig. 2), suggesting that Genista mutation can affect TLR7 signaling without necessarily affecting TLR7 expression.

Contrary to the increased numbers and activation of DCs in Genista mice, previous studies have shown that Gfi1 deficiency leads to global reduction of all DCs in primary and secondary lymphoid organs, as well as, incomplete DC maturation and function [39]. Moreover, Gfi1 -/-hematopoietic progenitors are unable to develop into DCs in vitro and instead they differentiate into macrophages [39], leading to the belief that Gfi1 is a unique factor governing DC versus macrophages development.

However, we found that GM-CSF treated Genista hematopoietic progenitor cells were able to give rise to DCs, suggesting that the hypomorphic Genista mutation (C318Y substitution at the third zinc finger domain of Gfi1) does not affect the ability of Gfi1 to control DC development and function. Additional studies that are out of the scope of the Accordingly, in Genista mice we found an augmentation of autoantibody titers for RNA, DNA and smRNP, as well as frequences of spontaneous GCs, which were due to increased TLR7 signaling since these phenotypes were absent in Genista/TLR7 -/- mice. Our findings further confirm recent observations that B cell-intrinsic TLR7 signaling, through detection of self-nucleic acids and/or retroviral elements, is essential for the development of spontaneous GCs, and that in the presence of autoimmune conditions TLR7-dependent spontaneous GCs produce pathogenic autoantibodies [41]. In addition, we observed that the B cell subset of ABCs that is driven by TLR7 signaling, secretes autoantibodies and arise during autoimmune disease both in mouse models and humans [23,24,42,43], was also increased in Genista mice through a TLR7-dependent manner. Nevertheless, we noticed normal B cell proliferation in Genista mice, which is in accordance with previous studies on Gfi1 -/- B cells [44]. However, stimulation of Genista splenocytes with TLR7, TLR9 or TLR4 agonists promoted increased B cell differentiation to plasmablasts. Accordingly, Genista mice showed increased percentage of plasmablasts, while in Genista/TLR7 -/- mice this cellular compartment was normal, suggesting that plasmablasts differentiation is disturbed in Genista mice, due to increased TLR7 function.

Marginal zone and B1 B cells have the capacity to respond to foreign antigens more rapidly than conventional B cells, providing early immune responses to blood-borne pathogens, and both subsets are often altered in lupus prone mice [37]. In various transgenic mice in the C57BL/6 background that develop lupus due to increased TLR7 signaling, such as mice that carry the Yaa locus [45], transgenic mice that overexpress TLR7 [29], mice that carry the Unc93B1-D34A mutation [46], or TLR8deficient mice [21,22], the increased TLR7 signaling leads to reduced MZ B cell compartment, probably due to increased activation of the MZ B cells and their exit from this compartment [47]. Surprisingly, the MZ B cell population was markedly expanded in Genista mice, and this phenotype was not altered in the absence of TLR7 signaling, suggesting that Gfi1 independently of TLR7 might act as a transcriptional regulator responsible for controlling the differentiation and/or lifespan and survival of the MZ B cells. B1 B cells are located mainly in the peritoneal and pleural cavities and play critical roles in immediate defense against viruses and bacteria, as well as housekeeping removal of cellular debris, through the spontaneous and constitutive secretion of polyreactive IgM antibodies [48]. B1 B cells express various TLRs and upon detection of microbial components TLR signaling induce a massive egress of B1 B cells from the peritoneal cavity, which is associated with coordinated down-regulation of integrins and CD9 [49]. Thus, the dramatic reduced numbers of peritoneal B1 B cells in Genista and Genista/TLR7 -/-mice could be due to increased TLR signaling (other than TLR7) in B1 B cells that leads to rapid mobilization of these cells out of the peritoneal cavity or the Genista mutation leads to defects of the factors that are critical for the development and migration of B1 B cells and/or precursors in the peritoneal cavity.

To avoid inappropriate inflammatory responses, many negative TLR regulators exist that act at different levels in the active TLR signaling pathways and use different mechanisms such as dissociation of adaptor complexes, degradation of signal proteins or transcriptional regulation [28]. Based on its biochemical function as transcriptional repressor and recruiter of histone modifiers, Gfi1 is considered as an epigenetic regulator that modify chromatin structure mostly to repress transcription [50]. Thus, Gfi1 in contrast to most known negative regulators of TLRs, does not affect the proximal cytoplasmic components of TLR signaling pathways but rather acts at the downstream end of the pathway [9]. Indeed, in macrophages Gfi1 negatively controls the TLR4-mediated inflammatory response by antagonizing NF-κB p65 [9], but its implication to the signaling pathway downstream of other TLRs and in DCs has not been addressed. Our data clearly show that upon TLR7 or TLR4 stimulation the Genista mutation leads to increased production of cytokines (TNF, IL-6, IFN-β) and IFN stimulated genes (Isg15 and IRF7) by DCs, which is accompanied by increased NF-κB p65 and type I IFN signaling. Thus, Gfi1 seems to be a general negative regulator of the TLR signaling pathway.

The functional contribution of neutrophils in SLE pathogenesis versus protection remains controversial. In SLE patients, impaired neutrophil extracellular trap (NET) clearance and/or aberrant NETosis has been suggested to play a prominent role in the perpetuation of autoimmunity and the exacerbation of disease, as well as the induction of end-organ manifestation [51]. However, several recent studies revealed that neutrophils can inhibit systemic autoimmunity [52,[START_REF] Trigunaite | Gr-1(high) CD11b+ cells suppress B cell differentiation and lupus-like disease in lupus-prone male mice[END_REF]. Our results show that the neutropenic Gfi1 -/-and Genista mice develop spontaneous lupus-like autoimmunity, rendering these mice as new mouse models of lupus autoimmunity that is accompanied by neutropenia.

In conclusion, our findings demonstrate the critical role of Gfi1 in the regulation of myeloid cells, and prevention of spontaneous lupus autoimmunity by negatively controlling TLR7 signaling. It still remains to be seen if cases of human SLE can be attributed to Gfi1 gene deficiency or modifications.

Materials and Methods

Mice

Genista, Gfi1 -/-, TLR7 -/-and TLR8 -/-mice were generated as described previously [16,18,19,21]. Genista/TLR7 -/-and Genista/TLR8 -/-mice were generated by intercrossing Genista with TLR7 -/-or TLR8 -/-mice. All mice were backcrossed to the C57BL/6 background for more than 10 generations. Wild-type (WT) C57BL/6 mice were purchased from the Charles River Laboratories. Mice were housed under specific pathogen-free conditions. All animal experiments have been approved by the local Ethical Committee of Marseille for animal experimentation (04364.03).

Reagents

R848, LPS from E. coli 0111-B4, Pam2CSK4, CpG ODN 1826 and poly I:C were purchased from Invivogen.

Cell culture

Bone marrow cells were extracted from mouse femur and tibias and cultured in the presence of GM-CSF or M-CSF for the production of bone marrow-derived DCs (BMDCs) or bone marrow-derived macrophages (BMMFs) as described previously [13,21]. Erythrocyte-depleted splenocytes were cultured in RPMI medium supplemented with 10% FCS, 1% MEM Non-Essential Amino Acids, and 50 µM βmercaptoethanol.

Serological analysis and ELISA

Evaluation of IgM and IgG2a, and IgG autoantibodies against DNA, RNA and smRNP in serum samples was performed as described previously [21]. Cell culture supernatants were assayed for IL-6 by ELISA (eBioscience) according to manufacturer's instructions.

Flow cytometry

Cell suspensions were incubated with 24G2 hybridoma supernatant (to block Fc receptors) and stained using immunofluorescence-labeled antibodies against the following antigens: B220/CD45R, CD3, CD19, CD11b, Ly6G, CD21, CD5, CD44, CD86, CD38, CD138, CD24, CD172a (SIRPα), CD69, CD43, CD40, CD45.2, CD93, IgM, TLR7 and CD62L from BD Biosciences, IA/IE (MHC class II), GL7, CD23, Tbet, CD4 and CD8 from eBioscience and CD11c and SiglecH from Biolegend. For intracellular TLR7 staining, cell suspensions were stained for the indicated extracellular antigens, before fixation with Cytofix (BD Biosciences). Cells were then permeabilized with saponin 0.1% and stained for TLR7. Intracellular T-bet staining was performed using the FoxP3 Fixation/Permeabilization buffer (eBioscience).

Flow cytometry was conducted using an LSR2 (BD Biosciences) and data were analyzed with FlowJo (Tree Star). The full gating strategy that used for flow cytometry analysis is presented in Supporting Information Fig. 6. For splenocyte proliferation experiments, erythrocyte-depleted splenocytes were labeled with CellTrace Violet (Life technologies) according to manufacturer's recommendations.

After 4 days of stimulation, cells were harvested and stained with antibodies for analysis by flow cytometry.

RNA isolation and quantitative real-time PCR

Total RNA was isolated by TRIzol reagent (Gibco, Invitrogen) and reversed transcribed with SuperScript II reverse transcriptase (Invivogen). Q-PCR was performed with the SYBR Premix Ex Taq (Takara) using the primers shown on Supporting Information Table 2, on an Applied Biosystems PRISM 7700 Sequence Detection System, and the amount of target was calculated relative to the calibrator by 2 -ΔCT .

Immunohistology

Murine kidneys or spleens were embedded in OCT compound and frozen in liquid nitrogen. Cryostat sections were rehydrated in PBS, incubated with blocking solution (2% BSA in PBS) for 2h at room temperature. Kidneys were stained with anti-IgG Alexa Fluor 488 (Invitrogen) and IgM PE (BD Biosciences) and fluorescence images were acquired using a Zeiss LSM 780 laser scanning confocal microscope and analyzed with the Zen software.

Western blot

For whole cell protein extracts BMDCs were washed with ice-cold PBS and cells were lysed in RIPA buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1% Triton-X100, pH 7.5), while for isolation of cytosolic or nuclear protein extracts BMDCs were treated in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA) or buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl 1 mM EDTA), respectively. Protein samples were then loaded onto 10% SDS-PAGE gels, electrophoresed and transferred onto Immobilon P membrane (Millipore). Blotting was performed using the following antibodies: p65 and phospho-p65 S311 (Santa Cruz), IRF7 (OriGene) and beta-actin (Sigma). The bound antibodies were detected using the Pierce ECL Western Blotting substrate (Thermo Scientific).

Statistics

Statistics were calculated using Prism 5 (GraphPad Software) by the Mann-Whitney U test with p values indicated throughout as *p < 0.05, **p < 0.01, and ***p < 0.001. Table 1. Body weight, spleen weight, total cell count, and major splenic cell populations of WT, Genista and Genista/TLR7 -/-mice a) .

WT

Genista Genista/TLR7 .. 9 .. Supporting Information Table 1. Activation status of WT, Genista and Genista/TLR7

  current manuscript are needed to further clarify the implication of different Gfi1 variants in DC development. B cells contribute to lupus pathogenesis by autoantibody secretion and GCs are an important B cell tolerance checkpoint in the periphery. Thus, increased autoantibodies and spontaneous GCs are characteristic of several autoimmune prone mice [40].

a)

  Data are from 5 or 6-7 ( * ) months old male mice. Values shown are average of at least four mice per genotype ± SD. a p < 0.05 versus WT and b p < 0.05 versus Genista determined by the Mann-Whitney U test.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Genista mice develop TLR7-dependent lupus autoimmunity. Sera from 9

Figure 3 .

 3 Figure 3. Spontaneous B cell activation and enhanced B cell responses to TLR

Figure 4 .

 4 Figure 4. Immune cells and activation status in WT, Genista and Genista/TLR7 -/-

Figure 5 .

 5 Figure 5. Increased responses of Genista DCs to TLR agonists. (A-B) BMDCs from

  Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Table 2 .

 2 -/-splenic DC subtypes < 0.05 versus WT and b , P < 0.05 versus Genista determined by the Mann-Whitney U test. Values shown are average of at least three mice per genotype ± SD. Primers used for qPCR

		Activation
	DC subtype		WT	Genista	Genista/TLR7 -/-
		marker
		CD86	236.0 ± 102.1	375.0 ± 59.8	188.3 ± 104.4 b
	CD11b-like
		CD40	302.8 ± 20.4	365.0 ± 10.6 a	330.3 ± 14.8 b
		CD86	482.3 ± 207.7	654.2 ± 52.5	354.7 ± 205.3 b
	CD8α-like	CD40	247.0 ± 33.4	372.3 ± 26.0 a	310.8 ± 37.8
		CD86	677.0 ± 249.9	1243.0 ± 121.5 a 286.7 ± 123.7 b
	pDC
		CD40	213 ± 31.6	312.5 ± 84.1 a	176.8 ± 28.3 b

a , P

Acknowledgements

We thank the members of the animal facility, histology and flow cytometry core facilities of the CIML for help and assistance. These studies supported by a grand from Arthritis Fondation Courtin to L.A. and institutional grants from CNRS and INSERM. A.B.M. was supported by a fellowship from Région Provence-Alpes-Côte d'Azur co-financed by InnatePharma. D.O.R. was supported by fellowships from FRM and ARC.

Conflict of interest

The authors declare no commercial or financial conflict of interest.

Supporting Information Figures and legends

Supporting Information Figure 1. Increased lupus phenotype in Genista/TLR8 -/-versus Genista or TLR8 -/-mice. (A and B) Sera from 3 to 4 months old male WT (n = 8), TLR8 -/- (n=7), Genista (n = 8) and Genista/TLR8 -/-(n = 7) mice were used for the evaluation of (A) serum levels of IgM and IgG2a and (B) IgG autoantibodies against RNA, DNA and smRNP Supporting Information Figure 2. Expression of TLR7 by splenic B cells and DCs at steady state. Splenocytes from 6 months old female WT (red line), Genista (blue line) and Genista/TLR7 -/-(green line) mice were stained for B220, CD11c, SiglecH, MHCII and TLR7. Plots represent the intracellular expression of TLR7 after gating on B cells (B220 + SiglecH -), pDC (B220 + CD11c int SiglecH + ) or cDC (B220 -CD11c hi MHC II hi ). Supporting Information Figure 5. Expression of Gfi1 by WT macrophages and DCs, DC activation status and BMMFs cytokine production upon TLR stimulation and DC responses upon TLR activation. (A) BMMFs or (B) BMDCs from WT mice were left untreated or stimulated with Pam 2 CSK 4 (TLR2/6 ligand) (30 ng/ml for BMMFs and 50 ng/ml for BMDCs), 50 µg/ml poly I:C (TLR3 ligand), 10 ng/ml LPS (TLR4 ligand), 50 nm R848 (TLR7 ligand) or CpG (TLR9 ligand) (30 nm for BMMFs and 50 nm for BMDCs) for the indicated time points. Total RNA was extracted from the cells and the expression of Gfi1 was assessed and normalized with the 2 -ΔCT method to the β-actin internal control by Q-PCR. Data are from a single representative experiment of 3 independent experiments. (C) Increased activation status of Genista splenic DC subsets at steady state. Splenocytes from 7 months old male WT (red line), Genista (blue line) and Genista/TLR7 -/-(green line) mice were stained for CD19, CD11c, SiglecH, CD11b, CD8, MHC-II and CD86. Plots represent the expression of CD86 after gating on pDC (CD19 -CD11c int SiglecH + ), CD8-like cDC (CD19 -CD11c hi MHC-II hi CD11b -CD8α + ) and CD11b-like cDC (CD19 -CD11c hi MHC-II hi CD11b + CD8α -). (D) BMMFs from WT (n=3) and Genista (n=3) mice were left untreated or stimulated with R848, LPS or CpG. After 8 h, the concentrations of IL-6 and TNF in the culture supernatants were assessed by ELISA. (E-F) Densitometry quantification related to figure 5C. Densitometry was determined for the P-NF-κB p65, NF-κB p65, IRF7 and β-actin bands. Graphs represent the ratio of (E) P-NF-κB p65 to NF-κB p65 and (F) IRF7 to β actin band intensities, respectively. Data (A-F) are representative of at least 2 independent experiments (n = 3-5 per group).

Supporting

Supporting Information Figure 6. Gating strategies used for immunophenotyping cell populations in different compartments. Cells from (A) blood, (B) spleen or (C) peritoneal lavage were gated via FSC/SSC on live cells and debris and doublets were excluded. (A) In the blood, T cells (CD5 + ) and B cells (B220 + ) were excluded from CD45.2 + hematopoietic cells and the remaining cells were separated into neutrophils (Ly6G + CD11b + , fraction a) and monocytes (Ly6G -CD11b + , fraction b). (B) In the spleen, plasmablasts were determined as CD138 + B220 lo/-cells (fraction c), while GC B cells were determined as B220 + GL7 + CD38 - cells (fraction d). CD19 + cells were divided into MZ (CD21 + CD23 -/lo , fraction e), follicular (CD21 + CD23 + , fraction f) and immature (CD21 -CD23 -, fraction g) B cells. For the analysis of DCs, CD19 + cells were gated out, pDCs were then defined as CD19 -SiglecH + CD11c int (fraction h) and cDCs as CD19 -MHCII hi CD11c hi cells (fraction i). cDCs were further stained with CD24 and CD172a to obtain CD8-like (CD19 -MHCII hi CD11c hi CD24 + CD172a -, fraction j1) and CD11b-like (CD19 -MHCII hi CD11c hi CD24 -CD172a + , fraction j2) DC cells. Moreover, splenocytes were stained for CD3 and separated into CD8 T cells (fraction k) and CD4 T cells (fraction l), which were further divided into naïve (CD62L hi CD44 lo/-, fraction m), central memory (CD62L hi CD44 hi, fraction n) and effector memory (CD62L lo CD44 hi, fraction o) subpopulations. (C) For peritoneal cells, CD19 + B cells were stained with CD5 and CD43 to obtain B2 (CD43 -CD5 -, fraction p), B1a (CD43 + CD5 + , fraction q) and B1b (CD43 + CD5 -, fraction r) cells.


Philippe M Dondon-J
email: philippe.dondon@enseirb.fr

J Micouleau-

Legall-P

rue A Schweitzer- Kadionik Enseirb

Talence

Design of a low cost BPSK modulator/demodulator for a practical teaching of digital modulation techniques

Keywords: digital transmission and modulation, BPSK, education, practical lessons

Teaching the digital transmission techniques-like other fields of electronic-is not always easy : There is classically theoretical courses and practical lessons. The link between the two is often the most important difficulty that appears for the students. An abundant literature on digital wireless, including high mathematical level approaches is available; since a few years, theoretical courses are easily illustrated by the 2D computer simulations. But simple descriptions or analysis are rarely found and simulations do not replace a concrete true design. In order to improve the efficiency of our teaching, we present here a practical and pedagogical low cost BPSK modulator/demodulator model kit to teach modulation during practical lessons.

Introduction

Since a few years, we observed some major changes in student's behaviour in our engineer school: They are less interested in theory and "reject" all courses which have no concrete application. So, one of our challenge is how to motivate these new students generation to the fundamental courses. Moreover, even if simulations tools like SIMULINK are helpful and "funny to use", they do not fit perfectly the behaviour of a true circuit. The use of an intuitive and practical approach seems to be more efficient than traditional courses for these students. The job we present here is a try to create a didactical link between theoretical course and practical lesson. The example of a hand made low cost BPSK modulator/demodulator is used to illustrate this approach.

This kit is used by students during practical lessons without necessity of expensive and sophisticated test equipment. The schematic diagram and pictures are given and explained in the next paragraph.

Digital modulations background 2.1 Generalities

A complete digital radio link is a complex system [START_REF] Ph | Cours internes de faisceaux hertziens numériques société T.R.T[END_REF], [START_REF] Escrig | Cours systèmes de communications numériques[END_REF], [START_REF] John | Digital Communications[END_REF], [START_REF] Glavieux | Communications numériques[END_REF] which consist of several main blocks as indicated on the figure 1. The subset which interests us here is the modulator/demodulator on intermediate frequency. Among all the possible numerical modulations, we chose to start with the students, by simplest ie BPSK modulation. This allows obtaining a modulated signal with two states of phase 0° and . The phase shift then occurs when the IF modulated signal crosses zero. Without the preliminary synchronization of signal NRZ, the phase shifts would happen in a random way compared to the carrying signal and would have a harmful effect on the spectrum (more marked transitions). Around 90% of the total energy is contained in the main lobe.

BPSK demodulation principle

The demodulation is known as coherent when that one has with the reception a local oscillator synchronized in frequency and phase with the emitted frequency. We obtain then at the output a two level signal: 1/2 AB and -1/2A.B corresponding to the two level of the NRZ emitted code.

To demodulate the signal, it is obviously necessary to reconstitute locally the carrier wave one not presents in the emitted spectrum. That can be carried out according to two principles: rise squared in carrying or Costas loop [START_REF]Application note n° HSAP2031 HYPERCEPTION "Costas Loop Implementation with Hypersignal Block Diagram/RIDE[END_REF]: we chose to use the first, simpler of approach, the Costas loop being approached in a more complete handling on modulation QPSK. The signal modulated received BPSK is applied simultaneously to the 2 entries of a multiplier. At the output, one finds the product: Cos 2 t or Cos 2 (t + ), depending on carrier phase.

As Cos 2 a = (1+cos 2a)/2, it results from it a DC voltage and a signal at frequency 2 without phasing. A simple filtering and a division by 2 are enough to restore locally the carrier wave in phase with the emitted IF carrier. The corresponding block diagram is given in figure 6.

Practical design

To simplify the design and to reduce the costs of realization, we chose to work at the standard frequency of 455 Khz, with an adjustable binary rate from 10 to 20 kBits/s. The use of integrated circuits of the trade traditional easy to supply ensures the perennially of our models and an easy maintenance service.

General schematic diagram

The board consists of a pseudo random pulse train generator which can (or not) be integrated in a FPGA. This generator drives the IF 455 kHz modulator whose essential component is an analogical multiplier AD 835. The 455kHz carrier wave is generated by a sinusoidal integrated oscillator MAX038 which can be possibly replaced by an oscillator with door and with ceramic resonator 3,64 MHz, a divider by 8 with D latches and one ceramic filter narrowband, tuned on 455 Khz, for more stability.

The modulator is directly connected to the demodulator primarily made up of a multiplier AD835. The recovery of carrier frequency is it also based on multiplier AD835 wired as a squared associated with a divider by 2 and one filter ceramic SFZ 455 A. The demodulated signal is given in form by a fast comparator AD790 then a simple logical gate. Because of the differences in power supply voltages between circuits, a voltage level adaptation is necessary between Xilinx and the other circuits.

In the two following paragraphs, we detail the constitution of the blocks integrated in a CPLD.

Pseudo random pulse train generator

In order to observe the characteristics of the modulated signal, we included in the design, a digital pulse train generator [START_REF] Kadionik | Personal ENSEIRB WEB site[END_REF] which simulates the data to be transmitted, based on the following diagram of figure 8. The more important the number of D latches used is, the longer the random pseudo sequence will be and the spectrum observed nearer to reality. In our case, we used 8 D latches and a sequence of 219 bits was selected. Its length is sufficient to obtain a "quasi" continuous spectral concentration. The generator can be designed starting from discrete D latches or integrated in a small standard XILINX XC 9572 programmed under "HDL designer" for example.

Scrambler /Descrambler

To better match the reality of a modulation system, we integrated a scrambler/descrambler whose role is double: to code information and to increase the number of transitions in the signal to facilitate the recovery of the binary rate in the receiver.

M(n)= E(n)  [Q3(n)  Q4(n)] (6) or : M(n) = E(n)  [Q2(n-1) Q3(n-1) (7)] Then : M(n) = E(n)  [Q1(n-2) Q2(n-2)] (8)
And finally : Where E is the input and Qn, are the outputs of the different latches.

M(n) = E(n)  [M(n-3) Q1(n-3)] (9)
It yields:

M(n)=E(n) [M(n-3) M(n-4)] (10)
The descrambler looks almost like the scrambler (figure 10). With M applied to the input of the descrambler, we can write the logical equation at the n th period of clock between the output S and the input :

S(n)=M(n) [M(n-3) M(n-4)] (11)
If we associate the two circuit scrambler/descrambler, we obtain the logical equation:

S(n)=M(n) [M(n-3) M(n-4)] (12)
With:

M(n)=E(n) [M(n-3) M(n-4)] (13)
And it comes:

S(n)= E(n) [M(n-3)  [M(n-3) M(n-4)] ( 14
)
Finally :

S(n)= E(n) (15)
The descrambler-scrambler system is known as auto synchronizing: After a maximum of n period, (with n = a number of latches stages, here 4), the system is locked and the exit of the descrambler restores the signal accurately. The unit must be correctly initialized with the powering to avoid any blocking of the D latches.

Gaussian filtering

The Gaussian filtering of the numerical train before modulation normally requires a DSP and a sophisticated algorithm. For reasons of cost, one can advantageously replace this filtering by an integrated filter for example of the 8th order, called of "improved Bessel" [START_REF]Linear technology Application[END_REF] to which the effect will be very close on the spectrum to the transmitted signal. For teaching questions and of facility of provisioning of the components, we propose also a simple filter RC which will already make it possible to the pupils to include/understand and compare the effect of these filters on the signal and the obstruction spectral. The selection of one or other filter is done by a simple switch.

Main components

For information, we give the list of the principal components and their suppliers:

AD835

Experimental

The experimental results that we give here are those which the students can do during the practical lesson, on our BPSK board. A simple oscilloscope equipped with FFT function, or an oscilloscope and an analyzer of spectrum are enough for the practical observations of Work. Figure 11 shows the chronograms raised with an oscilloscope Agilent "Megazoom" mixed analogical numerical, in run/stop mode synchronized on the data flow. The phase shift is well observed in the passing to zero of frequency FI. Then, one checks by bringing into service a simple R,C filter, on the bit stream 24Kbit/s (figure 14), the reduction of the secondary lobes. This observation has only a pedagogical goal, that to show the impact of filtering on the spectrum width of the modulated signal.

As already indicated this filtering is naturally far from sophisticated Gaussian numerical filtering really used but is sufficient from a teaching point of view.

A filtering closer to reality can, at this step, be introduced on the electronic board by "switching on" the improved Bessel 8 th order filter LT 1164-7. The "widening" of points 0° and testifies of phase vibration on modulating signal NRZ due to the electronic circuits.

Figure 16 : BPSK vector analysis

According to the operating mode of the analyzer, one can visualize (figure 17) the eye diagram (to be compared with figure 14), the constellation and the parameters of amplitude and phase errors in the same window.

Fig. 17 : Eye diagram

In the opposite case (no vector analyser available), the constellation can also be observed on a simple oscilloscope in X,Y mode.

Possible improvements

The design and the use of these low cost models , (cf photograph of figure 18) constitute above all, an initiation with the numerical modulation and a practical complement with the theoretical lesson: the reaction of the receiver in the presence of a noisy transmission channel, the measurement of error rate and the oscillators jitter, the extraction of the binary rate (clock recovery) in reception are currently not presented on the model, but that can constitute an evolution or a later possible extension.

Conclusion

We showed that with reasonable techniques and financial tools l (as well in the field of the components as the equipment measurements), it is possible to create a very cheap practical lesson which illustrates perfectly the modern concepts of digital transmission of information. In complement of the traditional courses and work on standard simulator MATLAB which makes it possible to simulate complete connections, Our models complete the "often difficult transition" between abstract and concrete with which our students are confronted during their schooling.

After this experiment, we are introducing, and on the same way, "hand made" board for QPSK modulation/demodulation with Costas loop, and soon FSK which will supplement the "numerical communications" lessons given in ENSEIRB.

Figure 18 : BPSK board view

Fig 1 :Fig

 1 Fig 1 : Digital transmission block diagram

Fig 3 :

 3 Fig 3 : Modulation BPSK

Fig 4 :

 4 Fig 4 : BPSK modulated signal spectrum

Fig. 5 :

 5 Fig. 5 : Demodulator principle

Fig. 6 :

 6 Fig.6 : IF carrier recovery principle

Fig 7 :

 7 Fig 7 : Schematic diagram

Fig 8 :

 8 Fig 8 : Pseudo random generator principle

Fig 9 .

 9 Fig 9. : Scrambler and generator

  Fig 10 : Descrambler

Fig 11 :Fig 12 :

 1112 Fig 11 : BPSK modulated signal BPSK Trace 1 : IF 455 kHz modulated D14 : random pulse train 11Kbits/s , D15 : bits clock 11 KHz Zoom Trace 1 : phase shift with D14 changes The spectrum of the modulated carrier is given in figure 12.

Fig 13 :

 13 Fig 13 : Spectrum with a simple R ,C filter

Figure 14 :

 14 Figure 14 : Digital signal after 8 th order Bessel filtering (cutoff frequency tuned at 9kHz)Lastly, if an expensive test equipment such as vector signal analyzer (Agilent E4443 for example[START_REF]Digital Modulation in Communications Systems -An Introduction[END_REF]) is available, one can visualize, the phase diagram, and eye diagram (figure 15, 16,)