Gazengel Bruno
email: bruno.gazengel@univ-lemans.fr

Ayrault Christophe
email: christophe.ayrault@univ-lemans.fr

SCIENTIFIC CONCERTS OR LEARNING ACOUSTICS WITH ARTS

Keywords: acoustic teaching, concerts, real time demonstration

This paper presents the concept of "scientific concerts" developed at Le Mans University by two acoustics teachers-researchers and professional musicians, with the collaboration of three professional musicians.

This project was born in 2009 and aims to introduce the basic concepts of acoustics by mixing science and arts. The originality and core principle of a scientific concert is to combine projected animations and spoken explanations with real-time experiments and demonstrations that are being given and played by both physicists and musicians. Learning is also eased by alternating musical themes and scientific content. Two different shows have been developed and given either in high schools, in popular science events or in concerts halls welcoming teenagers or general audience. The first show deals with the three main characteristics of a sound (intensity, pitch, timbre), while the second introduces the physics of musical instruments using the principles of elemental resonators like ducts and strings.

The presentation will describe the science-to-music back-and-forth process that has been followed so as to achieve this project, including examples of experiments and auditory demonstrations. A quantitative and qualitative assessment of the concerts given in France and Canada will be finally provided.

Introduction

Today, many projects mixing arts and science are proposed in different areas [1,2]. Particularily, acoustics is a very good candidate for mixing science and arts, since music is very near from many physical problems such as room acoustics and musical acoustics. Some examples of project using art for presenting science exist, such as the "scientific concert'' organized at EPFL (Switzerland) [START_REF]ScientificConcert[END_REF] presenting research projects by alternating music and scientific presentations. Another project mixing acoustics and music is also presented in France for preventing young people from ear damage. This project, untitled "Peace and Lobe" [START_REF]Peace and Lobe[END_REF] presents the history of amplified music and the danger of high levels.

The "scientific concerts" presented in this paper differ from this approach by presenting the physical aspects of acoustics which includes intensity, pitch and timbre and physics of musical instruments. They were created by acousticians and musicians working together at Le Mans Université (France). The aim of this project is to present acoustics fundamentals during a concert for young students or non scientist people. For this, the mix between scientific presentation, real time demonstration and musical interlude enables the public to alternate between focus and entertainment. The music is played by a quintet composed of three saxophones, a double bass (or electric bass) and a drum. Moreover, many sound accessories are used in order to make real time auditory and visual demonstrations.

Scenario contents

This section presents the two scenarios built for the "scientific concerts". The first scenario was designed to present the three usual characteristics of a sound (intensity, pitch, timbre) and the second one to present the physical principles of musical instruments.

Intensity, pitch, timbre

The scenario designed to present the three characteristics of a sound is presented in this section.

Sound loudness

Scenario of the first part (sound loudness) is presented in figure 1 and show how oral presentation, sound demonstration and musical excerpt alternate to make the concept clear for listeners. This scenario is built in three major parts. First part aims at discovering the phenomenon with perception, second part presents physical aspects of sound loudness, then last part is dedicated to decibels and danger of high levels.

The most important aspect of this part is to understand that acoustic pressure is a very low variation of atmospheric presssure and that these very low pressure variations lead to high acoustic levels which can damage strongly the ear. For this, we use a analogy between atmospheric and acoustic pressure as shown in figure 2.

Real time measurements of acoustic levels while musicians playing are performed in the middle of room and at the drummer ears. The effect of the listening duration is also presented in order to prevent young people to listen music at high level during a long time. A original tool, called "acoustical head" developed by the Engineering School in Electronics ESEO (Angers, France) is used in order that young people measure the level of their own music player (mp3).

Pitch

The aim of this part is to show the relation between pitch perception and frequency. Pitch is illustrated by listening to different musical instruments and simpler sounds, such as a tuning fork or a glass excited by friction. Drum brushes which produce a random noise which pitch can not be identified are also singed. Specific phenomena such as the missing fundamental are not presented. The signals produced by the pure tone sources are observed on a real time oscilloscope presented to the public. Real time signals enable to introduce the period and the fundamental frequency of a simple signal (sinusoid). The comparisons of two different sounds (tuning fork at 440 Hz and singing glass at 600 Hz) enable the public to link between the perception of pitch and the fundamental frequency represented by the number of oscillations per second shown by the real time oscilloscope. An example of Flash animation explaining the sinsuoidal signal is given in figure 4. The timbre is illustrated by musical examples generated by two different instruments (flute, and soprano saxophone) playing the same tune at the same loudness. Then differences in the sounds emitted by different instruments (tuning fork, flute, saxophone) are analysed in real time using the RTSPEC software [5] or AudioXplorer for Mac OS X. The analysis of the real time spectra of periodic sound (tuning fork, flute, saxophone) gives to the listeners the understanding of the fundamental frequency and harmonics. Differences in the spectra enables to show the differences in timbre. Finally, the signal produced by drum brushes and applause are analysed in real time, which permits to show the concept of non tonal sources like random or pseudo-random noise.

Physics of musical instruments

This section presents the general principle of the scientific concert dedicated to the physics of musical instruments.

General principle of musical instruments

The general organization of musical instruments, splited into three parts (exciter (called transmitter), resonator and emitter) is first presented with different instruments (double bass, cajon and transverse flute). For each part we show the different elements described in Table 1. Music is played by the three instruments in the background allowing to focus on each element described by the speaker.

Instrument

Principle of string resonance

First, the sound provided by a string vibration of a double bass is observed in the time domain (oscilloscope). The periodic effect is highlighted. Then, in order to observe the cause of this periodicity, a long string (8 meters) of elastic trap type is plucked. This allows to see the wave travelling along the string. The experiment is repeated twice for two different values of the string tension, which enables to observe a difference in the wave speed.

This periodic phenomenon leads to talk about resonance. The Single Degree of Freedom (SDOF) resonator is then observed and explained, first, with a mechanical system. Experiment is realized with a mass-spring system using the string as a vertical spring (figure 5). Measurement of the period for two different tensions of the string are realized. Explanation of the role of mass and spring for calculating the resonance frequency are provided. Then, an analogous acoustical system, the Helmholtz resonator, is presented with bottles. Demonstration of the effect of different bottle size on the fundamental frequency when blowing into the bottle is presented. Three musicians are playing a musical sequence with three bottle as a musical background and illustration.

The first resonance frequency is then explained in a string by viewing a wave traveling in a string with a video at low speed [START_REF] Russel | Motion of Plucked String[END_REF]. Explanation of relation between distance, period and speed and relation with resonance frequency are given. Influence of different parameters (length L, tension T , mass per unit length m) on pitch is illustrated wit the double bass.

Finally, the explanation of harmonics is made using the long string with a weak tension, providing a fundamental frequency weaker than 1 Hz. Two operators are showing the 6 first modes of a string (figure 6). In parallel musicians are playing a rhythm with percussion at the same tempo (we assume that the quarter note corresponds to the fundamental period). This experiment can be criticized because the harmonics of the string (expressed in frequency domain) are illustrated by percussive musical excerpt (in time domain). Musical illustration of the harmonics (which has of course another meaning) is played by the double bass, introducing a new tune.

Principle of ducts resonance

After the string instruments, wind instruments are presented only for cylindrical ducts. First, duct is opened at both ends, like for the flute. An animation shows a pressure wave travelling in the duct and radiating at both ends (see figure 7). The relation between distance, period and speed and resonance frequency is explained. The influence of length L on pitch is illustrated with two musical ducts from boomwhakers [7]. Sound speed is assumed to be constant. In particular, for a lengths ratio of 2, octave is introduced using physical and musical approaches (frequency doubling, singing the octave) The equation f = c 2L is used to design an instruments with many ducts and to explain briefly the role of side holes in a resonator. It also shown, like in the string, that harmonics can be emitted with an homemade harmonic flute Secondly, cylindrical duct opened at one end and closed at the other one, like for the clarinet, is exhibited. Clarinet and transverse flute, playing a melody separately and then together, show that the clarinet plays lower than the flute with a different timbre. Simplifying the experience with a cylindrical tube (boomwhaker), the effect of the end closing, leading an octave below, is shown. Real time analysis of clarinet and flute spectra, with the same tube length, shows that the fundamental of the clarinet is indeed an octave lower than the flute fundamental. Physical explanation of this phenomenon is made with a theatrical demonstration of the wave propagation in an opened-closed duct. The duct is mimicked by a string hold by two persons and the presenter mimics the positive and negative pressure traveling into the duct. This shows that the wave needs to travel along 4L (and not only 2L for the flute) at speed c so that the period is twice greater and the fundamental frequency twice lower than in the flute.

Last aspect concerns the building of strange instruments using demonstration made by Joe Wolfe [START_REF] Wolfe | Music Acoustics[END_REF]. Mouthpieces of flute and clarinet are inverted with suitable plastic pieces and played. It has to be said that it is difficult to build quickly (without any simulation) instruments with good intonation.

Design process

The development of the different shows have followed the following steps. Scientists first listed all items that could be concerned by the topic of the show with some demonstrations ideas (animations, videos or real time experiments ...). A selection of the most relevant and easiest elements to understand and to illustrate was made and a first scenario, with slides, was presented to musicians. Musicians asked a lot of questions to understand the physical concepts and obliged scientists to simplify the scenario. Musical illustrations that can explain the main concepts were thought together. A try error process to create sequences with explanations, demonstration, musical illustrations and music was used. Then, a complete and precise scenario with speech, and suitable music, was written. Finally, a work with an artistic coach was realized in order to simplify and transform the conference into a real show (work on motions, attitudes, expressions, intentions in the music, voice ...) with sound and lights creations.

The first show on intensity -pitch -timber was developed in one year (2009) but modified during several years with the experience of the concerts, particularly in high schools, with the pupils' evaluation. The second show on instruments, more complex, was developed during 2 years (2013-2014) and modified in 2018 with the professional stage direction and sound and lights creations.

Project assessment

These shows were presented 75 times (in total), for around 10 000 listeners, mainly in high schools (around 70 % of the public), in France but also in Canada (2018). The satisfaction of the public was measured after each concert from 2014 to 2016, with a satisfaction inquiry filled by teachers and pupils. A few points on the complexity of the scientific speech and the show were highlighted. However, this assessment was not systematically provided and can not enable to bring out strong tendencies expressed by this young public. However, one can cite for instance a 14 years-old pupil saying after the concerts: "I usually don't like science, but it was really interesting".

Assessment was also achieved with informal discussions with public in real concert rooms for instance. It appears that the professional stage direction with sound and lights creations has improved greatly the perception of the show. Particularly, the smoothing between music and science is appreciated. It is considered now as a real show that could be distributed in professional cultural network. The initial idea to avoid a musical conference (dichotomous separation between music and scientific speech) seems to be achieved. Therefore, it is easy to interest the public to a science (here acoustics), which is the main goal (the idea is not to understand everything, like in a lesson). However, this point could be even more developed by removing all slides (and so the few equations included) with only real-time experiences, speech and music, leading to a real live show.

Conclusion

Two shows, mixing science (dealing here with acoustics) and music, were developed from 2009 to 2019 by a group of teachers -researchers -musicians and professional musicians. Is is based on different media, mainly real time demonstrations, supported by music and staged. The collegiate work between scientists and non scientists made scientists adapt the scientific presentation and speech to a general non experienced public. Thereby, this educational approach has influenced our own lectures at the university. The professional stage direction has been essential to make easier the perception of the show and so to go easily into the science, even for uninterested people. The show can be given today in high schools but also in show rooms with general public. Prospects of theses scientific concerts can concern the realization of MOOC [START_REF]Les bases de l'acoustique : la voix dans tous ses états -session 1[END_REF], educational booklets for teachers wishing to work on acoustics before the concerts, transmission of this experience to musicians or scientists acting in scientific mediation ...

Figure 1 :

 1 Figure 1: Sound loundness scenario.

Figure 2 :

 2 Figure 2: Analogy between atmospheric pressure and acoustic pressure. (a) View of the atmospheric effects. (b) schematic view of the acoustic effect.

Figure 3 :Figure 4 :

 34 Figure 3: Acoustic head used for measuring acoustic level of music players. Initial green light becomes orange above 85 dBA, for one ear, and red above 100 dBA

Figure 5 :

 5 Figure 5: View of the experiment made for measuring the fundamental frequency of a spring mass system, the spring being the horizontal string.

Figure 6 :

 6 Figure 6: View of the experiment made in order to shows higher modes of the string in forced excitation.

Figure 7 :

 7 Figure 7: View of the animation made to illustrate the propagation of a pressure wave in a open-open cylindrical duct. The green color represents the negative acoustic pressure while the red color represents the positive acoustic pressure (Animation made by André Almeida).

Table 1

 1

		Exciter	Resonator	Emitter
	Double bass	finger	String	soundboard
	Cajon	hand	wood plate	wood plate + Helmholtz resonator
	Transverse flute Air jet cylindrical air column	mouthpiece + first opened hole

: Examples used for describing the classification of musical instruments

ICSV26, Montreal, 7-11 July 2019

ICSV26, Montreal, 7-11 July 2019

Acknowledgments

The authors thanks their partners for the financial and logistical supports:

André Almeida for animations (flute, clarinet) • Le Mans University: Acoustic Laboratory (LAUM), technical and industrial culture center, artistic culture center, numerical resources center, Institute Le Mans Acoutics, • Maine Sciences: scientific, technical and industrial culture center in Sarthe, • Sherbrooke University • Région Pays de la Loire


Pierre-André Taillard
email: taillard@hispeed.ch

Jean-Pierre Dalmont

Bruno Gazengel

Alberto Muñoz Arancón

Analysis of Nonlinear Characteristics of the Clarinet Exciter Obtained via a New Measurement Method

A new method for measuring the nonlinear characteristic of the clarinet exciter, which binds the air flow entering into the clarinet with the pressure drop (∆p) across the reed, is described. It uses a clarinet mouthpiece equipped with a reed and an artificial lip whose position ψ is controlled by a micrometer screw. The mouthpiece is connected to a bottle in which a moderate vacuum is generated at the beginning of the experiment. After a short time lapse, the opening of the reed occurs. The thermodynamics of the volume in isochoric conditions enables the calculation of the volume velocity entering the mouthpiece from the pressure measurement. 13 reeds with 10 different embouchures are measured. The measurements enabled the estimation of the equivalent aeraulic section S(∆p, ψ). We propose a model of S as a convex function of ∆p and ψ, defined as the sum of two 1D stiffening springs plus a porosity constant. The mean standard error of the model is 0.2%.

INTRODUCTION

Since the invention of the instrument, about 300 years ago, the clarinettists complain about the difficulty to find musically suitable reeds (reeds with a "good vibration"). Many scientific studies are devoted to this topic, for instance [START_REF] Casadonte | The Clarinet Reed: An Introduction to its Biology, Chemistry, and Physics[END_REF][START_REF] Van Walstijn | Discrete-time modelling of brass and reed woodwind instruments with application to musical sound synthesis[END_REF][START_REF] Guimezanes | Etude expérimentale et numérique de l'anche de clarinette[END_REF][START_REF] Taillard | Statistical estimation of mechanical parameters of clarinet reeds using experimental and numerical approaches[END_REF]. Some authors measure the nonlinear characteristics of the clarinet exciter (reed+mouthpiece+lip), for instance [START_REF] Dalmont | Nonlinear characteristics of single-reed instruments: Quasistatic volume flow and reed opening measurements[END_REF]. This paper describes a new, precise measurement method of these characteristics. We are convinced that the main musical differences between clarinet reeds are related to the quasistatic aeraulic behavior. The "vibration" of the reed is probably not the major point, but the ability of the reed i) not to interfere negatively with the sound production of the clarinet, ii) to enable an efficient sound control by the lip and iii) to allow an operating blowing pressure which is comfortable for the clarinettist.

This paper is organized as follow: in Sec. 2 the setup of the measurement method is described and its thermodynamical behavior is modeled in Sec. 3. Sec. 4 illustrates an application of the method to clarinet reeds and proposes a quasistatic model of the aeraulic section as a function of the pressure drop and the lip pressure.

DESCRIPTION OF THE SETUP

The measurement is divided in 2 phases: 1) calibration of the diaphragm, 2) measurement of the nonlinear characteristics of the reeds. The underlaying thermodynamic problem must be solved for each phase.

b) d) a) c) b) d) a) c) g) h) f) k) e) i) j) 1)
2)

Figure 1. Schematic setup of the problems 1) and 2). a) diaphragm, b) piezoelectric pressure sensor (bottle), c) thin tube (about 1 mm inner diameter), d) cock valve, e) adaptation barrel with absorbing foam (damping the acoustic oscillations), f) piezoelectric pressure sensor (mouthpiece), g) clarinet mouthpiece, h) artificial lip (silicon 10 mm thick), i) steel beam (diameter 3 mm) glued to the artificial lip, j) micrometer screw (controlling the position ψ of the artificial lip), k) clarinet reed

Problem 1): discharge through a diaphragm

A diaphragm is connected to a hermetically closed, rigid volume (bottle, 3.178 liters, well isolated thermally and opaque to the light). A moderate vacuum (about 15-20 kPa) is generated in the bottle trough a cock valve at the beginning of the experiment (while the diaphragm is closed). After about 1 minute (allowing the observation of the heat exchange with the bottle), the diaphragm is quickly opened and the discharge starts (the air comes back into the bottle). The pressure in the bottle is measured with a piezoelectric sensor (Endevco). See Fig. 1.

The purpose is to determine the effective aeraulic section of the diaphragm S dia from the pressure measurement (via the computation of temperature and flow rate).

Problem 2): discharge through a varying aeraulic section

The setup of Problem 1) is completed by a clarinet mouthpiece and a reed. An artificial lip (silicon) compresses the reed against the lay of the mouthpiece. Its position (normal to the table of the reed and denoted as ψ) is controlled by a micrometer screw. The reed is not moistened before measurement, in order to avoid a bias in the measurement due to drying. The purpose is to determine the effective aeraulic section of the channel (slit between reed and mouthpiece) in quasistatic conditions from the pressure measurement inside the mouthpiece p and the aeraulic section of the diaphragm S dia calibrated with Problem 1). The measurement of the pressure in bottle serves only as a control for the computations. This way the delicate problem of pairing between sensors can be avoided and the precision and the reproducibility of the measurements is increased.

Typical discharge measurements for problems 1) and 2) are depicted on Fig. 2. The total duration of one experiment is about 90 s.

THERMODYNAMIC MODEL

Laws of thermodynamics

This subsection recalls some laws of thermodynamics, using mainly the traditional notations.

Constants and parameters

Ideal gaz constants : R = 8.314J/mol/K γ = 1.4 (for diatomic gazes), Density of air : ρ, Pressure : P Atmospheric pressure : P 0 Pressure drop : ∆P = P -P 0 Volume : V Flow rate : U Aeraulic section : S R×number of moles of gaz : N = nR Absolute temperature : T Ambient temperature : T 0 Heat capacity at constant volume :

C v = 1/(γ -1)N Internal energy : E Work : W Heat : Q Sample rate, time step : f s = 1/t s (typically f s = 5000 Hz)
Parameters with uppercase generally refer to values inside the bottle. In lowercase the same parameters refers to the value inside the mouthpiece.

Laws

Summary of thermodynamical laws: (1) Ideal gas law, (2) First principle of thermodynamics, (3) Newton's law of cooling (thermostat) and (4) Bernoulli's law:

P V = N T (1)
dE = dW + dQ (2)
dT th /dt = -r(T -T 0 ) (3)
U = sign(∆P ) S 2|∆P |/ρ (4)
Remarks:

Newton's law of cooling: r is a positive constant, which has to be determined experimentally. Bernoulli's law: valid for incompressible fluids and large ducts, comparatively to the aeraulic section S, for Reynolds numbers Re U/(ν √ πS) > 2000 (ν = 15.6 × 10 -6 for air at 25 • C). For compressible fluids the equation is approximately valid for the conditions at the output of the jet.

Isochoric model

The heat variation due to the thermostatic effect of the bottle is:

dQ = C v dT th (5)
The work of small air volume dV leaving the jet outgoing the diaphragm with a temperature T jet and a pressure P is dW = dV P = dN T jet . On the other side, the variation of energy is dE = 1/(γ -1)((N + dN )(T + dT ) -(dN T jet + N T )). Applying Eqs. 1, 2, 3 and 5, we obtain the equation of our thermodynamic model in isochoric conditions (i.e. at constant volume):

dN = dP V + N r (T -T 0 )dt γ T jet (6)
Considering an adiabatic expansion in the jet, the temperature of the air leaving the jet is T jet = T 0 (P/P 0 )

γ-1 γ .

Discrete time scheme for Problem 1

During an experiment, the pressure in the bottle P [t] is measured with time steps t s : P m = P [m t s ]. We have to deduce from the equations above the temperature T m and the number of moles (×R) of air in the bottle, N m . Initialization At time step m = 0 (before generating the vacuum), the air in the bottle is at ambient temperature T 0 and atmospheric pressure P 0 . Applying Eq. 1, the initial quantity of air in the bottle is: A validation of the model was performed with a series of chamfered diaphragms of nominal diameters 1, 1.5, 2, 2.5, 3 and 3.5mm. Eq. 4 allows the computation of the equivalent aeraulic section S from U , ρ (in the bottle) and ∆p, from which we deduce the equivalent aeraulic diameter of the diaphragms.

N 0 = P 0 V /T 0 . Iterations for m > 0 dN = V (P m -P m-1 ) + N m-1 r t s (T m-1 -T 0 ) γ T jet N m = N m-1 + dN T m = P m V /N m U m = dN T m /(P m t s ) (7
Fig. 3 depicts the results. The following values of r were determined by optimization (among 3 tests with different initial depressions for each diaphragm): 0.25, 0.32, 0.39, 0.46, 0.54, 0.61, for the diameters 1 to 3.5mm. This accounts approximately for the greater heat exchange with the bottle when the flow rate through the diaphragm is high. Before the discharge r = 0.20 (measured value in static conditions).

The aeraulic section of the diaphragm S dia used in Problem 2) is calibrated with this method (in our case 2.9mm). The adaptation barrel with absorbing foam (damping the acoustic oscillations) belongs formally to the diaphragm (like every aeraulic resistance downstream the mouthpiece sensor). dN ch /(p dt) and its corresponding aeraulic section S ch is deduced with Eq. 4.

Subproblem A) The mouthpiece is treated with Eq. 7 in which the variables of the mouthpiece replace those of the bot-tle (P → p, U → u, and so on). An adiabatic approximation can be used for this case : dQ = 0, thus r = 0.

Subproblem B) the combination of Eq. 6 with Bernoulli's law Eq. 4 enables the calculation of the pressure in the bottle P m (at discrete time m) from the corresponding measurement of the pressure in the mouthpiece, denoted p m .

Initialization Like Problem 1), additionally: p 0 = P 0 .

Iterations for m > 0

δp = p m-1 -P m-1 , ρ = (M N m-1 )/V dN = sign(δp) P m-1 S dia |2δp/ρ|) t s /T m-1 dP = (N m-1 r t s (T 0 -T m-1 ) + dN γ T jet )/V N m = N m-1 + dN P m = P m-1 + dP T m = P m V /N m U m = dN T m /(P m t s ) (8)
with M = 0.028965/R = 0.00348388 for the air. for the reeds J03 and J12, ψ = 1 . . . 9 (color lines). Below the transition to turbulent flow, S is approximated from the optic sensors inside the mouthpiece.

MEASUREMENTS OF CLARINET REEDS

A series of 14 clarinet reeds was measured with the described method (Rigotti and Rico, strength 3 and 3 1/2). 10 The observed behavior can be approximated as a sum of 2 1D stiffening springs and a porosity constant:

S(∆p, ψ) S 1 (∆p + k 1 ψ) + S 2 (∆p + k 2 ψ) + k p (9)
The following values were determined by optimization for our setup : k 1 = 1374.2 and k 2 = 670.3 (same values for all reeds). The porosity constant k p accounts for a residual flow which do not vanish for high values of ∆p and must be determined individually for each reed.

This model implies that S(∆p, ψ) is a convex function of ∆p and of ψ. In other words, the second derivatives of S are non-negative. The stiffening springs can be implemented with non-negative, reproducing kernels (for instance gaussian kernel) as bandlimited functions. We used a kernel allowing a real-time implementation of the measured exciter for synthesis purpose (but this is beyond the scope of this paper).

The mean standard error of the model is around 0.02mm 2 . This represents a height of 1.5 µm for a rectangular channel, 13mm wide, or 0.2% of the maximal measured aeraulic section.

CONCLUSIONS AND PERSPECTIVES

The precision of the method enables a detailed study of the nonlinear characteristics of clarinet reeds. A great variety of quasistatic behaviors could be measured, confirming the every-day experience of the clarinetists with their reeds and the importance of the non-linear contact between reed and mouthpiece. A comparison with 3D simulations about the dichotomic sensitivity of the reed to the lip pressure shows that k 1 accounts mainly for air coming from the middle of the channel while k 2 concerns mainly the air entering from the sides of the channel.

This short paper could only demonstrate the main features of the method, but could not investigate the musical consequences of the measurements for reed makers and clarinettists, explaining relationships with geometric and flexural measurements, nor demonstrate the ability of the model for a real-time simulation of the measured reeds.

Aknowledgments We thank the engineering school HE-Arc (Neuchâtel, Switzerland) for the facilities granted.

Figure 2 .

 2 Figure 2. Typical measurements of ∆P during the discharge (zoom) for Problems 1) and 2): 1) diaphragm of 1.5mm diameter. 2) reed #J03 with embouchure ψ = 7 [arbitrary units]

Figure 3 .

 3 Figure 3. Color lines: aeraulic diameter of the tested diaphragms computed with the isochoric scheme Eq. 7. Each diaphragm is measured 3 times with different initial conditions. Thin dark lines: nominal diameter of the diaphragms.

3. 5 .

 5 Discrete time scheme for Problem 2 This problem is subdivided in 2 subproblems: A) compute the net mass flow dn entering into the mouthpiece B) compute the net mass flow dN entering into the bottle The total mass flow entering trough the channel is dN ch = dn + dN , from which U ch = T 0 (p/P 0 ) γ-1 γ

Figure 4 .

 4 Figure 4. Aeraulic section S(∆p, ψ) determined with Eq. 8 for the reeds J03 and J12, ψ = 1 . . . 9 (color lines). Below the transition to turbulent flow, S is approximated from the optic sensors inside the mouthpiece.

Figure 5 . 2 S ∂∆ψ 2 .

 522 Figure 5. Contour plot of the second derivative of the aeraulic section ∂ 2 S∂∆p 2 for the reeds J03 (Rigotti 3) and J12 (Rico 3 1/2), ψ = 1 . . . 9, according to the proposed model Eq. 9 (arbitrary color scale)

Proceedings of the 2017 International Symposium on Musical Acoustics, 18-22 June, Montreal, Canada Peer Reviewed Paper

Proceedings of the 2017 International Symposium on Musical Acoustics, 18-22 June, Canada

Proceedings of the 2017 International Symposium on Musical Acoustics, 18-22 June, Montreal, Canada


Jessica Varilh

Jennifer Bonini

Magali Taulan-Cadars

Role of Non-coding RNAs in Cystic Fibrosis

Keywords: CFTR gene expression, Cystic Fibrosis, non-coding RNA, lncRNA, miR-NA

Cystic Fibrosis (CF) is a common autosomal recessive disorder, caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. CFTR gene expression is tightly controlled by transcriptional and post-transcriptional regulatory factors, resulting in complex spatial and temporal expression patterns. Here, we describe an overview of the findings about the contribution of ncRNAs, especially miRNAs, in physiological CFTR gene expression and in CF. Determination of mechanisms governing its expression is essential for developing new CF therapies. ncRNAs, including lncRNAs and miRNAs, could also contribute to CF progression and severity and their dysregulation in CF opens new perspectives for patient followup and treatment.

Introduction

Cystic Fibrosis (CF) is a common autosomal recessive disorder. Although in its classical form CF affects several organs, including the pancreas and the gastrointestinal and reproductive tracts, its morbidity is mainly due to pulmonary damages. This disorder is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. This gene displays great mutational heterogeneity, depending on the ethnic and phenotypic background, with almost 2,000 referenced CFTR alterations (genet.sickkids.on.ca/). The most common mutation is the p.Phe508del, whereas other mutations, located both in coding and non-coding regions, are rare or private. The p.Phe508del mutation induces aberrant protein folding, leading to endoplasmic reticulum (ER)-associated degradation, atypical intracellular trafficking and reduced stability of the CFTR protein at the apical membrane. Dysfunction or lack of the CFTR protein causes an obstructive lung disease characterized by impaired ion transport in the airway epithelium, accumulation of sticky mucus in the air space and chronic airway inflammation. Physiological CFTR expression is tightly controlled by transcriptional, post-transcriptional, translational and post-translational regulatory mechanisms, resulting in complex spatial and temporal expression patterns. Notwithstanding the importance of CFTR transcriptional regulation [START_REF] Mccarthy | The CFTR gene and regulation of its expression[END_REF][START_REF] Viart | Phosphorylated C/EBPβ influences a complex network involving YY1 and USF2 in lung epithelial cells[END_REF][START_REF] Viart | Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis[END_REF], CFTR expression can be modulated through other mechanisms. Indeed, epigenetic changes, such as DNA methylation or histone acetylation, also influence CFTR gene expression in different tissues [START_REF] Bartoszewski | The mechanism of cystic fibrosis transmembrane conductance regulator transcriptional repression during the unfolded protein response[END_REF][START_REF] Li | Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREB-binding protein (CBP)-associated factor and CBP[END_REF][START_REF] Paul | The epigenetic signature of CFTR expression is co-ordinated via chromatin acetylation through a complex intronic element[END_REF][START_REF] Bergougnoux | A balance between activating and repressive histone modifications regulates cystic fibrosis transmembrane conductance regulator (CFTR) expression in vivo[END_REF]. Post-transcriptional controls also regulate its expression, for instance via the usage of upstream open reading frames (uORFs) encoded within the CFTR 5'UTR [START_REF] Wl | Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel exon 1 usage to produce a unique amino-terminal protein[END_REF] and the 3'UTR that controls CFTR mRNA stability through ARE sequences (AU-rich elements) [START_REF] Baudouin-Legros | Cell-specific posttranscriptional regulation of CFTR gene expression via influence of MAPK cascades on 3ʹUTR part of transcripts[END_REF]. An emerging area of research is focusing on the role played by non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), in CFTR gene expression. Starting from 2011, a few studies have shown the involvement of miRNAs in the physiological control of the complex spatio-temporal expression pattern of CFTR mRNA [START_REF] Gillen | MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene[END_REF][START_REF] Megiorni | Synergistic Post-Transcriptional Regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 Specific Binding[END_REF], including a recent work by our group [START_REF] Viart | Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis[END_REF]. Moreover, the implication of long non-coding RNAs (lncRNAs) and miRNAs in human diseases is well documented [START_REF] Bartels | MicroRNAs: novel biomarkers for human cancer[END_REF], including in inherited disorders [START_REF] Twayana | Biogenesis and function of non-coding RNAs in muscle differentiation and in Duchenne muscular dystrophy[END_REF] and lung diseases [START_REF] Moffatt | Genes in asthma: new genes and new ways[END_REF][START_REF] Sp | Lung microRNA: from development to disease[END_REF][START_REF] Mestdagh | Non-coding RNAs and respiratory disease[END_REF][START_REF] Booton | Emerging role of MicroRNAs and long noncoding RNAs in respiratory disease[END_REF]. However, only few studies, described below, have hitherto been carried out on the role of ncRNAs in CF. This chapter is an overview of the findings about the role of ncRNAs in physiological CFTR gene expression and in CF.

What are non-coding RNAs?

Large expanses of the genome are transcribed into RNAs, but only a small portion of these RNAs encode proteins [START_REF] Morris | The rise of regulatory RNA[END_REF][START_REF] Derrien | The long non-coding RNAs : a new player in the « dark matter[END_REF]. Many fundamental cellular processes rely on conserved ncRNAs, particularly on ribosomal RNAs (rRNAs), the ribosome RNA components that allow mRNA translation into proteins (Figure 1). Other roles are the transport of amino acids via transfer RNAs (tRNAs) and mRNA splicing through the implication of small nucleolar RNAs (snoRNAs). miRNAs and their crucial role as key modulators of post-transcriptional gene regulation were discovered more than 20 years ago [START_REF] Lee | The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementary to lin-14[END_REF]. In the last few years, lncRNAs have been identified as new modulators of key biological processes [START_REF] Hu | Regulation of mammalian cell differenciation by long non-coding RNAs[END_REF][START_REF] Fatica | Long non-coding RNAs: new players in cell differenciation and development[END_REF][START_REF] Michalik | Long noncoding RNA MALAT1 regulates endothelial cell function and vessel growth[END_REF][START_REF] Morris | The rise of regulatory RNA[END_REF]. Currently, ncRNAs are divided in two classes, based on their length; long ncRNAs (lncRNAs, > 200 nt) and short ncRNAs (<200 nt), such as miRNAs, small nucleolar RNAs (snoRNAs) and PIWI-interacting RNAs (piRNAs) [START_REF] Santosh | Non-coding RNAs: biological functions and applications[END_REF].

Ribosomal RNA (rRNA) and transfer RNA (tRNA) are the most represented ncRNAs in humans. Long non-coding RNAs (lnc or long ncRNA) are longer than 200 nt and are subdivided in five categories based on their genomic localization: pRNA (promoter-associated RNA), eRNA (enhancer-associated RNA), gsRNA (gene body-associated RNA), lincRNA (intergenic RNA) and NAT (Natural Antisense Transcript). Short non-coding RNAs (short ncRNAs) are smaller than 200 nt and are subdivided in four classes based on their size and function: siRNA (small interfering RNA), miRNA (microRNA), piRNA (PIWI-interacting RNA), snoRNA (small nucleolar RNA) and derived snoRNA (sdRNA).

Long non-coding RNAs

lncRNAs include all ncRNAs longer than 200 nt (except rRNA and tRNA). They constitute the bulk of the non-coding transcriptome [START_REF] Derrien | The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution and expression[END_REF].

lncRNA biogenesis

It is thought that most lncRNAs originate within a 2-kb region surrounding the Transcription Start Site (TSS) of protein-coding genes (65% of lncRNAs overlap with a promoter and are called pRNAs), or map to enhancer regions (19%; named eRNAs), or derive from antisense transcripts that overlap with annotated gene bodies (5%, called NATs), or are associated with the bodies of protein-coding genes (gsRNA, gene body-associated lncRNAs) [START_REF] Bonasio | Regulation of transcription by long noncoding RNAs[END_REF][START_REF] Sigova | Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells[END_REF]. The remaining lncRNAs originate from more distal (>2kb) unannotated regions (11%) and are commonly referred to as long intervening or intergenic ncRNAs (lincRNAs) [START_REF] Guttman | Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals[END_REF][START_REF] Ulitsky | Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution[END_REF] (Figure 2). a-Promoter-associated RNAs (pRNA), b-Enhancer-associated RNAs (eRNA), c-Intronic and gene body-associated (sense) RNAs (gsRNA), d-Natural Antisense Transcripts (NAT), e-Long Intergenic RNAs (lincRNA). In the lower part of the figure are described the main lncRNA functions. The finding that a large number of lncRNAs arise from loci close to protein-coding genes is consistent with previous genome-wide analyses of lncRNAs [START_REF] Van Bakel | Most "dark matter" transcripts are associated with known genes[END_REF]. Although all studies agree that the 5′end of lncRNAs, like for mRNAs, is capped by methylguanosine, their splicing status and their 3′-end processing have not been fully defined [START_REF] Bonasio | Regulation of transcription by long noncoding RNAs[END_REF]. It is likely that splice site recognition occurs at low frequency at most lncRNA loci and that lncRNAs may be predominantly mono-exonic and non-polyadenylated [START_REF] Bonasio | Regulation of transcription by long noncoding RNAs[END_REF]. Most lncRNAs are not translated [START_REF] Guttman | Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals[END_REF] and their localization is predominantly nuclear [START_REF] Derrien | The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution and expression[END_REF].

lncRNA functions

lncRNAs have regulatory functions in different biological processes (Figure 2). Many of their functions are related to their capacity to bind to RNA, DNA and proteins. The founding member of the lncRNA family is Xist (~17 kb). Xist originates from the silent X chromosome in female cells and coats this chromosome during the early stages of development to establish epigenetic X inactivation [START_REF] Clemson | XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure[END_REF]. lncRNAs can be used as indicators of the transcriptional activity of a locus or a gene [START_REF] Derrien | The long non-coding RNAs : a new player in the « dark matter[END_REF]. Their roles as scaffolds for nuclear processes, guides for ribonucleoprotein complexes or decoys have been described in the literature. Similarly to miRNAs, they can act as activators or repressors of protein expression. lncRNAs are considered to be more species, tissue and developmental stage-specific than mRNAs [START_REF] Djebali | Landscape of transcription in human cells[END_REF]. A growing number of studies show that lncRNA deregulation has a role in various diseases [33,34; for reviews : 35,36], including pulmonary disorders. Several reviews have discussed the role of lncRNAs and miRNAs in respiratory diseases [START_REF] Mestdagh | Non-coding RNAs and respiratory disease[END_REF][START_REF] Booton | Emerging role of MicroRNAs and long noncoding RNAs in respiratory disease[END_REF][START_REF] Vencken | Non-coding RNA as lung disease biomarkers[END_REF] and a recent work reported lncRNA involvement in CF (detailed in section 4.2.1).

MicroRNAs

miRNA localization and biogenesis

Animal miRNAs derive from the nuclear genome. In humans, the majority of canonical miRNAs are encoded by introns of non-coding or coding transcripts, but some miRNAs are encoded by exonic regions (Figure 3). Often, several miRNA loci are in close proximity, thus constituting a polycistronic transcription unit [START_REF] Lee | MicroRNA maturation: stepwise processing and subcellular localization[END_REF]. Most miRNAs use their host gene transcripts as carriers, but separate transcription from internal promoters remains possible. Generally, miRNAs in the same cluster are co-transcribed. Most miRNA genes located in introns of protein-coding genes share the promoter of the host gene [START_REF] Monteys | Structure and activity of putative intronic miRNA promoters[END_REF]. miRNA genes often have multiple transcription start sites [START_REF] Ozsolak | Chromatin structure analyses identify miRNA promoters[END_REF]. miRNA loci in intergenic regions apparently have their own transcriptional regulatory elements, thus constituting independent transcription units.

miRNA gene

Gene 1 Gene 2

Intergenic

Intronic Exonic Intragenic Cluster Isolated OR Figure 3. Genomic locations of miRNAs. miRNA genes, isolated or in clusters, are located in intergenic (ex: miR-494) or intragenic genome regions, including exons of non-coding (e.g. miR-155) or coding (e.g. miR-985) genes and introns of non-coding (e.g. the miR-15a ~16-1 cluster) or coding (e.g. miR-126) genes. miRNA biogenesis includes several steps. First, the gene coding for a given miRNA is transcribed by RNA polymerase II into a long primary transcript (pri-miRNA, ranging from 100 nt to several kilobases). Some miRNA genes, especially those located in Alu elements, are transcribed by RNA polymerase III [START_REF] Borchert | RNA polymerase III transcribes human micro-RNAs[END_REF]. When several miRNAs are in a cluster, pri-miRNAs can contain multiple miRNAs. Transcription factors, such as p53, MYC, C/EBP, FOXA positively or negatively regulate miRNA transcription [START_REF] Viart | Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis[END_REF][START_REF] Kim | Biogenesis of small RNAs in animals[END_REF][START_REF] Krol | The widespread regulation of microRNA biogenesis, function and decay[END_REF]. Epigenetic control, such as DNA methylation and histone modifications also contribute to miRNA gene regulation [START_REF] Bn | Mechanisms of control of microRNA biogenesis[END_REF].

Then, several maturation steps are necessary for miRNA processing. Indeed, the long pri-miRNAs (typically over 1kb) contain stem-loop structures in which mature miRNA sequences are embedded. The nuclear RNase III Drosha acts by cropping the stem-loop to release small hairpin-shaped RNAs of 65 nt in length (pre-miRNA) from the pri-miRNAs. To do this, Drosha, together with its cofactor DiGeorge Syndrome Critical Region 8 (DGCR8), forms the Microprocessor complex. As Drosha cleavage defines one end of the mature miRNA and thereby determines its specificity, it is important that the Microprocessor complex precisely recognizes and cleaves each pri-miRNA. Importantly, Drosha-mediated processing of intronic miRNAs does not affect splicing of the host pre-miRNA [START_REF] Kim | Processing of intronic microRNAs[END_REF]. Multiple auxiliary factors could contribute to pri-miRNA maturation [START_REF] Auyeung | Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing[END_REF]. For example, three primary sequence determinants (the basal UG, CNNC and the apical GUG motifs) contribute to efficient processing of human pri-miRNAs. At least one of these three motifs is present in almost 80% of human miRNAs [START_REF] Auyeung | Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing[END_REF]. The splicing factor SRp20 (also called SRSF3) and the RNA helicase DDX17 bind to the CNNC motif and increase processing of human pri-miRNAs by Drosha. Moreover, the terminal loops of miRNA precursors are enriched in cis-elements that recruit regulatory proteins. For example, the splicing factors HNRPA1 and KSRP bind to the conserved terminal loops of some pri-miRNAs and facilitate Drosha-mediated processing [START_REF] Guil | The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a[END_REF][START_REF] Trabucchi | The RNAbinding protein KSRP promotes the biogenesis of a subset of microRNAs[END_REF][START_REF] Michlewski | Posttranscriptional regulation of miR-NAs harboring conserved terminal loops[END_REF].

Following Drosha processing, pre-miRNAs are exported in the cytoplasm where they are cleaved by Dicer near the terminal loop, liberating a small RNA duplex. Dicer, like Drosha, belongs to a family of RNase III-type endonucleases that act specifically on double-stranded RNA.

RNA duplexes include two mature miRNAs: one derived from the 5ʹ strand and the other one from the 3ʹ strand of the precursor (e.g. miR-27a-5p and miR-27a-3p). One is also called the 'guide' (miRNA) and is usually more biologically active than the other one (the 'passenger', often referred to as miRNA*). The passenger is normally degraded, but, in some cases, it can be functional [START_REF] Bartel | MicroRNAs: target recognition and regulatory functions[END_REF]. The mature miRNA strand is subsequently incorporated in the RNAinduced silencing complex (RISC, or miRISC for miRNA-containing RISC, or miRNP for microribonucleoprotein), where it directly binds to a member of the Argonaute (AGO) protein family (four AGO members, AGO1 are the most frequently used).

To date, about 1,900 miRNAs (1,881 precursors and 2,588 mature miRNAs; GRCh38 human genome assembly) have been reported in the miRbase database (http://www.mirbase.org/). A substantial number of these miRNAs have dubious annotations and for nearly one-third of miRNA loci, there is no convincing evidence concerning the production of authentic miRNAs (miRbase).

miRNA roles

miRNAs are small ncRNAs that can act in the nucleus and in the cytoplasm [START_REF] Salmanidis | Direct transcriptional regulation by nuclear microRNAs[END_REF] through binding to RNA, DNA and proteins. They play an important role in the negative regulation of gene expression by base-pairing to partially complementary sites on the target mRNAs, usually in the 3' UTR part. Binding of an miRNA to its target mRNA, within the RISC complex, typically leads to translational repression and exonucleolytic mRNA decay. However, highly complementary targets can be cleaved endonucleolytically. Several miRNAs have a role in lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (see Table 1). The studies reporting the involvement of miRNAs in CF are detailed in section 3.2.2.

Disease

MicroRNAs

Targets References Asthma miR-106a IL-10 [START_REF] Sharma | Posttranscriptional regulation of interleukin-10 expression by hsa-miR-106a[END_REF] miR-21 IL-12 [START_REF] Lu | MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression[END_REF] miR-133a RhoA [START_REF] Chiba | Down-regulation of miR-133a contributes to up-regulation of Rhoa in bronchial smooth muscle cells[END_REF] miR-26a Glycogen synthase kinase 3ß [START_REF] Mohamed | Mechanical stretch up-regulates micro-RNA-26a and induces human airway smooth muscle hypertrophy by suppressing glycogen synthase kinase-3β[END_REF] let-7 IL-13 [START_REF] Polikepahad | Proinflammatory role for let-7 microRNAS in experimental asthma[END_REF] COPD miR-181d Interferon γ, collagen XVI αI [START_REF] Christenson | MicroRNA as regulators of gene expression changes that occur with the progression of emphysema[END_REF] miR-30c Proto-cadherin [START_REF] Christenson | MicroRNA as regulators of gene expression changes that occur with the progression of emphysema[END_REF] miR-146a Prostaglandin E2 [START_REF] Sato | Reduced miR-146a increases prostaglandin E in chronic obstructive pulmonary disease fibroblasts[END_REF] Idiopathic pulmonary fibrosis miR-21 SMAD7 [START_REF] Liu | miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis[END_REF] miR-155 KGF [START_REF] Pottier | Identification of keratinocyte growth factor as a target of microRNA-155 in lung fibroblasts: implication in epithelial-mesenchymal interactions[END_REF] miR-let7d HMGA2 [START_REF] Pandit | Inhibition and role of let-7d in idiopathic pulmonary fibrosis[END_REF] COPD, chronic obstructive pulmonary disease.

Table 1. Examples of pulmonary diseases in which miRNAs have a role

Role of non-coding RNAs in the physiological regulation of CFTR gene expression

As the function of lncRNAs has not been studied yet, we only present findings on the miRNA roles in the regulation of CFTR gene expression.

miRNAs and CFTR gene expression

CFTR gene expression is spatially and temporally regulated. Several studies have demonstrated the differential use of transcription start sites, depending on the tissue type or the developmental stage [START_REF] Chou | Characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene[END_REF][START_REF] Yoshimura | The cystic fibrosis gene has a ''housekeeping''-type promoter and is expressed at low levels in cells of epithelial origin[END_REF][START_REF] Koh | Characterization of the cystic fibrosis transmembrane conductance regulator promoter region. Chromatin context and tissue-specificity[END_REF][START_REF] White | Tissue-specific in vivo transcription start sites of the human and murine cystic fibrosis genes[END_REF]. In the lung, CFTR transcripts can be detected early during embryo development (12th week of pregnancy) and their level progressively increases up to the 24th week of pregnancy. Thereafter, CFTR expression in the airways decreases and is repressed until after birth and remains very low during adult life [START_REF] Harris | Expression of the cystic fibrosis gene in human development[END_REF][START_REF] Marcorelles | Evolution of CFTR protein distribution in lung tissue from normal and CF human fetuses[END_REF]. The changes in CFTR protein expression in human foetuses are consistent with CFTR mRNA temporal pattern of expression [START_REF] Marcorelles | Evolution of CFTR protein distribution in lung tissue from normal and CF human fetuses[END_REF]. In a recent study, we showed that miRNAs (miR-101, miR-145, miR-384) regulate the switch from strong foetal to very low CFTR expression after birth. Specifically, miR-101 and miR-145 negatively regulate the level of CFTR transcripts in adult lung cells, while they have no effect in foetal lung cells. miR-101 directly acts on its cognate site in the 3'UTR-CFTR in combination with an overlapping AU-rich element. Other studies showed that CFTR expression is also post-transcriptionally regulated by miRNAs, such as miR-145 and miR-494 [START_REF] Gillen | MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene[END_REF][START_REF] Megiorni | Synergistic Post-Transcriptional Regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 Specific Binding[END_REF].

Gillen et al. demonstrated that miR-145 is expressed in primary adult human airway epithelial cells, where CFTR expression is low, and directly acts on CFTR stability [START_REF] Gillen | MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene[END_REF]. In addition to its specific role in mature lung cells, miR-101 decreases luciferase activity in an embryonic kidney cell line [START_REF] Megiorni | Synergistic Post-Transcriptional Regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 Specific Binding[END_REF], whereas it does not affect CFTR mRNA stability in pancreatic cell lines [START_REF] Gillen | MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene[END_REF], suggesting a potential role as a tissue-specific factor.

Methods to investigate miRNA role in the regulation of CFTR gene expression

Different approaches can be employed to investigate miRNA role in the regulation of CFTR gene expression, as depicted in Figure 5A.

Predictive tools are freely available

Predictive tools are necessary to assess the putative presence of miRNA binding motifs. A nonexhaustive list, including information about each program, is proposed in Table 2. Databases collecting all information on miRNAs are listed in Table 3. Although miRBase (http:// www.mirbase.org/) is the most used database and collects links for several predictive pro-grams, others exist. These tools have been developed to predict miRNA targets or the miRNAs that putatively bind to a selected gene. Some of them propose the possibility to input several miRNAs and/or several genes to identify integrated networks. and TargetScan to identify miRNA targets in the 3'UTR of target genes.

[100]

Cystic Fibrosis in the Light of New Research

Functional approaches

To evaluate in vitro the impact of miRNAs on CFTR gene regulation, several approaches can be used (Figure 5 A). a) First of all, in silico analysis by using bioinformatics tools can be performed (see 3.2.1). b) The role of specific miRNAs could be assessed in vitro by using airway cell lines and luciferase gene reporter assays, in which the luciferase coding sequence is under the control of the 3' UTR of CFTR, as previously described [START_REF] Megiorni | Synergistic Post-Transcriptional Regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 Specific Binding[END_REF][START_REF] Viart | Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis[END_REF]. c) By transfecting mimics or precursors, miRNAs can be overexpressed and the relative level of luciferase expression reflects the effect of a given miRNA on the 3'UTR-CFTR. Inhibitors may also be used to confirm the specific effect of the miRNA under study. d) To validate the direct effect of the identified miRNAs (for instance, miRNA-494 and miRNA-101), the cis element for miRNAs binding in the 3'UTR-CFTR can be mutated by site-directed mutagenesis (created mutation). Target-site blockers may also be used to inhibit binding to the tested motif [START_REF] Viart | Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis[END_REF]. e) To validate these effects, the endogenous level of CFTR mRNA following miRNA overexpression or down-regulation can be assessed using different techniques.

Role of non-coding RNAs in CF

4.1. What is the impact of mutations in microRNA target sites on the CFTR gene?

Mutations in the 3'UTR of the CFTR gene

Assessing the putative impact of single nucleotide polymorphisms (SNPs) in the 3'UTR of CFTR is essential to define the pathological importance of these cis-regulatory motifs in noncoding regions, especially after the development of Next-Generation Sequencing (NGS) technologies. Recent work identified the SNP c.*1043A>C on the 3'UTR of the CFTR gene in one patient with a CFTR-related disorder and congenital bilateral absence of vas deferens [START_REF] Amato | Gene mutation in microRNA target sites of CFTR gene: a novel pathogenetic mechanism in cystic fibrosis?[END_REF]. This SNP is located in a region predicted to interact with miR-433 and miR-509-3p. Expression analysis demonstrated that the c.*1043A>C mutation increases the affinity for miR-509-3p and slightly decreases that for miR-433. In vitro, these two miRNAs reduces CFTR protein expression. The authors suggested that the very low expression of miR-509-3p in normal human bronchial epithelial (NHBE) cells could explain the mild phenotype caused by this mutation. Thus, the c.*1043A>C mutation, by acting as a mild CFTR mutation that enhances the affinity for an inhibitory miRNA, could represent a novel pathogenic mechanism in CF.

Methods to investigate the impact of CFTR gene mutations

Bioinformatics tools, such as miRNA binding site prediction programs (listed in Table 2), could be used to predict whether at a mutated position there is a cis-regulatory motif and whether it corresponds to an miRNA binding site. Moreover, tools like RNAhybrid could allow predicting the binding energy of the mutated motif. In vitro luciferase reporter gene assays together with mutagenesis approaches to create degenerate 3'UTR-CFTR sequences can be used to study the different variants (Figure 5Ad).

Deregulation of non-coding RNAs in CF

Clinical manifestations of CF are various including chronic pulmonary inflammation and infection that strongly contribute to the morbidity and mortality of these patients [START_REF] Bonfield | Inflammatory cytokines in cystic fibrosis lungs[END_REF]. Overinflammation precedes chronic infection that is then amplified by pathogens. Notably, the protease-antiprotease balance, which is responsible for lung remodelling, is disrupted in CF airways early in life and then this imbalance is chronically maintained [START_REF] Birrer | Proteaseantiprotease imbalance in the lungs of children with cystic fibrosis[END_REF]. Pulmonary tissues in patients with CF are usually infected by antibiotic-resistant Pseudomonas aeruginosa.

Interleukin-8 (IL-8), IL-6 and Tumor Necrosis Factor alpha (TNF-alpha) are pro-inflammatory cytokines that are highly expressed in CF lung epithelial cells and allow the recruitment of neutrophils [START_REF] Bonfield | Inflammatory cytokines in cystic fibrosis lungs[END_REF]. Lipopolysaccharide (LPS) and IL-1beta, which bind to Toll-like receptor 4 (TLR4) and IL-1R, respectively, are also involved. The identification of the molecular events involved in lung epithelium injury and repair is essential for understanding CF physiopathology. Expression levels of miRNAs physiologically vary greatly among tissues. Recent advances explored their effects on influencing signaling pathways in CF. Identification of deregulated miRNAs may offer possible future directions for clinical applications.

Deregulated lncRNAs in CF

To date, only one publication has reported aberrant expression of specific lncRNAs in CF bronchial epithelium in vivo [START_REF] Mckiernan | Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium[END_REF]. To establish the lncRNA profiles of CF and non-CF bronchial epithelium, 10 CF and 12 non-CF (controls) bronchial brushing samples were analyzed using a human lncRNA Array v3.0 (ArrayStar). In this way, more than 30586 lncRNAs and 26109 protein coding transcripts were evaluated. Overall, 1063 lncRNAs, most of which were intergenic, were differentially expressed in non-CF and CF bronchial brushing samples. RT-qPCR analysis of the differential expression of well-known ncRNAs (XIST, MALAT1, HO-TAIR, and TLR8 antisense) did not confirm the down-regulation of MALAT1 and HOTAIR in CF samples compared to controls. Interestingly, MALAT1 (lnc-SCYL1-1*) and HOTAIR (lnc-SMUG1-7*) have been described as oncogenic lncRNAs in lung cancer (*asterisks meaning the existence of several isoforms).

Deregulated miRNAs in CF

miRNA profiling studies identified various miRNAs with altered expression in CF (summarized in Figure 6). For instance, in vivo the expression of miR-145, miR-223 and miR-494 is increased in CF bronchial brushing samples (individuals with at least one p.Phe508del CFTR allele) compared to non-CF controls and correlates with decreased p.Phe508del CFTR expression. Moreover, these three miRNAs inhibit CFTR mRNA expression [START_REF] Oglesby | Regulation of cystic fibrosis transmembrane conductance regulator by microRNA-145, -223, and -494 is altered in ΔF508 cystic fibrosis airway epithelium[END_REF]. These authors also highlighted a relationship between their regulation and CFTR chloride channel activity. Specifically, they showed that treatment with inh-172, a specific inhibitor of the CFTR chloride channel, significantly increases the level of miR-145, miR-223 and miR-494 in 16 HBE14orespiratory epithelial cells. These data are in agreement with the hypothesis by Wenming Xu et al. [START_REF] Xu | MicroRNAs and cystic fibrosis-an epigenetic perspective[END_REF] that CFTR chloride channel alteration affects the miRNA profile. Another study reported that miR-509-3p and miR-494 are increased in well-differentiated primary cultures of human CF but not of non-CF airway epithelia, [START_REF] Ramachandran | Posttranscriptional regulation of cystic fibrosis transmembrane conductance regulator expression and function by microRNAs[END_REF]. Other miRNAs have been found to be deregulated in CF, such as miR-155, miR-126 and miR-31 [START_REF] Bhattacharyya | Elevated miR-155 promotes inflammation in cystic fibrosis by driving hyperexpression of interleukin-8[END_REF]. miR-155 is overexpressed in IB3-1 CF cells compared to IB3-1 control cells and in ex vivo CF cells (bronchial brushing samples versus normal human bronchial epithelial cells and CF neutrophils CD66+ versus control cells). Moreover, in IB3 cells, miR-155 up-regulation is related to defective CFTR chloride channel activity (following exposure to inh-172). This leads to repression of SHIP1, a well-known effector in the regulation of inflammation, and consequently to activation of the PI3K/AKT pathway that stabilizes IL-8 mRNA through MAPK. Thus, miR-155 up-regulation contributes to the maintenance of a pro-inflammatory phenotype. Furthermore, miR-155antagomir leads to IL-8 down-regulation in IB3-1CF cells and could represent a candidate treatment for CF. The biogenesis of miR-155 has been in part elucidated and involves the inflammatory RNA binding proteins KSRP and TTP [START_REF] Bhattacharyya | Regulation of miR-155 biogenesis in cystic fibrosis lung epithelial cells: antagonistic role of two mRNA-destabilizing proteins, KSRP and TTP[END_REF]. KSRP promotes miR-155 production, while TTP down-regulates, via miR- Conversely, miR-126 down-regulation has an anti-inflammatory role to compensate the immunity response. Oglesby et al. [START_REF] Oglesby | miR-126 is downregulated in cystic fibrosis airway epithelial cells and regulates TOM1 expression[END_REF] reported that miR-126 is consistently decreased in CF compared to non-CF airway epithelial cells and this reduction correlates with up-regulation of TOM1, a negative regulator of TLR2, TLR4, IL-1, IL-1β and TNF-alpha [START_REF] Katoh | Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes[END_REF][START_REF] Yamakami | Tom1 (target of Myb 1) is a novel negative regulator of interleukin-1-and tumor necrosis factor-induced signaling pathways[END_REF]. TOM1, which also negatively regulates NF-κB, may play an anti-inflammatory role in CF lung. The authors hypothesized that the observed reduction in miR-126 expression in CF cells may be due to ER stress induced by accumulation of misfolded p.Phe508del CFTR proteins. A complete analysis of the pathways triggered in CF cells seems essential to identify/develop novel treatments for CF.

Another study assessed the miRNA profile, by using Agilent microarray, of CF IB3-1 cells infected or not with Pseudomonas aeruginosa, a model mimicking the inflammatory response observed in pulmonary tissues of CF patients [START_REF] Fabbri | Expression of micro-RNA-93 and Interleukin-8 during Pseudomonas aeruginosa-mediated induction of proinflammatory responses[END_REF]. Two miRNAs (miR-93 and miR-494) were found to be strongly deregulated in infected cells. These two miRNAs are predicted to interact with the 3'UTR of IL-8 mRNA. Down-regulation of miR-93 was confirmed in two other bronchial epithelial cell lines (CF CuFi-1 and non-CF Nuli-1 cells). In non-infected CF cells, miR-93 is strongly expressed and is involved in IL-8 down-regulation combined with low NF-κB recruitment to the IL-8 promoter. In CF cells infected with Pseudomonas aeruginosa, the combined effects of high NF-κB recruitment to the IL-8 gene promoter and miR-93 downregulation lead to high IL-8 expression [START_REF] Fabbri | Expression of micro-RNA-93 and Interleukin-8 during Pseudomonas aeruginosa-mediated induction of proinflammatory responses[END_REF].

Finally, miR-31 is down-regulated in CF bronchial brushing cells compared to non-CF cells [START_REF] Weldon | miR-31 dysregulation in cystic fibrosis airways contributes to increased pulmonary cathepsin S production[END_REF]. In CF epithelial cells, miR-31 negatively modulates the expression of IRF1, a transcription factor that regulates the level of cathepsin S (CTSS). CTSS is overexpressed in CF airways cell lines, such as bronchial (CFBE), tracheal (CFTE) and CF primary bronchial epithelial cells (CF-PBECs) and has been detected in CF lung secretions. CTSS activates the epithelial sodium channel and cleaves and inactivates antimicrobial proteins such as surfactant A, lactoferrin and members of the β-defensin family, thus contributing to lung inflammation in patients with CF [START_REF] Birrer | Proteaseantiprotease imbalance in the lungs of children with cystic fibrosis[END_REF][START_REF] Mckiernan | Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium[END_REF][START_REF] Oglesby | Regulation of cystic fibrosis transmembrane conductance regulator by microRNA-145, -223, and -494 is altered in ΔF508 cystic fibrosis airway epithelium[END_REF][START_REF] Xu | MicroRNAs and cystic fibrosis-an epigenetic perspective[END_REF]. Moreover, in a cohort of paediatric patients with CF, it was found that CTSS level correlates with the decline of lung function. Thus, miR-31 is a potential regulator of CTSS expression via IRF1 in CF epithelial cells [START_REF] Weldon | miR-31 dysregulation in cystic fibrosis airways contributes to increased pulmonary cathepsin S production[END_REF].

Methods to study deregulated non-coding RNA

Methods to quantify miRNAs in CF and non-CF samples are depicted in Figure 5B. Approaches to identify dysregulated lncRNAs in CF samples are detailed in Figure 7 and databases for lncRNAs are listed in Table 4.

Impact of mutations in lncRNA or miRNA genes

To date, no ncRNA mutation has been described in CF. However, alterations in RNA sequence and/or structure can affect the synthesis, maturation and turnover of ncRNAs. Changes in RNA molecules can be introduced in different ways. For instance, SNPs may affect miRNA biogenesis. miRNA tailing can modify pre-miRNAs and mature miRNAs. RNA editing can modify nucleotide sequences of RNA transcripts. NGS technologies, including exome sequencing and complete re-sequencing of the CFTR gene, could reveal mutations in lncRNA sequences that may affect CF severity and outcome.

Targeting miRNA as new putative therapeutic tool

Could miRNAs help in improving CF treatment?

A recent work demonstrated that miR-138 mimics might restore CFTR-Phe508del expression and functional chloride transport. However, the authors stressed that miR-138 mimics may also have undesired effects, because miR-138 targets SIN3A, a highly conserved transcriptional repressor that regulates many genes [START_REF] Ramachandran | Posttranscriptional regulation of cystic fibrosis transmembrane conductance regulator expression and function by microRNAs[END_REF]. Another anti-miRNA agent has been exploited as inhibitor of miR-509-3p, which is involved in the regulation of the CFTR gene. Recently, we reported that miRNA function can be blocked by targeting the CFTR gene with blockers. We designed blockers to prevent the binding of several miRNAs specifically to the 3'UTR-CFTR and tested them in well-differentiated primary human nasal epithelial cells from healthy individuals and patients with CF carrying the p.Phe508del CFTR mutation. These molecules rescued CFTR chloride channel activity by increasing CFTR mRNA and protein levels. This is in agreement with previous studies showing that complementation of just 6-10% of CFTR transcripts leads to the production of enough CFTR to maintain normal chloride transport in epithelia [START_REF] Broackes-Carter | Temporal regulation of CFTR expression during ovine lung development: implications for CF gene therapy[END_REF]. These data are supported by findings that the presence of a naturally occurring sequence variation in the CFTR promoter, in cis of a severe mutation, increases transcription. This allows the production of enough CFTR protein to reach the apical membrane cells and partially restore CFTR channel function, thus leading to a moderate CF phenotype despite the presence of a severe disease-causing mutation [START_REF] Romey | A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression[END_REF]. Similarly, stabilization of p.Phe508del CFTR protein has been associated with increased p.Phe508del CFTR channel activity [START_REF] Hutt | Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis[END_REF].

Assays and molecules

As depicted in Figure 5Ad, inhibitors or target-site blocker oligonucleotides have been previously used to restore CFTR expression. Tests have been performed by incorporating inhibitors that induce degradation of the targeted endogenous miRNA or with oligonucleotides that block miRNA binding to the 3'UTR of CFTR in cell lines, primary cultures and reconstituted epithelium

Conclusion and remarks

The identification of cisand trans-regulators and pathways involved in CFTR gene expression is essential for developing new CF targeted therapies. Over the past four decades, therapies for CF have focused entirely on symptoms to improve patients' quality of life. The first treatment (VX-770) targeted the basic defect in p.Gly551Asp-CFTR (1.6% of patients with CF worldwide) [START_REF] Ramsey | VX08-770-102 Study Group. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation[END_REF]. The new molecule VX-809 has been evaluated in patients carrying the p.Phe508del CFTR mutation; however, on its own it does not have clear effects [START_REF] Clancy | Personalized medicine in cystic fibrosis: dawning of a new era[END_REF] and clinical trials testing the combination of different molecules are in progress. The mechanisms responsible for the phenotype severity are not well understood yet. Mutational heterogeneity and complex alleles influence CF severity. Moreover, the role of few modifier genes has been established [START_REF] Knowles | The influence of genetics on cystic fibrosis phenotypes[END_REF]. ncRNAs could also contribute to CF progression and severity and their dysregulation in CF opens new perspectives for patient follow-up and treatment.

Figure 1 .

 1 Figure 1. Non-coding RNAs.

Figure 2 .

 2 Figure 2. lncRNAs, from biogenesis to functions.

Figure 4 .

 4 Figure 4. Main miRNA roles. a. Translation block by inhibiting cap and poly(A)-binding protein recognition. b. Elongation inhibition by slowing down elongation or ribosome 'drop-off'. c. Degradation by deadenylation and decapping. d. Proteolysis. Degradation of a nascent peptide. e. mRNA storage in P-bodies that contain exonucleases, RNA helicases, decapping enzymes, DCP1/2, exosomes, deadenylases.

Figure 5 .

 5 Figure 5. Approaches for miRNA study. A. Strategies used to study the involvement of miRNAs in the regulation of CFTR gene expression. B. Strategies to identify miRNAs that are deregulated in CF samples compared to non-CF samples. Chips for global miRNA profiling are commercialized by Agilent, Affimetrix and Exiqon. miR-seq (miRNA sequencing) is usually performed by Illumina platforms. Luc, luciferase; TLDA, TaqMan Array Micro Fluidic Cards (Applied Biosystems); WT, wild type.

Figure 6 .

 6 Figure 6. Deregulated miRNAs in different CF models. The schematic lists miRNAs that have been reported to be upregulated or down-regulated in CF (tissues, cell lines, primary cultures) versus (vs) non-CF samples. *ALI: Air-Liquid Interface.

  1, miR-155 mature expression in CF lung epithelial cells.

	IB3-1 CF vs IB3/S9	miR-155 miR-126	miR-101 miR-138	CF vs non CF bronchial
				miR-145	brushing
					miR-223
			miRNA	miR-494
	CF vs non CF	miR-145 miR-494	dysregulation in CF airways	miR-126 miR-146-a
	Nasal cavity
	brushing
					miR-145
				miR-223
			miR-494	miR-494
			miR-509-3p	miR-126
			miR-138
					CFBE41o-vs
					16HBE14o-
		ALI* culture of CF vs non CF
		airway epithelia
		(trachea and primary bronchi)

Cystic Fibrosis in the Light of New Research

Role of Non-coding RNAs in Cystic Fibrosis http://dx.doi.org/10.5772/60449

Name

Website

Characteristics References

MiRonTop http://www.microarray.fr: 8080/miRonTop/index Identification of miRNAs from DNA microarrays and high-throughput sequencing data.

Uses several existing miRNA target prediction approaches.

[101]

MIR@nt@n http://maia.uni.lu/ mironton.php

Integrative approach for searching miRNAs target sites, to build networks including motifs as feedbacks and feedforward loops from lists of molecular actors. Contains genomic location, overlapping small ncRNAs, associated repeat elements and imprinted genes and lncRNA promoter information.

[119]

Table 4. Free databases for lncRNAs Few techniques allow the quantitative analysis of lncRNAs in biological samples. RNAseq: global sequence screening is currently an easy option thanks to NGS and small RNA sequencing. This method allows detecting and quantitating in a biological sample all non-coding transcripts. However, as lncRNAs are very weakly expressed, this strategy might not be fully appropriate. To obtain a significant profile of ncRNAs, more than 180 million reads are required, whereas for protein-coding transcripts, less than 30 million are needed. Therefore, microarray analysis remains a powerful tool for global profiling of lncRNAs. In total, more than 30,586 lncRNAs have been analyzed based on the last lncRNA databases. AONs: antisense oligonucleotides.

Author details

Jessica Varilh 1 , Jennifer Bonini 2 and Magali Taulan-Cadars 2* *Address all correspondence to: magali.taulan@inserm.fr 1 Laboratoire de Génétique Moléculaire, CHU Montpellier, France 2 Laboratoire de Génétique de Maladies Rares, Université Montpellier, UFR de Médecine, Montpellier, France


E Sferlazza

Jean-Frédéric Calas

Brun

Effects over one year of low-intensity endurance exercise

targeted at the level of maximal lipid oxidation