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MARIE SKLODOWSKA-CURIE ACTIONS

Co-funding of regional, national and international programmes (COFUND)

DOC2AMU THESIS PROJECT 2018 CALL FOR APPLICATIONS

Active response of Red Blood Cells to mechanical stress in splenic filtration

1. GENERAL INFORMATION

Call 2018-23

Topic Nano-health

Keywords Human red blood cells, splenic filtration, active volume regulation, microfluidics, live microscopy, computational modelling

2. THESIS DIRECTOR(S), RESEARCH UNITS AND DOCTORAL SCHOOLS

Thesis director Emmanuèle HELFER

Research Unit Centre Interdisciplinaire de Nanoscience de Marseille

Doctoral school ED 352 - Physique et Sciences de la Matière

Thesis co-director Catherine BADENS

Research Unit Génétique Médicale et Génomique Fonctionnelle

Doctoral school ED 062 - Sciences de la Vie et de la Santé

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MARIE SKLODOWSKA-CURIE ACTIONS

Co-funding of regional, national and international programmes (COFUND)

DOC2AMU THESIS PROJECT 2018 CALL FOR APPLICATIONS

Active response of Red Blood Cells to mechanical stress in splenic filtration

(ActiveRed)

1. DESCRIPTION OF THE PHD THESIS PROJECT

1.1 OBJECTIVES OF THE PROJECT BASED ON THE CURRENT STATE OF THE ART

Blood consists in a highly concentrated suspension (45% in volume) composed mainly of red blood cells (RBCs,

99%) and few other blood cells (leukocytes and platelets, 1%). The efficient and sustainable circulation of RBCs

is an outstanding physical tour de force. During their 120-days lifespan, they continuously circulate through our

intricate microvascular network composed of slits, capillaries, bifurcations, etc. During such cycles they undergo

very strong deformations: for example, the RBC passes through blood vessels as small as 4 µm in diameter or

through submicron slits located in the spleen. The RBC being a biconcave disk around 2 µm in thickness and 8

µm in diameter, it cannot go through such constrictions if not highly deformable, and highly robust as well. This

is in part due to its complex double envelope that encloses the viscous haemoglobin solution. This double shell

is made of an incompressible fluid viscous lipid bilayer at the outside and a 2D elastic network of cross-linked

spectrin filaments at the inside, connected to the lipid bilayer by protein complexes. Additionally, a

mechanosensitive ion channel was recently discovered whose activation is triggered by a mechanical stress

applied on the RBC membrane [Coste 2012]. A second ion channel ion is then activated in cascade, leading to

water release out of the cell, as a way to control the RBC volume [Rapetti-Maus 2015]. A new hypothesis thus

arised that these ion channels could play an active role in RBC volume changes, in order to rapidly adjust the cell

deformability under a mechanical constraint.

The purpose of the ActiveRed project is to understand quantitatively the physical mechanisms of large

deformation and the molecular mechanisms of volume regulation in the specific case of the passage through

the splenic submicron slits. Recent studies indeed suggest that the spleen senses RBC deformability and

spheroidicity thus defining the size and shape of RBCs allowed to remain in the microcirculation [Pivkin 2016]. A

current hypothesis is that RBCs have to pass a ‘physical fitness test’ in the spleen, the submicron slits (Fig. 1A),

to be allowed to remain in the blood flow. Yet, no known physical mechanisms rationalize this hypothesis as

experiments are strongly lacking.

From a physics point of view, the process of splenic filtration raises basic physical questions: what is the link

between RBC mechanical parameters and passage/sequestration in splenic slits? What mechanical properties of

RBCs are most crucial to go through submicron splenic slits? Is the selection mechanism of RBCs by the spleen

based on mechanical criterions only? Or are indeed additional active phenomena, such as volume change,

needed to avoid RBC rupture under large deformation?

From a clinical point of view, there is a strong need for understanding the clearance process by the spleen. It

normally occurs as RBCs age and lose their deformability, to eliminate older RBCs and renew the RBC pool in the

blood stream. However, the spleen is also a major player in a number of diseases, whether infectious (malaria)

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or genetic (sickle cell disease (SCD), hereditary spherocytosis (HS), hereditary xerocytosis (HX)…). In all these

diseases, the RBC deformability is altered and RBCs get massively sequestered in the spleen. It thus causes severe

hemolytic anemia due to an accelerated splenic destruction of RBCs. Therefore, we expect that physics

experiments leading to understanding how an RBC passes the spleen fitness test and how the process is affected

in RBC genetic disorders will have a major impact in haematology.

Figure 1. A) An RBC (highlighted in red) squeezing through a splenic slit (≈0.5 x 2 x 5 µm3). B) SEM image of

the silicon master of a typical microfluidic device with a series of slits. C) Optical images of RBCs squeezing

through 0.8 x 1.9 x 5 µm3 biomimetic slits. The bottom one displays a tip while exiting the slit. Scale bar: 5 µm.

The ActiveRed project is based on the recent technological breakthrough we made in 2017, by fabricating a

microfluidic device with slits of physiological splenic dimensions (Fig. 1B) [Gambhire 2017]. This device allowed

us to observe human RBCs passing through these biomimetic slits and revealed new modes of deformation due

to high confinement (Fig. 1C, bottom). This is the first device that reproduces the dimensions of splenic slits (≈0.5

x 2 x 5 µm3) in comparison with previous biomimetic devices that could not reach such small dimensions [Rigat-

Brugarolas 2014, Deplaine 2011]. We will study the RBC behavior as they are submitted to controlled mechanical

stress (the slit dimensions and the flow pushing the RBCs). To investigate whether the RBC volume actively

changes in response to the stress, we will target the two ion channels which are thought to act together, the

mechanosensitive Piezo1 and the Ca2+-sensitive Gardos (Fig. 2A).

Figure 2. A) Schematics of Piezo 1 and Gardos interplay that controls ion fluxes: mechanical stress, e.g. RBC

stretching, activates Piezo1 which become permeable to cations, including calcium. The Ca2+ influx activates

Gardos leading to K+ and Cl- exit concomitantly with water. It results in a decrease in RBC volume, thus in an

increase in area-to-volume ratio, and presumably an increase in healthy RBC deformability. B) Activators

(arrows) and inhibitors of the two channels that will be used in the study.

Most studies on RBC ion channels are done on non human cells, mostly murine ones [Cahalan 2015], neglect

physiological flow and usually focus on one or the other channel. Here, modulation of Piezo1 and Gardos channel

activities will be studied in combination and not separately, in human RBCs, and in the physiological situation of

RBCs flowing though splenic slits. A recent work by the group of Kaestner studied their interplay using 3-µm wide

constrictions, they observed a response even at such small RBC deformation [Danielczok 2017]. We thus expect

a stronger response by using our biomimetic splenic slits.

The channels’ activity and interplay will be modulated via various combinations of known inhibitors and

activators (Fig. 2B). Healthy RBCs as wells as RBCs with disordered channels will be studied. Indeed, HX disease,

A B

ℓ= 1.9 µm 𝓌 = 0.8 µm

C

A B

Ca2+

Piezo 1 Gardos K

+

RBC

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due to various mutations in either Piezo1 or Gardos channels, is characterized by abnormal cation leak and cell

dehydration, leading to cell fragility and hemolytic anemia [Badens 2016]. We have access to a pool of patients

with some of these mutations. Their RBCs will be assayed in the biomimetic slits, untreated and treated with the

biochemical blocking/activating agents.

Our quantitative results will be combined with 3D computations (from international collaborator) that take into

account the RBC dynamics and the channels’ activity to derive the physical mechanisms responsible for RBC

active response to mechanical stress applied. Our findings will highlight which physical parameters can be used

as a new read out to follow disease evolution or treatment effect, and potentially lead to novel therapeutic

targets.

References:

Badens C and Guizouarn H. Advances in understanding the pathogenesis of the red cell volume disorders. Review.

Brit J Haematology 174:674-685 (2016)

Cahalan SM, Lukacs V, Ranade SS, Chien S, Bandell M, Patapoutian A. Piezo1 links mechanical forces to red blood

cell volume. eLife 4:e07370 (2015)

Coste B et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.

Science 330:55–60 (2010)

Danielczok et al. Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca2+-mediated

Adaptations. Front Physiol 8:979 (2017)

Deplaine et al. The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro.

Blood 117:e88–e95 (2011)

Gambhire et al. High aspect ratio sub-micron channels using wet etching: Biomimetic spleen slits for red blood

cell studies. Small 13:1700967 (2017)

Pivkin IV et al. Biomechanics of red blood cells in human spleen and consequences for physiology and disease.

PNAS 113:7804–7809 (2016)

Rapetti-Maus et al. A mutation in the Gardos channel is associated with hereditary xerocytosis. Blood 126:1273-

1280 (2015)

Rigat-Brugarolas et al. A functional microengineered model of the human splenon-on-a-chip. Lab Chip 14:1715

(2014)

1.2 METHODOLOGY

The PhD program is pluridisciplinary and the student will learn the different required skills in the groups of the

two supervisors. The different tasks and milestones of the ActiveRed project are:

Task 0 (UMR_S910): Blood collection – Healthy and pathological blood sample collection will be obtained in the

context of regular clinical follow up at Hospital La Timone, and characterized.

Task 1 (UMR_S910): Characterization of Piezo1 and Gardos channel activity – Inhibitors and activators of Piezo1

and Gardos will be used to modulate RBC permeability (healthy and mutated). RBC ion content will be measured

under the various treatments to define the optimal combinations that will be assayed in the physics experiments.

Task 2 (CINaM): Relationship between RBC mechanical properties, RBC volume and channel activity as a function

of the applied mechanical stress – Prior to the microfluidic experiments, RBCs will be submitted to

meso/macroscopic deformations using atomic force microscopy (AFM) and optical tweezers. These techniques

allow applying forces in the pN-µN range. The resulting Ca2+ influx will be tracked using the commercial Fluo-4

calcium probe. Healthy and channel-deficient RBCs will be studied. From these measurements the

mechanosensitive characteristic response times will be extracted for healthy and patients’ RBCs.

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Task 3 (CINaM): Design and fabrication of new biomimetic devices – In the current device both the main wide

channel and the thin slits have the same height, thus RBCs are horizontally constrained before reaching the slits.

To get a geometry closer to that of the spleen, the device will be improved to have a main channel of larger

height (> 10 µm) with slits of 5 µm height.

Task 4 (CINaM + UMR_S910): Physical fitness test: relation between RBC deformation, transit time, flow rate,

channel activity, and slit size – Microfluidic experiments on individual RBCs flowing through biomimetic slits

(current and future devices) will be performed using ultrafast-videomicroscopy (> 1,500 fps) to observe RBC

deformation combined with standard fluorescence videomicroscopy (25 fps) to track calcium influx.

Experimental conditions (flow rate, slit dimensions) will be optimized so that the transit time is higher than the

response time derived from mesoscopic experiments and to be able to observe the calcium entry. The passage

of RBCs from healthy donors and from patients will be studied in absence and presence of the channel’s

modulators. The goal is to establish the laws of behaviour between the severity of the fitness test (given by slit

dimensions and flow rate) and RBC dynamics (shape deformation and velocity, ion fluxes).

Task 5 (CINaM + UMR_S910): PhD thesis writing and defense

Task 6 (Collaborator, Univ Notre Dame, USA): Modelling of the RBC active deformation under mechanical stress

– During the total duration of the project, we will communicate with our collaborator in the USA who will develop

a 3D model of the RBC that integrates the double envelope components, the channels, the resulting ion

transport, and the effect of the channel regulators. The coupling of experimental and numerical approaches will

lead to identification of the critical factors that cause RBC deformation, entrapment or damage, and investigate

how molecular mutations influence these critical factors. We expect to provide a complete physical

understanding of the dynamics of RBCs in spleen-like slits that will allow to predict the RBC behavior and the risk

of damage in case of altered mechanical properties. Moreover, we will conclude on the existence of an active

volume regulation in response to applied stress during spleen filtration.

1.3 WORK PLAN

1.4 SUPERVISORS AND RESEARCH GROUPS DESCRIPTION

Supervisor 1 – Centre Interdisciplinaire de Nanoscience de Marseille (AMU/CNRS, UMR7325, Marseille)

Emmanuèle HELFER joined CINaM in 2014, and now belongs to the newly created Physics and Engineering of

Living Systems (PIV) Department. The CINaM is a multidisciplinary structure, composed of approximately 180

persons, which hosts a nano/micro-fabrication platform that allows design and fabrication of complex

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Task0 Blood collection

Task 1 Characterization of channel's activity

Task 2 Characterization of RBC response

times (AFM, optical tweezers)

Task 3Design and fabrication of new

microfluidic devices

Task 4 Microfluidic experiments and analysis

Task 5 PhD writing and defense

Task 6

(collab)

Modelling of the RBC active

deformation under mechanical stress

DescriptionTasks

Months 1-12 Months 13-24 Months 14-36

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microfluidics devices. PIV department is equipped for cell biology, optical and near-field microscopies,

micropipette experiments, and microfluidics. E. Helfer works in the interdisciplinary field of physics of

macromolecular assembly since her PhD. She is an expert in in vitro biomimetic reconstitution of biological

processes, focusing on interactions between the actin cytoskeleton and cell membranes. In CINaM she goes on

developing a biomimetic approach of actin-based regulation of intracellular traffic. Since her arrival in Marseille,

she also worked in close collaboration with scientists from PIV Department to develop a microfluidic setup with

features mimicking closely the submicron-sized slits of the spleen that are dedicated to red blood cell (RBC)

filtration and selection. She is now interested in the molecular mechanisms occurring during RBC passage

through these biomimetic slits, i.e. the active regulation of the cell volume while it undergoes such a strong

deformation to squeeze in through the slit.

Supervisor 2 – Medical Genetics & Functional Genomics Unit (AMU/Inserm, UMR_S910, Marseille)

The UMR_S910 is presently staffed by approximately 100 people organized into 8 teams and is located at the

Campus Santé Timone (Medical School) of Marseille. Catherine BADENS is the head of the Molecular Genetics

Unit at the children hospital La Timone. She has a longstanding experience in medical biology and molecular

diagnosis of RBC genetic diseases, among which Sickle Cell Disease (SCD) and Hereditary Spherocytosis (HS).

These two diseases alter the mechanical properties of the RBCs leading to their sequestration in the spleen and

subsequent anemia. Recently, she has also characterized mutations in the Gardos ion channel, which result in

RBC dehydration. She now works on the combined action of this Ca2+-activated channel and the

mechanosensitive ion channel PIEZO1. She is designing various combinations of specific inhibitors of the channels

activity to modulate the hydration level of normal RBCs.

The project follows up a research program funded by A*Midex (RedPath, 2015-2017, coordinator: A. Viallat)

which allowed the fabrication of the biomimetic splenic-like slits.

2. 3I DIMENSIONS AND OTHER ASPECTS OF THE PROJECT

2.1 INTERDISCIPLINARY DIMENSION

The ActiveRed project involves two teams with the two supervisors attached to two Doctoral Schools of Aix-

Marseille University: ED352 (Physics and Matter Sciences) and ED62 (Life and Health Sciences). The PhD deals

with the fabrication of innovative microfluidic devices mimicking splenic slits, combination of high-speed video-

microscopy and fluorescence microscopy, image analysis, and manipulation of biological material. The PhD

student will participate in all the phases of the project, from the development of the devices, the microscopy

setup for dual observation, and the various combinations of biochemical treatments on RBCs to patients’

recruitment and sample collection.

E. Helfer has longstanding expertise in physics of the cytoskeleton and of cell membranes, and in using

biochemistry, cell biology and optical microscopy techniques to derive molecular interactions between actin

networks and cell membranes. She will lead the microfluidic, AFM and optical tweezers experiments and

subsequent analysis to extract RBC behavior changes under mechanical stress. C. Badens will provide molecularly

characterized healthy blood samples and samples affected with mutations in the Piezo and Gardos channels. She

will design various combinations of specific inhibitors and activators of the channels’ activity so as to modulate

the volume of normal RBCs in circulation. She will measure the cell ion content changes under the various

treatments to define the optimal combinations that will be assayed in the physics experiments. The experimental

results from RBCs under stress will be interpreted thanks to the input of the international collaborator (Z. Peng,

Univ Notre Dame, USA) who will inject the molecular elements of RBC in his computational model.

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The project is interdisciplinary by essence as it proposes a combination of know-how, expertise and knowledge

from different scientific fields and cultures: medical research, mechanics, nanotechnology and computation.

From our results we expect major advances in the global understanding of increased haemolysis and subsequent

anemia in RBC volume disorders induced by genetic mutations of Piezo 1 or Gardos channels.

2.2 INTERSECTORAL DIMENSION:

The non-academic partner is the Centre Hospitalier Universitaire La Timone, a hospital from Assistance Publique

des Hôpitaux de Marseille (APHM). La Timone is the most important hospital in PACA region and considered as

the 3rd european hospital from its activity, equipment and staff.

Dr Emmanuelle Bernit, member of the medical team of Department of internal medicine of APHM, focuses her

activity on hereditary anemias such as hemoglobinopathies and red cells membrane disorders. Around 200

patients with such blood genetic diseases are regularly monitored at the department she belongs to. Dr Bernit

will provide access to the cohort of patients, and specifically to those carrying mutations in the Piezo/Gardos

channels. The PhD student will participate in the recruitment process, i.e. meeting the patients and obtaining

their consent for providing their blood for the study. This clinical experience will be of benefit for her/him as it

will extend her/his understanding of the project from the fundamental research aspect to the clinical and ethical

one as well.

This ActiveRed project fits particularly with the Nano-Health axis of the SRI-S3 objectives. It aims at deciphering

the role of ion channels’ molecular interplay in the deformability and cell volume homeostasis of RBCs. Such

understanding is indeed of importance for chronic hemolytic anemia as it can open the way to development of

future treatments and diagnosis tools.

2.3 INTERNATIONAL DIMENSION:

We collaborate with Zhangli PENG, assistant professor at University Notre Dame (Indiana, USA), Within the PhD

period, the student will go visit his lab for one month to extend her/his knowledge to computing skills. Moreover,

she/he will attend once a year the annual APS DFD (Division of Fluid Dynamics) Meeting, the annual meetings of

either the British or the American Societies of Hematology, and at the national level the annual congress of the

Club du Globule Rouge et du Fer.

Dr Z. Peng has ten years’ experience in computational modeling of RBCs. Using advanced computational methods

he successfully predicted the bilayer-cytoskeletal interaction strength at the molecular level in RBCs and

highlighted the biomechanical mechanisms of hereditary spherocytosis and malaria transmission. He is currently

developing a multiscale model of RBCs with mechanosensitive channels flowing through splenic slits. In this

project, he will adapt his model to include the Ca2+-sensitive Gardos channel and compute RBC dynamics and

Ca2+/K+ transport at the cellular level. He will take into account the presence of agonists/antagonists or genetic

mutations of the two channels and explore their effects on RBC volume control. He will compare the predicted

cell deformation and ion transport with experiments for validations. The synergistic effects from our

complementary skills in microfluidic experiments on RBCs and multiscale simulations will enable the realization

of the ActiveRed project.

3. RECENT PUBLICATIONS

Relevant publications and patents of Supervisor 1: Dr Emmanuèle HELFER

1. Ghambire P, Atwell S, Iss C, Badens C, Helfer E, Viallat A, Charrier A. High aspect ratio sub-micron channels

using wet etching: Biomimetic spleen slits for red blood cell studies. Small 13:1700967 (2017)

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2. Rommelaere S, Millet V, Rihet P, Atwell S, Helfer E, Chasson L, Beaumont C, Chimini G, do Rosário Sambo M,

Viallat A, Penha-Gonçalves C, Galland F, Naquet P. Serum pantetheinase/Vanin levels regulate erythrocyte

homeostasis and severity of malaria. The Am J Pathol 185:3039-3052 (2015)

3. Helfer E, Harbour ME, Henriot V, Lakisic G, Sousa-Blin C, Volceanov L, Seaman M, Gautreau A. Endosomal

recruitment of the WASH complex: active sequences and mutations impairing interaction with the retromer. Biol

Cell 105:1-17 (2013)

4. Derivery E, Helfer E, Henriot V, Gautreau A. Actin polymerization controls the organization of WASH domains

at the surface of endosomes. PLoS One 7:e39774 (2012)

5. Delatour V, Helfer E, Didry D, Lê KHD, Gaucher JF, Carlier MF, Romet-Lemonne G. Arp2/3 controls the motile

behavior of N-WASP-functionalized GUVs and modulates N-WASP surface distribution by mediating transient

links with actin filaments. Biophys J 94:4890-4905 (2008)

Relevant publications and patents of Supervisor 2: Prof Catherine BADENS

1. Rapetti-Mauss R, Picard V, Guitton C, Ghazal K, Proulle V, Badens C, Soriani O, Garçon L, Guizouarn H. Red

blood cell Gardos channel (KCNN4): the essential determinant of erythrocyte dehydration in hereditary

xerocytosis. Haematologica 102:e415-e418 (2017)

2. Rapetti-Mauss R, Soriani O, Vinti H, Badens C, Guizouarn H. Senicapoc: a potent candidate for the treatment

of a subset of hereditary xerocytosis caused by mutations in the Gardos channel. Haematologica 101:e431-e435

(2016)

3. Badens C and Guizouarn H. Advances in understanding the pathogenesis of the red cell volume disorders.

Review. Brit J Haematology 174:674-685 (2016)

4. Rapetti-Mauss R, Lacoste C, Picard V, Guitton C, Lombard E, Loosveld M, Nivaggioni V, Dasilva N, Salgado D,

Desvignes JP, Béroud C, Viout P, Bernard M, Soriani O, Vinti H, Lacroze V, Feneant-Thibault M, Thuret I, Guizouarn

H, Badens C. A mutation in the Gardos channel is associated with hereditary xerocytosis. Blood 126:1273-80

(2015)

5. Patent. Badens C, Guizouarn H, Thuret I. Diagnostic and treatment of Hereditary Xerocytosis. Property: APHM,

AMU. Patent # EP 15305921.7

4. EXPECTED PROFILE OF THE CANDIDATE

The applicant must have a master degree in experimental physics or biology, with a strong interest towards

biophysics. A background in optical microscopy and/or basic knowledge in biology, though not required, will be

welcome.

The PhD student is expected to be sociable and eager to broaden her/his interdisciplinary knowledge. She/he

will have to interact with physicists, biologists, biophysicists and physicians. Skills in image analysis and

programming (Image J, Matlab, Python) will be acquired during the PhD period.

5. SUPERVISORS’ PROFILES

Supervisor 1:

Emmanuèle HELFER was born in Nancy (France) in 1973. She did her studies at the University of Strasbourg

where she obtained her PhD in 1999 in Biophysics, on the viscoelastic properties of actin-coated vesicles, under

the supervision of D. Chatenay and L. Bourdieu. In 2001 she obtained a CNRS position in the 'Cytoskeleton and

Cell Motility' group, in the Laboratoire d'Enzymologie et Biochimie Structurales (LEBS, Gif-sur-Yvette, France)

where she worked on biomimetic reconstitution of actin-based motile processes. In 2010 she moved to the

'Cytoskeleton and Cell Morphogenesis' group (LEBS) where she studied the role of actin structures on

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intracellular endosomal membranes by combining cell biology and biochemical approaches. End of 2014, she

moved to Marseille (France) to join CINaM, where a new department, 'Physics and Engineering of Living Systems'

(PIV), was being created and she actively participated in its establishment (official start in January 2018). In PIV

department she develops her biomimetic approach to decipher further the role of endosomal actin in

intracellular traffic. On the other hand, she started collaborations with the other scientists of the group on red

blood cell dynamics and mechanics, and on cell adhesion. Her involvement in the RBC project has been increasing

since then and she is now taking the leadership in aspects related to molecular regulation.

E. Helfer has published 20 papers in peer-reviewed journals and 2 book chapters, in the domain of actin-

membrane interactions and actin biochemical regulation. She gave 3 invited talks, 4 oral presentations at

international conferences, and 2 at national ones. She has been funded an ANRJCJC (COORDACTIN, 2010-2012),

a local grant from PACA Région (BioMimWASH, 2017-2018) and one from A*Midex in which she is leading the

CINaM participation (MecaLam, 2018-2020; coordinator: C. Badens). She has been requested to participate in

committees for faculty selection (Paris-Diderot Univ, 2009-2011) and laboratory evaluation (LJP Hcéres

committee, 2018), and reviewed 2 PhDs in 2017 (ENS Chimie, Paris; Institut Jacques Monod, Paris). At the

national level, she is member of the National committee of the CNRS (2016-2021), in Section 11 (“Supra and

macromolecular systems and materials: design, properties, functions”) where she is one of the experts in the

field of physics-biology interface.

She obtained in 2014 her Accreditation to Supervise Research in Biochemistry and Cell Biology (Paris-Sud

University), on The interactions between the actin cytoskeleton and membranes, before she moved to Marseille.

She has been co-supervising 1 PhD thesis (2003-2007, Vincent Delatour) leading to 2 publications [New J Phys,

2008; Biophys J, 2008] and 1 review [Biophys Rev & Lett, 2009]. V. Delatour obtained a position in 2008 in

Laboratoire National de Métrologie et d'Essais (LNE, Paris). Since October 2017, she is co-supervising a PhD

student with Dr A. Viallat (PIV) on the vaso-occlusion mechanism in the pathological context of Sickle Cell Disease

where RBCs become less deformable and adhesive, and thus form aggregates. She will supervise a PhD student

from October 2018 on a new project on cell mechanics in premature aging diseases funded by A*Midex. She (co)-

supervised 12 master students since 2001, among them 3 were supervised and 3 were co-supervised since her

arrival in CINaM.

Supervisor 2:

Catherine BADENS is Pharm D, DES Biologie Médicale (1991), PhD(1996) and did her studies at Aix Marseille

University.

She is the head of the Molecular Genetics Unit at the children hospital La Timone and head of the Laboratory of

Biochemistry in La Conception, both hospitals located in Marseille city center. She has a longstanding experience

in medical biology, molecular diagnostic in rare diseases, patients registry and translational research projects.

Since October 2011, she is Professor of Biological Sciences, at the Faculty of Pharmaceutical Sciences, Aix

Marseille University, France. She is the scientific coordinator of the French Registry for Thalassemia and a

member of the board of the French Centre of reference for Thalassemia (http://www.chu-lyon.fr/web/2652 ).

She has authored or co-authored 104 papers in peer-reviewed journal and is co-inventor of 3 patents (pending).

She has supervised 3 PhD thesis, all 3 are now working at APHM (1 PH, 1 engineer) or AMU/APHM (1 MCU/PH).

She has recently been funded a grant from A*Midex as the coordinator: MecaLam, 2018-2020.

DD EE PP AA RR TT EE MM EE NN TT DD EE MM EE DD EE CC II NN EE II NN TT EE RR NN EE

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Docteur Emmanuelle BERNIT

Praticien Hospital ier

RPPS : 10003428785

Docteur Nicoléta ENE

Praticien Hospital ier

RPPS : 10004415906

Docteur J. François LAMARCHI

Praticien Hospital ier

RPPS : 10003431458

Docteur Christine NICOLINO

Praticien Hospital ier

RPPS : 10003346698

Docteur Véronique VEIT

Praticien Hospital ier

RPPS : 10003376133

Docteur Laure SWIADER

Praticien Hospital ier

RPPS : 10003370011

Docteur Estelle JEAN

Praticien Hospital ier

RPPS : 10100410215

Docteur Mikaël EBBO

Maître de Conférence des Universités

PH -RPPS : 10100287019

Docteur Pauline BELENOTTI

Assistant Chef de Cl inique

RPPS : 10100613701

Docteur Jacques POZZO di BORGO

Assistant Chef de Cl inique

RPPS : 10100686459

Docteur Aurélie GRADOS

Assistante Chef de Cl inique

RPPS :10100842276

Docteur M. Pierre DI COSTANZO

Vacataire

RPPS : 10003358172

Accueil Consultations Externes

Tél : 04 91 38 60 33

NUMADIC : Numéro d’Aide au Diagnostic

Du lundi au vendredi de 9 h à 18h Tél : 04 91 38 79 00

Marseille, le 06/01/2018

As a member of the medical team of Department of internal medicine of the

“Assistance Publique des Hôpitaux de Marseille”, I am supporting a biological physics

project entitled:

“Active response of red blood cells to mechanical stress in splenic filtration”

The physics student will conduct this project under the co-supervision of a physicist, Dr

Emmanuele HELFER and a biologist, Prof. Catherine BADENS. I am deeply convinced

that in the context of his/her doctoral training, an experience in clinical laboratory and a

reference center for rare diseases would be very beneficial for his/her project by enabling

a global understanding of the topic.

Our department participates in several research projects including clinical trials with

industrial or academic sponsors. My main activity focuses on hereditary anemias such as

hemoglobinopathies and red cells membrane disorders. A cohort of a bout 200 patients

are monitored regularly at our hospital. Anemia observed in such diseases is partly due to

splenic sequestration of red cells whose deformability is altered for further destruction.

An unmet need does exist for a better understanding of red cells deformability in chronic

hemolytic anemia and of the implication of red cells volume homeostasis via ion

channels. Advances in this field will certainly lead to the identification of new targets for

future innovative treatments and to more powerful diagnosis tools. This could be achieved

by interdisciplinary projects such as the one we are supporting here. The project aims in

deciphering the mechanisms at play during the passage of red cells through splenic

filtering slits, and more specifically the ones involving ion channels regulating the cell

volume.

The success of this project depends in part on the access to the largest number of blood

samples. These samples are collected in our department on clinical research participants

after their appropriate consent.

I deeply think that the student should participate in this essential step of the research:

meeting the medical team and discussing about his/her project and the expected outputs

will surely motivate him/her. Furthermore, it would raise the student’s awareness about

ethical, legal and clinical aspects of translational research. The student time dedicated to

this aspect of the project with the medical staff, will be approximately one day per month.

Thank you in advance for your attention to this project,

Sincerely,

Dr Emmanuelle Bernit