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BIOPHYSICS AND IMAGING 2015
by FdV and AIV students
Centre de Recherches Interdisciplinaires
Wednesday, April 08, 2015
9h15 – 9h30
Registration and Reception
9h40-10h00
Ultra-rapid imaging of a drosophila memory center :
Danae, M2
Analysis of the response to an olfactory stimulation
10h00-10h20
Henri, M2
10h20-10h40
Flore, M2
10h40-11h00
Juanma, M2
11h00-11h20
11h20-11h40
Anita, PhD
Breakage and repair of the nuclear envelope in confined cell migration
Preparation and characterization of dense fibrillar collagen matrices
mimics connective tissues.
Enabling localization microscopy in thermoacidophiles archaea
Break
Force Measurement at B cell Immune synapse
11h40-12h20
Dr. E. Farge,
Institut Curie
Mechanics and Genetics of embryonic development
12h20-13h40
Lunch
13h40-14h00
Ophélie, PhD
14h00-14h20
Sushmitha, PhD
14h20-14h40
Mariela, M2
Characterization of cortical folding during early development in ferret by
MRI
Highly sensitive voltage-sensitive dyes emitting in the near infrared
(NIR-VSD)
Quantification of prostate cancer perfusion using dynamic contrastenhanced sonography
14h40-15h20
Dr. JM Correas,
Langevin Institute,
Prostate cancer diagnosis: Current limitations
Necker Hospital
15h20-15h40
15h40-16h00
Adrien, M2
16h00-16h20
Xavier, M2
16h20-16h40
Yara, M2
Break
Innovative orthopedic cast for the treatment of bone fractures
Stochasticity and double-strand DNA repair.
Neuronal circuits underlying locomotion in the spinal cord of the
Zebrafish Larva model
16h40-17h20
Dr. Chavez & Dr.
De Vico Fallani,
ICM
17h20-17h40
17h40-18h00
Mélanie, PhD
18h00-18h20
Perrine, PhD
18h20-18h40
Gerardo, PhD
Models and methods for images and signals of the brain
Break
Gradients in the mechanical properties of auditory hair cells.
Heterogeneity of nucleation: deciphering social rules of epithelia
stability using optically patterned EMT activation
Title to be announced
18h40-19h00
Fabien, PhD
Title to be announced
Ophélie (PhD Pasteur Institut, R. Toro)
Characterization of cortical folding during early development in ferret by MRI
There is a striking, largely unexplained, relationship between neocortical organisation and
cortical folding. Indeed, many neurologic and psychiatric disorders (autism, for example) are
related to changes in cortical folding. The ferret is an ideal animal model for studying cortical
folding. Many processes likely to be involved in cortical folding, such as cytoarchitectonic and
connective development, occur during the first few weeks of postnatal life. We aim at
characterizing the development of the neocortex organisation and folding anatomy in ferrets,
based on structural MRI data. We found that during the first two weeks the ferret brain goes
from ~250mm3 (the size of a small mouse brain) to more than 1400mm3, a 5-fold change.
During the same period the cortical surface area goes from 300mm2 to almost 1100 mm2, a 3.7
fold change. We also observed a steady increase in folding length, meaning that folds are not
only becoming deeper (which would not change folding length), but also longer. Surprisingly
the folding pattern appears instantly before the post-natal day 8, then the folds go deeper and
longer but the pattern does not change.
Henry (M2 - Curie, Piel)
Breakage and repair of the nuclear envelope in confined cell migration
Several cell types must migrate long distances to fulfill their function, notably dendritic cells
which migrate from the peripheral tissues to lymph nodes where they present antigens to other
immune cells. However, along the path of their migration, these cells must crawl around and
between many obstacles (cells and extracellular matrix), and must transverse throw narrow
pores to enter lymphatic vessels. Similarly, cancer cells that metastasize must enter the blood
stream and enter foreign tissues which may present occurrences of highly confined
environments. Here, we observe migration of cells both in ex vivo and in vitro micro-channel
systems and observe rupture of the nuclear envelope (NE). We have systematically quantified
nuclear rupture during migration of different cell types with varying dimensions of constrictions
in micro-channels. We also discovered that same protein machinery used for endosome sorting
and repair of plasma membrane (ESCRTIII) also is recruited to sites of nuclear breaching,
presumably for the repair of the NE. This process of repair seems required to allow cell
survival. The consequences from this nuclear rupture is to be further explored but already we
have observed breakage of DNA during confined migration in live imaging.
Flore SALVIAT (M2, Chimie de la Matière Condensée de Paris, Collège de France,
N.Nassif)
Preparation and characterization of dense fibrillar collagen matrices mimics connective tissues.
The objective of my research is to design and develop innovative materials with optical and
mechanical properties dedicated to tissue engineering. Our research shows that collagen type I
has particular organizations in a concentration range that was not previously described.
The quality of the reconstituted fibrils and fibril 3D network is evaluated in transmission
electron microscopy, scanning electron microscopy and polarized light-microscopy. The
transmittance of the matrices is measured by the ellipsometer.
In the biomedical context, we wish to overcome the deficiencies of the human body by
proposing "custom-made" of biomimetic matrices of connective tissues structured at different
scales and for a less costly development.
Anita KUMARI (PhD, Institute Curie,Paolo PIEROBON and Ana-Maria LENNON)
Force Measurement at B cell Immune synapse
B lymphocytes are the cells that produce antibodies and are therefore essential effectors of
adaptive immunity. In vivo, their activation is mostly triggered by the engagement of their B cell
receptor (BCR) with antigens exposed at the surface of neighboring antigen presenting cells.
This leads to the formation of the immune synapse that coordinates the signaling and
cytoskeleton rearrangement events that are essential for B cells to extract and process the
antigen. To acquire antigen from the surface of an antigen presenting cell, B cells first spread
around it and then contract, however the contraction phase has never been characterized. Our
aim is to measure forces at immune synapse by traction force microscopy using antigen coated
polyacrylamide (PAA) gels with embedded fluorescent beads. We characterized that these
forces showing a remarkable periodicity and that are triggered specifically by BCR engagement.
B cells from Myosin II KO mice show a decrease in contractile energy; therefore we deduce that
these contractile forces could be Myosin II dependent. These results open interesting
perspectives on the role of mechanics in the acquisition of specific antigen.
Mélanie TOBIN (PhD, Institut Curie, P. Martin)
Gradients in the mechanical properties of auditory hair cells.
Hearing is based upon the capacity of hair cells in the inner ear to detect and amplify mechanical
vibrations evoked by a sound stimulus. Hair cells behave as cellular microphones capable of
mobilizing biochemical energy in order to actively resonate with sound-evoked mechanical
vibrations. To understand speech and enjoy music, our auditory system relies on acute frequency
discrimination over a range of frequencies covering three orders of magnitude, from 20 Hz to 20
kHz in humans. In the auditory organ, the cochlea, the mechano-sensitive hair bundle
that protrudes from the apical surface of each sensory cell shows a morphology that varies
regularly along the cochlear axis. Correspondingly, each cell is maximally sensitive at a
characteristic sound frequency that decreases exponentially from the base to the apex of the
cochlea. This observation suggests that the mechanical properties of the hair bundle control the
frequency selectivity of each hair cell. The mechanism underlying this regulation, however,
remains an unresolved question in auditory physiology. We propose to conduct a systematic
study of passive (stifness, friction) and active (motility, tension) components of hair-bundle
mechanics along the cochlear axis.
Yara ALCHEIKH (M2, ICM, C. Wyart)
Understanding neuronal circuits underlying locomotion in the spinal cord of the Zebrafish Larva
model
How circuits of diverse neurons with dynamic activity give rise to a complex motor output is a
key question today. Locomotion relies on the spinal cord neuronal networks termed the central
pattern generators (CPGs). CPGs are comprised of segmentally-organized motor neurons as well
as excitatory and inhibitory interneurons able to generate rhythmic movements in the absence of
the descending input from the brain and local sensory feedback. The precise function of each
cell type and how specific cells are recruited is not fully understood. However studies in the
zebrafish larva have provided important insights into these questions because of its genetic and
optical accessibility. The larva is simple model system of 100 000 spinal neurons, transparent,
and we are able to recognize each type of cell and observe locomotion at only 5 days old. I will
discuss about the methods we use in the lab to modulate neurons in order to understand the role
of specified classes of neurons from measurable behavioural observations.
Perrine MIQUEL (PhD, Institut Curie, François Amblard)
Heterogeneity of nucleation: deciphering social rules of epithelia stability using optically
patterned EMT activation
Cancer is caused by the loss of homeostatic regulation of cells to extracellular signals. The
behavior of an individual cell results from both the complex interplay of external influences
received from its neighbors, and its internal state (genome, transcriptome, proteome) giving rise
to the idea of community control. Phenotypic homeostasis within a tissue is most likely a
consequence of the interplay between these two congruent sources of influence. However, when
the internal state of a given cell is destabilized, the faith of this cell is subjected to the
competition between normalizing effects impinged onto the affected cell by the community, and
the ease of propagation of this destabilization from the given cell to the neighbors. Our project
aims at quantitatively assessing the extent to which the epithelial phenotype relies on internal
determinants vs. social control. Using the epithelial mesenchymal transition (EMT) as a
perturbation, our goal is to study how the expected transition nucleates or not when its cause is
locally distributed among cells. Because a cell can only be epithelial if it has neighbors, EMT
can simply not be a single cell process. We have established a model where EMT can be
optically induced using photoactivatable cyclophen. We are using MDCK cells expressing a
stable fusion protein made up of Snail1, a key inducer of EMT, and the ERT2 estrogen receptor
moiety that activates Snail1 upon cyclophen binding. Using holographic control, EMT will be
induced according to various geometrical patterns to look for social rules of nucleation. Results
show that cyclophen induces EMT as revealed by qPCR and immunofluorescence, and
conditions for cyclophen photoactivation are being established. From this, we hope to
understand the extent of cell interdependence vs. cell autonomy, and measure the ability of an
epithelium as a cell community to collectively resist to the onset of carcinogenesis.
Juan Manuel GARCIA ARCOS (MRC LMCB, University College London, Buzz BAUM)
Enabling localization microscopy in thermoacidophiles archaea
Thermoacidophiles often appear at very basal parts of phylogenetic trees at very short distances
from the main branch, which suggests that in general they might have evolved very little and that
they are the closest living forms to the LUCA (last universal common ancestor). One of the main
model organisms of Archaeal thermoacidophiles is the Crenarcheaota Sulfolobus acidocaldarius,
which grows optimally at 76C and pH 3.2. Molecular biology is possible in these organisms, are
there are known transformation vectors and methods, but imaging becomes a very hard task
when the growth conditions are replicated. Moreover, the size of S. acidocaldarius requires super
resolution microscopy to image subcellular structures. In this work we propose to study the
feasibility of both fixed and live cell imaging of S. acidocaldarius with different super resolution
techniques such as PALM, n-STORM or SIM. The optimization of staining and imaging
protocol described during this work open a new field of exploration for evolutionary biology.
Sushmitha RAJA (PhD, Centre de Psychiatrie et Neuroscience, Alan URBAN)
Highly sensitive voltage-sensitive dyes emitting in the near infrared (NIR-VSD)
Whether for basic research or drug discovery, precise measurement of voltage changes at the
cell membrane is essential for understanding function, pathology, and potential therapeutic
effects in electrically active cells.
Voltage-sensitive fluorescent dyes provide bright, membrane-localized signal but they suffer
from poor signal to noise, secondary side effects and difficulties to achieve single-cell
resolution. Moreover, capturing transient, millisecond events such as action potentials requires
an indicator that responds very quickly to voltage changes.
We developed novel near-infrared (NIR) VSDs based on 3 different heterocyclic fluorophores
that offers advantages of high sensitivity, deep photon penetration, reduced light scattering and
minimal autofluorescence from living tissues, rendering them valuable for noninvasive in vivo
imaging of cardiac and neuronal activity.
The properties of these dyes were first assessed in vitro using patch clamp and high-speed
fluorescence imaging (1 KHz). We evaluated voltage sensitivities of these NIR-VSDs in both
HEK cells and primary neuronal cultures and demonstrated that sensitivity curves for all dyes
are well fit by a log-normal function with nonlinearity at the spectral edge. Moreover, we
observed that NIR-VSD are fast and sensitive enough to resolve single action potentials without
averaging.
Then, NIR-VSDs were used ex vivo in Langendorff perfused rat hearts. Spectral properties were
determined in these conditions and we observed optimal peak emission wavelengths for all dyes
were found between 700 nm and 750 nm. These results are in good agreement with values
observed in vitro.
Finally, SNR and kinetics were measured in epifluorescence imaging experiments. We showed
that NIR-VSDs have sensitivity ranging from 6.5 % to 20 %, compared to 5.5% for the
reference di-4-ANEPPS dye. Large Stoke shift up to 250 nm were observed. Moreover, NIRVSDs show reduced internalization and photo-bleaching compared to di-4-ANEPPS as
confirmed by the enhanced stability of the signal during optical recordings.
These properties suggest that NIR-VSD voltage sensitivity could extend the capabilities of
modernelectrophysiological techniques for probing brain and heart function, and allow for the
investigation of previously inaccessible research studies.
Keywords: voltage sensitive dye, near-infrared, in vivo imaging; fluorescent dyes.
Xavier ZAOUI (M2, University of Edinburgh, M. El Karoui)
Stochasticity and double-strand DNA repair.
E. coli cells possess a complex reparation system dealing with double-strand DNA breaks, hence
avoiding information loss and/or cellular death. This system is formed of proteins present in such
a small amount that any variation in their concentration could possibly lead to disastrous
consequences. Our question is ‘how sensitive this system is to stochasticity’. In other words, can
random molecular processes slightly disturb the production of such necessary proteins? And
then, what about the consequences regarding induced DNA damage?
Using Single-Molecule Microscopy and DNA-damaging genetic constructions, we explore the
potential part of randomness inherent to a major feature of cellular fate.
Mariela SKENDI ( Master 2, Langevin Institute)
Quantification of prostate cancer perfusion using dynamic contrast-enhanced sonography
Prostate Cancer (PCa) is the most commonly diagnosed malignancy in men with an estimated
incidence 340,000 cases in 2012 and a mortality of 71,000 men every year in Europe.The
incidence has doubled in the last 15 years, mainly due to increased use of serum Prostate
Specific Antigen (PSA) testing. An elevated PSA or an abnormal Digital Rectal Examination
(DRE) triggers TransRectal UltraSound (TRUS) guided prostate systematic biopsies. TRUS has
limited sensitivity and specificity ranging from 40% to 50% for PCa detection, explaining why it
is not used for cancer diagnosis but mainly to direct biopsies into the gland.
The purpose of this study will be to provide quantitative perfusion information about prostate
cancer, normal peripheral and transitional prostate tissue using TRUS-CE imaging, in
correlation to prostate biopsies. Data acquired prospectively was collected in order to improve
prostate cancer diagnosis and management. For each patient, TRUS-CE was performed after
intravenous administration of SonoVue® (BR1, Bracco SA, Milano, Italy). The transducer was
maintained still at the most suspicious level in order to acquire a 30 to 40 sec cineloop during
the transit of the microbubbles. The time-intensity curves acquired on this single plane are
processed using perfusion parametric software, such as prototype VueBox® (Bracco, Geneva,
Switzerland).
This work describes the advances of ultrasound contrast agents and signal processing and
techniques for perfusion estimation using a bolus model based upon time-intensity curves.
Perfusion parameters including peak signal intensity, area under the curve, time to peak,
regional mean transit time, perfusion index, wash-in and wash-out slopes were correlated to the
pathology results of targeted prostate biopsies including the Gleason score, a cancer
aggressiveness marker. This work is the preliminary step to further developments for PCa
detection, including biomarker functional imaging as aggressiveness is correlated to vascularity
and perfusion. Further steps, including 3D/4D CE-TRUS and Shear Wave Elastography will be
discussed.
Danaé DAVID (Master 2, ESPCI)
Ultra-rapid imaging of a drosophila memory center : Analysis of the response to an olfactory
stimulation
Is it possible to link a cellular activation pattern to memory formation?
Drosophila melanogaster is a commonly used model for the investigation of memory formation,
it grows rapidly and a huge library of genetic tools exists for this model. In adult flies, memory
is usually studied with behavioral tests in different conditions for different genetic background.
The project I worked on aim at using imaging technics and informatics tool together with
behavioral tests to answer the following question: Is it possible to link a cellular activation
pattern to memory formation?
In drosophila, memory formation is processed in the same center that integer olfactory sensing
and other sensitive signals. As we use olfactory stimulation to assess memory in the behavioral
tests, the first step of this project was to measure, the response of the cells to an olfactory
stimulation.
During my three-month internship I studied this olfactory response, using a special dissection
method, imaging technic and a hand-made odor-presentation system. In a second part, the
images obtained have to be submitted to further informatics treatment to extract the relevant
parameters for a given response (exact number of cells in the observed area, their coordinates
and intensity changes over time).
Adrien GLOMAUD (Master 2, Openlab )
Innovative orthopedic cast for the treatment of bone fractures
Orthopedic cast (or plaster cast) is a medical device that is commonly used in the treatment of
bone fractures. It consists in plaster bandages assembled to form a shell encasing and stabilizing
the broken limb until the bone is completely healed. However, a plaster cast has many defaults :
it is unbreathy, heavy, prevents the wearer from washing his broken limb, and needs to be set up
by a skilled doctor. Recent works have thus tried to create 3D printed, custom-made, and
alveolated casts which are lighter and washable. However, they are costly, patient-specific, take
time to craft, need cutting-edge technologies (3D printers, 3D scans, modeling softwares) and
trained professionals for each patients. Here, we will show that we can overcome these problems
by creating a universal, alveolated, and self-hardening plaster cast by using a net-structured
polyurethane cast and perform strength tests. Our results will demonstrate that we can create a
universal, innovative, breathy, easy-to-make, easy-to-put, and washable orthopedic cast that
improves both patient comfort and medical support.