CENTURI multi-engineering platform
Ongoing projects

BIOINFORMATICS

#B1| IBDM | In silico search of novel components of vertebrate deuterosomes

Applicant: Laurent Kodjabachian & Camille Boutin

Institute: IBDM

Engineer: Thomas Vannier


In eukaryotes, universal cellular functions are compartmentalized in organelles, such as transcription in the nucleus, energy production in the mitochondrion or glycosylation in the Golgi apparatus. However, specialized organelles may also achieve unique tasks in specific cell types. This is the case of the deuterosome that serves as a platform for massive centriole synthesis in multiciliated cells (MCCs). MCCs harbour myriads of motile cilia, which rest on modified centrioles, and beat synchronously to generate polarized hydrodynamic forces (Boutin & Kodjabachian, Curr Opin Genet Dev, 2019). MCCs are present throughout metazoan evolution and serve functions ranging from locomotion of marine larvae and flatworms, to brain homeostasis, mucociliary clearance of pathogens and transportation of gametes in mammals. The deuterosome, however, is found only in the vertebrate phylum, which begs a fascinating question: What did it take for this organelle to emerge through evolution ? In other words, how many new proteins were necessary and how do they interact with more ancestral components to build up the deuterosome ? Recent studies suggested that deuterosomes are built from ancestral cell-cycle proteins combined to MCC-specific cell-cycle paralogs that evolved only in the vertebrate phylum (Zhao et al., Nat Cell Biol, 2013; Al Jord et al, Science 2017; Revinski et al, Nat Commun, 2018). A major goal of our research is to reveal how many such genes were shaped by evolution to make up the vertebrate deuterosome. To this aim, we wish to conduct a bio-informatic screen to identify potential deuterosome components extracted from available MCC transcriptomes. The rationale will consist in focusing on genes expressed in human, mouse and Xenopus MCCs, which are absent in non-vertebrate genomes.

Keywords: Single-cell RNA sequensing

#B2| INMED | Long term effect of oxytocin treatment in early life on transcript and protein expressions in a mouse model with « autism-like » disorder

Applicant: Françoise Muscatelli

Institute: INMED

Engineer: Thomas Vannier


Oxytocin (OT) is a social neurohormone associated with autism spectrum disorder (ASD). MAGEL2 is a gene mutated in pathologies associated with ASD. Previously we have shown that social behavioral alterations in Magel2 tm1.1Mus KO mice are rescued by an OT-treatment in the first week of life. Such positive effect of an OT treatment has been reported in different genetic rodent models of ASD. However, we do not know how an OT treatment in the first week of life can have a long-term effect. Here using transcriptomics and proteomics analyses of a critical brain structure we aim to reveal the differences of transcripts and protein expression between the mutant and wild-type neonates and adult mice having been treated or not with oxytocin after birth.

Keywords: Proteomics, RNA-seq, autism spectrum disorder

#B3| CIML | Mechanisms involved in VNN1-dependent control of sarcomas

Applicant: Philippe Naquet

Institute: CIML

Engineer: Thomas Vannier


We have identified and demonstrated the role of the VNN pantetheinase in the control of the extracellular degradation pathway of Coenzyme A. VNN1 is a regulator of metabolic adaptation in tissues exposed to damage. In mouse, VNN1 behaves as a tumor suppressor in part through its ability to inhibit the Warburg effect and restore mitochondrial activity. However, additional tumor-intrinsic or extrinsic mechanisms are probably involved. The goal of this project is to obtain bioinformatics help to screen human and mouse tumor databases, ii) identify VNN1-dependent signatures involved in tumor control and iii) test whether similar mechanisms occur in other types of human tumors.

Keywords: bulk RNA-sequencing, data mining, human tumors

#B4| IBDM | Epithelial integrity controls the differentiation of hiPSCs into Primitive Streak by regulating TGF-b receptor accessibility

Applicant: Rosanna Dono & Diane Rattier

Institute: IBDM

Engineer: Thomas Vannier


Summary

Human induced pluripotent stem cells (hiPSCs), considered as in vitro equivalent of the human epiblast, are a powerful model to study mechanisms underlying embryonic germ layer differentiation. We explore the impact of physical cues on hiPSC fate acquisition. By taking advantage of hiPSCs with reduced levels of the morphogen regulator, GLYPICAN4 (GPC4; line namely shGPC4) we have highlighted that differentiation of hiPSCs into Primitive streak (PS) like cells relies on an interplay between hiPSC morphology and perception of morphogens. Briefly, the shGPC4 line show altered ,morphological organization of the hiPSC epithelial sheet as highlighted by the presences of cell patches with abnormal tight junction formation and apical constriction processes. Through our cellular and molecular analyses, we have showed that disruption of the hiPSC epithelia integrity enhances competence of these cells to activate TGF-b signaling and to differentiate into PS. At present, we aim to understand whether epithelial integrity directly regulates gene expression in hiPSCs through RNA-sequencing analyses.

Requested task

In order to understand whether the modification of the epithelium integrity, observed in shGPC4, primes hiPSCs for PS differentiation, we performed a bulk RNA-sequencing analysis of WT, shCTRL and shGPC4 hiPSCs (in triplicate each) at the undifferentiated stage. For this project, we need to analyze the raw data from this sequencing experiment. To this aim, the requested tasks are: 1) Clean the raw data, 2) Map the cleaned data on the human genome to obtain a list of expressed genes in each sample, 3) Compare the different samples to obtain a list of differentially expressed genes: WT/shCTRL, WT/shGPC4 and shCTRL/shGPC4.

Keywords: bulk RNA-sequencing, hiPSCs

DATABASE MANAGEMENT AND DATA CURATION

#DM1| CENTURI | Microscopy DataBase for the CENTURI community

Applicant: CENTURI community

Institute: CENTURI multi-engineering platform

Engineer: Guillaume Gay


Summary

Data preservation and sharing, beyond publication, is a common issue for the labs of the CENTURI
community.
In this project, we will setup and compare two software solution adapted for the storage and
publication of microscopy data,
- OMERO : https://www.openmicroscopy.org/omero/institution/
- CYTOMINE https://cytomine.be/

Requested task

- Setup both database servers
- Retrieve data from a former omero installation at IBDM
- Retrieve example data from the various labs of CENTURI
- Redact a comparison between the two solutions
- Plan and dimensioning of a future campus-wide platform

#DM2| CENTURI | Automated metrology of instrument quality

Applicant: CENTURI community

Institute: CENTURI multi-engineering platform

Engineer: Guillaume Gay


Summary

Assessing microscope quality over time ideally requires a regular evaluation of the optical quality through a dedicated characterization protocols, for example PSF characterization with fluorescent beads, or illumination homogeneity for wide field microscope. Unfortunately, such protocols are time consuming and not adapted to current microscopy platform, where a single engineer or technician must maintain many microscopes.

In this project, we explore the direct use of the images produced by the platform users to automatically assess instrument quality over time.

Requested Task

To achieve this, we implement various blind measure algorithm and setup a workflow that :

1. systematically apply those algorithms to all the images in a collection
2. display the measure for each instrument over time on a dashboard

The project should be mainly database agnostic (meaning that it should work with minimal effort for e.g. omero and cytomine).

#DM3| LAI | Force Data Classifier

Applicant: Pierre Henri Puech

Institute: LAI

Engineer: Guillaume Gay


Code and GUI to analyze and classify Atomic Force Microscopy data

Requested task

Shaping up of the code base, debugging, setting up of Continuous Integration

IMAGE DATA PROCESSING

#IDP1| CINaM |Microcirculation of Red Blood Cells in biomimetic splenic slits

Applicant: Alexis Moreau

Institute: CINaM

Engineer: Benoit Dehapiot


The scientific project is to understand the mechanisms of red blood cell passage through the interendothelial slits of the spleen. Red blood cells pass a physical fitness test through these submicron wide slits and undergo extreme deformations. The objective is to answer the following questions. What are the rheological properties of red blood cells required to pass this test and what are the deformation mechanisms? Are mechanotransduction mechanisms involved, in particular by cascading activation of ion channels (including PIEZO1) leading to a reduction in red blood cell volume? We have developed an in-vitro micro-nanofluidic system that reconstructs slits of physiological dimensions and we are conducting quantitative in-vitro experiments. We observed large populations of red blood cells flowing in these devices and want to extract quantitative data.
To this end, we need to develop an image processing program to recognize the contour and position of individual red blood cells on videos

Keywords: Live segmentation and tracking

#IDP2| IBDM |Development of an automated nuclei segmentation (mouse spinal cord neurons) and a user interface for cell selection

Applicant: Lucie Pepino

Institute: IBDM

Engineer: Benoit Dehapiot


Pain research has been carried on male rodents for decades. Yet, several studies showed that women and men experience pain differently. The use of female subjects to understand the mechanisms explaining sex differences in pain neurobiology is of high interest. A complex network of excitatory and inhibitory interneurons allows the spinal cord to modulate pain by controlling peripheral inputs and spinal cord projection neurons outputs. Few studies investigate sexual differences in the modulation of pain at the spinal cord level.

One part of my project aims at determining whether males and females activate the same spinal neural network following a painful stimulus. I used a double in situ hybridization strategy, on male and female spinal cord sections at 5, 15 and 45 minutes after painful stimulation (n=3 per sex per time-point). I used cFos probe as a read-out for neuronal activity, in combination with specific probes for 6 spinal interneuron subsets identified as responsive to a specific painful stimulation. To this aim, we wish to develop an automatized pipeline allowing: 1) cell nuclei identification 2) positive cells selection 3) positive cells number and fluorescence intensity 4) positive cells localization within the spinal cord 5) males versus females data statistic analysis.

Keywords : Nuclei segmentation

#IDP3| IBDM |Segmentation of nuclei and analysis of expression markers

Applicant: Camille Boutin

Institute: IBDM

Engineer: Qinzong Tseng


To study different aspects of multiciliated cells biology, we apply drug on Xenopus embryos. In these kind of experiments we analyze the fate specification of epidermal cells by double fluorescent in-situ hybridization. I would like to develop an automated pipeline to analyze my confocal acquisitions that would recognize all nuclei of the epidermis and analyze for each nuclei their status (positive/negative) for different cell type markers.

Keywords : Nuclei segmentation

#IDP4| IBDM |Investigating the role of mechanical tension during sarcomere self-organisation in human iPSC-derived muscle fibers

Applicant: Qiyan Mao

Institute: IBDM

Engineer: Benoit Dehapiot


During skeletal muscle development, hundreds of micrometre-long sarcomeres assemble simultaneously into millimetre-long myofibrils. How are correct numbers of sarcomeres determined? Our previous work in Drosophila indirect flight muscle indicates tension as a mechanical compass to coordinate simultaneous sarcomere self-organisation across the entire length of the myofibril. In this study, we investigate whether the tension-driven myofibril self-organisation mechanism is fundamentally conserved in human myofibers.

We have obtained high resolution immunofluorescence images for overall cultures and individual myofibers during the first two weeks of differentiation. We aim to characterize overall myofiber differentiation by systematically quantifying fiber length, width, fusion index as well as axial coordination between different myofibers. We will further characterize individual myofibers by analyzing sarcomere periodicity. This will offer us crucial inputs to undersand whether myofibrillogenesis takes place simultaneously or sequentially in hiPSC-derived skeletal myofibers. Finally, we have successfully ablated sarcomeric TTN-GFP myofibrils with a femtosecond pulsed IR laser (in collaboration with the Lenne lab). Our preliminary results show clear recoils of myofibril free ends, demonstrating presence of tension. We wish to quantify the recoil of myofibrils post ablation by kymograph analysis.

Keywords : Sarcomere spatial frequency analysis

#IDP5| CIML |Neurons dictate LN development

Applicant: Serge van de Pavert

Institute: CIML

Engineer: Qinzong Tseng


Lymph nodes are the cross roads of our immune system, connecting the innate and adaptive immunity. They orchestrate effective responses to pathogens and host peripheral tolerance control. Secondary lymphoid organs form during embryonic development in a specific period on specific locations. In this project, we study the role of the neurons in the initiation of embryonic LN formation.

Keywords : LTi, ILC, neuron, lymph node, embryo

#IDP6| IBDM |Identification and quantification of pulsed contractions in a moving invaginating tissue (Drosophila endoderm)

Applicant: Claudio Collinet

Institute: IBDM

Engineer: Benoit Dehapiot


Project summary :
Pulsed contractions drive many cell and tissue shape changes during animal morphogenesis. In the Drosophila posterior endoderm a polarized cell- and tissue-level wave of Rho1/MyoII activation and cell invagination drives the invagination and its associated anterior movement. MyoII activation in each cell occurs with a characteristic sequence. First, cells spread onto the vitelline membrane and MyoII speckles appear at low levels apically (pre-activation). Then, discrete steps of MyoII activation at high levels occur from the posterior to anterior of cells as they detach from the vitelline membrane.

Requested task : 
In order to characterize quantitatively this process we wish to develop an image analysis framework to semi-automatically detect MyoII steps of activation in segmented cells and quantitate their features (intensity, duration, association with actin cortex deformation, position in the cell, etc.). The analysis framework will have to interface with another framework developed in the lab to quantify local MyoII rates of change, cortex advection and MyoII activation reaction kinetics.

MECHATRONICS

#M1| CIML |Focus drift correction

Applicant: Mathieu Fallet

Institute: CIML

Engineer: Mathias Lechelon


Summary

Intravital organ microscopy allows visualization of B cells in the lung after viral infection (Mauro Gaya team). This phenomenon is complex both in terms of locating the area to be imaged and controlling the focus, and requires recordings lasting more than an hour. As a result, the image must be stabilized throughout the recording in order to be able to visualize the movements of the cells over fairly long periods of time. Commercial microscopes (here a Leica SP5) are not open about the possibility of external focus control; autofocus is sometimes available but is not effective for this project. We would like to compensate for the Z-drift due to the crushing of the sample over time by controlling the microscope's remote control knob with a motor. The speed should be calculated beforehand and easily changed during the experiment, and a stop should be set to prevent the objective from being crushed on the sample. An easily accessible STOP button would also be very useful in case of problems.

Requested task

The idea is to build a motor that will fit the Z-axis knurl of the two-photon microscope in order to change the focus constantly to correct the drift, due to the crushing of the sample (that will have to be calculated beforehand). This will not prevent the use of the joystick. A stop should be set so the objective does not damage the specimen, depending on the working distance of the objective used (here a 25x Leica), and an easy to use STOP button could be placed next to the computer.

#M2| CIML |Automated image capture

Applicant: Jérôme Belougne

Institute: CIML

Engineer: Mathias Lechelon


Summary

Optimize the automation of the image capture already present ( DMS300 / Workstation Tecan gemini).
The aim of the optimization will be to replace the currently used automaton with an X-Y automated platform system.
Create a plexiglass enclosure to control the environment (humidity, temperature) around the camera and the X-Y platform.
Optimize the imaging instrument, to obtain higher quality images.

#M3| CIML | Study of T cell activation with a new class of photocontrolable agonists

Applicant: Rémi Lasserre

Institute: CIML

Engineer: Mathias Lechelon


Summary

In vivo, T cell activation is initiated by sequential and short-lived interactions with Antigen Presenting Cells (APCs). Different dynamics of T cell/APCs interactions (duration, frequency) have been associated to different activation outcomes (Immunity vs. tolerance). However, due to technical limitations, the causal link between signal dynamics and activation outcome is not formally demonstrated yet.
We recently developed an efficient recombinant optogenetic system to reversibly trigger T cell Receptor signaling with light. Our goal is now to generate a versatile illumination toolbox to provide specific stimulation patterns to T cells and analyse how they influence their activation outcome.

Requested task

We would like that the Centuri mechatronician help us for the design and the realization of an illumination tool box able to generate specific temporal pattern of illumination to T cells in cell culture plates (24 well plates for exemple). This box should have the capacity to provide an independent control for each well of :
- The light power
- The temporal pattern of illumination
- The light wavelength by using 2 type of LEDs (660nm for light induced TCR aggregation, and 730 nm for the reversion of this process).

#M4| Fresnel |Rackmount integration of multi-photon endoscope

Applicant: Victor Tsvirkun

Institute: Fresnel

Engineer: Mathias Lechelon


Summary

Creating a demonstrator prototype for using multimodal flexible imaging (FIP) probe, we have to design, assemble and interconnect different modules (optics, electronics, etc.) within the rackmount system. Such a design would allow for a compact system while keeping the modularity and being able to integrate into different existing experimental set-ups or to operate in a completely standalone mode.

Requested task

We outlined several tasks after a briefing with our associated CENTURI engineer, including both the design of the internal layout of the abovementioned rackmount modules and fabrication of the parts (employing 3D printing and CNC milling) required for the final assembly of all the components inside the modules. An additional effort would be allocated to prototyping the working surface situated at the top of the rackmount system, first adapting an existing breadboard and next designing and milling a dedicated fully-customized piece in-house.

OPTICS AND BIOPHOTONICS

#O1| Fresnel |Flexible multimodal Imaging Probe

Applicant: Hervé Rigneault

Institute: Institut Fresnel

Engineer: Sophie Brustlein


This Tech Transfer project deals with the development of a flexible probe that can be handheld or inserted into a small animal and that performs multimodal imaging (1P and 2P and 3P fluorescence, fluorescence lifetime imaging, second harmonic generation (SHG), third harmonic generation(THG) and coherent anti-Stokes Raman scattering (CARS). The proposed project aims at developing an engineer grade version of the multimodal flexible imaging (FIP) probe that has been in development at the Fresnel institute over the last 5 years. The current development has reached TRL4. Most of the technical steps have been demonstrated and validated but the full device remains a demonstrator that cannot be exported in application labs and imaging platforms, neither it can be operated easily with a user friendly interface. We have identified several mechanical, optical, electronic and computer programming steps that need to be addressed to bring the FIP technology to the level of a useable device for the CENTURI community.

Keywords: optical fibered probe, 1P 2P 3P fluorescence, Ca2+ imaging, nonlinear label free imaging

#O2| CINaM |Optical tweezers setup

Applicant: Emmanuèle Helfer

Institute: CINaM

Engineer: Sophie Brustlein


The project requires the help of an engineer in optics for finalizing the implementation of an optical tweezers experiment. The setup consists in focusing an infrared (IR) laser in the sample via the microscope objective, the laser beam will be deviated by acousto-optic deflectors (AODs) at the back focal plane of the objective, leading to a translation of the optical trap in the focal plane. AODs allow fast switch between several positions thus simultaneous trapping of multiple objects.
The experiment will be used for several projects in the group: 1) to apply forces on single cells and nuclei and measure their mechanical properties, in the frame of premature aging diseases (nuclei with altered mechanics); 2) to pull membrane tubules from artificial vesicles and determine conditions required for tube fission. The setup can be used by CENTURI colleagues for other projects, for example to investigate flows generated by coordinated cilia cells.

Keywords: optical tweezers, microscopy, acousto-optic deflectors, mechanical stress/strain

#O3 | CIML |Accurate 3D focus-locking correction for single-molecule resolution

Applicant: Sebastien Mailfert

Institute: CIML

Engineer: Sophie Brustlein


The super-resolution technique requires accurate correction for the mechanical motion of the sample and setup during acquisition. We aim at implementing a controller to actively stabilize the system during SMLM acquisition and consequently to reduce the drift across all three dimensions.

Requested Task

The engineer will help us to develop the optical part of the system based on an active three-dimensional real-time drift correction in collaboration with Sebastien Mailfert who will develop the optoelectronic part that will control the system.

#O4 | CIML |Description of T cell plasma membrane dynamic using advanced solvatometric lipid probes

Applicant: Tom Borianne

Institute: CIML

Engineer: Sophie Brustlein


Summary

Upon interaction with the peptide-Major Histocompatibility Complex (pMHC), the TCR rapidly shifts from an apparent homogeneous distribution to a dense cluster at the junction between a T cell and an antigen-presenting cell, termed the immunological synapse. It has been speculated that a remodeling of the plasma membrane organization mediates the signal transduction across the bilayer. Using a variety of solvatometric lipid probes, we would like to describe potential changes in the plasma membrane order inside TCR clusters and test what is the function of these changes in signal transduction.

Requested task

Our chemist collaborator Andrey Klymchenko in Strasbourg is sending us new probes that requiere 2-photon excitation. However, this probe can only be used on live cells and the current 2-photon microscope in the CIML is not adapted for such experiments (because it's not an inversed microscope so the objectives do no fit into small imaing chambers). Instead, we would like to use the 2-photon excitation of the ABLA microscope, currently non-fonctionnal.

SOFTWARE DEVELOPMENT

#S1| Fresnel |4POLAR Imaging Method Software

Applicant: Sophie Brasselet

Institute: Institut Fresnel

Engineer: Masoud A. Sharbaf


The 4POLAR is a fluorescence microscopy imaging method that determines the organization of  molecules in a 2D plane of sample. For this to happen, the fluorescence emission from the sample is projected along four different linear polarization angles of light (0, 45, 90 and 135 degree polarization) and an image is recorded for each direction. The orientation at each particular pixel of the image is then reported with three angles that indicate planar orientation, wobbling and off plane orientation. The goal of this project is to develop the 4POLAR method as a plugin for the ImageJ (Fiji) platform.

Keywords: 4POLAR imaging, ImageJ, Fiji

INTERDISCIPLINARITY PROJECT

#IP1| IMM |Photosensitive magnetoreception

Applicant: Long-Fei Wu

Institute: LCB

Engineer: Sophie Brustlein & Mathias Lechelon


Summary

Illumination with UV and violet light can change swimming velocity of multicellular bacteria in magnetic field. We need implement a custom-made microscope to determine the wavelength (energy) and irradiance (number of photons) require for triggering the photokinesis behavior.

Requested task

Need the help of optics and biophotonics engineer to modify the current design and construct the microscope, and mechatronics engineer to 3D print condenser and sample holders.