Doctoral Research Project Proposals
Interface Biology and…
Acetylcholine/Glutamate co-transmission in the striatal network. Anatomical and functional heterogeneity of synaptic vesicles in cholinergic interneurons (Bernard Véronique & Pietrancosta Nicolas)
Abtract: Striatal activity is regulated by cholinergic interneurons (CINs) that use acetylcholine (ACh) and glutamate (Glu) as neurotransmitters. The ACh/Glu cotransmission depends on the action of the vesicular transporters of ACh (VAChT) and Glu (VGLUT3) in axonal varicosities of CINs. Our hypothesis is that CINs varicosities contain three pools of SVs expressing either VAChT, VGLUT3 or both releasing ACh, Glu or both.
We will explore this vesicular heterogeneity and its functional implications using a multidisciplinary approach combining super resolution microscopy, pharmacological tools targeting VGLUTs and optogenetics stimulation coupled to measures of ACh/Glu release to :
- Characterize the heterogeneity of SVs in CINs axonal varicosities using high resolution microscopy to detect organelles with a resolution compatible with visualization of unique SVs (super resolution microscopy and electron microscopy)
- Develop new chemical tools to locate VGLUT with an increased spatial resolution. We will continue to develop small size fluorescent sensors to detect VGLUT3.
- Analyze the impact of various discharge profiles of CINs on ACh or Glu release coupling optogenetics to stimulate CINs and fluorescent ACh & Glu sensors to evaluate ACh & Glu release.
Dissecting glutathione signaling pathways in Chlamydomonas reinhardtii using a bioorthogonal chemo-biological strategy (Lemaire Stéphane & Vauzeilles Boris)
Abstract: Glutathione is a crucial tripeptide (γGlu-Cys-Gly or GSH) present in almost all living organisms. GSH can form a disulfide bond on protein cysteines, a redox post-translational modification favored under stress conditions and called S-glutathionylation. This dynamic modification is mainly controlled by glutaredoxins (GRXs) and constitutes a molecular switch with regulatory and signaling roles. By engineering a Chlamydomonas strain able to synthesize metabolically a clickable analog of GSH, we will be able, after Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction, to enrich efficiently and selectively proteins undergoing glutathionylation. Combined with quantitative mass spectrometry, this click-chemistry based approach will allow to profile the in vivo dynamics of S-glutathionylation. This strategy will allow us to determine the specificities of this redox modification in response to physiological stresses. Selective inactivation in this engineered strain of genes coding for the major GRXs, will allow to decipher their specific roles. Overall, this innovative chemo-biological approach will allow a better understanding of the structural determinants governing both glutathionylation and de-glutathionylation processes and will shed light on the importance and the role of glutathionylation in eukaryotes.
Novel KCC2 enhancers for epilepsy and related disorders: Molecular modeling and virtual screening for drug discovery. (Poncer Jean-Christophe & Acher Francine)
Abstract: Altered neuronal chloride regulation is a hallmark of many neurological disorders including epilepsy. Perturbed expression/function of the cation-chloride cotransporters KCC2 and NKCC1 impair GABA signalling and neuronal excitability. Whereas inhibiting NKCC1 function lacks CNS specificity, acting to promote KCC2 function is a highly promising therapeutic strategy in epilepsy and related disorders. Candidate KCC2 enhancers have been proposed based on screening of drug-like compound libraries. However, their mechanisms of action, specificity and therapeutic potential in epilepsy remain unknown. Our project aims to develop a totally novel approach based on molecular modelling and virtual screening to design new molecules with improved specificity and bioavailability. This goal will be achieved by combining advanced medical chemistry approaches (F Acher) together with cellular imaging and electrophysiological techniques (JC Poncer) in order to design and optimize specific KCC2 allosteric modulators, test their potency and specificity as well as evaluate their therapeutic potential in epilepsy.
Abstract: Cell-cell communication participates in regulating and synchronizing cellular functions. In tissues, the transport of ions and molecules from a cell to another occurs in particular through protein nanometric pores across the cells membrane. Such nanopores can be inert (simple nano-holes) or mechanosensitive with an ionic/molecular permeability that depends on the stress acting on the membrane. This project proposes a biomimetic approach of cell-cell communication in tissues. Tissues will be mimicked with 2D arrays of aqueous droplets connected by lipid membranes decorated with transmembrane proteins, inert or mechanosensitive. Mechanosensitive proteins will be synthetized directly within the droplets using cell free Transcription Translation (TxTl) reactions. For inert networks, we will probe with epifluorescence microscopy how the diffusion of Ca2+ ions depends on the network topology and the pores concentration. For mechanosensitive networks, we will probe with a rheo-microscope how controlled deformations of the network affect the transport properties. Our results will be modeled using random walks in nanoporous media, in which the opening gate probability depends on the local stress.
Development and characterization of bio-inspired scaffolds with advanced electromechanical properties for cardiac tissue engineering (Li Zhenlin & Labdi Sid)
Abstract: This PhD project aims to produce 3D bio-inspired scaffolds for cardiac tissue engineering. We showed that nanofibrous scaffold mimicking the fibrillar structure of extracellular matrix can be produced by electrospinning. Specifically, our goal is to develop electrospun scaffolds based on piezoelectric nanofibers to obtain bio-inspired materials with improved electro-mechanical properties to explore cardiomyocyte mechano-sensitivity. Indeed, the heart is a mechanically active organ able to sense and respond to its environment. Cardiac cells transduce mechanical forces into electric currents, which modulate heart activity. However, the mechanisms through which forces are converted into electric signals and vice-versa are not fully understood. Through this project, we will investigate the electromechanical forces that modulate cell behavior in the cardiac muscle. Our objective is to untangle the complex interplay between matrix electromechanical properties, cell adhesion, differentiation, and the mechanical properties of cardiomyocytes. First, we will develop and deeply characterize a new cell-matrix platform that mimic the in vivo environment; second, we will determine the optimal culture conditions to promote organotypic assembly of seeded cardiomyocytes.
Abstract : The role of physical cues in shaping the development of multicellular eukaryotic organisms is now firmly established. Even though it is now also appreciated that bacteria mostly live within dense multicellular communities called biofilms, the understanding of the role of physical cues within these communities is still in its infancy. This doctoral project will aim at studying the role of physical forces in the early biofilm formation of species of the Neisseria genus. Focusing on a pair of species, one pathogen and one commensal, the PhD candidate will combine molecular biology, genetics, biophysics and microscopy to tackle the role of physical cues in the physiology of these members of the human microbiota in order to both unravel the fundamental role of mechanical cues in bacterial physiology and understand how to use these cues to control the spread of these bacteria.
Studying extracellular matrix assembly in a multi-species model biofilm using micro- Raman spectroscopy (Henry Nelly & Arbosa de Aguiar Hilton)
Abstract : Our project aims at studying the assembly of the polymer extracellular matrix of a multispecies bacterial biofilm. This is a central organ of these communities, widespread in human and natural organizations. Our objective is to perform an in situ chemical and functional spatiotemporal imaging of the matrix. To this purpose, we will combine new advances in compressive Raman microspectroscopy which will provide a high speed, label-free, molecular signature of the material — and a microrheology mapping of the matrix which will provide the functional imaging. The experiments will be performed in a recently developed laboratory model of multi-species biofilm growing in a millifluidic device. This project is expected to bring about significant instrumental advances in Raman imaging and provide a unique vision of the mechanisms underpinning the assembly of the biological living material formed by bacteria.
Dissecting how excitable ependymal cells participate to locomotor rhythms using an all-optical approach (Mangin Jean-Marie & Debregeas Georges)
Abstract: From peristaltic crawling in earthworms to bipedal walking in humans, animal locomotion relies on repeated sequences of muscle contractions triggered by a rhythmic central pattern generator (CPG) located in the ventromedial spinal cord. Despite decades of research, the identity of the spinal neurons constituting the CPG remains unclear. In the present project, we will investigate the original hypothesis that the spinal CPG is not exclusively made of neurons but also rely on a unique group of ventromedial ependymal cells recently found by our team to generate non-neuronal action potentials. To address this question, we will use an all-optical method allowing us to both manipulate and visualize electrical activity in ependymal cells and spinal neurons during fictive locomotion in spinal cord explants. More specifically, this project will combine transgenic mice allowing the specific expression of optogenetic actuators and calcium sensors in the spinal cord with a next-generation 3D imaging and optogenetic system recently developed with the physicists at Laboratoire Jean Perrin. If successful, the proposed project will be a major milestone in understanding the spinal CPG in mammals and how it could be manipulated to restore locomotor function in various pathological conditions.
Early stages of Huntington disease: Spatiotemporal co-ordination of the cell reprogramming (Bonneau Stéphanie & Betuing Sandrine)
Abstract : To understand the mechanisms of cellular reprogramming in Huntington’s disease, we are studying the influence of multi-scale spatio-temporal dynamics of cells on biological activity. We focus on mitochondrial and plasma membranes, which are impacted from the early stages of the disease. We overcome two key problems: the experimental limitations for mesoscale observation of living cells on the one hand, and on the other hand the validation of theoretical models to understand these complex problems. Thus, in a systems approach, our efforts to question the multi-scale relationship between structures and functions constitute a challenge for systems biophysics of cells. Moreover, aging and neurodegenerative diseases are real public health problems. The development of our methodological approaches may open new diagnostic perspectives and, in the long term, may pave the way to mitochondrion-like bioinspired devices to balance energy deregulation.
Abstract: By generating multiple transcripts from the same gene, alternative splicing has the potential to greatly expand eukaryotic proteomes. In this doctoral project, we propose to leverage the growing body of available transcriptomics and proteomics data to generate new protein functional diversity. We will use the notion of evolutionary conservation to identify a set of alternative-splicing-induced sequence variations likely relevant to protein function. We will carefully cross this information with transcript expression and proteomics data. We will then map the identified sequence variations onto protein structures and interactions, at the level of protein domain families. We will build a probabilistic model that will learn « rules » from this curated resource to determine where and how to target a protein in order to modulate its function. We will represent the input data as graphs and will develop a suitable deep learning architecture (e.g., variational auto encoder). We will produce a knowledge base for alternative splicing and new methods for graph learning applied to proteins. The expected outcome will improve our understanding of protein functioning and help to guide protein design.
Deep learning to predict metabolic protein-protein interaction networks for environmental microbial communities (Carbone Alessandra & Bittner Lucie)
Abstract: Metagenomics provides a huge inventory of species present in environments and of metabolic functions performed by environmental communities. It offers enormous potential for discoveries, as more than 99% of microbial species cannot be cultivated in the laboratory. It has given rise to several large-scale projects to characterize the microbial diversity of the oceans (e.g., GOS , Tara Oceans , Malaspina , OSD ), of microbes in symbiosis with humans , of the composition of soils , urban environments , or subjected to extreme conditions (eXtreme Microbiome Project). Each metagenomics experiment generates large amounts of raw data (on the order of several terabytes of sequence per sample), the processing of which presents several algorithmic and data analysis / learning challenges.
The goal of this PhD project will be to develop new computational approaches based on deep learning to reconstruct protein-protein interaction (PPI) networks for metagenomic samples starting from sequence reads. The aim is to predict PPI networks that allow a community of microbes to perform their metabolic functions. Questions on biogeography and evolution of PPIs will be addressed with a comparison of PPI though samples from different ecosystems.
Abstract: Expanding populations, such as tumors, bacterial colonies, or invasive species, often evolve towards better dispersal. But complex intra-specific interactions can alter this trend. What control the robustness of dispersal evolution? We tested this question with bacterial swarming and saw a diversity of evolutionary outcomes: bacteria’s dispersal is sometimes improved and other times impaired. This project combines biophysical and evolutionary approaches to decipher how interactions between bacteria at a microscopic level can lead to changes at the whole colony scale, which will determine the collective spreading ability and ultimately, the individual evolutionary fate.
Rationalizing the use of prior knowledge in systems modeling and molecular neurosciences (Neri Christian & Gris Barbara)
Abstract : Huntington’s disease (HD), a disease caused by CAG expansion in huntingtin, is recognized as a model to understand the role of cellular resilience over senescence systems in neurodegenerative diseases (ND). As large omic datasets become available to study these systems in HD and other NDs, there is a critical need for biologically-precise systems-modeling methods. To this end, we developed a new and promising metric for precisely modeling the responses to mutant huntingtin on molecular and functional levels. This metric is based on a design matrix that contains the prior knowledge on HD that is introduced in the analysis. Understanding the influence of such prior knowledge on the information provided by in silico models is of major interest in systems modeling. The aim of the PhD project is to rationalize and automatize the use of prior knowledge in our metric. This will make our new method generalizable and easy to use for a wide range of applications while obtaining new insights into the dynamics of disease resistance systems in HD.
Regulation of gene expression coupled to cell growth and division in bacterial adaption processes (Sclavi Bianca & Cosentino Lagomarsino Marco)
Abstract : The DnaA protein can act as the regulatory link between the timing and level of gene expression and the different phases of the bacterial cell cycle. The cooperative binding of the DnaA protein to the origin of replication leads to DNA opening and the assembly of the replication forks. But DnaA is also a transcription factor, a highly connected node in the network of genes coding for proteins required for DNA replication and the repair of DNA damage. However, a description of the regulation of gene expression by DnaA as a function of the cell cycle is still lacking. The interdisciplinary approach used here will consist in the measurement in gene expression and cell growth parameters in real time at the single cell level by using microfluidics coupled to microscopy. This quantitative data will then be used for the creation of both stochastic and coarse-grained mathematical models that can be used to test different possible hypothesis on the regulatory links between gene expression and cell growth and division that permit rapid cellular adaptation to changes in growth rate.
Study of piRNA clusters: loci involved in genome stability (Teysset Laure/Carré Clément & Antoniewski Christophe)
Abstract: Transposable elements (TE) are a source of deleterious mutations. A first defense that comes into play is based on small RNAs, called piRNAs, which target TEs by sequence complementarity and block their transposition. This defense mechanism is often compared to an adaptive immune system. piRNAs come from discrete loci, the piRNA clusters. Composed of intertwined and inactive TEs, these clusters represent the library of mobile sequences that cells must repress in order to maintain genome integrity. The main goal of this iBio PhD project is to understand the dynamics of activation of these piRNA clusters and subsequent production of piRNAs that control TE expression.
RNA chemical modifications (mostly methylation) on the piRNA precursors transcripts will be also investigated in this project. Indeed those RNA modifications are probably involved in the correct funneling of the piRNA precursors transcripts to the piRNA processing factory localized in the cytoplasm. Thus, the PhD project required both biochemistry and chemistry knowledge. In addition, Drosophila genetics and bioinformatics sequences analysis and statistics will be an asset for the future recruited iBio PhD student.