Many sexually reproducing microorganisms are self-fertile, but in-breeding and out-breeding strategies have different outcomes on genetic diversity. Recent work from the Martin lab showed that self-fertile fission yeast S. pombe cells exhibit an exploratory polarity patch at their periphery, which allows them to preferentially pair with non-sister cells. However, the mechanism of this preference and its prevalence in nature are unknown. Here, I will study whether out-breeding is a general feature of fission yeast cell mating and dissect the molecular mechanism and genetic basis of this phenomenon. I will first develop a zygote FACS sorting method, which I will use together with high-resolution live-cell microscopy to study how pheromone concentration, cell size and transcriptional timing influence exploratory polarization and mate choice. I will then investigate whether out-breeding is a general feature amongst the recently sequenced collection of natural variants. If I detect pre-zygotic barriers amongst these strains, I will use pedigree analysis to probe for possible causative mutations. Finally, I will combine FACS sorting with experimental evolution to isolate strains favoring in- or out-breeding and uncover the genetic basis of the mating preference via comparative genomics/transcriptomics. Along the evolutionary experiment, I will also follow the exploratory polarization patterns by live-cell microscopy to relate the molecular mechanism of mating preference alteration with genetic changes. Finally, I intend to integrate the identified mutations into wild type lab strain to reconstruct the altered Cdc42 dynamic patterns and breeding preference.

To understand how cellular machineries work, we typically rely on reconstituted systems that often do not represent the complexity existing in vivo. We lack innovative methods to describe protein-protein interactions at high-resolution, specially the very transient ones, in their physiological environment. Chromatin is a good example of a system whose complexity cannot be fully reconstituted in vitro. Indeed chromatin is regulated by hundreds of chromatin remodelling enzymes and hundreds of possible combinations of histone post-translational modifications (PTMs) and variants. This complexity cannot be fully reconstituted in vitro.
We propose a novel combination of synthetic biology, mass spectrometry and high-resolution imaging to define the molecular details of how proteins function on chromatin in their physiological environment at high resolution. The challenge is to use photochemical traps installed by genetic code expansion in histones of living cells to “freeze” interactions of proteins bound to chromatin, especially the very transient ones that could be disrupted or missed by conventional purification or bulk chemical cross-linking. The trapped protein-chromatin complexes will be analysed by cryo-electron microscopy. By mass spectrometry we will map the interactions between remodelers and histones in vivo and we will quantitatively describe all chromatin PTMs associated to specific remodelers. This way, we will be able to analyse the spatio-temporal activity of chromatin-bound complexes at high-resolution at specific time points or upon specific stimuli.

Adult mammalian tissues are composed of heterogeneous cell types generated during development. To maintain homeostatic function, the identity of each of those cell types remains stable over time, and tissue renewal is normally sustained by dedicated stem cells. After injury, however, differentiated cells in many tissues can exit their quiescent state, acquire stem cell features and cross developmental identity boundaries to participate in the repair process. In the adult mammalian brain, injury-induced cellular plasticity (i.e. the acquisition of a new identity) remains poorly understood.
Here I plan to identify which brain cells exit their normally quiescent state upon stroke and study the molecular regulation of this process in situ. To this end I propose a novel strategy combining unbiased genetic fate mapping of stroke-generated cells with single-cell and spatially-resolved gene expression analyses. Specifically, stroke-generated cells will be marked with a genetic tag, identified using single-cell RNA-seq, and their contribution to repair traced over time. Next, we will use spatial transcriptomics to identify molecular triggers of cell plasticity, as well as their cellular source within their native environment. Finally, the identified molecules will be functionally tested for their capacity to enhance endogenous recovery after stroke by increasing cellular plasticity. This study will shed light on the cellular and molecular mechanisms governing the self-repair capacity of the brain.

Amino acid and peptide radicals have been implicated in a wide variety of biochemical processes and physiological disorders, including Alzheimer’s disease, arteriosclerosis and aging. At a molecular level, they are associated with protein damage and enzyme function, with the glycyl radical enzyme (GRE) family as important yet mostly unknown contributor. Using the high reactivity of peptide backbone radicals, GREs enable strict and facultative anaerobic organisms to catalyze a diverse range of chemically difficult reactions, including anaerobic toluene metabolism, the conversion of choline to trimethylamine (TMA) via C–N bond cleavage or glycerol fermentation. In taking advantage of the reactivity of the protein-based radical intermediates, tools will be developed to characterize novel GREs within intact microbial communities. For the first time, the concept of activity-based protein profiling will be expanded to include an entirely new class of enzymes. Furthermore, assigning functional and interactive relationships is suspected allow for the development of specific small molecules inhibitors to improve host health thereby opening up new opportunities in the way medicinal chemistry approaches certain disease. An additional key obstacle in studying GREs is the posttranslational mechanism of activation, which involves separate activating enzymes (AEs). The reconstruction of activase activity can be problematic and therefore, unnatural amino acid mutagenesis using designed glycine radical precursors to install a radical in the enzyme active site in a separate chemical step will facilitate studying GREs.

Some animals have great regenerative capability to regrow whole bodies even from small fragments. This ability relies on their pluripotent adult stem cells, which are capable of self-renewal and differentiating into any missing body part. During regeneration, positional signals such as Wnt signaling are crucial for stem cell regulation and specification. In planarians, inhibition of Wnt signaling promotes formation of head-specific cell types. However, the molecular control of stem cell fate by Wnt signals is unclear. In part, this is due to the limitation of lineage tracing in the well-established planarian system. Here I propose to use the acoel, Hofstenia miamia, as an alternative system to dissect this process. Hofstenia is an early diverging bilaterian and evolutionarily distant to planarians, and yet possesses whole-body regenerative ability and similar patterning pathways. In particular, Hofstenia produces large amounts of accessible embryos that are injectable, making lineage tracing of stem cells possible. I will first identify conserved Wnt signaling components by generating spatial transcriptomes along the anterior-posterior axis. Next, I will test the role of Wnt signaling in stem cell fate specification by using RNAi of Wnt pathway genes together with single-cell RNA sequencing. These data will reveal distinct transcriptional landscapes and identify Wnt-responsive cells within stem cell populations. Finally, I will develop lineage tracing methods for live imaging of stem cell differentiation and behavior. Together, this study will provide a basic understanding of how stem cell fate is regulated by positional signals during regeneration.

Cellular signalling pathways usually involve cascade processes. For some of these processes, ensemble average analyses cannot answer biological questions originating from stochastic biomolecular interactions. Though single-molecule analyses are sometimes helpful but still the oligomeric forms of many membrane protein complexes remain controversial. Protein or polymer scaffold lipid discs have emerged as platforms for monodispersed proteins. It is difficult to manipulate them to study protein-protein stoichiometric interactions. To overcome this, and motivated from DNA origami approaches, I propose to use DNA scaffolds to form lipid nanodiscs (DSN). Using DSN I will develop a novel programmable platform to study membrane protein-protein interactions in an isolated environment. First, I will create DNA nanorings, functionalised along their inner circumference with hydrophobic groups to hold a lipid membrane. Computational approaches will be used to optimise the designs along with experimental characterisation. The functional nature of DSNs will be established with an embedded P450 oxidoreductase enzyme. To develop a programmable platform, embedded proteins will be tethered at the edges of DSN with DNA linkers. Using DNA strand-displacement methodology proteins will be released to diffuse freely and interact in the membrane. As a proof of concept, an experiment will be set up using two formyl peptide receptors. Interactions will be analysed using AFM, single-molecule spectroscopy etc. Such platform, if achieved, could prove revolutionary for single-molecule analysis of protein oligomerization for studying, e.g., signal pathways, interaction energetics, metabolism etc.

Traditionally, model systems in experimental and theoretical condensed matter physics have provided a simplified/stylized version of target problems enabling fundamental advances. Over the years, soft condensed matter physics has undertaken the goal of explaining basic properties of living matter phenomenologically. This led to tremendous insights into specific phenomena such as behaviour of individual biological macro-molecules or mechanochemistry of biological forms, but the fundamental goal of understanding what the exact boundary is between living and non-living active matter remains elusive. Based on recent advances in soft-matter physics, here we propose an alternative approach to this question via developing an experimental abiotic (non-biological origin) physical model system capable of exponential self-replication and evolution (Aim 1a). With this framework in place, we intend to write down the fundamental properties of self-replicating and evolving matter in conjunction with the physical environment, laid out in terms of laws of physics (Aim 2), linking genotypes and phenotypes (Aim 3). These microfluidic experiments will be compared to robust hydrodynamic Molecular Dynamics (MD) simulations and theoretical Stokesian fluid mechanics, where hydrodynamic interactions and thermal noise are accounted for (Aim 1b). Together, these techniques could help formulate a theory for the essential ingredients of directed evolution and reproduction subject to physical constraints. We thus aim to discover new robust, abiotic architectures capable of self-replication of physical matter and information (Aim 4) and further exploit these findings in exponential manufacturing.

Sleep has roles in memory consolidation. Memory consolidation is thought to include systems consolidation which reflects communication among different brain regions, as well as synaptic consolidation at the local circuitry level. I found systems consolidation during sleep among separated regions in cerebral cortex (Miyamoto et al., Science, 2016), however, synaptic consolidation pattern is still unknown. Net cortical synaptic strength changes homeostatically through wake/sleep cycle: average synaptic strength increases during wakefulness and decreases during sleep (Tononi & Cirelli, Neuron, 2014). While skill learning is known to potentiate subsets of synapses in motor cortex for encoding skill memory (Hayashi-Takagi et al., Nature, 2015), plasticity of those synapses through sleep is unknown. If sleep depresses memory-encoding potentiated synapses, it may result in impairing memory consolidation. If sleep depresses other non-potentiated synapses relatively, it may enhance signal/noise ratio and memory consolidation. Here I will use complex-wheel task for studying synaptic mechanisms of sleep-dependent skill memory consolidation in mice. Through skill learning and sleep, I will perform in vivo two-photon imaging in motor cortex to visualize fluorescence tagged AMPA receptors, since the expression of these excitatory glutamate receptors is an established index of synaptic strength. To visualize plasticity in synaptic population, I will analyze time-course of fluorescence intensity change at single-synapse resolution. This study will help clarifying how sleep favors both memory consolidation and synaptic homeostasis.

The formation of higher order chromatin structure is crucial for DNA replication, transcription, recombination, repair and chromosome segregation. The cohesin-complex is thought to contribute to formation of chromatin loops and of topologically associating domains (TADs) by entrapping two different regions of the same chromatids. Its binding along genomic DNA is thought to be controlled by two of cohesin’s cofactors, CTCF and Wapl. Cohesin has been proposed to regulate the long-range chromosomal cis-interaction by a “loop extrusion” mechanism, in which cohesin can regulate chromatin loop formation by translocating along genomic DNA. However, how cohesin controls chromatin structure remains unclear. To address this question, I will first establish assays based on FISH, Cas9-FISH in living cells, oligopaint strategy and super resolution microscopy (SRM) to visualize chromatin loops and TADs. Second, I will employ these assays and Hi-C to analyze chromatin structure in cells in which the genomic distribution of cohesin is experimentally altered. For this, I will use knowledge from my host lab which recently discovered that the distribution of cohesin in mammalian genomes depends on the DNA binding protein CTCF, the cohesin release factor Wapl and transcription. Third, I will monitor the transition of cohesin re-location and of conformational change of chromatin induced by rapid degradation of Wapl and CTCF in real time. I will then test prediction of the loop extrusion model by comparing my experimental data with the simulations recently described in the literature.

How genetic programs regulate mammalian morphogenesis is one of the central problems in developmental biology. Digit patterning, a representative model to study mechanisms of morphogenesis, is regulated by multiple factors including cytokines and Homeobox-containing genes, or Hox genes, particularly Hoxa13 and Hoxd13. Recently, it has been illustrated that a reaction-diffusion model can explain this patterning in which the interactions of Sox9, a transcription factor, and WNT and BMP, cytokines, generate the periodic digit pattern. Interestingly, this periodic wave is modulated by FGF signaling and Hox genes. However, little is known about the molecular basis how Hox genes contribute to the proper digit patterning by regulating these interactions. In this project, to elucidate the role of Hox genes in mammalian digit patterning and autopod tissue identity, I will conduct integrative analyses including genetic, biochemical, and computational approaches. Firstly, single-cell RNA sequencing will be performed on both of wild type and Hoxa13 and/or Hoxd13 knockout mice to discover a gene network consisting the periodic wave pattern and analyze dosage effects of Hox genes on it. Secondly, HOXD11/D12/D13 will be epitope-tagged with genome editing tools. Thirdly, they will be immunoprecipitated to comprehensively analyze their binding specificity to the genome and other proteins. Lastly, these data are integrated with the reaction-diffusion model and investigate mechanisms of the modulation of BMP and WNT signaling by Hox genes to generate the periodic digit pattern, and consequently, establish the identity in autopod regions.

Controlling the direction of cell expansion is one of the key aspects of environmental and developmental signaling responses in plants. The direction of cell expansion largely relies on the microtubule network just beneath the plasma membrane. These cortical microtubules are dynamic and their organization is rearranged by a wide range of signals such as gravity, mechanical stress and light, but the molecular mechanisms that carry out these signal transmissions and microtubule array rearrangement are poorly understood. Our recent studies have demonstrated that Phototropin signaling regulates microtubule nucleation and severing activities. However, it is not clear how Phototropin regulates these activities. Phototropin is thought to have kinase activity and control wide range physiological responses. While our preliminary data with minus-end regulator suggest microtubule reorientation in response to blue light also requires the regulation of minus end dynamics, we have almost no information on how the remarkable minus end dynamics are orchestrated in plant cell. To tackle this linking question between plant physiology and cell biology, I propose to study about how the microtubule organization are regulated by transmitted blue light signaling through Phototropin. Specifically, I will propose following 3 projects: 1) identify factors being downstream of Phototropin kinase and upstream of microtubule organization, 2) investigate the molecular mechanism of the microtubule minus ends, and 3) introduce a new technique enabling us to conditionally manipulate target proteins to understand their spatiotemporal function during live-cell imaging.

A myocardial infarction in mammals typically displays limited regeneration and instead produces significant cardiomyocyte death. This loss of contractile cells is instead replaced with a permanent non-contractile fibrotic scar. In contrast, the pro-regenerative zebrafish displays increased cardiomyocyte proliferation and fibrotic scar resolution following injury. In order to find candidate genes, other labs have RNA sequenced whole mammalian hearts prior to and after injury. The spatial resolution however is lost, and other unrelated genes may mask candidate genes responsible for regeneration. This project will use a cardiac injury zebrafish model and at critical stages during regeneration, apply a novel genomic technique “RNA Tomography (Tomo-seq)” to spatially resolve gene expression at a genome-wide level. In addition, Tomo-seq concepts will be applied to develop a new genomic technique to examine the regulatory regions surrounding genes. Bioinformatic analysis will identify candidate genes and their regulatory regions associated with different phases of regeneration, and spatially correlate them with different areas in the injury site. Finally potential targets are functionally validated in zebrafish. By identifying pro-regenerative candidate genes at a spatial-temporal resolution in zebrafish, we can further understand and possibly modulate signals to increase the limited regenerative capacities in mammals.

The gut is under constant surveillance by immune cells, which ensures the homeostasis of commensal gut microbiota whilst conferring protection from invading enteric pathogens. B lymphocytes are central to maintaining balanced responses through production of huge quantities of the Immunoglobulins IgA and IgG. Ig production is the outcome of specialized germinal center (GC) responses, which form spontaneously in gut-associated lymphoid tissues in response to chronic exposure to microbiota. Despite being the precursor to Ig secretion, gut GC responses are entirely uncharacterized. Recently, when mesenteric lymph nodes (mLNs) were visualized using multicolor fate mapping (MCFM) large proportions of spontaneous GCs became monoclonal, an unexpectedly specific outcome in the face of exposure to such a diverse range of antigens. We intend to combine MCFM with in vivo models of commensal colonization to track the kinetics and clonal diversity of GC and Ig responses in mLNs through multiphoton microscopy. After GC isolation, we will use sequencing approaches to determine which bacterial species predominantly induce clonal GCs and map subsequent Ig specificity. Following this, we will investigate GC responses during enteric infection. We are especially interested in whether newly activated pathogen-specific B cells utilize pre-existing spontaneous GCs for their own affinity maturation, and how this is altered by composition of the current or past bacterial community. These data could have wide-reaching implications for both the fundamental biology of gut immunology as well as universal principles of GC dynamics in the presence of multiple antigens.

Previous studies have suggested that embryogenesis is robust to environmental, genetic, or stochastic perturbations. However, little is understood about the mechanisms that enable multicellular organisms to develop robustly, and about the extent to which – and the way in which – they changes their developmental patterns in response to harmful perturbations. Especially, how organisms adapt to environmental stresses during embryogenesis is not well defined, partly because in most cases experimental organisms have been cultured under favorable conditions. Considering that organisms usually live in harsh conditions in their wild habitat, it is highly likely that they have evolved the undiscovered ability to deal with environmental stresses.
In this project, I will employ a high-performance cell tracing system and sophisticated computational techniques to accurately track the positions of ALL cell nuclei in developing embryos of the nematode Caenorhabditis elegans. By this approach, I will comprehensively examine the effects of nutrition deficiency on cell division and differentiation during embryogenesis. Additionally, I will elucidate the developmental basis of transgenerational starvation response. After the detection of the developmental differences caused by nutritional stress, I will clarify how the differences are induced (or suppressed) at the molecular level using powerful molecular genetics and mathematical analyses.
The success of this project will provide fundamental insights into embryogenesis and might lead to understanding of the mechanisms underlying teratogenesis, spontaneous abortion, and neonatal death in nutritionally challenged humans.

The spectacular manoeuvres of flocking birds and schooling fish are among the most dramatic and mysterious sights in the natural world. How can hundreds or thousands of individuals coordinate their movements so perfectly, behaving almost as a single super-organism? The answer to this puzzle began to be uncovered through mathematical models showing that collective order can emerge as a by-product if all individuals within a group follow simple rules to align with and stay close to their neighbours. However, unlike the simulated agents in these models, real animals are not identical, and can differ both in their individual characteristics and in their relationships with one another. A group’s composition is therefore likely to affect its overall structure and cohesion as well as its ability to reach consensus decisions when responding to the environment. Understanding these effects has important implications, from determining how animal groups respond to threats, to mitigating the impacts of crop pests, managing crowd safety and developing intelligent systems in robotics. We will use mixed-species flocks of rooks and jackdaws (birds of the crow family, or corvids) to understand the effects of group composition on collective behaviour in nature. Combining field experiments with cutting-edge imaging and computational techniques, we will produce 3D reconstructions of the movements of every bird within flocks of varying composition and examine how a flock’s composition affects its structure and movements, and its responsiveness when avoiding or mobbing predators. Are more homogeneous groups better able to respond as a coherent unit, or does diversity enhance group responses as in human social institutions? Our 3D reconstructions will also allow us to determine the fine-scale internal structure of flocks. Do corvid flocks, like human crowds, contain sub-groups, reflecting flock members' social preferences? Finally, we will use our data to understand how individuals' flight decisions are influenced by who their neighbours are. By building mathematical models based on these measurements and testing the models using flocks of robot-controlled drones, we can find out how local interactions and social preferences among neighbours generate both internal sub-structure and collective order in complex societies.

The control of phenotypic variance, known as phenotypic robustness, is a fundamental characteristic of living organisms. This assures a relative stability of phenotypes even when organisms are exposed to stressful internal or external environments. However, it has been extensively documented that stressful conditions decrease organismal robustness resulting in an increase of phenotypic variance at the population level. Although ubiquitous in biology, the underlying genetic factors driving such shift in variance are poorly understood. This project specifically addresses this question by studying the genetic basis of gene expression variability in Drosophila melanogaster exposed to dietary stress. For this, genomic loci associated with transcriptional variability will be identified (variance-eQTL or v-eQTL); these are loci whose allelic state predicts the amount of variability around the expected mean. Recently developed outbred Drosophila populations will be used; this will allow the exploration of more genetic diversity than ever before. Thousands of flies will be exposed to two dietary conditions, standard food and high-sugar diet. v-eQTL maps will be generated for both conditions and will be compared with two objectives: 1) to reveal cryptic genetic variants only relevant under stress; and 2) to explore how stress disturbs the co-expression networks present in normal conditions. The analysis of this data will reveal the genomic loci associated with variation in transcriptional robustness between individuals, and will ultimately offer a deeper evolutionary and medical understanding of phenotypic robustness.

Synaptic vesicle (SV) is a vital cell organelle in neuronal communication via the exocytosis process and it corresponds to the basis of cognition, memory and neural diseases. The overall objective of this project is to develop new micro- and nano-analytical approaches to discover how cognitive drugs (CDs) modulate chemical communication processes of neurons by SVs. At first, I aim to use the electrochemical cytometry (EC) to realize the changes in the neurotransmitter content of SVs after taking individual CDs. But the orientation of vesicle opening in EC is unclear. Realizing this orientation is important as it lets us accurately measure the vesicle content, and it is also a model for the pore opening at the membrane in exocytosis. I propose two new methods, a nanogap electrode and nanohole system, to trap and measure vesicle contents with EC. Then I will carry out intracellular EC inside living cells and in Drosophila Melanogaster after administration of CD, by ultrasmall nanometer electrode tips. I hypothesize that a vital part of drug action in the brain, beyond to the change in the synaptic vesicle content, can be alteration of lipid distribution and change in the neuron membrane composition following the fusion of SVs to the cell membrane via the exocytosis. I propose imaging experiments by the state of the art instruments like nano secondary ion mass spectrometry, to prob the entire contents of vesicles and cell membrane; and to compare the changes observed with the dynamic electrochemical detection of exocytotic release in intracellular EC. All these experiments lead to fascinating new ideas about how the release of transmitters from vesicles is regulated by CDs.

Tissue morphogenesis is a process by which the embryo is reshaped into the final form of a developed animal. Tissues are constituted by cells that are interconnected one another: local changes of cell mechanical properties and shape drive consequent tissue shape change. Nevertheless the knowledge per se of the mechanisms and mechanics at the cell level which drive cell shape changes is insufficient to explain how tissues change their shape. Emerging properties arise at higher scales resulting from the interaction of cells within tissues and of tissues coordinating and interacting with one another. Studying this is a great challenge both technologically and conceptually. From the technological perspective, new tools are needed to be able to visualize cells and to provide quantifiable data at high temporal and spatial resolution over large regions and across the entire embryo. New techniques are required to manipulate tissues with temporal and spatial specificity and to measure mechanical properties in different regions of an embryo. Conceptually, it is challenging to understand how different sheets of cells with different mechanical properties interact. I will use the Drosophila embryo as a model system and focus on the process of tissue folding, process that is vital for the animal since folding defects can impair neurulation in vertebrates and gastrulation in all animals which are organized into the three germ layers. Thanks to my interdisciplinary background and expertise in biology, engineering and physics, my work will provide new knowledge on how biophysical forces sculpt functional living organisms.

Despite its significance, the transgenerational inheritance adaptive behaviors through germline modifications are poorly established and understood in terms of the underlying biological mechanisms. Here, by employing a novel social learning paradigm in Drosophila I aim to understand how predator-threat information is successfully inherited by subsequent generations through an epigenetic reprogramming of parental germline cells. In fruit flies, exposure to an endoparasitoid wasp elicits preferences for alcohol containing food and oviposition depression. Visual input mediated predator presence induces germline apoptosis resulting in both acute and learned oviposition depression in wasp-exposed females. Strikingly, daughters of these exposed females are preprogrammed to prefer egg-laying in alcohol enriched media even in the absence of predator. In addition, the altered food preference behavior in response to an anticipated predator-threat persists for up to five generations. These interesting observations set us to understand the mechanisms for predator-threat induced social learning in terms of (i) cellular and molecular signals, which trigger apoptosis in developing oocytes of wasp-exposed females, and (ii) epigenetic reprogramming in exposed flies that alters germline information conferring transmission of behaviors to subsequent generations. While the current proposal questions the classical conception of learning, the outcomes of this study in terms of the neurophysiological basis for inheritance of social behaviors will radically transform the way we think about learning, memory and behavior.

Learning to associate fear with stimuli predicting aversive outcomes is key for adaptation and survival. However, experiencing fear in maladaptive contexts can be crippling and disabling. Around 4.5% of the global population suffers from anxiety disorders such as post-traumatic stress disorder, generalized anxiety disorder and specific phobia. One of the most influential ways to overcome feared associations is extinction, which consists of exposure to previously feared stimuli in neutral circumstances, thus reducing the fear response. Extinction, frequently manipulated in the laboratory context, putatively stands at the base of effective psychological exposure therapy. In this proposal, I plan to adapt a new experimental paradigm, targeted memory reactivation (TMR), to strengthen fear extinction in humans. TMR, which has effectively facilitated several different types of memory, involves the subliminal presentation of previously associated cues during slow-wave sleep. Employing the lab's expertise in TMR procedures, I will try to strengthen extinction in a controlled setting and reveal the underlying neural mechanism by which TMR affects learnt memories using neuroimaging techniques (electroencephalogram, functional MRI) and electrophysiological recordings (electrocorticography). With this project, I aspire to bridge between the cognitive literature on the features and limitations of human memory and the more mechanistic animal-based literature on consolidation and memory formation. This can only be achieved by combining a well-controlled and manipulated behavioral paradigm together with state-of-the-art high-resolution brain-imaging techniques.