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.

Recent genome-wide methods of mapping functional DNA elements have uncovered a vast landscape of genomic loci that have the characteristics of enhancers. However, the identification and mapping of this complex putative enhancer landscape does not link specific genes to their enhancers, nor does it prove any causal relation between the putative enhancers and gene activation or repression. In this project I will combine a powerful screening platform that harnesses CRISPR-cas9-based screens to test the function of hundreds of enhancers, in isolation and combination, which putatively regulate gene expression of genes associated with ricin sensitivity. In the first part of the project I will identify all potentially "active" enhancers during the cell response to ricin. In the second stage I will generate a CRISPR-cas9 library to disrupt the activity of all enhancers in isolation or in designed combinations, and measure the change in the cell response to ricin. In the third phase I will focus my attention on dozens of individual enhancers that affect the cell response to ricin, and using ATAC-seq, RNA-seq and 4C-seq methods I will determine the regulatory mechanism for these individual enhancers. In the final part of the project I will build a model trying to explain enhancer behavior in gene regulation. This work will provide the first genome-wide, high-throughput investigation of enhancer function on gene regulation, and will begin to unravel the combinatorial effects of enhancers by modulating them in their native genomic location. In short, this work will put regulatory genomics on firm functional or mechanistic footing.

Precise and coordinated regulation of tyrosine phosphatases and kinases is crucial to mount an effective immune response and to prevent autoimmune diseases. Lyp, a lymphoid-specific phosphatase, is a negative regulator of T-cell receptor (TCR) signaling. Some studies point to a gain-of-function when Lyp interacts with the tyrosine kinase Csk, while others suggest that Lyp/Csk complex limits its activity. This project aims to elucidate how the spatiotemporal coordinated arrangement of Lyp, Csk, and the transmembrane adaptor phosphoprotein PAG to which both associate, down-regulates TCR signaling by exploiting the potential of super-resolution fluorescence microscopy. Super-resolution imaging has already proven to be a powerful tool to study the immunological synapse. Still, multiplexed interrogation of distinct target proteins remains challenging. I will implement a recently reported multiplexed super-resolution imaging technique named DNA-PAINT (Points Accumulation for Imaging in Nanoscale Topography) to image the target proteins with high spatial and temporal precision during T-cell activation using a single-laser. I will further develop a 3-color co-localization clustering analysis for unveiling the complex pathways involved in signaling termination. Given the strong association of a single-nucleotide polymorphism in the gene encoding Lyp with various autoimmune disorders, I will also address the regulatory mechanism involved in the disease-associated Lyp variant.

Large-scale monitoring of the brain demonstrates that neuronal computation arises from interactions of numerous neuronal populations. Yet, methods that allow for large-scale recording of activity at high spatial resolution and for extended time periods are lacking. To meet this challenge, we propose to design, develop and test novel large-scale neural interface devices that will allow long-term, high spatiotemporal resolution, stable recording and stimulation of neural activity by injecting polymers into the scalp. These devices, and the data generated by them, will be used to address key questions in systems neuroscience. We aim to investigate the coupling mechanisms of neural oscillations between functionally distinct brain regions, and identify how different oscillations generate unique spatial and temporal patterns of activity. We will establish electrical stimulation protocols to modulate the occurrence rate, power, and frequency of specific brain oscillations to be able to assess the function role of such oscillations. Lastly, we will combine these recording and stimulation interfaces in form of closed-loop electrophysiological experiments to be able to manipulate and observe the effect of these oscillations on physiological and behavioral levels. The ability to acquire, stimulate, and analyze neural network simultaneously from multiple brain regions will enhance comprehension of neural network processes and has implications for brain disorders characterized by disordered network function.

I seek to understand how the patterns of animal movement seen in nature emerge from the combined actions of many individuals. For group-living species, this process occurs over two scales of organization: individual decisions emerge into coordinated group movement, and the movements of groups drive population-level patterns of space use. To understand how these processes connect, we need to collect and analyze data at both of these scales, yet doing so within a single system has remained a challenge. I will address this challenge using the meerkats of the Kalahari Desert as a model system. Meerkats (Suricata suricatta) live in stable groups that remain together as they forage over large distances. Leveraging recent technology, I will collect data on the fine-scale movements and vocalizations of all individuals within meerkat groups. Through probabilistic modeling, I aim to determine what rules govern where individuals move, and how these rules give rise to coordinated group movement. Key questions include how meerkats’ calls allow groups to maintain cohesion and decide where to go, and whether certain individuals have disproportionate influence over group movements. At the group level, I will use movement and demographic data collected over 20 years at the Kalahari field site to determine what factors drive group movement, including habitat features, memory, and interactions with other groups, to understand how aggregate space-use patterns emerge. Bringing together a wealth of data from a long-term field study with newly-available high-resolution tracking methods will enable us to link the multiple spatial and temporal scales driving animal movement patterns in the wild.

The ability to precisely edit and manipulate the genome is critical for biological research and the treatment of human disease. The power of such tools is highlighted by advances in CRISPR-Cas9 mediated gene editing, but there is continual need to develop new tools. The discovery of diverse genome manipulating enzymes throughout nature highlights the importance of basic biological findings and the wealth of natural systems that may be amenable for repurpose in human cells.

One compelling biological system is the programmed genome rearrangement of the ciliate Oxytricha which involves the fragmentation and joining of over 225,000 DNA pieces to form functional genes. Remarkably, gene coding sequences are often scrambled and must be reordered during reassembly, a process thought to be mediated by RNA templates. This phenomenon warrants further investigation and hints at the existence of remarkable genome manipulating tools. Despite our knowledge of the outcome of rearrangement, how this process happens and the proteins involved is not well understood.

The objective of my research is to identify the key proteins involved in the programmed genome rearrangements of Oxytricha and to elucidate the molecular mechanism of this process.
I aim to:
1) characterize changes in the transcriptome during programmed genome rearrangement,
2) develop functional genetic tools to screen for required factors, and
3) to recapitulate genome rearrangement in vitro using cell extracts.

This work aims to elucidate the identity and basic biological properties of the enzymes involved in this process with the long-term objective to adapt and harness identified components for use in human cells.

Neurons are characterized by their unique morphology, which requires efficient sorting and transport of cargoes over long distances. One class of molecules that affect neuronal development, function and survival are neurotrophins. Neurotrophic growth factors are target tissue-derived cues that bind to their cognate receptors on the pre-synaptic membrane and are transported retrogradely to the soma, where they induce signalling events which control transcriptional programs governing neuronal differentiation and survival. Despite their importance in neuronal homeostasis, the regulation of uptake and transport of neurotrophins and related growth factors is presently unknown. Recently, the host laboratory reported that the tetanus neurotoxin requires the synaptic basement membrane protein nidogen for its entry and axonal transport in mammalian motor neurons (Bercsenyi K, et al., 2014). Importantly, the tetanus toxin is known to enter the same intracellular trafficking pathway entered by neurotrophins (Salinas S, et al., 2010). In this context, this proposal is aimed at investigating the question: Does the synaptic basement membrane play a role in regulating the uptake, transport and biological activity of neurotrophins and related growth factors? As part of the research plan, I intend to identify growth factors that bind to nidogens and other components of the basement membrane at the mammalian neuromuscular junction, genetically modulate this binding and study its effects on neurotrophic factor endocytosis and trafficking, with a view to understanding the physiological relevance of this biological process.

Immunogen specific elicitation of HIV-1 broadly neutralizing antibodies has been proposed as the key step of an AIDS vaccine. Unfortunately, such vaccination strategy remains elusive. Priming B cell receptor germline precursors with specific engineered immunogens has been already proposed to be a potential strategy to generate bNAbs. Hence, characterizing and identifying potential germline precursors of bNAbs in the B cell repertoire of patients that has been exposed to native HIV-1 or a HIV-1 mimicking immunogen for a short period of time is a necessary step for the iterative design of successful bNAbs generating HIV-1 immunogens. In this project, using the PGT121-class germline-targeting Env SOSIP trimer, I will isolate low-affinity binding germline precursors of the bNAb PGT121 via particulate presentation from samples from (1) HIV-1 freshly infected patients and from (2) HIV-1 vaccinated patients. Using single cell RNA-seq, I will compare the antibody sequence of the isolated PGT121-class germline precursors with the sequence of their corresponding mature bNAb PGT121. I aim to obtain unprecedented information about the potential generation of germline precursors of bNAbs in patients after short exposure to HIV-1. The information obtained in this project will be crucial to design vaccine immunogens that can generate immune protection against HIV-1.

Our understanding of how central and peripheral processes interact and affect balance control is limited. This project will elucidate the interplay of brain and brawn in balance control. It will use a comprehensive multi-faceted approach of laboratory and daily-life assessments using body-worn sensors, to test the hypothesis that central processes can compensate for a slowing down of peripheral processes. The overall goal of this project is to advance current knowledge by disentangling central and peripheral contributions to balance control.
Specifically, the goal of the project will be reached by addressing two major aims:
Aim 1: Establish how the interplay of central and peripheral impairments affects balance control;
Aim 2: Identify whether prefrontal brain areas govern this interplay.
This project is novel in that it will be the first to use body-worn sensors to study underlying mechanisms for balance problems in the home environment during every day life. The outcomes are expected to inform personalised interventions to prevent and restore balance problems. The novel sensor-based techniques developed in this project provide tools to identify individuals that may benefit from such interventions and can also be used to evaluate the effectiveness of these interventions.

The presence of immune cells in decidua during early to mid-pregnancy creates a specific environment at maternal/fetal interface, which is crucial in preventing rejection of the fetus. Although unique receptors and interactions have been identified for decidual immune cells, the full spectrum of their responses is still not completely understood.
In my post-doctoral research, I will study the cell types, their transcriptional regulation, and their cellular interactions established in maternal/fetal immune cross-talk. This research will be addressed by analysing the transcriptomes of decidua-infiltrating immune cells at a single-cell level. With these results and using bioinformatics tools, I will reconstruct the specific niche required to ensure success at this first stage of pregnancy.
I will pay special attention to the unique cell-cell communication occurring in this particular context. To study cellular interactions, I will develop “CellCommDB”, a curated database of known ligands and receptor proteins involved in cell-cell communication. The innovative part of “CellCommDB” will be the integration of the multiple levels of complexity found in protein receptors, like the fact that many cell surface receptors consist of multiple different protein chains.
Overall, my results will help to decode the paradigm of maternal/fetal allogenic co-existence, and to design novel, targeted therapies to increase pregnancy success. Moreover, it will pave the path for further studies to analyze the unique cellular interactions that defines the function and regulation of tissues.

Microbes are key players in global carbon and nitrogen cycles that fuel the biosphere and modulate atmospheric composition. The ‘household’ economy of energy budgeting in a microbial cell strongly affects its fitness, and thus its rise and fall in ecosystems. However, cellular energy intake and spending cannot be quantified by current approaches.

I propose to develop a novel experimental framework to dissect the energy budgeting strategies of different microbes based on their growth-dependent heat dissipation. I will first use calorimetry and isotope labeling to respectively measure heat and anabolic rates of a laboratory strain for establishing a strategy to extract energy usage for maintenance and growth. I will apply this approach to studying environmentally-relevant microbes with different anabolic capacities from diverse geographical origins, including archaea and bacteria that produce and consume methane (a potent green-house gas) and that fixes atmospheric nitrogen gas. Furthermore, I will develop a novel microfluidics-based calorimeter and combine it with nanoscale secondary ion mass spectrometry to enable parallel, long-term, single-cell analyses on microbial metabolism in environments that differ in nutrient abundance, nutrient composition, temperature, and acidity.

The goal is to unveil: (i) the fitness tradeoffs between microbial assimilations of different carbon, nitrogen, and energy sources, and (ii) different energy budgeting strategies that govern bacterial and archaean adaptation. The obtained knowledge will advance our prediction on how microbe-based natural carbon and nitrogen cycling accommodates to manmade global geochemical and climate changes.

In Hh signaling pathway, activity of Smo, a seven-transmembrane (7-TM) protein belonging to G-protein coupled receptor (GPCR) family, is repressed by Patched (Ptch). After Hh binds to Ptch, the repression of Smo is relieved and the derepression consequently results in the activation of transcription factor Ci/Gli and then transcription of Hh target genes related to embryo development and postnatal tissue homeostasis. Smo plays a central role and mediates almost all biological activities of Hedgehog proteins. Dysfunction of Smo can lead to phenotypes ranging from severe malformations of the head and limbs to embryonic lethality, and even various tumors. The molecular mechanisms of Smo activation and its interactions with the downstream effector are still not fully understood, albeit with the available structures of full-length Smo and isolated domains bound to the respective small-molecule ligands. Compelling evidences suggest coupling of Smo to G protein during activation, specifically Gi protein. Inspired by recent technological breakthrough in single particle cryo-electron microscopy (cryo-EM), I propose to determine the structures of Smo-Gi protein complex by single particle cryo-EM. I will use conformational specific fragment antigen binding (Fabs) as fiducial marks to facilitate cryo-EM image analysis. Furthermore, I will use lipid nanodisc or salipro system to stabilize the Smo-Gi complex and aim to analyze the roles of lipids on Smo activation. The ultimate goal of this study is to facilitate structure-based drug design with better treatments for Smo-related cancers.

Cancer is not a single disease and individual tumors have different molecular profiles. Glioblastoma multiforme (GBM), the most aggressive form of glioma, can be grouped into four different molecular subtypes including proneural, neural, mesenchymal and classical, based on distinct gene expression signatures. GBM has an invariably terminal prognosis. Most patients die within 14 months following diagnosis, even with surgery, radiation and chemotherapy, stressing the urgency for new therapies. Non-cancerous cells in the tumor microenvironment (TME), including different populations of immune cells, fibroblasts and endothelial cells, represent genetically stable therapeutic targets since they are less likely to develop acquired resistance as a result of genetic changes, in contrast to tumor cells. One immune cell type that has been successfully targeted in preclinical trials of proneural GBM are tumor-associated recruited macrophages and resident microglia (TAMs). However, it remains to be determined whether targeting TAMs also results in clinical responses in other GBM subtypes with different genetic alterations. As a consequence of a distinct molecular profile it may be expected that different populations and/or amounts of stromal cells are recruited and differentially educated in the TME. Therefore, I will determine how specific genetic alterations in different GBM subtypes sculpt the TME. I also aim to assess the effect of targeting TAMs in these GBM subtypes and to gain insights into potential resistance mechanisms. To achieve this, I will employ the latest genetic mouse models for GBM, extensive flow cytometry analyses, RNA sequencing and intravital microscopy.

Although apparently simple, transparency is a complex coloration strategy. Long viewed as exclusively for camouflage (obeying the ‘being invisible to go undetected’ principle), it has recently been proposed to also play a role in communication. While morphological solutions for transparency are diverse, the physical challenges and properties are poorly known, and developmental and biophysical mechanisms at work to build transparent structures remain poorly understood. Previous studies of transparency are sparse and devoted to aquatic organisms, as transparency is frequent in water but extremely rare on land. Furthermore, understanding transparency requires working at the interface between physics, evolutionary biology and developmental biology. We propose an intercontinental collaborative project that aims to elucidate the adaptive functions of transparency in clearwing butterflies and the generative processes leading to modified structures in transparent wings, bridging the gap between development, function and evolution. First, by conducting physical measurements, we will characterize structural, optical, thermal and hydrophobic properties of transparent wings. Notably, we will characterize light transmission efficiency as well as optical patterns, such as iridescence, that may be involved in communication. Second, by examining and experimentally manipulating pupal wings at various developmental stages we will identify the cellular modifications that distinguish transparent areas from opaque areas of the same species and homologous regions between species, and define genetic pathways that underlie these distinctions. Third, by analyzing physical and developmental data in a comparative phylogenetic and ecological context we will reconstruct the evolution of transparency to test functional hypotheses (camouflage, communication, thermoregulation, water repellency) and to assess the contribution of history and selection to the evolution of transparency. This project will bring significant advances in our understanding of animal coloration strategies and terrestrial transparency.