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2018 -
Long-Term Fellowships - LTF

Projection-specific dopamine-dependent synaptic mechanisms in the striatum

MATIAS Sara (PORTUGAL)

Department of Molecular and Cellular Biology - Harvard University - Cambridge - USA

UCHIDA Naoshige (Host supervisor)

The role of midbrain dopamine neurons in learning has been extensively studied over the last two decades, mainly focused on the commonly observed reward prediction error(RPE) activity, i.e. the discrepancy between expected and obtained reward. Still, the field is going through a critical turning point as increasing evidence shows heterogeneity of dopamine neurons’ responses, opening the possibility that dopamine could modulate learning in multiple ways besides through a RPE.
This proposal aims to understand if distinct dopamine projections to the striatum, a structure involved in motivation and action selection, play differential roles in learning. For example, dopamine projections to the ventral striatum signaling a RPE could drive the encoding of value observed in this area, while those to the dorsal striatum could influence the encoding of performance vigor, and those projecting to the tail of the striatum could involve this area in attention. I will also test the hypothesis that corticostriatal plasticity follows common rules across the striatum, but striatal activity will vary across regions due to heterogeneous information conveyed by dopamine innervations. I will use molecular techniques to image simultaneously the activity of dopamine axons and of principal neurons in various striatal regions with two-photon microscopy while mice perform a sensorimotor go/no-go task to achieve rewards and avoid punishment by modulating their gait.
This proposal will elucidate fundamental mechanisms of dopaminergic control of associative learning and motor activity, providing relevant results for the understanding of neurodegenerative diseases such as Parkinson and Huntington’s.

2018 -
Long-Term Fellowships - LTF

Structural and functional investigations of the Trypanosoma brucei mitochondrial RNA editing complex

MATTEI Simone (ITALY)

Department of Biology - ETH Zurich - Zurich - SWITZERLAND

BAN Nenad (Host supervisor)

Trypanosomatids are parasitic protozoa causing a range of devastating diseases. Many mitochondrial genes of these pathogens are transcribed as precursor mRNAs that require insertion and/or deletion of uridylate residues to become functional. The RNA editing reactions are catalysed by the editosome, a large molecular machinery present in the trypanosomatids mitochondria. Editosomes are parasite-specific complexes representing ideal therapeutic targets, however their structural organisation and mode of action are not fully understood. The aim of the proposed research is to obtain a comprehensive structural and functional characterisation of the trypanosomatids RNA editing machinery. To achieve this, I will first produce genetically modified Trypanosoma brucei cell lines expressing tagged editosomal proteins that will allow for rapid and efficient purification of editosome variants under native conditions. I will then use cryo-electron microscopy and single particle analysis to determine high-resolution structural models describing the organisation and the interaction network of the purified editosomes. By determining the structure of the complexes actively engaged in the editing reactions, I also aim at elucidating the conformational states associated with the individual steps of the enzymatic cascade. The proposed studies will provide a broad characterisation of the molecular architecture of the editosomes that will be crucial both for the fundamental understanding of RNA editing and the development of therapeutic strategies against trypanosomatids pathogens.

2018 -
Long-Term Fellowships - LTF

Microbial metabolic networks: the hidden key to resilience of coral algal endosymbionts

MATTHEWS Jennifer (UK)

Climate Change Cluster (C3) - University of Technology Sydney - Sydney - AUSTRALIA

SUGGETT David (Host supervisor)

Bacteria play a ubiquitous role in the ecological success of many plants, algae, and animals. Much of this success reflects the capacity for bacteria to provide resources to their hosts that are otherwise scarce or inaccessible in the immediate environment. Microalgae of the genus Symbiodinium are found globally in tropical and temperate coastal ecosystems, forming symbioses with many invertebrates but notably reef forming corals. Over 50 years of research has shown immense functional divergence amongst the genus Symbiodinium. This functional divergence has commonly been ascribed to fundamental differences in gene acquisition and expression of resource exchange and stress tolerance. However, I hypothesize that obligate associations with bacterial consortia regulate Symbiodinium fitness and functional diversity over space and time. As such, mutualistic bacteria may act as essential “resource surrogates” for Symbiodinium when they live transiently as free-living cells outside of their host corals. The type and health of the algae–bacterial community interactions are entirely unrecognized but in fact, determine the survival of Symbiodinium, and hence the future for reef systems worldwide as they are subjected to accelerating anthropogenic stress. To address this complete unknown, I will use a combination of state of the art techniques (e.g. NanoSIMS, chemotaxis flow imagers and bioreactors) to characterize key ecologically-relevant Symbiodinium-bacterial associations and identify key metabolic networks that promote Symbiodinium ecological resilience. This study will transform our understanding of what makes a healthy coral symbiont, and hence the fitness of reef ecosystems.

2018 -
Long-Term Fellowships - LTF

Do cortical feedback connections store statistical knowledge of the environment?

MAZO Camille (FRANCE)

Cortical Circuits Laboratory - Champalimaud Center for the Unknown - Lisbon - PORTUGAL

PETREANU Leopoldo (Host supervisor)

Perception is shaped both by sensory information and internal variables. Our brains uses regularities of the environment to make predictions, and combines them with sensory stimuli to form percepts. However, how and where is statistical knowledge of the environment stored and learned remains unknown. Visual perception arises from a set of hierarchically-organized cortical areas. Abundant feedback inputs project from higher to lower areas but their role in perception is unknown. In this proposal, I will test if knowledge of the world is stored in the connectional specificity of feedback projections. I hypothesize that the tuning-specific wiring pattern of feedback projections terminating in mouse primary visual cortex reflects spatiotemporal visual statistics of the environment. First, I will assess if sensory experience plays a role in visual feedback wiring. To do so I will visually-deprive mice or raise them in artificial environments with altered visual statistics. Using a novel combination of dual-color optical recordings, I will measure how connectional specificity of functionally-characterized feedback axons in primary visual cortex relates to the animal’s visual experience. I will also determine if spatiotemporal patterns of correlated neuronal activity are sufficient for instructing the observed wiring organization. These experiments will unveil if feedback inputs are a neural substrate for storing learned regularities of the world. By doing so, they will shed light on the computational role of this enigmatic connections while providing a mechanistic description of the internal factors shaping perception.

2018 -
Long-Term Fellowships - LTF

Elucidating the molecular assembly of a pioneer transcription factor with nucleosomes using cryo-EM

MICHAEL Alicia (USA)

Quantitative Biology - Friedrich Miescher Institute for Biomedical Research - Basel - SWITZERLAND

THOMÄ Nicolas (Host supervisor)

Specific regulation of genes is essential for cellular life. In mammals, transcription factors play a critical role in this process as they distinctly mark regions of the genome for expression and are the pillars of cell identity. Reprogramming of a terminally differentiated cell into a pluripotent cell can be accomplished by mere expression of a few transcription factors. These transcription factors regulate developmentally silenced regions of the genome and initiate reprogramming. Fundamentally, how a transcription factor is able to target specific DNA sequences within chromatin is still largely a mystery. Oct4 is one of the key factors in this ‘pluripotent cocktail’ and it can directly interact with nucleosomes. Most transcription factors cannot interact with nucleosomes because of the inaccessibility of DNA wrapped around histones. Pioneer transcription factors, such as Oct4, are able to access these restricted regions of DNA, yet we still do not know how. I propose to use biochemical and structural techniques to elucidate the molecular assembly of Oct4 and chromatin. The results of my proposed work aim to uncover the molecular determinants of a transcription factor for association with nucleosomes and the mechanism by which a transcription factor complex may initiate nucleosome rearrangement/eviction to recreate cell identity.

2018 -
Long-Term Fellowships - LTF

Neural population dynamics during flexible feedback control

MICHAELS Jonathan (CANADA)

Neural Prosthetic Systems Laboratory - Stanford University - Stanford - USA

SHENOY Krishna V. (Host supervisor)

All of our movements require the coordination of dozens of muscles to ensure that we reach our desired goal. Indeed, the ability to perform complex motor corrections that depend on the context, termed flexible feedback control, is a fundamental aspect of human behavior. Three brain areas essential for the preparation and execution of reaching movements are the interconnected dorsal premotor, primary motor, and primary somatosensory cortices. These areas modulate their activity to reflect the context of upcoming actions, but the roles of each area and the underlying computational principles are poorly understood. To address this question, macaque monkeys will be trained to reach targets in virtual reality using a haptic feedback device in response to various mechanical loads and sensory expectations while neural activity is recorded from over 1000 sites simultaneously. To dissect the computational principals of this complex control problem, a modular neural network model of the circuit will be trained to complete goal-directed tasks by learning to control a 50-muscle biomechanical model. Finally, neural projections between these brain areas will be selectively manipulated using optogenetic stimulation to dissect the roles of each of these areas during feedback control. The results of these three experiments will provide a rich bi-directional transfer of information between theory and experiment and significantly improve our understanding of motor control.

2018 -
Long-Term Fellowships - LTF

Exploring cell type-specific variation in the cell division machinery

MIERZWA Beata (AUSTRIA)

Ludwig Institute for Cancer Research - University of California - San Diego - USA

OEGEMA Karen (Host supervisor)
DESAI Arshad (Host supervisor)

Cell division mechanisms are highly conserved, yet mechanistic requirements vary between species and cell types. For example, while epithelial cells round up for mitosis, hematopoietic cells grow in suspension, maintaining their round shape throughout the cell cycle. Division of cells with distinct morphologies likely requires specialized machineries, yet cell type-specific mitotic mechanisms remain largely unexplored.
Understanding specialized cell division machineries might profoundly improve anti-cancer therapies. Many chemotherapeutic drugs target universal cell division pathways, resulting in toxicity due to mitotic perturbation of hematopoiesis, yet the majority of cancers are of epithelial origin. Thus, exploiting cell type-specific vulnerabilities has the potential to increase the specificity of chemotherapy.
The aim of this work is to explore how mitotic mechanisms adapt to diverse challenges in different human cell types and growth geometries. I plan to uncover cell type-specific mitotic pathways by genome-wide CRISPR/Cas9 screening, complemented with a candidate-based approach. First, I will use two screening approaches to compare cellular machineries required for chromosome segregation and cytokinesis. Second, I will explore how epithelial and hematopoietic cells accommodate different demands for shape changes during division by investigating cell type-specific regulation of cortex contractility during mitotic entry and cytokinesis.
Overall, this project will shed light on how cell division mechanisms accommodate specialized requirements in multicellular organisms, with the tremendous potential to uncover tissue-specific targets for cancer therapy.

2018 -
Long-Term Fellowships - LTF

Mechanical stress response and adaptation at the nuclear-cytoskeletal interface

MIROSHNIKOVA Yekaterina (USA)

Helsinki Institute of Life Science - University of Helsinki - Helsinki - FINLAND

WICKSTRÖM Sara A. (Host supervisor)

Skin is a remarkable tissue requiring dynamic and robust behaviors. It acts as a tight bi-directional barrier while being constantly exposed to mechanical stress. The epithelial layer of the skin, the epidermis, acts as load-bearing layer. The presence of tension in the epidermis is critical for its function, however it also constantly threatens the integrity of this tissue which quickly degenerates in the presence of impaired mechanical reinforcement of cell adhesions or the cytoskeleton. Thus it is critical to uncover the poorly understood mechanisms of mechanical stress protection and mechanoadaptation in the epidermis specifically. It is becoming evident that the rheological changes in the nucleus, nuclear envelope, and modifications in chromatin architecture are critical for cellular adaptation and recovery from external mechanical cues, but the mechanisms and precise functional implications of this regulation are unclear. This project aims to investigate the mechanisms and functional consequences of dynamic remodeling of perinuclear actin and heterochromatin in regulating nuclear rheology and thereby the mechanical stress response of epidermal stem cells. Second, based on the preliminary data from the host laboratory, I will test whether strain-induced poly-ADP ribosylation is a mechanopreotective mechanism which, by placing the DNA damage repair machinery in close proximity to the sites of DNA damage enhances the efficiency of repair upon mechanical stress. These studies will unravel molecular mechanisms that allow cells to adapt to mechanical stress through dynamic regulation of nuclear rheology.

2018 -
Long-Term Fellowships - LTF

What does it mean to be modified? From RNA modification to phenotype

MOOIJMAN Dylan (NETHERLANDS)

- EMBL - Heidelberg - GERMANY

EPHRUSSI Anne (Host supervisor)

RNA modifications have been proposed to affect various aspects of RNA biology, including translation and RNA stability. However, how these effects are mediated is not fully understood.
Drosophila melanogaster embryonic development is an excellent model to study the effects of RNA modifications, as RNAs with critical roles during development are highly post transcriptionally regulated. I propose to develop and apply a new method to detect the RNA modification 5-methylcytosine (5mC) transcriptome wide at single nucleotide resolution in the Drosophila embryo. This strategy relies on reverse transcription of RNA and subsequent digestion of the RNA-DNA hybrid with a modification dependent restriction enzyme. The digestion sites are ligated to an adapter containing a T7 promoter, facilitating amplification of the RNA-5mC containing regions. Next, RNA library preparation and Illumina sequencing will reveal the site of modification at the start of the sequence read. The application of this technology will identify the modified RNA species together with the exact location of the modification in Drosophila embryos. In combination with spatial transcriptomics and RNA localization data, in a second step I will elucidate the role of RNA modification status on RNA localization and translation. To reveal the functional consequences of RNA-5mC, I will use affinity purification in combination with mass spectrometry to identify RNA-5mC reader proteins. The abundant genetic tools available in Drosophila will allow me to assess the functional consequences of the removal of each of the reader proteins on the modified transcripts and on a whole organism phenotypical level.

2018 -
Long-Term Fellowships - LTF

Dissecting the functional role of layilin on tumor-infiltrating T cells

MOREAU Joshua (CANADA)

Department of Dermatology - University of California - San Francisco - USA

ROSENBLUM Michael (Host supervisor)

Tumor-infiltrating T cells (TILs) are critical participants in controlling tumor growth, metastatic potential, and ultimate lethality of many cancers. These immune cells act contextually within the tumor microenvironment to either promote tumor clearance or facilitate tumor growth. This is exemplified in the recent successes of the immune checkpoint inhibitors, anti-CTLA-4 and anti-PD-1. In these treatments, subsets of patients experience remarkable tumor regression mediated by rescue of ‘partially exhausted’ CD8+ T cells (peTILs) and negation of the immunosuppressive functions of regulatory T cells (Tregs). However, the dominant molecular pathways preventing TIL-mediated tumor rejection remain obscure. Identification and detailed investigation of these pathways is of fundamental importance in order to understand immune responses to aberrant tissue growth. Recently, the gene LAYN has been identified as highly expressed in the tumor microenvironment and associated with the suppressive signature of Tregs, as well as the exhausted state of CD8+ T cells. Strikingly, high LAYN expression on Tregs correlated with poor clinical outcomes in multiple cancers. Our laboratory has independently observed high LAYN expression on TILs in melanoma patients. As there is a lack of knowledge regarding layilin biology (only 17 publications to date) we propose to comprehensively dissect the functional role of layilin on tumor-infiltrating T cells. We hypothesize that layilin signaling acts to augment Treg suppressive capacity while attenuating CD8+ TIL cytotoxic activity. To address this hypothesis we have generated novel tools to delete or over express layilin in both human and mouse TILs.

2018 -
Career Development Awards

Studying real-life conscious vs. unconscious processing: a novel experimental approach

MUDRIK Liad (ISRAEL)

School of Psychological Sciences and Sagol School of Neuroscience - Tel Aviv University - Tel Aviv - ISRAEL

Consciousness is probably the biological construct we are most intimately familiar with, yet our understanding of its possible functions remains quite poor. Various – sometimes contradicting – accounts range from denying it from having any functional role to considering it a prerequisite for all high-level functioning. Here, I propose a new experimental approach which will hopefully enable us to make the much-needed leap towards a genuine theory of consciousness. I argue that most current methodologies have drifted away from the studied phenomenon: conscious vs. unconscious processing of information. While the latter is held to occur regularly in everyday lives, it takes on a very different form in the lab, when probed using psychophysical techniques which commonly rely on artificial operationalizations having little resemblance to real-life processes. And the stimuli are typically highly degraded, arguably evoking weak signals (and, in turn, non-robust findings). Instead, I suggest two novel ways to get closer to real-life unconscious processing of stimuli with which subjects can interact: the first – a unique method we developed to suppress, for the first time, real-life, actual 3D objects. The second – a virtual reality protocol which mimics unconscious processing of stimuli in real-life environments (e.g., driving or walking). These two experimental paths enable us to examine the processing of strong, non-degraded, real-life/real-life-like stimuli. I propose 7 experiments that – if successful – promise to fundamentally change the way consciousness and its functions are studied and open a new branch of study, with far-reaching theoretical and practical implications.

2018 -
Long-Term Fellowships - LTF

Is gene body methylation under selection in plants? A test using population data in Arabidopsis

MUYLE Aline (BELGIUM)

Department of Ecology and Evolutionary Biology - University of California - Irvine - USA

GAUT Brandon S. (Host supervisor)

Some epigenetic marks are heritable in plants, such as cytosine DNA methylation in the CG dinucleotide context. Because epigenetic marks evolve at rates that are much faster than DNA, they are a potential agent for rapid adaptation to a changing environment, provided they impact fitness. Recent advances in sequencing technology have generated valuable resources, including genomes and more recently methylomes. These data have furthered our understanding about the repressive role of DNA methylation on transposable elements. Surprisingly, however, the role of gene body methylation (gbM) remains enigmatic. gbM is characterized by DNA methylation in the CG context within the body of genes – i.e., between the transcription start site and the transcription termination site – and it is associated with constitutive genes that have moderate to high expression levels. There are two explanations for this phenomenon. One is that gbM is a neutral consequence of transcription without any functional significance. Alternatively, gbM may be functional, in which case it will be subjected to natural selection. This project will search for footprints of selection acting on gbM levels, utilizing ~1000 methylomes available for Arabidopsis thaliana. I will adapt classical population genetic approaches, such as the McDonald-Kreitman test and analyses of the site frequency spectrum, to the study of gbM. By doing so, I will determine whether gbM is under selection in plants and also extend analytical approaches to population methylomic data. If selection on gbM level is detected, the study of the affected genes and their expression patterns may offer clues about gbM function.

2018 -
Long-Term Fellowships - LTF

Integration of multiple diverse microbiota-derived signals by dendritic cells

NAGASHIMA Kazuki (JAPAN)

Department of Bioengineering - Stanford University - Stanford - USA

FISCHBACH Michael (Host supervisor)

The human microbiota produces a wide range of diffusible and cell-associated molecules, many of which are crucial for immune and metabolic homeostasis. The human immune system contains dendritic cells (DC), which sense bacteria-derived molecules through innate immune receptors and initiate an immune reaction that can be immunogenic or tolerogenic in tone. Previous studies have examined the effect of just a single or a few bacterial molecule(s) on the immune system. However, it is not yet understood how the immune system integrates a complex combination of numerous and diverse microbiota-derived molecules and 'decides' what kind of an immune response to mount. In this research, I will systematically elucidate the combinatorial effects of multiple microbiota-derived factors on DCs and identify genes in DCs underlying the integration of multiple signals.
In Aim1, a large library of molecules from the microbiota and microenvironment will be generated. The library includes novel unpublished microbiota-derived molecules, in addition to commercially available microbial molecules and microenvironmental factors (i.e., tissue-specific cytokines).
In Aim2, DCs will be cultured with combinatorial sets of molecules, and their transcriptional profiles and cytokine expression will be determined by multiplexed RNA-seq analysis.
In Aim3, sets of genes required for DC responses will be identified by genome-wide CRISPRi/a screening and comprehensively analyzed using computational techniques.
In Aim4, the results of the in vitro analysis will be tested in vivo using mouse experiments in which germ-free mice are colonized by genetically modified bacteria expressing multiple molecules.

2018 -
Long-Term Fellowships - LTF

Harnessing protein homeostasis for the rejuvenation of aged cells

NAVARRO NEGREDO Paloma (SPAIN)

Department of Genetics - Stanford University - Palo Alto - USA

BRUNET Anne (Host supervisor)

The goal of my proposal is to harness protein homeostasis (proteostasis) to restore adult neural stem cell (NSC) function. Ageing is associated with a striking decrease in adult stem cell function in all tissues. In the nervous system, the number of NSCs and their ability to exit quiescence and to produce new neurones decline dramatically during ageing, which may underlie age-dependent decline in cognitive function. Similarly, disruptions in the proteostasis machinery are associated with accelerated ageing and neurodegenerative diseases. Thus, understanding the molecular mechanisms NSCs use to maintain proteostasis, and how they change with age, may help uncover ways to preserve their regenerative potential during ageing. Recent evidence in the Brunet laboratory has revealed that quiescent NSCs (qNSC) accumulate more aggregates than their activated counterparts, with even higher aggregation seen in old qNSCs. In the proposed project, I will carry out a genome-wide genetic screen to identify proteins involved in the formation of aggregates in qNSCs during ageing and will perform cutting-edge mass spectrometry to identify the proteins present in these aggregates. I will then use this knowledge to determine the function of the aggregates in NSCs, with the goal of restoring old NSC function. Completion of these studies will provide unique mechanistic insights into the regulation of protein aggregation during ageing in regenerative cells and their differentiated progeny. This work will pave the way for building new methods for the ‘rejuvenation’ of old cells and the restoration of proteome solubility, crucial steps in countering age-related decline and pathologies.

2018 -
Long-Term Fellowships - LTF

Torpor as a defence mechanism against pathogens

NIKKANEN Joni (FINLAND)

Cardiovascular Research Institute - University of California - San Francisco - USA

CHAWLA Ajay (Host supervisor)

This project takes a novel approach to study how host organisms combat against pathogens. In general, hosts use resistance to attack and eliminate pathogens, but an alternative method is to employ tolerance, which protects tissues from damage without affecting pathogen burden. The molecular mechanisms of resistance are well established, but the regulation of tolerance is poorly known. Mammals activate tolerance mechanisms when they confront energy-constraining periods, such as infection or starvation. They temporarily abandon the maintenance of body temperature and engage in torpor, which protects organs from tissue damage. Since torpor enables organs to sustain stress without tissue damage, understanding the protective mechanisms might lead to discovery of novel treatment strategies against infections, which might be applicable to other human conditions as well. In the proposed work, I aim to study 1) how animals enter torpor, 2) what are the metabolic signatures of torpid animals and 3) how torpor affects organismal fitness during infections. To accomplish these goals, I will use lipopolysaccharide (LPS) to induce torpor in mice. First, I aim to identify which tissue senses LPS to induce torpor using tissue-specific knock-out mice for TLR4, which is the receptor for LPS. Next, I will perform untargeted metabolomics and transcriptomics to identify metabolic regulators of torpor. Finally, I will ask if torpor affects tissue damage during E. coli infection by assessing immunopathology of the infected animals. The proposed work will expand our understanding of tolerance mechanisms and will, possibly, provide novel strategies to treat infections.

2018 -
Long-Term Fellowships - LTF

Dissecting the molecular mechanisms of arrestin recruitment by G-protein coupled receptors

PAKHARUKOVA Natalia (FINLAND)

Department of Chemistry - Duke University - Durham - USA

LEFKOWITZ Robert J. (Host supervisor)

G-protein coupled receptors (GPCRs) regulate virtually all physiological processes and represent the most common drug target. Apart from the classical signaling, involving the activation of heterotrimeric G-protein, GPCRs also initiate parallel signaling events by interacting with ß-arrestins. ß-arrestins serve as key junctions that receive input from receptors and route the signal to various effectors within the cell. The discovery of signal transduction through ß-arrestins underscores the multifaceted functionality of GPCRs and opens new horizons for future studies. Mapping the extensive GPCR-ß-arrestin-mediated signaling networks offers a possibility to design drugs with greater specificity of action and fewer side effects. However, a detailed structural understanding of interactions between GPCRs and ß-arrestins is still lacking. In this project, I will elucidate the molecular mechanisms of ß-arrestin-1 recruitment by the clinically important ß2-adrenergic receptor (ß2AR). To achieve this goal, I will employ a combination of structural and biophysical methods, such as X-ray crystallography, cryo-electron microscopy and double electron-electron resonance. The project will be performed in the laboratory of Professor Lefkowitz (Duke University, Durham, North Carolina, USA), a leading expert in GPCR biology. I aim to obtain the conformational snapshots of ß2AR-ß-arrestin complex at high resolution, identify the binding interface of the receptor and ß-arrestin and elucidate the dynamics of the complex upon ligand binding. These results will provide a holistic understanding of ß-arrestin recruitment by ß2AR and facilitate the development of novel pathway-specific drugs.

2018 -
Long-Term Fellowships - LTF

Polyphenism, obesity and cancer: searching for alternate states of tumor susceptibility

PANZERI Ilaria (ITALY)

Epigenetic Department - Max Planck Institute of Immunobiology and Epigenetics - Freiburg - GERMANY

POSPISILIK J. Andrew (Host supervisor)

Phenotypic plasticity describes how a single genotype can generate a range of alternative phenotypes. The concept is exemplified by complex trait divergence between monozygotic co-twin pairs. Recently the Pospisilik lab identified a genetic circuit that regulates phenotypic plasticity by buffering against the emergence of alternate epigenetic states. The original Trim28(+/D9) line used for that work represents the first in vivo model for exploring the impact of mammalian polyphenism on complex trait biology and disease.
Intriguingly, both gain- and loss- of function mutations in Trim28 have previously been associated with cancer. Here, I propose focusing on Trim28(+/D9) haploinsufficiency to assess for the first time the impact of bi-stable, epigenetically determined phenotypic variation on cancer. I will monitor tumour latency, progression and spectrum using in vivo imaging and assess tumour heterogeneity down to the single-cell level. In order to gain mechanistic insight, I will use unbiased in vivo shRNA-mediated oncosuppressor screening followed by molecular dissection using human and mouse organotypic cultures in vitro.
My overarching scientific goal is to understand the non-genetic roots of cancer. The unique tools, knowledge and environment of the Pospisilik lab are ideal for assessing how non-genetic variation regulates cancer susceptibility, triggering and progression. If successful, these data will not only drive me much closer to these goals, they will reveal a first gene-gene-environment framework for understanding cancerogenesis in the context of phenotypic variation and epigenetic bi-stability.

2018 -
Long-Term Fellowships - LTF

Measurements of the impact of extinction and invasion on microbial community structure and stability

PEARL MIZRAHI Sivan (ISRAEL)

Physics Department - Massachusetts Institute of Technology - Cambridge - USA

GORE Jeff (Host supervisor)

In recent years there is a growing realization that many species are at risk of extinction due to habitat loss, while the spread of invasive species increases. These changes could influence the stability and function of ecosystems. Our ability to predict how perturbations, such as species extinctions and invasions, affect stability would help protect biodiversity, for example by restoring "keystone species" to sustain ecosystem structure and stability. The aim of the proposed project is to quantify and predict the impact of removal and addition of species on communities. Using a well-defined set of microbial species, we will construct synthetic communities composed of subsets of this set. We will study the effect of removal/addition of species from/to each subset on the species’ abundances. Specifically, the extinction of species as a result of the removal of other species would be of great interest since it will allow the identification of keystone species. These measurements would form a large dataset of experimentally validated co-occurrence networks, and would enable us to identify properties that make communities more vulnerable to the addition or removal of particular species. Next, we will test whether species’ pairwise interactions can be used to predict the community structure. For this aim, we will develop an experimental framework to map the pairwise interactions between all species, their directions and strengths. Finally, we will develop a mathematical model to predict extinction and invasion effects on a given ecosystem.

2018 -
Long-Term Fellowships - LTF

Structural studies of the minor spliceosome

PETER Daniel (GERMANY)

- European Molecular Biology Laboratory - Grenoble - FRANCE

GALEJ Wojciech (Host supervisor)

Non-coding segments - introns are removed from precursor messenger RNAs (pre-mRNAs) through a process known as pre-mRNA splicing, which is catalyzed by two large ribonucleoprotein (RNP) complexes, the ‘major’ and the ‘minor’ spliceosome. While recent structural studies of the major spliceosome have tremendously advanced our understanding of the basic splicing mechanism, the structure and mechanism of the minor spliceosome remains largely elusive.
The minor spliceosome differs in its composition and specificity and no comparable structural insights are available. Therefore I propose to use cryo-electron microscopy (cryo-EM) to study the assembly of the minor spliceosome on its specific pre-mRNA substrates. To achieve this, I will extract endogenous complexes directly from human cells. Affinity tags that I will introduce into the genome by CRISPR/Cas9 gene editing techniques will enable me to purify this large RNP machine from nuclear extracts. I will reconstitute the minor spliceosome on substrate RNAs and analyze them for their composition and homogeneity by mass spectrometry and negative stain EM. Ultimately, I want to obtain atomic models of those complexes by cryo-electron microscopy.
My studies will close the gap in knowledge between both spliceosomes in terms of structural insights and thus i) reveal new aspects about the evolution of the splicing mechanism, since the minor spliceosome might constitute a more simple system that is closer related to lower eukaryotes, and ii) provide a molecular understanding on diseases related to malfunctions of the minor spliceosome as well as opportunities for site-specific targeting of the major spliceosome in cancer.

2018 -
Long-Term Fellowships - LTF

Coordination of mesendoderm fate specification and internalization during zebrafish gastrulation

PINHEIRO Diana (PORTUGAL)

Life Sciences Department - Institute of Science and Technology Austria - Klosterneuburg - AUSTRIA

HEISENBERG Carl-Philipp (Host supervisor)

Embryonic development and organ morphogenesis entail both the correct differentiation of cells into given lineages and their proper positioning along the body axes. During gastrulation in the zebrafish embryo, specification of the germ layers is concurrent to internalization movements, which reposition the future mesendoderm precursors underneath the prospective ectoderm. Nodal/TGF-ß signalling is important for both processes, raising the possibility that cell differentiation and tissue shaping may be coupled. Yet the existence, nature and spatiotemporal scale of the mutual feedback between these processes is still poorly understood. To address this fundamental question, I will follow three lines of research: (1) characterize with high spatiotemporal resolution the dynamics of Nodal-mediated mesendoderm fate acquisition and internalization along the dorsal-ventral margin of the germ ring; (2) dissect the molecular mechanism by which Nodal-mediated cell fate specification is linked to mesendoderm internalization; and, finally, (3) explore whether cell internalization feeds back onto mesendoderm fate specification, via mechanotransduction pathways. This exploratory research project will provide a map of cell specification with unprecedented cellular and tissue resolution and establish a framework to understand how gastrulation movements are coordinated with ongoing fate acquisition and embryonic axes establishment. Since there are striking similarities between the molecular mechanisms underlying tumour growth and metastasis and those governing gastrulation, this work is likely to provide key insights to the understanding of both embryonic development and cancer progression.