Autophagy is a cellular process to degrade cytoplasmic material either for nutrient regeneration during starvation or elimination of unwanted proteins during certain biological events. It represents the cells’ response to maintain homeostasis under conditions of stress. Therefore deregulation of this process is tightly associated with many diseases including cancer, neurodegeneration and aging etc. In general, autophagy is characterized by the engulfment of cytosol and organelles by double membrane vesicles termed autophagosomes. Since its discovery, the cellular origin of the autophagosome has remained controversial. Here, I set out to establish an in vitro pre-autophagosome generation system in order to explore the molecular events that occur during the initial stages of autophagosome formation with special focus on the following issues: 1) the membrane that gives rise to the pre-autophagosome, 2) the organelle membranes supporting pre-autophagosome generation, 3) characteristics of the isolated pre-autophagosome, 4) molecular requirement for pre-autophagosome formation. I believe that a cell-free approach will provide a new way to understand the mechanism and regulation of autophagy.

The human body harbors 100 trillion microbes on its internal and external surfaces—tenfold the number of human cells. Adaptive human coevolution with these microbial inhabitants over millennia has created a complex network of inextricable symbiotic bonds. The bacteria make essential contributions to human metabolism, immune system development, and overall health and in turn benefit from a nutrient-rich intestinal niche. Emerging evidence indicates that microbial species communicate with one another and interact with the host via unique, naturally produced biological molecules. I propose to study the interactions of commensal bacteria with the host immune system and to characterize the molecules and mechanisms they use to confer protection against diseases. Prof. Kasper has discovered that polysaccharide A, a natural molecule produced by an abundant commensal organism, drives immunomodulatory and anti-inflammatory activities promoting mammalian health. This discovery provides a prototype for the development of potential therapies based on beneficial commensal microorganisms. We hypothesize that intestinal bacterial species produce a wealth of immunomodulatory molecules with a largely untapped therapeutic potential. Combining systems- biology, biochemistry, and mathematical models, I propose to mine the microbiota for these molecules, to characterize them, to reveal the design principles underlying host-microbiota relationships, and to evaluate these natural molecules in preclinical studies. This strategy represents a fundamental shift in the paradigm of drug development, exploiting molecules evolutionarily designed to harness the immune system.

The well-evidenced theory of Ramón y Cajal states that neurons are individual entities that communicate through chemical and electrical synapses without any membrane continuity between cells, denying the existence of a neuronal syncytium (multinucleate cells). However, how this neuronal individuality is ensured is still unknown. Recently, it has been shown that in the nematode Caenorhabditis elegans, neuronal self-fusion (fusion within the boundary of a single neuron) occurs during normal development, with neurite branches fusing while remodelling a neurite; similarly, after axonal truncation, the two separated halves of an axon can rejoin by fusion and restore the original axonal tract. These events are highly specific suggesting that molecular machinery is in place to ensure correct fusion (self-fusion) and to prevent fusion to occur between neurites of different neurons (non-self-fusion) that would compromise neuronal identity. This project aims at identifying the key molecules that ensure neuronal individuality, using a genetic approach and C. elegans nervous cells as an experimental system. I will use C. elegans transgenic strains expressing different fluorophores in adjacent neurons as starting material for genetics screens aimed at identifying mutant animals where neurites of different neuron fuse (non-self-fusion), as evidenced by mixing of the fluorophores. I will identify the mutated genes responsible for the loss of neuronal identity and study their function and mechanism of action. These results will provide the first molecular evidence to the neuronal theory of Ramón y Cajal.

Mimicry of host egg colors by cuckoos is a prime example of sensory deception through arms races between hosts and parasites. Parasitic cuckoo chicks impose grave fitness costs upon hosts by destroying progeny and usurping parental care. Hosts respond by recognizing and rejecting foreign eggs and chicks from their nests. In turn, parasites respond through secretive egg-laying and mimicking the appearance of host eggs and young. For example, female common cuckoos (Cuculus canorus) specialize on parasitizing particular host-species by laying an egg-morph whose colors match closely the host egg colors. Although this visual mimicry has been studied extensively from ancient to modern times, the physical, chemical, and structural basis of the eggshell color and pattern mimicry of hosts by parasites remain largely unknown. We aim to use an integrative approach analysis of eggshell pigments and the nano-fabrication of biomimetic materials to generate diverse egg colors. This project will thus provide answers for the age-old question: do cuckoos achieve visual similarity by replicating host egg-pigments and -structure OR is mimicry a result of convergence using different pigments and structure to achieve the same perceptual effect?

Plasmodium falciparum, a Malaria parasite, evades our immune system by antigenic variation. Var is the best characterized gene family undergoing antigenic variation. Monoallelic expression ensures only a single var gene is transcriptionally active, whereas the others are silenced. Factors involved specifically in var gene activation remain unknown. Hence, I will develop an in vivo plasmid pull-down approach combined with quantitative proteomics. A plasmid containing var regulatory sequence elements controlling expression of a selection marker will be used to generate cell pools with either active or silenced plasmid expression. Plasmid-bound proteins will be pulled-down and analyzed by quantitative mass spectrometry to detect candidate proteins specific for transcriptional activation and repression of var genes. More generally, this will provide an attractive assay to study specific DNA-protein interactions in vivo for any virulence gene family in P. falciparum or even in other organisms. Another intriguing aspect of var gene regulation is distinct subnuclear localization of active and inactive var genes at the perinuclear rim. I want to test whether nuclear pore association plays a role in var gene activation using colocalization studies and super-resolution microscopy. Further, I will develop a correlative fluorescence in situ hybridization and electron microscopy approach to study precise var gene positioning within the ultrastructural context of the nucleus. This project will generate a range of new tools to study gene regulation in P. falciparum, improve our understanding of antigenic variation, and potentially provide new drug targets.

Differentiation promoting gene expression programs are maintained in a repressed state in stem cells. This repression is released at the onset of differentiation. One example is the repression of microRNA biogenesis in mammalian stem cells which is released when cells differentiate. Recent data suggest that a similar phenomenon exists in plants, as several miRNAs have been identified in maize whose precursor is transcribed but not processed in the stem cells of the shoot apical meristem (SAM). A potential advantage of this regulation is that differentiation promoting miRNAs are quickly produced when cells start to differentiate, without depending on new transcription, thus quickly activating the appropriate cell fate programs. However, little is known about how miRNA accumulation is regulated. During my postdoc I will investigate the generality and mechanisms of miRNA repression in plant stem cells. First I will identify miRNAs with an inhibited processing in the stem cells of the Arabidopsis SAM by sequencing precursors and mature miRNAs accumulating in different functional domains of the meristem. I will also look for new regulators of the miRNA pathway which determine miRNA accumulation in the SAM. One approach I will use is a reverse genetic screen to test the function of plant homologs of animal miRNA pathway components which are differentially expressed in the SAM. I will complement this strategy with a forward genetic screen to find mutants which release the repressed accumulation of an artificial miRNA in stem cells. This project will significantly contribute to our understanding of stem cell homeostasis and the spatial regulation of miRNA accumulation.

Half of the global primary production is carried out by algae and cyanobacteria in the ocean. The major carbon products are carbohydrates, or glycans, and heterotrophic bacteria effect their removal by binding to and enzymatically degrading them. However, despite their paramount importance in the global carbon cycle, the molecular mechanisms of bacterial interaction with marine glycans remain largely unknown. Here I propose to develop and test a high-throughput method to characterize bacteria and the molecular machinery with which they interact with marine glycans. I will mimic natural carbohydrate particles in the sub-um range, which represent the dominant particle fraction in the ocean, by designing glycan coated magnetic beads [glyco-nanoparticles (GNPs)]. Focusing on four chemically distinct and highly abundant glycans from marine eukaryotes (chitin, laminarin, alginate and fucoidan), these GNPs will serve as an affinity tag since microbial species with complementary binding proteins/enzymes can be captured, identified by sequencing approaches [marker genes and (meta)genomics], and the specific carbohydrate binding proteins then purified and characterized. These GNPs will thus enable for the first time realistic in situ sampling of key players from diverse bacterioplankton communities that execute the degradation of different types of organic particles. This study will foster our quantitative understanding of bacterial interaction with marine carbohydrates by testing how specifically and thus predictably different microbial species interact with marine carbohydrates. We also expect insights into more efficient exploitation of algae as alternative bioenergy sources.

Learning is a key feature of brain function. The cellular foundation of learning lies in synaptic plasticity, which is the ability of synaptic connections between two nerve cells to change in strength. Many synaptic connections are modulated by neurotransmitters that act on a family of receptors called NMDA receptors. Our limited knowledge of the spatial and temporal action of neurotransmitters in general, and those involved in learning in particular, prevents us from fully understanding complex cognitive processes. In this project, we will create Designer Optical neuroTransmitter Sensors (DOTS) that can directly visualize neurotransmitters in intact brain tissue. With these sensors we will be able to ‘watch’ neurotransmitters that activate NMDA receptors at the level of individual neuronal connections in real-time. Our goals are to understand the cellular mechanisms of learning and to establish a general role for designer sensors in biological research.

Neurons in the brain continuously undergo rhythmic oscillations in neuronal excitability. Interconnected neuronal groups synchronize their excitability oscillations, and they do so at different frequencies depending on behavioural state and task demands. Because excitability state determines whether neurons respond to sensory inputs, these grouped oscillations are critical to brain operations. Under high attentive conditions with sparse stimulation, neuronal groups synchronize activities at higher frequencies compared to awake quiescent states. However, many important environmental stimuli are themselves organized in rhythmic streams (e.g., the sounds of people and other animals walking and vocalizing), and in these cases, the input rhythm of the attended stream seems to dictate the frequency at which brain oscillatory rhythms entrain. Such cases have received little attention to date. Similarly the contribution of the brain’s neurotransmitter systems to attentional control of oscillatory entrainment is largely unknown. I aim to: 1) study rhythmic sensory processing in the auditory system of the monkey, and 2) describe how neurotransmitters contribute to attentional control of oscillatory entrainment, and thus to adaptive sensory processing and perception. Monkeys will be trained to alternate attention between concurrent rhythmic streams of stimuli, modelled after naturalistic events streams. Multielectrode laminar arrays will be used to record from numerous brain areas simultaneously to examine how specific neurotransmitter manipulations affect attention-related entrainment within localized neuronal ensembles, as well as between ensembles in different areas

While sleep is essential for survival, its functions remain elusive. Increasing evidence suggests a role for sleep in learning and memory consolidation. Although each learning task uses specific brain regions, sleep has been regarded as a whole brain phenomenon. Thus, sleep seemed to serve neural plasticity at the systems-level, for instance by shutting down external stimuli. However, a recent study reported that in sleep deprived rats, small areas of the cerebral cortex can fall into a sleep-like state while other areas do not (local sleep). When this occurs the animals appear behaviorally awake, and their scalp EEG is typical of waking. The observation provides us with a unique opportunity to explore whether local uses of specific brain regions lead to local sleep.
By implanting multi-unit arrays in brain regions of interest, we can record activity of individual neurons in freely behaving rats and ask whether greater plasticity in one area results in higher probability of local sleep in that area, and greater performance deficits in tasks relying on that area. First, we will study the effects on local sleep of electrically-induced long-term potentiation (LTP) in frontal cortex. Next, we will use physiological stimuli, tasks that rely on motor or frontal cortex. When local sleep is observed specifically in the used region, we will test the correlation between local sleep and performance in these tasks.
This study will allow us to assess the direct link between synaptic plasticity (due to LTP or learning a task) and local sleep, as well as the molecular mechanisms underlying the homeostatic sleep pressure and the impaired performance due to sleep deprivation.

Plants and animals both make use of short and long-range signalling molecules to integrate growth and development, to shape organs and to coordinate stress responses. The chemistry of these hormonal signals is however radically different in plants and animals, as are the receptors responding to them. Receptor kinases with leucine-rich repeat (LRR) extracellular domains form the largest family of receptors in plants. LRR-receptor kinases are central signaling hubs controlling plant growth, development, innate immunity responses and symbiotic interactions. Their corresponding ligands range from small molecule hormones to peptides and entire proteins. There is accumulating evidence that many of these receptors might be activated by interaction with a small family of sequence-related smaller co-receptor kinases. Here, I propose a set of experiments that will inform us how LRR-receptor kinases have evolved to interact with such diverse ligands, and how a small set of co-receptor proteins can mediate receptor activation for all these different complexes. We hope to define a common receptor activation mechanism for LRR-RKs that relies on the interaction of the receptor with shape-complementary co-receptors. A validated activation mechanism for plant membrane receptor kinases will allow us to study cross-talk between different signaling pathways and to specifically interfere with receptor activity in vivo. Our ultimate goal is to translate our findings to the design of receptor agonists and antagonists, to the development of biosensors providing real-time signaling readout, and to the discovery of new receptor – ligand pairs.

Aim 1. To study the role of TPR and Nup153 in chromatin organization.
With this purpose, I will reduce the levels of TPR and Nup153 in human primary cells and subject the different populations to: first, high-resolution chromosome capture conformation (Hi-C) experiments to identify the influence of these proteins in genome architecture; second, replication-timing analyses by CGH-arrays that reflect chromosome architecture; third, epigenetic analyses to address their influence in chromatin compactation and; fourth, in situ hybridization experiments to identify their participation in chromosome territories arrangement. The combination of these methodologies will unequivocally unveil the potential function of TPR and Nup153 in 3D organization of the chromatin in the nucleus.
Aim 2. To characterize the influence of chromatin organization in genome function and stability.
Based on knowledge gained from Aim 1 I will examine how nuclear basket dependent chromosome organization influences DNA metabolism and chromosome stability. To see the influence of TPR and Nup153 in genome stability I will perturb their function and analyze by CGH-arrays and in-situ hybridization experiments numerical and structural chromosomal aberrations. Besides, I will study the impact on gene expression regulation by checking transcription levels of particular candidate genes (to be obtained from Aim1) and conduct expression arrays in the same cell populations. Finally, I will combine DNA replication organization and kinetics techniques to determine the potential mechanistic links between Nups and chromatin organization with DNA replication spatial-temporal regulation and dynamics.

Metastasis is a characteristic feature of malignant tumors and also an important factor to determine the prognosis of cancer patients. Preventing or eliminating metastasis is thus considered one of the most important steps to control cancers. Although previous microarray-based studies have identified crucial gene expression changes in tumor metastasis, the mechanisms that melanoma cells use to acquire metastatic potential remain poorly understood. Here, I propose to combine single nucleotide-level high-throughput analysis with computational and molecular pathological approaches to characterize the genetic changes that underlie melanoma progression from a slow-growing, surgically-curable lesion to aggressive metastatic disease. I will perform single nucleotide-level genome and transcriptome analysis on patient-derived melanomas that have metastatic potential and those that do not have metastatic potential to identify mutations associated with the acquisition of metastatic potential. Although previous methods such as gene expression profiling have focused only on gene expression changes, I will simultaneously analyze expression level and mutation status to pursue the primary causes of melanoma metastasis. I will also test whether mutations I identify functionally contribute melanoma metastasis in xenograft models of patient melanomas, and identify specific mechanisms that confer the potential for melanoma cells to form distant metastases. My work will bridge single nucleotide-level high-throughput analysis, computational modeling, and experimental validation and will reveal new insights into the biology of tumor metastasis that could enhance the management of melanoma.

During early mouse development, cells undergo reprogramming and programming events that are governed by epigenetic regulation. However, there is no knowledge as to how chromatin structure changes during these events. This knowledge is key to understanding developmental potential transitions and epigenetic reprogramming. Here I propose to apply an improved established method, which will provide the first information on global nucleosome positioning and DNA accessibility in the early mammalian embryo.
Secondly, I will determine the nucleosome positioning in the maternal versus the paternal genomes upon fertilisation, key to understand the earliest steps of genome reprogramming.
Thirdly, I will examine the functional relationship between the dynamics of nucleosome positioning by correlating positioning with histone modification by using chromatin-immunoprecipitation technique, a technique that has been recently established in embryos by the host laboratory. Lastly, I will manipulate selected chromatin modifiers known to be essential for early development and ask how nucleosome positioning is altered. This will reveal how nucleosome positioning and DNA accessibility are regulated as well as how this manipulation affects developmental processes.
Through these analyses, I will determine the local and global shifts of nucleosome positioning and DNA accessibility during early mammalian development. This will establish how transitions in chromosome structure occur during reprogramming and programming and will allow us to decipher the effects of chromatin organization on cell potency during developmental reprogramming and programming.

Our goal-directed behaviors critically depend on top-down control, as represented by working memory to hold the relevant information in mind while ignoring the irrelevant. The prefrontal cortex (PFC) is known to play a major role in top-down control of sensory processing. The basal forebrain (BF) also contributes to the modulation of sensory processing through its cholinergic projections to the entire cortices. Since the PFC uniquely sends direct projections to the BF, it is hypothesized that this pathway may constitute a critical top-down control network for sensory processing. However, this hypothesis has not been tested because targeting specific neural pathways was technically difficult. I propose a new paradigm to selectively perturb the pathway from the PFC to the BF using optogenetics in rats. I will express Halorhodopsin (NpHR) and Channelrhodopsin-2 (ChR2) selectively in the PFC cells projecting to the BF, through two virus injections: one virus encoding a fusion protein WGA (wheat germ agglutinin)-Cre into the BF, and another virus encoding Cre-dependent NpHR and ChR2 into the PFC. As a result, only PFC cells projecting to the BF will express NpHR and ChR2. While the rat performs a visual working memory task, I will selectively inhibit and activate these PFC cells by optical stimulation, and simultaneously monitor the activities in the visual cortex and the BF using silicon polytrodes. This study can test how selective perturbation of the neural pathway affects working memory performance and neuronal processing in sensory cortical areas. It will provide a novel paradigm to assess the causal relationship between specific neural pathways and cognitive operations.

Sperm DNA organization is remarkably different than that of somatic cells. This organization results in a ~20-fold condensation and has been suggested to help achieve a number of vital functions: Reduction of the sperm nucleus to form a hydrodynamic sperm head, halting of transcription, protection of inherited DNA from damage and poising of selected genes for activation early in embryogenesis. Moreover, abnormal DNA packaging in sperm has been linked to male infertility. Still, little is known about how the 3D organization attains its functions, mostly due to the inability to measure this organization.
Recently, parallelized Chromatin Conformation Capture (3C) technologies, developed by Dekker et al., have enabled measurement of millions of interactions between DNA regions that are in proximity to one another in the nucleus.
I propose to study the 3D genome organization of human sperm. First, I will experimentally measure DNA interaction maps. I will measure one low-resolution genome-wide map and one high-resolution map of a 1 mb locus. Next, I will develop computational methods for solving the 3D organization from the interaction maps, for searching the 3D organization for repeating substructures and for detecting associations between local structural features and other genomic features. Using these methods, I will study how the sperm genome is organized and how this organization aids in achieving its suggested functions. I expect that this will have a major impact on the understanding of 3D organization and on reproductive and developmental biology. Any developed computational methods will be widely applicable to 3C-based measurements in other species and cell types.

Host defense mechanisms evolved to protect eukaryotic genomes against the negative effects of transposable elements (TE) activity. The control of the TE activity is especially important in the germline, where exacerbated TE activity can impair germ cell development and induce pronounced alterations in the inheritable genetic information. Recently, the existence of an abundance of epigenetically inherited TE-derived small RNAs, known as piwi-interacting RNAs (piRNAs), was uncovered in the Drosophila germline. piRNAs are originated from a small number of discrete loci and are supposed to regulate the activity of homologous TEs in trans. However, mutations impairing the piRNA pathway disturb the development of gametes, thus hampering the study of piRNAs in the germline. To overcome these difficulties, I propose to study piRNA-mediated silencing in the germline by setting up a two-component transgenic system. In this system, a genetically engineered construct will be introduced in the fly genome to produce piRNAs that are homologous to a second, unlinked reporter transgene. Combining this reporter system with cellular and developmental tools, I plan to investigate whether and when piRNAs mediate silencing in trans by following the activity of the target reporter transgene during germline development. In addition, molecular and genetic analyses will be conducted to dissect the mechanisms of piRNA functioning as well as to access the importance of genetic and epigenetic inheritance to the establishment and maintenance of transgenerational silenced states.

The central hypothesis in cortical research envisages a modular, or “canonical” cortical microcircuit, built out of many cell types which are connected in specific ways to implement a defined circuit function. However, there is no unified theory of how the cortex works because exact connectivity diagrams do not exist. Although “connectomes” of whole brains are being sought by large anatomical projects, the actual operational connections and their relative weights will not be revealed until living tissue is studied with a systematic high-throughput approach. This was recently done on inhibitory interneurons which were revealed to be densely connected in the circuit.
The aim of this proposal is to find out how cortical excitatory cells (the pyramidal neurons, which constitute approximately 80% of cortical cells) are connected to each other by combining electrophysiological recordings with an optical method that will sample hundreds of possible synaptic connections in 10 minutes. Brain slices from transgenic mice will be used to genetically target specific cells. Two-photon stimulation of the new opsin C1V1 with spatial light modulator optics as well as uncaging of RuBi-caged glutamate will be used to find cells presynaptic to simultaneously patch clamp recorded neurons. I will also reveal synaptic weight matrices for pyramidal neurons in different layers of the cortex and chains of synaptically connected neurons. Morphology and immunohistochemistry of recorded neurons will be characterised to obtain further information of the circuits. The results will reveal the level of specificity in cortical connectivity and substantially facilitate understanding cortical function.

It is an open question how tumor cell heterogeneity is generated in vivo. The longitudinal (over time) imaging of the tumor development process in living animals is the most direct and straightforward approach to the problem. I propose to use the zebrafish system to perform long-term in vivo imaging, and to apply a novel optogenetic strategy for non-invasive and conditional gene induction. Combined, these tools offer a means to make endogenous single cells become oncogenic and traceable in a heritable manner. My experiments will extract quantitative parameters associated with cell behavior, morphology, and localization, providing novel insights into the properties of individual tumor cells descended from single defined cells. My experiments will introduce selected oncogenic signals into distinct subsets of epithelial cells, and the follow them over time to systematically evaluate the influence of oncogenic signals and initial cell types on heterogeneity. Cells showing distinct properties will be photoconverted during in vivo imaging, extracted ex vivo, and characterized by single cell-based gene expression analysis. The genes selectively regulated in the photoconverted cells will be further investigated by generating reporter models and light-induced over-expression (or suppression) models for revealing the expression dynamics and the molecular functions, respectively, of the target genes during tumor development process. With these new experimental tools, I will define tumor cell heterogeneity in terms of cell dynamics, and study the molecular mechanisms that play pivotal roles in individual tumor cells acquiring their specialized abilities.

A major goal of neuroscience is to identify neuronal circuits that generate specific animal behaviors. This requires methods to activate specific neurons inside a behaving animal. Opto-genetics combining genetics and visible light to specifically activate or silence neurons satisfies this need, at least partially, and has begun to revolutionize the analysis of neuronal circuitries. However, visible light penetrates tissue poorly requiring piercing an optical fiber into the brain. In contrast, magnetic fields penetrate tissue much more easily. However, development of in-vivo magnetic cell stimulation is hindered because the weak interaction between magnetic fields and biomaterial require a transducer to translate the magnetic signal into a biological stimulus.
We propose a truly contact-less, genetically targetable magnetic field stimulation based on a novel nano-transducer. The transducer consists of super-paramagnetic nanoparticles (MNP) which convert the energy of an applied radio-frequency magnetic field into local heat which opens temperature sensitive ion-channels and activates the neuron.
Major novel aspects of our proposal are (a) local subcellular heating of regions of the cell membrane of genetically selected neurons by targeting MNPs to these places via binding to engineered surface receptors; (b) local temperature determination by fluorescence thermo-sensation; (c) self-assembly driven nano-actuators in form of a MNP array tethered to the temperature sensitive ion-channel; and (d) transfer of bacterial magnetosomes synthesis to eukaryotic cells.
The four objectives of the proposed research are A: develop fast magneto-stimulation of neurons in vitro, B: genetically target the transducer to specific cells and cell areas, C: validate the method in vivo, and D: transfer genes to produce MNP into eukaryotic cells. We will follow an interdisciplinary approach that includes methods from synthetic biology, cell and molecular biology, physical chemistry bacterial genetics, circuit neuroscience, and advanced microscopy and optics.
If successful, this research will lay the foundation of a novel field termed “magneto-genetics”, the remote stimulation of genetically selected cells deep inside the body. The potential reaches beyond neuroscience to other stimulatory cells, i.e. insulin secreting island cells, muscle cells and others.