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.

Mapping of dynamic connectomes during fear processing holds promise for transforming our understanding of fear regulation mechanisms and for providing novel insight into the cause of anxiety disorders, in which fear deregulation is the core feature. To achieve this goal, we need to be able to manipulate and record, the activity of single neurons throughout the brain fear circuits in behaving animals. Optogenetics combined with holographic phase shaping enables in vitro and in vivo “all-optical” readout and manipulation of activity in neural circuits with single-spike and single-neuron precision. However, light scattering limits optical imaging and focusing in mammalian brain to shallow depths (<300 µm). Here, we aim at overcoming these limitations by combining the development of new ultratargeted opsins with innovative endoscopy techniques based on emerging concepts of wavefront shaping and wave propagation in complex scattering media. This novel toolbox will be developed through a unique synergetic approach will permit optogenetics and minimally-invasive imaging with single cell precision at unprecedented depths, and will be used to decipher the neural response dynamics underlying the learning and expression of divergent fear responses in the central amygdala circuits in behaving animals.

From the stunning aerial displays of large bird flocks to the collective coordination of human culture, how do the interactions among individuals give rise to such interesting and emergent group behavior, and how are these interactions constructed and controlled within the brain and the body? While collective animal behavior has long fascinated and engaged scientists across disciplines, quantitative studies in three-dimensional environments in which animals naturally live are rare and phenomenological descriptions of collective interactions, on which many models are based, have not previously been connected with their foundation in biological circuits. Here, we propose a novel, cross-disciplinary effort to measure and model the three-dimensional collective dynamics of shoaling in the zebrafish (Danio rerio) and to use genetic, pharmacological and neural perturbations to connect these dynamics to underlying biological mechanisms. We will combine expertise in zebrafish genetics and biology with precision 3D motion tracking and theoretical ideas drawn from statistical physics to quantify and elucidate the interactions that drive such collective motion. We will design a custom apparatus consisting of multiple, fast cameras and image-processing software to capture, with high-resolution in space and time, the simultaneous dynamics of interacting fish in a fully three-dimensional arena. Using this apparatus and leveraging the power of the zebrafish model system, we will quantify the collective dynamics of wild-type shoals and compare them to shoals from zebrafish deficient in their visual and lateral line sensory systems. We will also analyze shoals with disruptions to social neural circuits in the brain, focusing in particular on oxytocin and dopaminergic signaling, using optogenetic imaging. Exploiting this rich abundance of shoaling trajectory data, we will construct maximum entropy and agent-based models of group dynamics, and we will use the parameterization of these models to quantitatively characterize the collective shoaling state and changes of this state between conditions. While shoaling is the focused target of our initial efforts, zebrafish exhibit a variety of social behaviors that can also be studied with our proposed capabilities, both computational and experimental.

The paths we follow when we speak.

The analysis of language processes is traditionally based on discrete, categorical variables, such as noun phrase, suffix or phoneme, quite different in nature from the continuously varying neuronal variables (firing rates, or even spike emission times) that at a microscopic level necessarily underlie them. Forms of analog-to-digital conversion have then to be assumed to link linguistic phenomena, in particular in relation to memory, to cortical network operations. Other memory-related phenomena, however, such as navigation in rodents, are beginning to be understood in detail and to reveal computations that remain analog even at the cognitive level – e.g., the choice of a trajectory in space. Are there analog computations that are relevant to understanding language, in humans?

We address this question at two different scales of complexity. First, in the choice of successive phonemes while uttering a word, which we take to be produced by a well-localized network, perhaps in the left inferior frontal gyrus. It may be envisaged as a continuous trajectory on a ‘phoneme manifold’ which expresses, in the space of all possible vocalizations, the phonological memory of one’s own language(s). We ask how the structure of such a manifold would reflect the statistical learning process with which it is gradually acquired during development, and how it would itself be reflected in the patterns of errors observed when reading aloud. To this end, we shall use network models comprised of individual neuronal units and psycholinguistic tests.

Second, to contrast analog with digital computations within the same paradigm, we consider the memory devices that have culturally evolved to remember extended verbal material, and have crystallized in poetry. Some of them, such as meter, can be thought of as expressing a quasi-continuous trajectory, while others, such as rhyme, are more punctuate and essentially digital. We intend to assess the effectiveness of devices of different nature by manipulating them in network models and in psycholinguistic and EEG experiments involving poetry recall. Since meter and rhyme are embedded in complex constructs including meaning, syntax and other components, which are beyond our scope, we shall represent the whole cortex as a network of Potts units, effectively a model of interacting cortical patches.

Understanding the regulation of somatic stem cell (SSC) self-renewal and their regenerative capacity is essential to understand the decline in SSC function during aging. SSCs are thought to produce adenosine triphosphate (ATP) by glycolysis because of the hypoxic environment, but my hypothesis is that glycolytic energy production is actively switched on to minimize the generation of reactive oxygen species (ROS). ROS are important signaling molecules and I hypothesize they have a relevant role in the regulation of HSC self-renewal and differentiation. Accumulation of mitochondrial DNA point mutations during aging could lead to changed ROS levels as well as translational stress within mitochondria, inducing a mitochondrial unfolded protein response (UPRmt). My aim is to elucidate how mitochondrial function and ROS status change in HSCs and other hematopoietic cells during aging.
A growing amount of data suggest that metabolic regulation is important for SSC homeostasis. Mitochondria are central players in the cellular metabolic pathways, by producing metabolic intermediates and reducing equivalents, and recent data suggest that mitochondrial DNA integrity is essential for normal SSC function. Deciphering the function of mitochondria in SSCs, which has been studied only to a limited extent, particularly in the context of aging, will shed light on the mechanisms of SSC dysfunction underlying aging. I aim to understand how mitochondrial function changes with age in HSCs and how this affects the HSC properties over time and the capacity to regenerate the hematopoietic system.

The cross-talk between integrin-based adhesions to the extracellular matrix and dynamic microtubules plays a crucial role in cell polarity and migration. Integrin-based adhesions can promote cortical microtubule stabilization in their vicinity. In turn, microtubules can strongly affect the formation and turnover of the adhesion sites. However, the molecular basis of these connections remains enigmatic.
Here, we propose to decipher the molecular chain of events induced by mechanotransduction at integrin adhesions that leads to cortical microtubule stabilization and analyze the morphogenetic impact of this process. To achieve this goal, we will bring together four distinct sets of expertise: cell biology of the cytoskeleton (Akhmanova); structural biology, biochemistry and biophysics of protein-protein interactions (Goult); mechanobiology and single molecule analysis of force dependence of protein-protein interactions (Yan) and genetic analysis of morphogenesis in flies (Tanentzapf).
We will use advanced cell manipulation assays to directly test whether mechanotransduction at adhesions affects microtubule capture and stabilization at the adjacent cortical sites. To uncover the molecular basis of this process, we will focus on a recently discovered connection between a cortical adaptor protein, which constitutes a part of the microtubule stabilization complex, and an adhesion component. At the same time, we will perform experiments, which will allow us to identify and explore additional links between focal adhesions and the microtubule-stabilizing complex, and thus generate a comprehensive functional map of these connections. Given that the investigated proteins are highly conserved, we will combine genetics, imaging and modeling to address their impact on morphogenesis and tissue maintenance in flies. Our work will shed light on how cellular mechanics and cytoskeletal dynamics are integrated at the molecular level.

Our body consists of trillions of cells, which derive from a single fertilized egg. The divisions that generate these cells constitute a genealogical tree, with a single root (the fertilized egg) and trillions of terminal branches (each of our cells). Knowing the shape and branching order of this tree is important because it provides vital information about how we developed; cells that are on the same branch shared the same cell precursors and a common history of developmental decisions. The cell lineage tree of an individual is also important for understanding cancer; its origins and history of colonizing the body.
Discovering the cell lineage of an organism is a huge challenge that has been solved only in the simplest cases. In the nematode worm Caenorhabditis, whose body consists of approximately 1000 cells, the complete cell lineage was painstakingly determined by direct observation of each cell division under the microscope. In larger organisms we can only infer partial cell lineages by direct observation, or through a method called clonal analysis in which genetic marks are used to label the progeny of individual cells. Each of these methods has its limitations. Direct observation is only applicable to transparent, tiny organisms and to events that occur within hours or days. Clonal analysis can inform us about the rough structure of the lineage tree, but its precise branching patterns are unresolved.
Here we propose a new strategy. It is based on the idea that random mutations, which accumulate in cells during the lifetime of an organism, can reveal the structure of its lineage tree: cells belonging to a major branch will uniquely share mutations that occurred in their common ancestors, those that lie together on a finer branch will share additional mutations, and so on. In principle, if a sufficient number of mutations were detected, we could infer the complete lineage tree of the individual. In practice, this approach is limited by our ability to find those rare somatic mutations within the genome of individual cells. Our strategy resolves this problem by relying on two new technologies: a method that allows us to generate mutations at specific sites in the genome, combined with a method for detecting these mutations in the organism, with single-cell precision.

Dengue virus (DENV), a mosquito-borne RNA virus, is a leading cause of illness and death affecting up to 100 million people worldwide annually. Similar to all other viruses, DENV relies on cellular ribosomes to produce viral proteins and inhibit the innate immune response, and has therefore developed various strategies to seize control of the translation machinery. In response, host cells have evolved various mechanisms to subvert these attempts and prevent viral replication. The proposed project will combine different approaches to study how DENV interacts with the translation machinery and triggers changes in protein synthesis that facilitate or hinder production, folding and assembly of viral proteins. Specifically, I will use ribosome profiling and mass-spectrometry (MS) to monitor translation in infected cells and better characterize the dynamics of viral RNA translation and host response. Then, I will use CirSeq, which allows high-resolution sequencing of viral populations, to search for mutations in the viral genome that affect association with ribosomes and thus alter translation rates. Finally, I will identify host factors involved in the synthesis of DENV proteins by using quantitative MS to analyze the composition of translating ribosomes from infected cells. This will be followed by biochemical and functional characterization of selected candidates to investigate their possible role in viral replication. This work on DENV will expand our understanding of virus-host interactions and may inform approaches to prevention and treatment.