Cardiovascular disease (CVD) is the leading cause of global morbidity and mortality. While psychological stress is a known cardiovascular risk factor, the mechanism by which the brain translates stress into CVD is poorly understood. The underlying cause of CVD is a chronic inflammatory disease called atherosclerosis, the progression and exacerbation of which have been strongly associated with the immune system, including B cells. B cells perform many functions including production of antibodies that provide immunity against disease and cytokines that modulate leukocyte function. However, it remains unknown whether B cells mediate the effects of stress on CVD. Recent studies from the host lab show that stress profoundly affects the number and distribution of B cells in the body. This occurs via a mechanism dependent on the activation of the hypothalamic-pituitary-adrenal axis. In this proposal, I will test the hypothesis that stress aggravates atherosclerosis by modulating B cell function. To recapitulate stress, I will employ a combination of optogenetic and chemogenetic approaches available in the host lab to locally activate specific regions in the brain and characterize the subsequent impact on the phenotype and functional diversity of B cells. I will then identify the B cell-specific mechanisms that mediate the effects of stress on the progression of CVD. These studies will not only delineate potential therapeutic targets for immunomodulation of B cells in prevention and treatment of atherosclerotic CVD, but also provide a direct mechanistic link between stress and chronic inflammation, a general concept with implications beyond atherosclerosis.

The flow of information between different regions of the brain during decision making is a largely unknown process. This is mainly because there are no tools available to disentangle the complex interaction of myriad cell types in the brain, governed by gene expression, connectivity, spatial organization, and other properties. This proposal aims to investigate the anatomical substrate of inter-regional communication at single neuron level. We hypothesize that a specific group of neurons that project directly between brain areas provide the “scaffolding” for the correlation between structural and functional connectivity. Tools developed in the proposed host group, as well as their previous work on the topic, make this question accessible for investigation. We aim to identify this privileged subpopulation of neurons by combining two-photon imaging of activity in single neurons with anatomical projections extracted using Barcoded Anatomy Resolved by Sequencing (BARseq). Successful completion of this project would generate unprecedented datasets that bridge information at all levels–anatomical, genetic, physiological and behavioural. Gaining knowledge of the flow of information in the brain would push the frontiers of what it is one of the grand challenges of our time–to understand how behaviour arises from neural circuits.

With their multiple arms, three hearts, suckered tentacles, camera eyes, and fast colour-changing skin, cephalopods seem to come from an imaginary world. These iconic invertebrates play a major role in balancing the entire marine ecosystem, yet the 4 million metric tons caught annually have led to their global decrease. Cephalopod diet and feeding systems remain enigmatic. They have a ‘beak’ composed of two jaws with no direct contact, a radula, and specialised masticatory muscles. Unravelling the secrets of their feeding system can inform us about their diet and potentially have huge global impact by aiding their survival. In this project, I will bring together for the first-time expertise in cephalopod biology and biomechanics to quantitatively characterize the anatomy and function of the feeding system in different species representing all cephalopod families. I will use a range of techniques based on material characterization and computational modelling to test 4 hypotheses. I will investigate the (1) material content and mechanical properties of the jaws and correlate these with their bite force and diet; (2) shape and surface properties of cephalopod jaws and its potential correlation with their diet; (3) muscular jaw articulation of cephalopods and the mechanisms that control its motion considering a range of diet; (4) impact of overall jaw and muscular joint morphology on the level of mechanical strain across each component. A major part of cephalopod biology will be unravelled in this project, providing new foundations for cephalopod conservation and other areas such as bioinspired soft robotics.

The role of metabolism during developmental processes remains largely unexplored due to challenges in measuring spatiotemporal dynamics of metabolic activities. In the mammalian post-implantation embryo, pluripotent cells differentiate into three germ layers, the ectoderm, mesoderm and endoderm that undergo specific morphogenetic movements during a process known as gastrulation. While it is known that metabolites influence cell fate by modifying the epigenetic and transcriptional states, the question, whether metabolism plays a functional role in germ layer specification, has not been addressed in an in vivo context. The two main aims of the proposed project are to characterize the metabolic profiles that accompany the emergence of germ layers and their possible function during germ layer segregation i.e. symmetry breaking that establishes the primary body axis. To accomplish both aims, I will use gastruloids, aggregates of embryonic stem cells that recapitulate hallmarks of gastrulation, as a model system. The gastruloid system will allow me to combine metabolic measurements, live imaging of biosensors and metabolic state reconstruction, to uncover emerging differences in metabolism among cells of the different germ layers. Further specific manipulations with inhibitors and optogenetic tools will test if the activity of certain metabolic pathways regulates cell fate decisions and coordinates tissue scale cell movements and thus probe possible molecular mechanisms. The findings of this project will reveal fundamental mechanisms underlying the interplay between metabolism and cell fate determination and thereby change our view of how gastrulation is robustly regulated.

Broadcast spawning is a method of reproduction used by many species of coral, where the corals release their gametes in a perfectly timed, synchronous manner to maximise fertilisation. The timing and molecular mechanism behind this extraordinary event are not understood. Coral bleaching and the death of reefs is something that is now firmly anchored in the public consciousness. However, another threat is looming for coral, the asynchrony of spawning. This marginalised ecosystem now faces an even bigger threat, the possibility of not being able to reproduce, spelling disaster not just for marine life, but also for the communities that depend on them. An understanding of this asynchrony is key and I have therefore designed a research plan which examines the molecular mechanisms behind the spawning event. In particular, I aim to study the light biology of coral, as they use multiple light stimuli, including moonlight, to regulate reproduction. To this day, we do not know what photopigments corals employ, where these are located and whether the algal symbiont is key in initiating different spawning steps. Moonlight is the critical signal, however, the full moon is present every 29 days, so another gating mechanism must exist. I aim to find out exactly what this is by varying different parameters in a lab setting. Using temporal differential gene expression analysis around this critical moonlight signal will provide a basis for understanding the key cellular responses driving spawning. Pioneering a deep understanding of reproductive asynchrony will allow the implementation of improved conservation measures and help save the world’s reefs from destruction.

The group setting forms the basis for most social interactions. However, despite the importance of group social behavior in almost every aspect of animal’s lives, very little is known about its neuronal substrates. This can be attributed to challenges associated with reductionist approaches to capture the complexity of group social interactions. Thus, a paradigm shift is necessary. Specifically, we identify these major gaps where most studies have: (1)focused on one brain at a time, (2)often did not consider individual differences and (3)neglected the long-term dynamics of group social behavior, especially in the natural context. To overcome these gaps, I propose an approach that leverages the rich social interactions in groups of bats, a new animal model for group social interactions in neuroscience. Utilizing cutting-edge methodologies that enable simultaneous wireless recording and manipulating of neural activity across the brains of group members, I will study the neural mechanisms of group social interactions as these naturally unfold. I will begin by focusing on two core aspects of group social interaction and their manifestation in the frontal cortex of socializing bats: (1)neuronal representations of individual’s identity as related to social vocal communication, (2)interbrain coupling across group members. Thus, I will study the relationship between group social and neural dynamics extending from the single neuron level in the individual brain to the relationship in neural dynamics across the brains of group members. Combined, this project will allow, for the first time, a unique mapping of the group social network onto the neuronal network of its members.

In order to gain something, sometimes you have to lose something. This is true in our lives, and it is also true for cells to gain special functions. The individual component of the phloem sieve tube, the sieve element (SE), loses the cytoplasm, organelles, and even part of the cell wall to be able to transport photosynthetic products and signaling molecules over long distance. However, the molecular mechanisms behind these dramatic changes have been shrouded in mystery for more than 60 years since the discovery. The main reason for this is due to the small size of SEs and their deep location in the tissue, which makes them extremely difficult to observe with confocal live imaging. In this proposal, I aim to reveal the autolytic mechanisms of SEs by establishing a novel SE induction system that overcomes such technical hurdles. This research provides new insights into the coupling between autolysis and cell specialization. It also has the potential to expand the field of plant science, as plant development have rarely been studied in terms of degradation control.

Protein quality control systems have been perfected through evolution to guard the proteome. Their capacity, however, decreases during ageing, which in turn threatens proteostasis and exposes humans to plethora of age-related disorders. Currently, a vast majority of these diseases - including neurodegeneration - remain incurable. The next scientific frontier is to convert the broad protein quality control knowledge into tangible benefit to humans by harnessing this intrinsically protective cellular system to foster cellular and organismal resilience to pathogenesis. An unbiased search for compounds that promote cell survival in stress pointed at the phosphorylation of the eIF2a translation initiation factor as a potent modulator of cellular fitness. Thus, eIF2a phosphorylation emerges as a key player in cellular resilience with its manipulation being potentially beneficial in many conditions. This cross-disciplinary proposal aims at using thorough and unbiased set of orthogonal approaches to delineate the role of phosphorylated-eIF2a in human pathogenesis. While exploiting human and mouse genetics, cell biology and pharmacology, I will: i) study the mechanistic bases of diseases genetically linked to eIF2a phosphorylation and the cellular consequences of its alterations; and ii) genetically modulate levels of phosphorylated-eIF2a in neurodegeneration to unambiguously define its role in disease pathogenesis. Together, this project will provide a fundamental, mechanistic framework to define how to manipulate the cellular cost-benefit ratio of eIF2a phosphorylation in pathogenesis and point at conditions that may profit from its modulation.

Over the past decade, programmable gene editing has been transformed by the discovery and development of prokaryotic adaptive immune systems known as CRISPR-Cas. These systems, which evolved to eliminate foreign genetic elements such as plasmid and phage via RNA-guided nucleic acid degradation, have been powerfully redeployed for many gene editing applications. Recently, these systems have been expanded with the discovery of nuclease-deficient CRISPR-Cas systems that direct RNA-guided transposition. Transposon-encoded CRISPR–Cas systems can insert large genetic payloads into bacterial genomes without relying on DNA double-strand breaks or host repair factors, eliminating the major limitations of current integration approaches that rely on CRISPR-Cas9. Nonetheless, the molecular mechanisms of key steps in this newly discovered RNA-guided transposition pathway remain uncharacterized, restricting the applications of this potentially powerful technology. Specifically, the mechanisms through which the transposase machinery is recruited following DNA targeting, the integration orientation of donor DNA is selected, and the megadalton protein-DNA transpososome complex is disassembled, are yet to be elucidated. To determine these mechanisms, I propose to employ a powerful combination of genetic, biochemical, and single-molecule biophysical approaches that is made uniquely possible by integrating the expertise in the Sternberg and Gonzalez laboratories. The results of my studies will transform RNA-guided transposition into a highly tractable, precise, and efficient RNA-guided DNA integration tool for next-generation genome engineering.

Although diseases are clearly harmful, many organisms carry genetic variation that causes disease. Why is this the case? My research proposes to combine techniques from field ecology and population genomics to investigate the forces maintaining genetic variants that cause cancer in swordtail fishes. In Mexico, it was recently discovered that some populations of swordtail fish have high frequencies of genes causing melanoma. Yet, these populations show no signs of losing these genes. I propose to conduct a large field experiment where I will manipulate the two major forms of selection - natural and sexual selection - to identify mechanisms that lead to the preservation of melanoma-causing genes in these populations. I hypothesize that reduced survival due to melanoma is countered by with increased growth rate and attractiveness to mates. My experiment will involve the construction of artificial streams where we can manipulate factors such as exposure to predators, mate choice, and parasitism. Using cutting edge techniques in population genetics, I will quantify directly how our manipulations change selection on genes. I anticipate that the results of this interdisciplinary experiment will yield novel and surprising insights into the forces that maintain disease-causing genetic variation in natural vertebrate populations.

Central nervous systems constantly detect environmental stimuli and integrate them into long-lasting behavioral changes. Adaptations to experience, for example associating a context with a positive or negative outcome represents an important form of learning. Such contextual learning is achieved in part by the modification of transcriptional programs in individual neurons. A broad range of evidence supports that immediate early gene transcription factor (IEG-TF)-dependent induction of gene expression controls various aspects of synaptic plasticity, and learning and memory. However, functions of the downstream activity-dependent gene products themselves are still poorly understood. Behaviorally-induced neuronal activity of hippocampal excitatory neurons was recently discovered to result in a bidirectional adaption of neuronal inhibition by parvalbumin- and cholecystokinin-positive interneurons. This requires a neuropeptide precursor SecretograninII (Scg2), downstream of the IEG-TF Fos, highlighting an unexpected role for a little-studied neuropeptide. In this research proposal I will use a combinatorial approach of molecular, electrophysiological, behavioral and imaging methods to identify how Scg2 mediates behaviorally-induced control of synaptic plasticity and connectivity in the mouse hippocampal microcircuit. In parallel, I will investigate the involvement of Scg2 in establishing neuronal assemblies for encoding memories and behavioral plasticity. Together, these studies will enhance our understanding how gene expression programs and, in particular, neuropeptide signaling regulate synaptic specificity and memory traces in neuronal circuits.

Neurons can be studied by monitoring intracellular calcium ion levels using fluorogenic calcium ion sensors. However, current sensors produce transient signals, potentially missing integral neuron activity and making it challenging to measure accumulated neuron activity over time. Alternatively, genetically encoded calcium ion signal integrators accumulate and integrate neuron activity over a defined time. However, these signal integrators are dependent on a light-assisted photoconversion process and only integrate neuron activity over a short time period. Furthermore, imaging cannot take place during irradiation and even irradiation must be ensured. We propose a calcium ion signal integrator that functions independently of photoconversion for monitoring neuron activity that has accumulated over extended time periods to address these issues. This novel tool consists of a genetically encoded protein sensor (GEPS) that undergoes a conformational change from an opened state to a closed state upon binding to calcium ions. After binding to calcium ions and adopting its closed conformation, the GEPS brings together two cysteines, which both react with a complementary and fluorogenic di-acrylamide probe. Labelling the closed GEPS with this fluorogenic probe slowly produces an irreversible and accumulating fluorescent signal independent of photoconversion but depends on calcium ion concentration. The integrated signal can be later imaged by fluorescence microscopy to identify activated neurons and their relative activity. This novel calcium ion signal integrator will greatly contribute to the future understanding of neuron development and diseases such as stroke.

As the causative agents of toxoplasmosis and malaria, the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. are responsible for a massive burden of disease worldwide. However, our understanding of how these parasites function at a molecular level is incomplete, limiting development of therapeutic interventions. The apicomplexan ubiquitination system represents one such major gap in our knowledge. Ubiquitination is an essential eukaryotic post-translational regulatory system that mediates protein homeostasis and activity. In apicomplexans, ubiquitination is linked to many essential parasite processes, and represents a potential source of drug candidates. However, apicomplexans are far removed from humans and yeast, the species in which ubiquitination is best studied, precluding bioinformatic assignment of critical functions. Recent advances in T. gondii genome engineering provide the opportunity to systematically investigate the apicomplexan ubiquitination system. My goal is to systematically map the entire ubiquitination system of T. gondii and assess the contribution of each component to infection. I will further determine the interactome of components deemed essential, or conditionally essential during stress, and attempt to determine their specific role in regulating parasite growth and infectivity. This project will lay the foundation for research into potential treatments and improve our understanding of apicomplexan biology.

All species need to navigate, forage, and interact with other animals in a constantly changing threedimensional world. For such behaviors, vision is one of the most important senses as it allows animals to map their immediate surroundings from a distance which in turn helps coordinating ongoing and future motor actions. To construct such a map, the visual system uses a wide variety of depth cues such as object size, binocular disparity, texture, accomodation, shading, perspective, motion parallax, and occlusions. However, despite extensive research into the function of visual systems, currently, it still remains unclear what the exact perceptual algorithms and circuit mechanisms underlying depth perception are. Therefore, I propose to investigate how larval zebrafish process depth information and use such cues during hunting behavior. Given its small size, genetic tractability, optically accessible brain, and rapid development of visually driven behaviors, the larval zebrafish is an ideal vertebrate model system to tackle these questions. Starting at 5 days post fertilization, larvae visually hunt small protists. At the start of a hunting sequence, they almost invariably select the closest prey, which suggests that they posses a way of visually estimating distance. In order to investigate how larvae perform this feat, I plan to design and build a novel virtual reality system than can provide both monocular and binocular depth cues to a freely swimming or restrained zebrafish larva. Using this VR system, I then aim to determine which depth cues are utilized and investigate their neuronal representation using whole-brain two-photon imaging.

The dysregulation of protein synthesis in favor of oncogenic proteins is a hallmark of cancer. Recent discoveries revealed that translation of upstream open reading frames (uORFs) within 5´ untranslated regions (5´UTRs) of oncogenic messenger RNAs (mRNAs) contributes substantially to increased oncogene synthesis during cancer initiation. Although we begin to understand the molecular mechanisms underlying the specific programs of protein synthesis, we lack a general picture of the order of events that lead to the remodeled protein synthesis in cancer. Here, I propose to develop, validate and apply a split-fluorescent uORF/mainORF (mORF) reporter system that recapitulates preferential oncogene translation in a squamous cell carcinoma (SCC) model. I aim at developing and characterizing this reporter system in order to record for the first time the translational remodeling in an in vivo mouse cancer model during transition from healthy tissue to different stages of tumor formation. In addition, this approach will enable me to screen for factors involved in the induction of oncogenic drivers by 5´UTR-associated mechanisms. Finally, I will also reveal the molecular mechanisms by which the newly identified factors drive translational reprogramming in cancer. My findings will substantially contribute to our understanding of protein synthesis pathways during health and disease and provide a foundation for the development of novel cancer therapies.

Maternal immune activation (MIA) in pregnant mice has been shown to induce phenotypes resembling those of Autism spectrum disorder (ASD) in offspring, among those are deficiencies in social interaction. A recent study showed social behavior deficits can be temporarily rescued by eliciting an inflammatory response. Specifically, it was shown that administration of lipopolysaccharide (LPS) induces interleukin-17a (IL-17a) or direct administration of IL-17a into primary somatosensory cortex dysgranular zone (S1DZ), promoted sociability in these mice. These results raise the following questions: (a) what is the role of S1DZ in sociability? And (b) how does IL-17a affect the neuronal activity in this brain area that rescues social behavior deficits? In my postdoctoral studies I plan to answer these questions by recording neuronal activity in S1DZ in freely moving MIA-affected and control mice during social interaction and characterize the neuronal representations in this brain area. Moreover, by administrating IL-17a I will study how this cytokine act as a neuromodulator affecting the neuronal activity in S1DZ. Identifying the role of S1DZ in social behavior and the underlying mechanisms increasing sociability that are prompt by the presence of IL-17a, will pave the way to design potential treatments for behavioral abnormalities manifested in ASD phenotypes.

RNA structure plays a crucial role in regulating gene expressions such as translation and RNA degradation. Considering the advance of high-throughput sequencing, researchers are currently able to access both in vivo RNA structures and RNA degradation information over tens of thousands of RNAs in one single experiment. However, it is still unclear which RNA structure feature/motif is generally responsive to RNA degradation in living cells. It is challenging for traditional analysis methods to process the emerging " big data" of RNA structure in vivo, and they are not able to assess the complexity of RNA structure features for the whole RNA, which is over thousands of nucleotides long on average. In this project, I aim to develop novel artificial intelligence (AI) frameworks incorporating deep learning and omics approaches to determine in vivo functional RNA structure motifs in RNA degradation. By integrated multi-omics data, I will construct deep learning models to establish the relationship between RNA structure and RNA degradation and consequently extract the potential RNA motifs. The combination of dry-bench and wet-bench methods will allow me to develop a prediction-validation working scheme to solve this multidisciplinary problem. Based on the identified functional RNA structure motifs, I will also establish corresponding web servers for the community to predict/design the RNA structure motifs for the RNA of interest. The proposed research will make major advances in our fundamental understanding of the RNA structure functionality in RNA degradation in living cells and the AI framework will enable further applications to the complex genomics dataset.

For decades, the nuclear pore complex (NPC) has been considered the sole gateway between the nucleus and the cytoplasm, allowing transport of molecules across the nuclear envelope (NE). However, in recent years NE budding emerged as a non-canonical alternative route for nuclear export of viral nucleocapsids, as well as for large ribonucleoparticles (RNPs). Yet, very little is known about the significance of this unconventional export pathway. The project proposed here aims to elucidate the role of NE budding as a non-canonical route for exporting large-RNPs. The strategy I propose is to track and perturb NE budding transport of large-RNPs. In particular, I will study: (I) the relationship between NE budding and NPC canonical export pathway; (II) its potential involvement in nuclear quality control; and (III) I will test the hypothesis that lamin and Torsin protein families participate in NE budding of large cellular complexes. To follow RNP trafficking and image NE budding events, I will apply state-of-art RNA live-imaging techniques and use a combination of cryo-electron microscopy and tomography (cryo-EMT) and high-resolution live-cell microscopy. Moreover, I will apply biotin proximity ligation assay (BioID) to identify novel RNA and protein cargos transported through NE budding in a Torsin-dependent manner. The results stemming from the work proposed here will represent a significant step towards understanding NE dynamics, and may shift the current dogma of nuclear export.

Lipid bilayer membranes define the outer boundary of a biological cell, protecting its internal volume from elements of the extracellular environment. To gain entry through these membrane barriers, some pathogens fuse their membranes with the membrane of the host cell in a process commonly known as membrane fusion. It is largely believed that placing two membranes near one another can lead to the spontaneous formation of a neck (stalk) joining the two membranes. The stalk eventually resolves into an opening (pore) for material exchange, followed by complete fusion of the two membranes into one. Dedicated proteins (fusogens) found in viruses and cell organelles accelerate this process by facilitating transitions between several intermediate states of the membrane fusion process. While our knowledge of the proteins involved in membrane fusion has steadily accumulated, much less is known about the actual mechanics of membrane fusion, as these processes occur too fast for conventional experimental approaches to capture them with sufficient resolution. This project aims to develop a new approach for studying membrane fusion mechanics by utilizing recent developments in the field of DNA nanotechnology, where molecules of DNA are reprogrammed to fold into well-defined structures of the same size as fusogenic membrane proteins. Using such DNA nanostructures as a type of a molecular scaffold, two membranes with different chemical and physical properties will be placed a prescribed distance away from one another to determine the likelihood of spontaneous fusion. Elements of fusogenic proteins will be incorporated within the DNA nanostructures to generate on-demand membrane fusion events. Advanced microscopy techniques will be combined with computer simulations to verify that such artificial DNA nanostructures can indeed capture the functionality of naturally occurring membrane fusion proteins. Finally, the method will be applied to induce and arrest fusion of a virus with a lipid membrane, providing a highly detailed view of the first step of viral infection. The fundamental insights into the mechanics of membrane fusion as well as the DNA nanotechnology platform to study such processes will open new avenues of investigations of various biological processes, from neuroscience to drug delivery.

Gaining insight into the structure and function of biological membranes is important for understanding cell organization. Yet, despite a great deal of progress with identifying lipids and proteins on biological membranes, we lack an understanding of their organization and movements. We propose to look at how proteins change shape during binding events using two innovative techniques. One technique uses acoustic waves to extract information of the size and shape of proteins determined by their hydrodynamic properties. The other, called atomic force microscopy, uses a miniature detector to rapidly scan a surface, similar to a finger reading braille text, to deliver a microscopic 3D view of the proteins and lipids and how their shapes change during binding events. Models of these events will be created to interpret how they are regulated and controlled. Cells have a complicated network of vehicles, called endosomes, which transport proteins and lipids to wherever they are needed at a moment’s notice. We study this endosomal network in fine detail to get a better idea of how it’s regulated. We build a simplified synthetic “endosome” containing some of the key lipids and proteins found on native endosomes. We monitor protein-lipid and protein-protein binding events to see how the proteins change their shape upon binding. By changing the density and patterns of the proteins on our synthetic “endosome” we seek to understand how a native endosome controls such critical events within the cell. The endosomal network impacts many diseases for example, Alzheimer’s and cardiovascular disease, as well as playing a role the entry of certain pathogens into cells. Also, drugs can be delivered to cells via the endosomal network. Thus, a better understanding of its regulation could impact human health and welfare.