Summary: In the world of molecular biology, post-translational modifications (PTMs) play a crucial role in how proteins function within our cells. Among these, glycosylationthe addition of sugar chains to proteins has long been viewed as a process confined to the secretory pathway, specifically within the endoplasmic reticulum and Golgi apparatus. However, emerging evidence suggests that glycosylation may also occur in unexpected places, particularly in the nucleus. This research project aims to investigate the fascinating yet largely unexplored role of glycosylation within the nucleus of cells, a process that may hold significant implications for our understanding of gene regulation and cellular functions. Aims: Our research is structured around three primary aims. First, we will catalog the nuclear glycome—essentially, the complete collection of glycosylated proteins within the nucleus. To achieve this, we will extract nuclei from a diverse range of mammalian cell lines and tissues, using advanced techniques to ensure purity and accuracy. By employing mass spectrometry and glycan-binding assays, we will investigate the diversity and distribution of nuclear glycans, shedding light on their potential roles in cellular functions. Second, we will focus on understanding how these nuclear glycans are synthesized and transported. Despite the primary localization of glycosylation machinery in the secretory pathway, we will utilize innovative genetic techniques, including CRISPR/Cas9, to identify and validate the genes responsible for nuclear glycosylation. This exploration will reveal the mechanisms that allow these modifications to take place in an environment where they were previously thought to be absent. Finally, we will investigate the functional implications of nuclear glycosylation, particularly its impact on RNA-binding proteins (RBPs). Focusing on specific RBPs that play significant roles in gene regulation, we will identify exact sites where sugars attach and create mutant versions that cannot be glycosylated using gene editing techniques. By comparing glycosylated and unglycosylated forms, we will examine changes in protein interactions and cellular localization. This could reveal how glycosylation influences the movement of RBPs between the nucleus and the rest of the cell and their functional roles. In the second part, we will develop an innovative method called \"modification-CLIP\" to understand how glycosylation affects RBPs' ability to bind RNA. This allows us to selectively isolate these proteins along with their bound RNAs, enabling a detailed comparison of RNA partners between glycosylated and non-glycosylated RBPs. Outcome: The anticipated outcomes of this research include a comprehensive characterization of the nuclear glycome, insights into the biosynthesis of nuclear glycans, and a better understanding of how these modifications influence RBP functions. Our findings could redefine current paradigms in cell biology and open new avenues for research into disease mechanisms, particularly in cancers where glycosylation patterns are often altered. Impacts: The impact of this project extends beyond the immediate field of glycobiology. By challenging established concepts and providing new insights into protein regulation, our research could influence various scientific disciplines, including molecular biology, cancer research, and therapeutic development. In summary, this project seeks to illuminate a hidden layer of cellular regulation by exploring glycosylation within the nucleus. Through combining cutting-edge proteomics, bioinformatics, and AI-driven machine learning with cell and RNA biology, we will provide the first systematic study of nuclear glycosylation. This research will reveal, characterize, and map the biosynthesis of nuclear glycosylation on RBPs and uncover how these modifications effect their function.

Mosquitoes are often depicted as the deadliest animals of the world. Despite being a bit confusing, this sentence is partially true: mosquitoes which perform blood feeding on humans are a huge burden for our society, mainly due to their ability to transmit pathogens causing potentially deadly diseases, including plasmodium, which causes malaria, and mosquito-borne viruses, such as dengue, Chikungunya and Zika. Among blood feeding species, only female mosquitoes need to feed on a vertebrate host to acquire blood, which is used as a nutrient source to complete egg development. Indeed, immature females and males are innocuous, as they feed on nectar. Mosquitoes find a human host by exploiting visual, thermal, and olfactory cues; next, they land on the human skin and move their proboscis around until identifying, based on unknown mechanisms, an area to insert their mouthparts. The mosquito proboscis comprises the labium, which is retracted during blood feeding, and the needle-like labrum, which forms the alimentary canal and is inserted in the skin while biting. The labium and the labrum harbour sensory neurons which are thought to be involved in the perception of chemical and physical stimuli during feeding. The labium is committed to nectar perception but could have a role in identifying a good area to pierce while inspecting human skin before biting, while the labrum is involved in blood perception. While probing the skin, mosquitoes salivate and insert and move their stylets in this tissue, to find a blood vessel to pierce. Then, they start engorging with blood and, finally, they remove their mouthparts from the skin and fly away for digestion. Blood feeding is pivotal in mosquito biology and highly relevant for humans, due to the risk of pathogen transmission through the injection of pathogen-enriched saliva at the site of bite. Despite its importance, we know very little about this process and particularly about how chemical stimuli, such as blood-related compounds, or physical stimuli, including temperature or blood flow, are sensed and integrated by sensory neurons within proboscis. This lack of knowledge prevents us from identifying what makes mosquitoes such efficient feeders, impeding the design of targeted innovative tools to interfere with blood feeding and relieve the burden of vector-borne diseases. To fill this knowledge gap, I aim to understand the role of different neurons of the labrum and the labium in the perception of chemical, thermal and mechanical stimuli during blood feeding, by studying the yellow fever mosquito Aedes aegypti, an invasive species competent for the transmission of several arboviruses. To fulfil this objective, I will identify and select different populations of labrum and labium neurons, involved in three sensory modalities (chemosensation, thermosensation, and mechanosensation) during blood feeding. By exploiting genetic engineering, I will identify the chemical, thermal and mechanical stimuli which activate each selected population of neurons. Finally, I will use a behavioral assay for the quantitative evaluation of biting and feeding behaviors of individual mosquitoes, to investigate the effect on blood feeding of the elimination of populations of proboscis neurons. This approach will allow me to identify the role of different neurons in the mosquito proboscis in exploiting different chemical and physical stimuli to locate blood vessels in the skin. The results of this research project will provide new insights about mosquito neurosensory system in blood feeding and could also be exploited to study other processes in these and other insects. The pathways which I will characterize through this project can be targeted in the future to inhibit the blood feeding process in mosquitoes, leading to the development of novel strategies for mosquito management and control of vector-borne diseases.

Memory is one of the brain’s most essential functions, enabling us to recall past episodes and learn from our experiences. At the core of this process are memory engrams – groups of neurons believed to store or “bookmark” memories in the brain. These engram neurons work together through coordinated activity, but how these memory networks are formed and the plasticity mechanisms that govern them remain poorly understood. In the 1940s, Donald Hebb proposed a theory to explain engram formation, famously suggesting that “cells that fire together, wire together”. He posited that repeated co-activity strengthens the connections between neurons, allowing the activation of a subset of the network to reinstate an entire memory through pattern completion – where the network is driven into a state that recalls the full memory. In such systems, memory is distributed across many neurons, meaning the loss of some neurons does not entirely erase the memory. This theory raises intriguing possibilities: if repeated co-activity is sufficient to bind neurons into a memory network, could it be possible to artificially recruit non-engram neurons into an existing engram and have them share the memory? Moreover, if the original engram neurons were subsequently removed, would the memory still persist in these newly incorporated neurons? My project aims to directly test this hypothesis by using cutting-edge brain-machine interfaces (BMIs) to integrate non-engram neurons into existing memory traces, potentially transferring memories between neuron groups. BMIs are powerful tools that allow real-time control of neuronal activity. While their traditional use leveraged electrophysiological recordings to decode activity patterns for motor rehabilitation, recent advancements in calcium-based BMIs – which combine BMIs with in-vivo calcium imaging – have made it possible to monitor and control genetically defined neurons, such as engrams, with unprecedented precision. In this project, I will use a calcium-based BMI to synchronize the activity of engram neurons representing a contextual fear memory with non-engram neurons – binding them into a unified memory network. Afterward, I will selectively ablate the original engram neurons to test whether the memory is retained in the newly incorporated neurons. To achieve this, I will first use two-photon calcium imaging to longitudinally record the activity of neurons in the hippocampal CA3 region – a critical area for encoding contextual fear memories – before, during, and after fear conditioning. This will allow us to identify patterns of neuronal activity that prime certain neurons for engram allocation and strengthen their functional connectivity upon memory encoding. Next, I will use a BMI to induce co-activity between engram neurons and non-engram neurons, effectively integrating the non-engram neurons into the memory network. Following successful integration, I will use genetic tools to selectively ablate the original engram neurons and assess memory retention by evaluating memory-driven behavioral responses and monitoring neuronal coactivation during memory recall. This project holds the potential to reveal critical insights into the plasticity rules that govern memory networks. By demonstrating that memories can be transferred or expanded to include new neurons, this project could establish a biologically feasible technique using BMIs to restructure memory networks. Such advancements could lead to therapies for memory disorders, providing ways to restore lost memories or alter maladaptive ones.

Proteins are molecular machines that are necessary for us and our cells to survive as they are involved in nearly every process in life. For example, they enable uptake and digestion of nutrients, movement or perception of and communication with the environment. For many of these processes, proteins have to work together in large protein complexes to achieve a certain goal. This is comparable to how a car is assembled from multiple parts that all function together. During its lifetime, the protein complex as well the car experience wear and tear that will eventually cause individual parts to break. If the car owner wants to continue driving, either the broken part in the car has to be replaced or they have to buy a new car. Additionally, the damaged piece has to be discarded correctly as a cluttered environment impairs cellular processes comparable to how it is difficult to work in a messy garage. Like the manufacturing of a car, production of proteins is a complicated process that costs a lot of energy and resources. Therefore, the cell aims to maintain a balance between production and degradation of proteins to ensure that there are enough functioning protein complexes to survive without cluttering the cell or tying up too many resources. In this proposal, I am suggesting to study whether individual subunits of a protein complex can be replaced like a broken car part or whether the whole complex has to be exchanged. I will also determine whether damaging different proteins in the same complex can lead to different outcomes. Similarly, some parts in the car, like a broken bulb, can be easily replaced, while other parts like the engine or the car frame might not be worth exchanging. In the latter case, it might be more cost and time efficient to buy a new car. Like the car frame, proteins that will lead to degradation of the whole complex are most likely found in its core and are essential for stability. Furthermore, I will analyze whether specific proteins are needed for the replacement process. While there are general proteins responsible for the degradation of damaged proteins, sometimes specialized factors are necessary for more difficult tasks. This is comparable to the use of a car brand specific tool, when a normal screwdriver is not suited for the repair. After determining the identity of the necessary protein tools, I will analyze how they exert their function to improve understanding of why specialized repair proteins are required. Overall, the proposed study will increase knowledge of how damaged proteins in protein complexes are replaced. I will show whether the cell is able to perform cost effective repairs and which other proteins are necessary for the process.

Tertiary lymphoid structures (TLS) are immune cell aggregates resembling lymph nodes that form in conditions of chronic inflammation or cancer. Interestingly, tertiary lymphoid structures are widely observed in the clinics, and correlate with detrimental prognosis in autoimmune diseases, COVID infection and transplant patients, but with favorable therapeutic outcomes in several cancer types. While their prognostic value patients is widely recognized, the molecular mechanisms underlying these clinical associations remain elusive. Indeed, little is known about where, why and how TLS form. Most importantly, their immune function and contribution to disease course remain elusive. Here, I propose to study TLS formation and functions in the context of infection with hepatitis C virus (HCV), a virus that affects 1 to 3% of the worldwide population and causes liver damage and hepatocellular carcinoma. TLS are a diagnostic feature of chronic HCV infection, however their role in promoting viral persistence and disease progression is unknown. My aims are to (1) map cellular interactions in TLS arising upon HCV infection, (2) perturb TLS formation in order to functionally evaluate their contribution to disease progression to cirrhosis and hepatocellular carcinoma and (3) track the dynamics of TLS formation and the lymphocyte trafficking to and from these structures. To interrogate the cellular interactions that lead to TLS formation and maturation, this project will combine a mouse model of chronic hepatitis C infection with the LIPSTIC technology, recently conceived in the Victora laboratory. LIPSTIC (Labeling of Immune Partnerships by SorTagging Intercellular Contacts) is a method to study cell-cell interactions in the mouse based on enzymatic transfer of a labeled substrate from one cell to another. Like actual lipstick, this system allows tracking of cellular kiss-and-run events and can be applied to contact-trace immune and non-immune cells. Once identified, the cellular interactions occurring in the liver during hepatitis C infection will be tested to uncover which ones are promoting or suppressing formation of TLS. We will further track the trafficking of cells to and from TLS using photoconversion, a technique that allows to color cells at specific locations by shining a light on them. Cells can then be followed as they travel through the body. Finally, we will generate a mouse model in which TLS formation and location can be visualized in living mice, allowing for monitoring of these structures over time and greatly reducing the number of animals used for this study. This project, which leverages my expertise in in vivo screening in the liver and the Victora lab’s expertise in recording lymphocyte interactions and dynamics, will yield insights into the cellular events leading to TLS formation and maturation upon hepatitis C virus infection, with unprecedented resolution. It will further our knowledge of TLS function and uncover their role in regulating disease progression and tumorigenesis. Furthermore, it will generate tools to study TLS biology in other pathological settings such as infections by respiratory viruses, autoimmune diseases and cancer.

Cancer is one of the top threats to human health and a leading cause of death worldwide, taking away ~10 million lives each year. While significant progress has been made in understanding many aspects of cancer biology, the intricacy of how cancer arises, progresses, and interacts with the body remains inconclusive. Further research is essential for making breakthroughs in diagnosis, treatment, and cure. Emerging evidence has now linked cancer development with defects in the process that makes proteins, i.e., translation, and the levels of key cancer-driving proteins. However, little is known about the precise mechanisms causing these defects. This project is dedicated to providing new insights into the mechanisms that drive cancer development and progression, thus revealing novel diagnostic biomarkers for cancer types and potential targets for anticancer drug development. Here, I propose to expand my toolbox and learn an innovative platform of systems-level technologies to explore defects in the translation process across 50 Cancer Dependency Map (DepMap) cell lines from 5 tumor types (lung, glioblastoma, kidney, colon, and breast cancers; 10 cell lines each). This research is crucial because it has the potential to transform cancer biology by opening the door to systematic determination of the degree of dysfunction in the translation process in human cancers. This approach could also be useful for the study of over 100 other human diseases that have been connected with defects in translation. After completing all the cell analyses, I will learn and perform data processing, data mining, programming, and machine learning to comprehend the findings and identify patterns and signatures in the data. These signatures could be unique to each cancer type and useful for further development as diagnostic tools and therapeutic targets. Our team of experts in systems biology, computational biology, and cancer biology will also collaborate with leading medical institutions to extend the studies to dozens of human sarcoma samples to assess whether similar mechanisms and signatures can also be applied to clinical samples. Not only will this research add new and original knowledge about a frontier area of cancer biology, with creation of a database of defects in the translation process in cancer, but the results will also identify novel potential targets for drug discovery and development as well as the predictive power of the identified signatures for cancer type, prognosis, and drug sensitivity.

Sunlight is a crucial energy source that drives the functioning of aquatic environments. At the core of these ecosystems are phototrophic microbes—tiny organisms that “capture” sunlight and convert it into biochemical energy to support their metabolic needs. This vital process, known as light harvesting, is central to aquatic food webs, enabling microbes to funnel energy into the microbial community. Phototrophic microbes use specialized protein complexes to absorb light energy, initiating a series of chemical reactions that culminate in the production of adenosine tri-phosphate (ATP), a molecule commonly referred to as the universal energy currency of most organisms. Only two types of protein complexes are known to govern this capture of sunlight energy by microbes. One major group of microbes uses a form of chlorophyll to perform photosynthesis in a manner similar to plants. This process is well understood and plays a significant role in shaping microbial communities. However, during the past two decades, discoveries hint at another significant player in these light-harvesting processes: rhodopsin-bearing microbes. These microbes use the rhodopsin protein to pump protons (H+) across their membrane to generate a proton gradient, which is used to produce ATP molecules. Rhodopsin-bearing microbes are tremendously abundant in aquatic environments and can constitute more than half of the microbial population in marine and freshwater ecosystems. They may therefore represent a major source of energy entering microbial communities. However, due to a lack of sensitive methods to measure light harvesting mediated by rhodopsins, we know very little about their ecological contribution. Understanding how much light is captured by rhodopsin-bearing microbes will allow us to measure their ecological impact and offer insights into their roles in the flow of energy and elements in aquatic environments. This project is dedicated to developing tools to explore the contribution and impact of rhodopsin-bearing microbes to microbial light harvesting. The main aim of this project is to develop innovative measurement techniques to investigate how these microbes contribute to light-energy acquisition. We will develop cutting-edge technology to measure light-harvesting at the single microbe level, and to sort those microbes for identification and further analysis. Exploring the contribution of individual microbes is critical to understanding the role of different microbial species, which can vary across time and environments. This project will result in a deeper understanding of the fundamental process of light harvesting and its impact on aquatic microbial populations. This project aims to bring the ecological contribution of rhodopsin-bearing microbes to light harvesting out from the shadows and into the scientific spotlight. By uncovering their ecological significance and providing novel tools to measure their impact, this research holds the potential to make a significant contribution to our understanding of microbial ecosystems.

In Darwin’s tangled bank, organisms of different species make their living not only by exploiting, but also by cooperating with each other. Many species interactions are ecologically specialized, with partners engaging in cooperative and exploitative relationships with just a few other species, and evolving remarkable adaptations which they rely on to maintain those associations. However, these specialized interactions can also evolve in different ways, with parasitic interactions evolving into mutualistic interactions and vice versa, sometimes with surprising rapidity. This is particularly common in host-microbe interactions, in which microbes can rapidly evolve to become either pathogenic or beneficial for plants and animals. In the early 17th century, parts of Dutch society were transfixed by “tulip mania”, a cultural and financial craze over this exotic bulb that had been recently imported from the Ottoman empire. Driving this fad, in part, were tulips that spontaneously and unpredictably sported delicately feathered, multicolored streaks and spots in their flowers, which could not be inherited by seed and was associated with a gradual decline in plant vigor, making the plants that developed these traits particularly rare and valuable. Today, we know this phenomenon, known as color breaking, is a symptom of infection with a plant virus. Though viral infections weaken tulips and are often fatal, a few cultivars dating back as early as the era of tulip mania survive to this day: They harbor the virus, but nonetheless continue to be cultivated and asexually propagated as rare curiosities for their color-broken flowers. The careful maintenance of these cultivars coexisting with the virus living inside their tissues thus constitutes a possible artificial selection experiment on the evolution of viral mutualism, commencing centuries before viruses were even known to science. As a HSFP fellow, I will ask whether surviving color-broken tulips harbor viral mutualists that rapidly evolved during tulip domestication and breeding in Europe, and examine the genomic and ecological mechanisms underpinning viral mutualism. To do so, I will combine multiple approaches, ranging from the reconstruction of tulip virus evolution to the introduction of mutations into engineered tulip virus genomes. My research will address a fundamental question on the emergence of novel host-microbe mutualisms, which has consequences for our understanding of the ecological role of viruses in natural populations, and more broadly the evolutionary flexibility of host-microbe interactions. Beyond its importance for understanding how we can manipulate viruses to benefit agriculture and horticulture in a model organism of commercial & cultural value, my work also sits at the interface of biology and history, illustrating the role of evolutionary phenomena in shaping human history and culture.

Plants are often thought of as stationary and less responsive than animals, but recent research reveals they are much more perceptive than previously believed. Plants have a sophisticated way of sensing their perceptual world - “phyto-umwelt.” In the phyto-umwelt, plants can detect and react to a variety of signals, such as smells, sounds, and vibrations, which help them interact with and adapt to fluctuating environments in complex ways. This project focuses on understanding how plants perceive and respond to different and complex sensations, using apple trees as a study system. I will examine how apple trees react to environmental changes, such as attacks from herbivores, by integrating information from various sensory inputs, including chemical signals released by nearby plants, vibrations caused by insects, and other stimuli in their phyto-umwelt. By studying these responses, the project aims to uncover how plants have developed these sensory abilities and how they use them to adapt and survive. The research will begin with field studies in apple orchards to collect data on various sensory inputs. For example, plants and insects release chemicals called volatile organic compounds (VOCs) into the air and soil, which can signal different conditions or threats. I will also record environmental sounds and vibrations from the environment and nearby insects. By analyzing this data, I will investigate what apple trees experience in their phyto-umwelt and how these different signals might interact (e.g., buzzing sounds paired with insect-released chemicals). Follow-up lab experiments will investigate how apple saplings respond to these stimuli, using advanced molecular techniques to observe their reactions at the cellular level. In particular, I will investigate whether and how a plant’s response differs when exposed to single versus combined sensory inputs. For instance, saplings may show faster or more specific responses to a combination of signals from herbivorous insects than to just one signal alone. In the next phase, I will test whether a plant's ability to sense multiple signals is influenced by its environment, comparing wild and domesticated apple trees. There has long been a debate about whether domestication weakens plant defenses, but the evidence is mixed. It is possible that domesticated plants, living in controlled environments, have become specialized in responding to fewer types of stimuli, potentially losing their ability to handle a broader range of sensory events (e.g. invasive insects). To study this, I will recreate both natural and controlled environments for apple trees and expose them to various familiar and new stimuli to see how they react. By comparing their responses to familiar and new signals, this research will shed light on how plants adapt their sensory systems over time, especially in environments shaped by humans. The findings could have broader implications beyond plant biology. Understanding how plants process sensory information without a centralized nervous system, like the brain and spine, might offer new insights into information processing in technology, such as artificial intelligence and robotics. Additionally, this research could lead to new strategies for sustainable agriculture by improving plant resistance to pests and environmental stressors. Ultimately, this project could transform our understanding of how living organisms perceive their environment, potentially opening new avenues for research across the life sciences, from the molecular mechanisms of sensory perception to broader ecological and evolutionary contexts.

Antibiotic resistance is a growing global health crisis, making many bacterial infections harder to treat, leading to millions of deaths annually and threatening the success of routine medical procedures. The rise of resistant bacteria outpaces the development of new antibiotics, highlighting the urgency for improved treatment strategies. One reason for treatment failure is that genetically identical bacteria often react differently to the same antibiotic, where not all bacterial cells succumb; a few tolerant cells regrow, resulting in persistent infections. Understanding why certain bacteria survive antibiotic treatment, while others do not, is crucial for addressing this spreading pandemic of antibiotic resistance. Antibiotics typically target essential bacterial components like the cell membrane, DNA replication, or protein synthesis, which are encoded by essential genes and present in every cell. Thus, these drugs affect all the cells in a population, but not all cells in the population are killed. The underlying cellular mechanisms and behaviors in response to antibiotic treatment that lead to variability in cell death remain poorly understood. Interestingly, the cellular antibiotic response can either allow bacteria to survive or, paradoxically, contribute to cell death. While genetic resistance—which is passed down to subsequent generations—is well-studied, phenotypic resistance—where individual cells temporarily tolerate antibiotics due to environmental factors—is less understood and more challenging to study without advanced techniques. My proposed research aims to fill this gap by examining global gene expression fingerprints at the single bacterial cell resolution to identify key genes that can predict cell survival under antibiotics. Each bacterial genome acts as a codebook for producing thousands of proteins. It is the timing, levels, and lifetime of these proteins that dictate how individual bacteria behave under stress. I will probe the transcription of genes encoding every protein in the bacterial cell to identify the key genes that enable bacterial survival under antibiotics. The three essential developments to assay such a high throughput library include: a) time-lapse imaging to track lineage information b) single cell resolution assay to visualize individual bacteria, and c) high throughout scaling using custom built microscopes and genetic barcoding to assay the whole library in each experiment. This systematic approach will be made possible by harnessing technological advancement in the labs of my mentors – Johan Paulsson and Ethan Garner - at Harvard University and their collaborators. I will use a microfluidic based system with quantitative time-lapse imaging, which allows for the analysis of individual bacteria over many generations. This system has been advanced by my host labs, enabling the tracking of over 1 million bacterial lineages over time in independent growth trenches, which will allow me to capture an entire library of transcriptional reporter strains in an optical-pooled-screen approach, with each growth trench serving as a distinct experiment. By analysing individual lineages using machine learning, I aim to identify candidate genes whose expression differentiates live cells from dead ones under antibiotic treatment. Modulating the levels of protein products of the key genes associated with dead cells will aid in developing strategies to enhance bacterial susceptibility to antibiotics. Additionally, the findings will be applied to multidrug-resistant pathogens to evaluate if similar mechanisms can be targeted to improve treatment efficacy or whether the tolerance mechanisms are pathogen-specific. This project has the potential to make a significant impact beyond antibiotic resistance by offering valuable insights into other fields such as cancer research, immunology, and developmental biology, where understanding variability in cellular responses is vital.

Ultraviolet light (UV) from direct sun exposure can induce skin inflammation, sunburn and skin cancers in humans. Skin lipids (fats or oils) play an essential role in maintaining skin integrity and barrier function in protecting the body from foreign microbes and from the harmful effects of UV. Here, I hypothesize that natural skin lipids are altered by UV exposure and accumulate in the skin subsequently promoting inflammatory responses via a skin-specific novel (‘CD1’) pathway. Specifically, I will study the role of immune cells (‘T cells’) which are usually involved in the defence against infections, but in some cases can cause excess skin inflammation in chronic conditions such as psoriasis and other diseases. Despite research over many decades, we still know relatively little about why the T cells are active in inflamed skin. The proposed project therefore aims to analyse the T cell pathways driving skin inflammatory responses using high-throughput approaches, with the use of state-of-the-art technologies. Based on the functional and phenotypic readout of these approaches, will enable us to gain fundamental biological insights into alterations in human skin upon UV exposure in skin disease settings. Overall, these proposed studies have the potential to challenge existing ideas of how T cells function and will assess the effect of environmental UV on skin inflammation and disease.

In the human body, large structures such as tissues and organs are formed by the development of specialized structures made up of different types of cells. The cells need to adopt the desired cell identities for their different functions (e.g. muscle or neuron) and need to know when to divide or die. The goal of this project is to visualize a biological process called Notch signaling, which plays an essential role in human development by guiding many of these cell fate decisions. In normal Notch signaling, Delta or Jagged proteins on signal-sending cells bind to and pull on Notch proteins present on signal-receiving cells. By pulling on Notch, Delta or Jagged renders Notch susceptible to precise processing by molecular scissors. The processed Notch then enters the receiver cell nucleus, acting as an effector to stimulate changes in gene expression that specify cell fate. Aberrant Notch signaling, on the other hand, can result in cancer or other diseases. For example, mutations of the human Notch1 protein are causal in more than half of human T-cell acute lymphocytic leukemias. Mutations of human Notch2 or human Jagged1 also cause a devastating developmental disorder called Alagille syndrome that is often lethal at an early age. It has been recently discovered that the Delta and Notch molecules that perform Notch signaling are enriched on the cell surface at the sites of contact between signal-sending and receiving cells. These enriched sites are termed the “Notch synapse”, akin to the synapses that exist between cells in neuronal signaling. These synapses were observed with moderate magnifications that show the process at the cellular level. However, to see the finer details of the interaction at the subcellular level requires much higher magnification using the powerful new tools of electron microscopy. This project thus aims to use electron microscopy to view synapses between Notch and Delta cells at a resolution that can discern the molecular interactions taking place. By imaging the synaptic sites, information will be obtained about the arrangement of cell surface molecules and whether they cluster or use auxiliary systems for stability. The project will also study the role of the cell’s internal framework system, the cytoskeleton, in Notch signaling. The area directly inward from the synapse will be examined to determine the influence of the cell cytoskeleton in creating or maintaining synapses. In other cellular processes, the cytoskeleton is used as a railway network to move cargo around the cell. Lastly, the project aims to move beyond single cell pair interactions to organoid models. Organoids are three-dimensional clusters of cells that can be formed in a laboratory, outside of a host. Under optimized growth conditions, they can be stimulated to mimic human tissues and even the full complexity of an organ. It is not yet known whether Notch synapses form in organoids, and if so the extent of formation, as these cells have much more complex contacts with adjacent cells than the cell pairs. This project thus aims to utilize three avenues of research to increase our understanding of the Notch synapse, ranging from the molecular level to the complexity of an organoid. Because Notch signaling is required for the development and function of many tissues and organs (e.g. heart, brain, muscle, intestine, liver, immune system, etc.), the discoveries we will make in this project will have implications both for many areas of human biology, including developmental biology and immunology, and for the many diseases associated with aberrant Notch signaling, including cardiovascular disease and cancer.

Cells, the basic units of life, come in many shapes and sizes. Whether they're part of larger organisms like plants and animals or exist independently as single-celled organisms, their structure is essential to how they function. A cell's shape and organisation are largely determined by its internal skeleton, called the cytoskeleton. One of the key components of this internal framework is a structure known as microtubules. In most cells, microtubules form the backbone of important extensions called flagella or cilia. Flagella are whip-like structures that enable cell movement, as seen in sperm cells, for example. Cilia, present in nearly all cells, help with sensing the environment and communication between cells. Both flagella and cilia are assembled from a structure called the centriole, which also plays a critical role during cell division, helping ensure that genetic material (DNA) is properly separated and distributed to new cells. While typical animal cells have centrioles and flagella/cilia, many organisms have lost these structures over the course of evolution. Instead, their cytoskeletons are organized around alternative structures, such as those found in cellular slime moulds or yeasts, or through dynamic self-organisation, as seen in plants and many amoebae. The evolutionary origins of these alternative organising centres remain poorly understood. Given that the last common ancestor of all modern eukaryotes was almost certainly capable of forming both flagella and centrioles, organisms with non-centriolar organising centres must have evolved from ancestors that once possessed fully functional centrioles. This raises two questions: Did these organisms simplify their centrioles over time, or did they develop entirely new ways to organise their cells? Answering these questions is crucial as it will deepen our understanding of how life's complexity emerged and evolved. My research aims to address this by studying Meteora sporadica, a recently cultivated single-celled protist with an unusual cellular structure. Meteora moves using long protrusions and features several arm-like extensions that swing back and forth, all supported by bundles of microtubules. These microtubules converge at seven small organising centres that closely resemble parts of centrioles found in other cells. We believe these organising centres may be remnants of centrioles that have evolved over time. Notably, Meteora is closely related to other cells with flagella, which further supports the idea that these organising centres are related to centrioles. To explore this question, we will use cutting-edge imaging techniques to develop detailed models of Meteora’s organising centres. Meteora is an extremely small cell, and its organising centres are smaller than the resolution limit of visible-light microscopy. Therefore, we will use cryo-electron tomography, a revolutionary high-resolution imaging technique used to characterise macromolecular scale structures, to create a model of the organising centres. Moreover, we will search its genome to identify proteins of plausible centriolar origin, and generate antibodies to label these proteins with fluorescent tags. We will then use another advanced technique that bypasses the light resolution limit, called expansion microscopy, to pinpoint the location of these proteins in the cell. By using these state-of-the-art techniques, we aim to unlock the mysteries of Meteora’s organizing centers and illuminate the ancient pathways of evolution that shaped the complexity of life.

When we chew, speak, or drink our tongue moves precisely inside our mouth. How are these immaculate tongue movements controlled by our brain? Sometimes you bite your tongue or you choke – these unfortunate mishaps are caused by the uncoordinated action of breathing, tongue, and jaw movements. How do breathing and chewing movements coordinate with those of the tongue? To answer these questions, we need to monitor tongue movements with high temporal and spatial precision in animals that provide experimental access to the neural circuits that control these behaviors. Monitoring tongue movements inside the mouth and their precise millisecond coordination with other concomitant motor actions (e.g., breathing and chewing) is challenging. In my proposed project, I propose to integrate micro-X-ray reconstruction of moving morphology (micro-XROMM) with genetic and physiological approaches in mice to dissect, manipulate, and record brainstem neural circuits that control 3D intra-oral tongue movement during natural behaviors like chewing and drinking. Micro-XROMM offers access to detailed intra-oral 3D dynamics and kinematics of the tongue at millisecond temporal and submillimeter spatial resolutions. Moreover, mice offer unparalleled molecular-genetic selectivity for accessing and manipulating specific mammalian neural circuits. By combining these approaches, I aim to characterize the complex motor space of intra-oral tongue movements and how it is coordinated with chewing and breathing at the behavioral level. I will follow this precise behavioral characterization with anatomical and functional dissection of a putative brainstem neural circuit responsible for this coordination. Our work will lay the foundation for exploring the tongue as a unique sensory-motor system. I will introduce a new framework, based on biological insights, to model the circuit-level control of fine-scale complex dynamics of flexible elastic bodies using the tongue as a model, with implications that extend well beyond neuroscience and clinical medicine to soft robotics and continuum mechanics.

How did hydrogenase enzymes emerge, driving the first metabolic electron flow in early life? How did evolution bridge the transition from slow and unspecific geochemistry to the fast and specific biochemistry we know today? How were redox-active minerals first integrated into a surrounding protein shell? And what is the evolutionary rationale of the diverse hydrogenase groups and subgroups we know today, spanning more than 100,000 organisms? These and more questions will be explored in my research, focusing on the phylogenetic reconstruction, in vivo resurrection and biochemical characterization of ancestral hydrogenases. Overall, I will therefore (i) create a hydrogenase family-tree, (ii) infer the likely protein sequences of ancient hydrogenase enzymes, representing the predecessors of all extant hydrogenases, from the tree, (iii) produce them in applicable model organisms, and (iv) investigate their properties. Hydrogenases are the key drivers of hydrogen-based metabolism, catalyzing either the uptake of hydrogen, resulting in protons and electrons, or the reverse reaction, the production of hydrogen from protons and electrons. Furthermore, hydrogenases are metalloproteins, consisting of an apoprotein that is only built from amino acids, which then incorporates metal clusters that are crucial for enzymatic function, e.g. by allowing for an electron flow through the enzyme. Therefore, hydrogenases can be classified both by (i) their reaction directionality, driving either hydrogen uptake or production, and (ii) their metal cluster structure, resulting in [NiFe]-, or [FeFe]-hydrogenases. It is proposed that hydrogen served as the first electron donor during the origin of life. In evolution, hydrogenase gain-of-function therefore represents a critical step due to the rise of the first metabolic electron transport chain from hydrogen to CO2, resembling the electron flow between deep sea vents and their environment. Supposedly having originated in the transition from geochemistry to biochemistry, hydrogenases did not only exist in the Last Universal Common Ancestor, but further evolved in all three kingdoms of life. To date, hydrogenase genes have been discovered in >100,000 organisms. In addition, the occurrence of oxygen-tolerant hydrogenases illustrates that, even in geologically modern eras, hydrogenase activity is not limited to anaerobic environmental niches, but plays an important role in many aerobic microorganisms. Apart from interest regarding hydrogenase functioning, evolution and ecological relevance, these enzymes therefore gain industrial attention for their ability to consume and produce hydrogen under biotechnologically feasible conditions. However, many questions regarding this enzyme remain: the catalytic bias of hydrogenases, facilitating either hydrogen uptake or production, still lacks a mechanistic explanation. As hydrogenase enzymes are evolutionarily isolated from other enzyme classes and as these enzymes facilitated the first electron uptake in evolution, their gain-of-function remains to be unsolved. Since the abundance of hydrogenase sequences exponentially increased in recent years, the previously generated phylogenetic trees are outdated. More importantly, no efforts were so far undertaken to resurrect, produce and characterize such similarly novel and ancient enzymes. Addressing these points will be subject of my future research activities: (i) Analysis of hydrogenase diversity, (ii) thereby identifying the protein sequences of primordial hydrogenase ancestors and (iii) production of these ancestors in a model organism and (iv) characterization of the resurrected hydrogenases. Based on these findings, I can (v) propose an evolutionary rationale how hydrogenase classes emerged, (vi) model the transition from geo- to biochemistry, (vii) identify key requirements for hydrogenase activity that (ix) can pave the way for potential industrial applications.

As a parent, there’s nothing quite like the joy of watching your baby master a new skill. During the first year of life, a baby’s brain undergoes extraordinary changes to support the rapid learning of new abilities. One of the most fascinating questions in neuroscience is how the brain's structure and molecular composition develop to support its complex functions, from the formation of memories and thoughts to motor control and visual perception. Some of the most profound structural developmental changes occur in the cortex, the outer layer responsible for many cognitive functions. The cortex is organized into six distinct layers, each playing a specific role in processing information. As infants grow, these layers mature in a highly coordinated manner, with myelin and iron playing crucial roles in this development. Myelin, which insulates nerve fibers, ensures efficient communication between different brain regions. Iron supports the production of energy required for brain function and contributes to myelin production. My project focuses on investigating how myelin and iron accumulate and distribute in the cortical layers of the infant brain during the critical period of the first years of life. For hundreds of years, research on myelin and iron in the brain was limited to post mortem studies with histological techniques such as microscopy. Therefore, studying molecular changes in the living brain during development was not possible. In this project, I intend to design state of the art magnetic resonance imaging (MRI) techniques for studying myelin and iron dynamics non invasively in the brain of living infants. Traditional weighted MRI, generates images in arbitrary grayscale values, which can vary depending on scanning parameters and equipment. Quantitative MRI (qMRI) techniques have revolutionized the field of MRI by providing biophysical parametric measurements of brain tissue that can be measured in standardized physical units and compared across subjects, sites, and time points. Unlike the 'picture-like' images produced with weighted MRI, qMRI generates parametric brain maps that specifically quantify physical tissue properties. While qMRI is unlikely to ever fully resolve cellular makeup in living humans, it is extremely sensitive to the interaction of water molecules with their molecular environment. Therefore, qMRI has the potential to detect the statistics of the microstructure of brain tissue. I will develop novel biophysical qMRI models for encoding information about the iron and myelin composition in the cortex of infants. Based on these new qMRI techniques, I will map the distribution of myelin and iron across different cortical layers and monitor their changes over the first years of life. This will help in understanding whether changes in these substances are specific to certain layers or regions and how these changes influence overall brain development. Additionally, the research will investigate the relationship between structural changes and functional brain networks, assessing how alterations in myelin and iron impact cognitive development and network formation. The expected outcomes of this research include a deeper understanding of how myelin and iron contribute to functional brain development during infancy. These insights will address critical gaps in our knowledge of how structural changes in the brain support functional development.

Epigenetics refers to the biological processes that alter gene activity in a heritable manner without altering the DNA sequence. These mechanisms are key modulators of cell differentiation, during which a single cell divides, grows, and transforms into various specialized cells, such as neurons. Together, these processes guide the body’s development, shaping all the organs and tissues necessary for a living organism to function properly. Proper cell differentiation is essential for the development and health of any organism, but when this process goes awry, it can lead to pathologies like cancer. Cancer is often viewed as a genetic disease, characterized by mutations that compromise gene integrity and function. However, while adult cancers typically exhibit numerous mutations, pediatric cancers show few or no mutations but the extensive deregulation of epigenetic process. Of note, pediatric cancers seem to result from an aberrant differentiation process. This suggests that disruption of epigenetics processes could lead to an aberrant progression of cell differentiation. Yet, whether this is sufficient to induce cancer still remains elusive. This project aims to fill this gap in knowledge by disrupting these processes during cell differentiation in order to test whether cells transform into a tumoral fate and by providing a comprehensive characterization of the epigenetic profiles during and after these epigenetic perturbations. I will employ advanced techniques that manipulate the expression of epigenetic components in laboratory models of cell differentiation. I will deplete or overexpress proteins of the Polycomb group, epigenetic components whose deregulation was shown to be associated to progression of many types of cancer. I will test whether these manipulations are sufficient to transform cells into tumoral fates. In order to understand the molecular effects of the different perturbations, I will apply cutting-edge tools such as genome sequencing, epigenome profiling, 3D genome conformation mapping and super resolution imaging analysis. These techniques will provide a detailed view of the epigenetic changes occurring in individual cells and across various biological processes. Importantly, epigenetic mechanisms allow cells to acquire a \"memory\" of their previous states but, since epigenetic processes are inherently reversible, restoring epigenetic balance offers the potential to reverse their effects. In this context, I will apply transient perturbations of Polycomb protein levels in order to elucidate to what extent the alterations that they induce are reversible. In this last case, I will co-express a known activated oncogene in addition to applying a transient epigenetic perturbation, in order to test whether and how this additional insult aggravates cell transformation. By understanding the effect of Polycomb perturbations during cell differentiation and whether they are reversible upon restoration of the initial level of these components, we could gain insights into how cells maintain their identity and whether cancer might be reversed by manipulating epigenetics. In summary, this project aims to define cancer as a disease rooted in developmental cell differentiation processes and to deepen our understanding of cell differentiation and development.

The brain is one of the most complex structures in the universe and controls all our behaviors. This means that the immense variety of behaviors displayed by different animals – including ourselves – arose through the evolution of the complex neuronal networks within brains. However, we still do not know how central neuronal circuits change over time, integrating modifications within the brain's highly interconnected networks. Particularly puzzling is that evolution acts on genes but behavior arises through the activity of neuronal networks. How are these two levels integrated? How do evolutionary changes on the gene-level lead to changes in neuronal circuits that themselves lead to behavioral divergences across animals? Understanding the connection between genes, neuronal network function, and behavior will have far-reaching implications beyond the field of circuit evolution, as it will help us to better understand how brains work, and how they can be modified to precisely alter their function. To explore this question, we need a simple yet effective model. The larval olfactory system of the fruit fly, Drosophila, offers a powerful solution because: 1) different Drosophila species have evolved diverse odor-guided behaviors, 2) the olfactory system is numerically simple yet parallels more complex circuits, and 3) the availability of molecular tools and resources. I will use this system to pinpoint key areas of central neuronal circuit evolution, both in terms of function and genetics, by comparing two Drosophila species. One of the uniquely powerful methods I will use is comparative connectomics. Connectomics is a method by which we can create a map of all of the neuronal connections within a circuit in the brain. I will use the connectomic maps generated for two different species, and compare them to each other to identify what has changed in the brain of these two species. Then, I will make use of the powerful genetics available in Drosophilids to investigate how these connectivity changes alter the function of the brain circuits. In particular, using genetics, I will introduce tools that enable us to visualize and manipulate the neuronal activity of networks within the brain in the two species. This will help us to understand whether the observed changes in neuronal connections have a functional consequence. Thereby, I will be able to understand how evolution tinkers with neuronal circuits and changes lead to the differences in behavior between the two species. Because the networks in the brain are so complex, I will use mathematical modelling to help understand their function. Mathematical modelling will be extremely powerful because it allows to simplify complex interactions, and makes predictions for mechanisms, which can be tested in experiments. Next, since evolution acts on genes, I will investigate how the activation of specific genes in the neurons of one species but not the other lead to the observed connectivity differences. I will leverage these insights to re-engineer neuronal circuits across species, using the powerful genetic tools available in D. melanogaster. To re-engineer the behavior of D. melanogaster, I will take the genes from D. erecta and transfer them into D. melanogaster which will eventually lead to a change in neuronal circuitry and behavior. One prediction is that neurons responsible for processing and filtering inputs are hotspots of change and are responsible for the emergence of new behaviors. One indicator is that these neurons are likely to exhibit variations within a species, a crucial element for evolutionary changes to happen. Uncovering the genetic basis of these changes will enhance our understanding of the intricate interactions between genes, neuronal circuits, and behavior in the context of evolution. This will uncover fundamental principles of sensory processing in the brain and shed light on the most exciting question: how do brains evolve?

Mitochondria perform numerous cellular functions and form a highly dynamic network which rapidly modulates its structure through fission and fusion to meet cellular needs. Disturbances in mitochondrial dynamics have severe bioenergetic consequences in all cells but primarily manifest in neurological disorders. In neurons, mitochondria face particular challenges: thousands of mitochondria have to be distributed throughout the neuronal arbores to account for the high energetic demand of neurons. At the same time, neurons are not renewed over a humans' lifetime, meaning that oxidative damage and mitochondrial DNA mutations escaping quality control accumulate over decades. However, little is known about how mitochondrial dynamics ensure lifelong adaptation and rejuvenation of the mitochondrial network. Given that local protein synthesis is an important process that enables the polarized functions of neurons, we hypothesize that it also plays a role in mediating mitochondrial dynamics. However, barriers in spatial and temporal imaging resolution have made it difficult so far to study local protein synthesis in neurons. In this project we will investigate if local protein synthesis is responsible for the local regulation of fission, fusion and mitophagy and how it supports the maintenance of mitochondrial homeostasis in human neurons derived from induced pluripotent stem cells. We will develop a novel live-cell super-resolution imaging pipeline to characterize the dynamics of local mRNA translation in different neuronal compartments based on the SunTag reporter system. Concomitantly, we will image the mitochondrial dynamics events that are mediated by local protein translation in real-time in live neurons. Lastly, we aim to compare local protein translation and mitochondrial dynamics between healthy and aged neurons to identify possible mitochondrial dysfunctions that lead to aging or neurodegenerative diseases. Our proposed methodology will not only allow us to address so far unresolved questions in the mitochondrial field, but it can later also be expanded towards other fundamental cellular processes, potentially opening new research directions.

Imagine trying to recall events from your day: your brain naturally links moments that happen close together, like having lunch and then meeting a friend. But if more time passes between events, like your morning coffee and a meeting two days later, your brain keeps those memories separate. This ability to link or separate memories based on time is crucial for making sense of the world, but how does the brain decide which memories to link? What mechanisms track the time between events and determine how we remember them? My research aims to answer these questions by exploring the role of local molecular clocks, such as clock genes, in the brain. Clock genes are well-known for controlling circadian rhythms, the 24-hour cycle that governs sleep and wakefulness. However, preliminary findings suggest that these genes may also play a critical role in tracking time between experiences, helping the brain decide whether to link or separate memories. Clock genes may serve as internal timers, not just for day and night but for timing the gaps between important moments in our lives. I use Drosophila melanogaster, or fruit flies, as a model for studying how clock genes influence memory. Fruit flies offer a powerful system for brain research because of their genetic tools and the detailed maps we have of their brain’s memory centers. Though flies may seem far from humans, the basic principles of memory formation and time tracking are shared across many species, meaning that what we learn from flies could apply to humans too. We know that experiences occurring within a few hours of each other are more likely to be linked into a single memory, while events spaced further apart are stored separately. For example, in flies, a memory of a rewarding experience (like smelling a sweet odor in a room) can be erased or modified if the fly encounters conflicting information (like the absence of the sweet odor) within a specific time window. I think that clock genes, which are active in the brain's memory centers, may control this time window for linking memories. To test this, I’ll manipulate the activity of clock genes in neurons that form memory and observe how it affects memory linkage. By using advanced imaging techniques, I’ll watch neurons in real-time as the flies experience events spaced closely or far apart. This will help uncover how clock gene activity impacts memory circuits and their ability to link memories. Ultimately, this research will reveal how our brains use internal clocks to organize experiences. Understanding these processes could have important implications for treating memory-related disorders such as PTSD or addiction, where memories are either linked too strongly or not enough. By revealing the responsible genes, this work could lead to new approaches for treating these conditions.