Conscious perception is a core quality of human cognition. However, a comprehensive empirical account for consciousness remains one of the greatest challenges in modern neuroscience. Theoretical descriptions put forward mechanisms, but their realization in neural tissue is scarcely tested. One gap in knowledge preventing progress is in knowing where exactly to investigate. That is, the understanding of which brain areas are necessary for conscious perception remains elusive. Are there specific brain areas always engaged for conscious awareness and do any contribute more than others? Our objective is to map the functional connectome for stimuli entering and being maintained in conscious awareness. We hypothesize there is a core set of brain areas necessary for the conscious awareness of perceivable stimuli. To test this hypothesis, we will employ electromicrostimulation with functional magnetic resonance imaging (EM-fMRI) in behaving monkeys. Subjects will receive state-of-the-art cranial implants for unprecedented whole-brain access for neural stimulation. This method allows us to (1) probe a vast array of brain areas to determine their role in conscious perception and (2) identify the minimum configuration of brain areas that activate when a stimulus is perceived. Notably, where human fMRI falls short in causal experimental control and monkey electrophysiology in the spatial scope of measurement, the unique combination of EM-fMRI allows for comprehensive evaluation of neural circuitry for conscious perception. Crucially, we design the study to (3) dissociate circuitry for consciousness from that for attention, oft tightly coupled but rarely experimentally distinguished, and (4) test predictions of theoretical accounts for conscious awareness. The task designs orthogonalize attention and conscious access through multimodal electrical and sensory stimulation at the threshold for awareness. Moreover, the results will be explored within theoretical frameworks, including the Global Neuronal Workspace Theory and the Integrated Information Theory and the data can be used to test other theories, because we will publish them open access. The project is highly feasible, given the experience of the Roelfsema Group in probing neural mechanisms of consciousness (e.g., van Vugt et al., 2018, Science) and EM-fMRI (Ekstrom et al., 2008, Science). Beyond local institutional support, Profs. Stanislas Dehaene (Neurospin, FR) and Wim Vanduffel (KU Leuven, BE) are active collaborators whose expertise in consciousness and EM-fMRI will enhance the project and enhance my training. My experience in monkey cognitive neurophysiology has prepared me to pursue these objectives, yet the project affords substantial scientific growth in the form of methodological and conceptual training. Altogether, this project is designed to yield impactful findings that will propel the field of consciousness research forward and prepare me for a career as an independent investigator.

Phytoplankton species account for half of the worldwide CO2 fixation and determine several biogeochemical cycles. Characterizing these organisms' gene expression control mechanisms is critical to understanding their ecological role. Importantly, ocean microbial communities are synchronized by the effect of light fluctuations during the day:night cycle. Environmental and laboratory-based studies have shown different phytoplankton components such as RNA levels, metabolites, and lipids, cycle following diel patterns. Interestingly, the diel pattern also dictates metabolic interactions between single-cell eukaryotes and heterotrophic bacteria. Notably, phytoplankton was shown to have mutualistic and antagonistic interactions with bacteria. However, it is unclear if these interactions are diel regulated and how gene expression is controlled depending on the community composition and the light status. While the application of RNA-seq based protocols has allowed quantifying phytoplankton response at the mRNA level, the translational regulatory layer remains unexplored. Studies in several eukaryotes have shown that mRNAs get translated at specific rates, allowing fine-tuning gene expression, but its role in phytoplankton biology is unknown. In this work, I aim to characterize the phytoplankton translational regulatory landscape focusing on the impact of two key factors, the diel cycle, and bacterial interactions. I plan to use the centric diatom Thalassiosira pseudonana and the pennate diatom Phaeodactylum tricornutum as models, representing two subdivisions of an ecologically relevant phytoplanktonic group (Diatoms). I will measure translation efficiency using ribosome profiling in a culture experiment simulating the day:night light changes and in co-cultures with Ruegeria pomeroyi, a mutualistic bacteria, or Croceibacter atlanticus, an antagonistic bacteria. However, because laboratory models do not represent most species, it is also required to study natural populations directly. Yet, we lack a protocol to measure translational efficiency in them. To solve this constraint, I will develop a new method termed Meta-Riboseq inspired by the principles of ribosome profiling and metatranscriptomic protocols to quantify translational efficiency in natural phytoplankton populations. This combined approach will quantify the extent of translational regulation present in single-celled marine eukaryotes in a controlled experiment and in their environment. Specifically, this setup will allow identifying dynamically translated genes across the diel cycle and specific profiles triggered by mutualistic or antagonistic bacteria-algae interactions. Comparing the two different diatoms models and the diversity within natural populations will allow inferring evolutionary trends in this regulatory layer. In addition, this work will provide a new method and a relevant dataset contributing to further exploration and generation of new hypotheses in the field.

Cyclic nucleotides stimulate the immune response to pathogens and disease by acting as second messengers. In human cells, the enzyme cyclic GMP-AMP synthase (cGAS) recognizes double-stranded DNA from pathogens or tumor cells and produces 2'–5'/3'–5'-cyclic GMP-AMP (2'3'-cGAMP). 2'3'-cGAMP controls antiviral and anti-tumor responses by activating the receptor stimulator of interferon genes (STING) (Sun et al., Science 2012; Ablasser et al., Nature, 2013). Surprisingly, recent studies reveal that prokaryotic and eukaryotic genomes encode various cGAS-like nucleotidyltransferases named cGAS-like Receptors (cGLRs) (Whiteley et al., Nature 2019; Slavik et al., Nature 2021). Animal genomes each encode several cGLRs, and four cGLRs in human cells have no known function. These uncharacterized cGLRs suggest fundamental gaps in our understanding of nucleotide second messenger signaling. I hypothesize that cGLR enzymes in human and animal cells produce diverse nucleotide second messenger signals and control novel immune signaling pathways. My proposed research will combine biochemistry and structural biology approaches with an unbiased cell-based screen to elucidate how uncharacterized animal cGLRs recognize distinct ligands and to discover how cellular receptors respond to distinct nucleotide signals. First, I will use X-ray crystallography and cryo-EM to determine the structures of uncharacterized human cGLRs and a Drosophila enzyme named cGLR1 known to respond to double-stranded RNA. These structural and biochemical analyses will define how animal cGLRs evolved a conserved ligand-binding groove to recognize the various ligands, including viral nucleic acid and pathogen-derived metabolites. Second, I will conduct a cellular activation screen using nucleotide signals produced by prokaryotic cGAS-like enzymes to identify novel protein receptors stabilized in the presence of nucleotide signals. Given that human STING recognizes both prokaryotic cyclic dinucleotides and the endogenous cGAS product 2'3'-cGAMP, I predict that prokaryotic nucleotide signals including pyrimidine-containing cyclic dinucleotides and larger cyclic trinucleotide signals will enable me to discover new nucleotide signal receptors in animal cells. Supporting this notion, we have completed a pilot screen in human macrophages revealing new candidate receptors specifically stabilized in the presence of nucleotide second messenger signals. Analogs of 2'3'-cGAMP are rapidly developing as promising new therapeutic drugs, suggesting the importance of understanding nucleotide second messenger signals in cells. My results will reveal novel signals and nucleotide receptors that control the innate immune response and provide the molecular mechanism of how animal cells respond to viral infection.

Pain is a multidimensional sensory experience produced by complex activity in the nervous system triggered from the periphery through the spinal cord and on to the brain. Under healthy conditions, pain is essential for protecting us from harm, however, in the context of injury or disease of the nervous system, pain loses its defensive role and instead produces persistent suffering and disability, constituting a pathological disease state termed neuropathic pain that affects ~10% of the population worldwide (van Hecke et al., Pain, 2014). Neuropathic pain is categorized by both stimulus-evoked pain and spontaneous pain which possess distinct pathophysiological mechanisms. Of note, the majority of patients complain more of spontaneous pain than evoked pain (Bouhassira et al., Pain, 2005). However, to date preclinical studies are sharply geared only towards studying evoked pain. This is because while an evoked behavioral pain readout can be readily correlated with a stimulus, spontaneous neuropathic pain (SNeuP) arises without an external stimulus and possesses complex dynamics, rendering quantitative study very difficult, and in consequence the fundamental neurobiological mechanisms are essentially unknown (Mogil, Nat. Rev. Neurosci., 2009). I now aim to mechanistically interrogate the cellular circuits responsible for SNeuP in a rodent model of neuropathic pain developed in my host lab (Decosterd and Woolf, Pain, 2000). Importantly, we will leverage a novel behavioral readout in mice: brief awakenings from sleep, which is causally linked to, and a biomarker of SNeuP (unpublished data). We hypothesize that there are specific cortical areas whose neural activity are associated with brief awakenings and SNeuP. To test this, we will combine brain-wide functional imaging with miniaturized microscope-based neuronal calcium imaging in freely behaving mice to determine which cortical activity pattern is associated with brief awakenings. We will also examine whether this pattern is present only in specific cortical areas or occurs globally. Based on the spatiotemporal features of cortical activation, we will further classify the activity patterns in fully awake mice using support-vector machine algorithm, and determine which activation patterns are similar to those occurring during SNeuP-evoked brief awakenings in sleeping mice. Next, we will employ a spinal window to chronically image ascending neural pathways emanating from spinal cord nociceptive output circuitry that drive cortical activation. Electrophysiological alterations in the pathways will be studied to identify the source of the ectopic neural activity associated with SNeuP. Our goal is to link this neural activity to spontaneous pain behaviors, using optogenetics and machine learning-based behavioral analyses. In conclusion, this project aims, for the first time, to tease out the precise circuits underlying SNeuP, which will pave the way for exploration of novel therapeutic interventions.