Propagation of a-synuclein in the brain is the hallmark feature in the pathogenesis of Parkinson’s disease (PD). Patients with PD exhibit marked differences in the rate of a-synuclein propagation, but the reasons for this variability are unknown. I hypothesize that the rate of a-synuclein propagation varies because it is affected by various genetic modifiers. In addition, I hypothesize that individuals with PD are mosaics for a-synuclein mutations in the brain, a type of genetic variation that is missed by standard screening methods, which further impacts on a-synuclein propagation. In order to investigate these hypotheses, I am going to generate human iPSc-derived neuronal cultures expressing a construct in which mutant a-synuclein that is fused with mCherry on its C-terminus is also fused to a GFP tag on its N-terminus through a 2A self-cleaving peptide. Neuronal cultures expressing wild type (WT) a-synuclein fused with BFP on its C-terminus will also be generated. I am then going to mix these neuronal cultures to generate cultures of various mosaic percentages for the a-synuclein mutation. This system will enable the identification of single neurons that have uptaken WT or mutant a-synuclein through propagation based on their positivity to GFP, mCherry and/or BFP. I am then going to use a CRISPR-Cas9 high throughput screening system combined with high content imaging techniques developed at the host institution to assess the effect of thousands of genetic factors on the rate of propagation in the mosaic neuronal cultures, followed by RNA sequencing to determine molecular signatures that underlie the propagation process and that are specific for each genetic modifier.

Monitoring membrane potential is a necessary step in understanding neuronal activity. Invasive methods cannot provide the spatial resolution and coverage required for such information. On the other hand, optical methods that provide high spatio-temporal information demand optical sensors with large signal amplitude, photostability, live-cell compatibility, and fast response to voltage variations. Existing optical voltage sensors do not match all these requirements. Recently, semiconductor nanorods (NRs) with asymmetric structures have been shown to exhibit prominent voltage sensing capabilities. Their high luminescence and photostability will also allow for nanoscale resolution and reduction in photodamage of live cells. Therefore, here I aim to explore ways for functionalizing these particles with peptides of DNA origami for membrane insertion. I will evaluate the insertion mechanisms by measuring their orientations and accurate positions with respect to the lipid bilayer using single-molecule Metal-Induced Energy Transfer (smMIET) imaging. After stable insertion, I aim at characterizing their voltage sensing properties and performance with fast optical imaging techniques. In the end, I will explore their application in neuronal cultures by measuring action potentials and subthreshold events in individual cells and across the network. Membrane potentials in subcellular sections such as dendrites and spines will be monitored with high spatial resolution as a function of their morphology and structural dynamics, which has not been achieved to this date.

My project aims to identify neuronal circuits that are spared with severe spinal cord injury and function as motor command relay pathways during reactivation of the lumbar spinal cord. I will use a neuroprosthetic platform that supports bipedal stepping and implant an electro-chemotrode over the lumbar spinal cord of mice. First, I will optogenetically activate motor cortex projections in severely contused mice and assess whether immediate will-powered motor control is regained over completely paralysed hindlimbs during spinal stimulations (Aim 1). Next, I will use a combination of conventional tracers, such as Fast blue, and viral tracers that are regulated by a Cre promoter in vGluT2 Cre or 5-HT Cre transgenic mice to label and identify brainstem projections to the lumbar spinal cord. Moreover, I will prove these brainstem projections are required to rely motor cortex commands by chemogenetically silencing them with the DREADD technology, which should abolish any leg motor function (Aim 2). Mice will undergo 8 weeks of actively engaging neurorehabilitation with spinal electrochemical stimulation. I will assess recovery of will-powered voluntary motor function and remodeling of spared pathways (Aim 3). I will laser-microdissect cell bodies of motor command relay neurons in the brainstem and assess the transcriptional profile with RNA sequencing to identify which molecular cues are involved in circuit reorganization (Aim 4). Taken together, these results will establish the causal relationship between circuit reorganisation of residual descending projections and voluntary motor control with electrochemical neurorehabilitation in severe spinal contusion injuries.

Cell division and differentiation are strictly coordinated during development. Although it is known that differences in S-phase length are essential for embryogenesis our understanding of its causes and consequences is extremely poor. The second cell division of C. elegans is stringently asynchronous and is well-suited to study developmental control of replication regulation in a multi-cellular organism.
Studying replication initiation is limited by the availability of specific assays. I will develop a novel live assay to visualise the recruitment of replication factors towards origins in single cells within the C. elegans embryo. With the LacO/LacI system I will generate functional origins and measure the binding of fluorescently tagged replication factors by confocal imaging dissecting consecutive steps of DNA replication. This assay will permit to follow the dynamics of replication factors in real-time and to reveal changes in S-phase control during development. On this basis, I will perform an RNAi-based high-throughput screen for novel regulators of genome duplication in the C. elegans embryo. This unique screen implemented in a whole organism will reveal novel interactions between DNA replication and developmental signalling pathways. In a complementary approach I will generate mutants bypassing the normal cell cycle regulation in the embryo. I will examine potential changes in cell fate adoption through transcriptional analysis and will measure differences in their sites of replication. This approach will clarify if replication control plays a direct role in differentiation and illustrate a physiological role for S-phase differences in development.

It has been postulated that cancer stem cells (CSC), an identifiable subpopulation of cancer cell that can differentiate and repopulate cancer, exist. In this context, cancer cells follow strict hierarchy with CSC being at the apex of it. However, depending on the subtype and malignancy of cancer, cell fate plasticity had been observed where non-CSC dedifferentiates to acquire CSC function. However, the nature and regulation of CSC formation and plasticity has been difficult to understand in vivo. For example, what kind of niche signaling is responsible for cancer cell plasticity? What is the epigenetic or transcriptomic changes in cancer cell plasticity? In this project, I aim to address these questions in vivo using highly novel technique involving transgenic mice carrying recombinase-based state machine (RSM), which enables each individual cell within cancer to keep a memory of its history and sequence of changes in cell fate. The proposal will involve generation and validation of mammalian RSM that can label each cell with different cell fate history (e.g. original CSC and non-CSC that had dedifferentiated to CSC) with different fluorescent proteins. I will then cross the transgenic mice harboring RSM with mouse models of breast cancer and colon cancer can accurately identify heterogeneity in individual cancer cell fate in vivo and in situ. By examining the relative locations of cancer cell fate plasticity in intact tumor mass, and FACS sorting of cells with different fate history for transcriptomic or epigenetic analysis, I aim to dissect the extracellular factors and intracellular signaling events that govern cancer cell plasticity in vivo.

Spatial organization of chromatin and related genes is of key importance for genome expression and maintenance. Unlike stem cell differentiation, for T-cell activation gene regulation happens on a much faster timescale as the immune response is critical for the bodies defense mechanism. The chromatins’ three-dimensional organization as well as its interaction with nuclear lamina and the consequential impact on gene regulation is poorly understood. This proposal will in its first step focus on how mechanical forces can directly influence cellular functions such as the T-cell activation and furthermore elucidate how they ultimately change gene expression. We hypothesize that mechanical force triggers structural/conformational changes of the nuclear lamina network; hence genes encoded in chromatin proximal to lamina can be up/downregulated. Here, we propose to develop 3D and 4D Super Resolution Microscopy (SRM) technologies to enable us to determine the 3D structure of lamina associated chromatin domains, resolve how such domains are organized in 3D during the fast process of T-cell activation. We specifically aim to:
1. Establish imaging methods to measure tension and the dynamic structural re-organization of cytoskeleton, nuclear envelope and chromatin during T-cell activation.
2. Study the effect of perturbations of the mechanobiological pathway that links external cues to activation.

3. Link these perturbations to gene expression and spatial location of genetic loci and transcripts.
This project requires synergistic interplay between the two team members, which brings together early career researchers in T-cell biology, single molecule biophysics, structural biology and SRM.

Cytoskeleton networks are the main vehicles of cell mechanics. Coordinated network dynamics is responsible to processes as diverse as cell motility and cytokinesis. Understanding the principles governing motion in these networks requires studying in vitro assemblies of purified components which can be subjected to finely tuned conditions. Recent works in purified systems have shown them to be unstable: either extremely contractile to the point of network collapse; or extensile and turbulent. This proposal delineates a research plan for studying active gels of microtubule filaments and kinesin motors as a prototypical cytoskeleton. Its main goal is to understand how asymmetric contractile and extensile modes of deformation emerge from inherently symmetrical network elements. My experimental approach will be to develop tools for modifying and controlling network dynamics on three length scales. At the filament scale, I will use the SNAP-BG crosslink to decorate filaments with motors. I expect that motors crosslinked at the minus end of a microtubule will encourage contractile motion and that plus-end crosslinked motors will encourage extensile motion. I will elongate the minus-end crosslink with photosensitive short DNA. Irradiating a mixed sample of minus-end and plus-end crosslinked motors with UV, I will be able to spatiotemporally tune contractility on the micron scale. Macroscopic stresses in the network will be measured and applied through a patterned soft substrate. The multiscale approach combined in a single set-up will allow us to uncover mechanisms responsible for contractility in cells and provide us with novel tools for designing biomimetic systems.

Direction selectivity is a feature of selected neurons in the visual cortex that is conserved in evolution. The emergence of cortical direction selectivity has been extensively studied but its circuit mechanism is still debated. My goal is to understand the circuit mechanism of cortical direction selectivity that is independent of retinal direction selectivity. I plan to approach this by using two animal models in which retinal direction selectivity has been disrupted. I will investigate the neuronal circuits of those cortical direction-selective neurons which retain their specificity in these animal models by performing single-cell-initiated functionalized rabies tracing and single-cell electrophysiological recordings. I will ask the three following questions: Do axon terminals of the principal cells of the lateral geniculate nucleus (LGN) fully lose direction selectivity after the abolishment of retinal direction selectivity, or could cortical feedback from layer 6 entrain direction selectivity in a subset of LGN cell axon terminals? What is the synaptic input profile underlying retina-independent cortical direction selectivity in pyramidal neurons? What is the contribution of the local presynaptic networks to the direction-selective tuning of pyramidal neurons with retina-independent cortical direction selectivity? This work will provide insights into how cortical selectively for environmental features arises.

Eukaryotic genomes are organized into a highly-compacted nucleoprotein complex known as chromatin. Hi-C and high-resolution microscopy studies reveal organization of chromosomes at multiple levels, from chromosome territories, to MB-scale functional domains that are spatially separated from each other, to shorter contact domains often called topological associated domains (TADs). While genetic studies in cell culture coupled with Hi-C or high-resolution microscopy reveal key roles for a variety of factors in higher-order chromatin folding, our understanding of chromatin fiber folding largely derives from biochemical studies in vitro. Mostly, these in vitro studies rely on artificial, homogenous chromatin templates that do not reflect the heterogeneous local folding properties of in vivo chromatin.
The aim of this project is to approach higher-order chromatin folding and chromosome organization biochemically. Micro-C detects internucleosomal interactions, which refer to local chromosome folding, by identifying nucleosomal DNA sequences and is therefore equally suitable for in vivo and in vitro studies. I will use chromatin prepared from cells (ex vivo) and in vivo-like in vitro reconstituted chromatin for chromatin compaction studies. With Micro-C I will be able to measure autonomous salt-dependent chromatin folding driven by all endogenous factors (ex vivo chromatin) or solely by histone-DNA interactions. Biochemical manipulation will allow to remove or test individual candidate factors.
This biochemical approach will help to decipher the regulatory role of higher-order chromatin organization on DNA templated processes, such as gene regulation.

Synthetic approaches toward redesigning cellular circuits and signaling networks have accelerated our understanding of sophisticated cellular behaviors. In particular, exploiting optogenetic approaches on neurons to manipulate neuronal activities have significantly expanded our comprehension on the nature of neuronal circuitry, whereas those approaches were applied much less in immune cells. Indeed, repurposed and/or engineered immune cells have been successfully used as cancer treatments, but they are only sometimes successful and can have harmful side effects. We hypothesize that these attempts have been limited because they have been restricted to a limited number of wild-type like signaling inputs. Here, through evolution-based approaches we intend to engineer chimeric antigen receptors (CARs) in T cells as a model system to answer a simple, yet, fundamental question regarding finding the maximal potency and unique phenotypes of T cell-based immune responses. Also, we offer a generalizable design scheme for safely controlling CARs through an optogenetic module. Newly engineered CARs could be of great use in eradicating cancer, and unique combinations and sequences of T cell signaling components could provide unexplained mechanisms and further understanding of the evolutionary nature of designed immune system. In order to achieve the goal, we build CAR intracellular domain libraries, and with a properly designed activity-based selection strategy we anticipate to find novel classes of CARs with extraordinary potency of immune responses.

It has been a long-standing question what mechanisms counter antibiotic resistance in nature. Are there any widespread multidrug strategies in soil bacteria for avoiding, inhibiting, or bypassing resistance mechanisms against antibiotics, whose biosynthetic pathways evolved a hundred million years ago, but still exist across natural microbial communities? One of the well-known examples of naturally occurring combination strategies is the co-production of beta-lactam antibiotics and beta-lactamase inhibitor in Streptomyces spp. which has led to the development of a potent drug combination for clinical use known as Augmentin. However, our knowledge on this issue is extremely limited due to the shortage of systematic investigations. It is important to emphasize that the extraordinary capacity of soil bacteria to produce multiple natural products may reflect an ecological advantage for them. Therefore, I hypothesize that a systematic investigation of the combinatorial biosynthesis of secondary metabolites, including antibiotics and non-ribosomal antimicrobial peptides, could reveal novel drug combinations. First, using state-of-the-art computational approaches I will explore the co-occurrence of biosynthetic genes of known secondary metabolites and antibiotics in the genome sequence of soil microorganisms which presumably co-evolved under strong selection pressure. Second, I will explore the capacity of novel candidate combinations i) to restore susceptibility of the resistant bacteria, ii) to synergistically inhibit bacterial growth or iii) to invert the selective advantage of resistance mutations.

Plants have evolved sophisticated mechanisms to recognize non-self molecules, allowing them to deploy effective immune reactions against a myriad of pathogens. Sometimes, however, plants mistakenly identify their own molecules as foreign and induce autoimmunity, causing severe growth defects including plant death. Autoimmunity is a hallmark of a syndrome that is seen in certain intra- and interspecific hybrids and that is known as hybrid necrosis. A particularly interesting case in Arabidopsis thaliana is that of the RPP7 and RPW8 loci, where three different pairs of alleles from different wild strains interact in F1 hybrids to cause hybrid necrosis. RPP7, which confers resistance against oomycetes, encodes members of the well-studied family of NLR immune receptors. The biochemical function of the RPW8 coiled-coil domain proteins that primarily confer resistance to fungi is less well understood than that of NLRs. I propose to reveal the mechanisms by which interactions between RPP7 and RPW8 proteins activate immune responses, taking advantage of the allele-specificity of these interactions. By interpreting these findings in the context of normal immunity, my work will not only advance our understanding of genetic incompatibility and autoimmunity, but will also contribute to optimizing combinations of favorable immune alleles in crop breeding.

Cells are constantly making millions of proteins that need to be synthesized correctly, folded, trafficked, and assembled to their functional state. All of these steps are monitored by the cells for mistakes or failures, and these failed products are degraded to avoid their accumulation. These quality control (QC) pathways are emerging as major contributors to neurodegeneration. The newly discovered ribosome-associated quality control (RQC) pathway represents one of the most cleverly imposed QC pathways to allow cotranslational surveillance of erroneous translation products. Recent studies have identified the factors involved in RQC pathway and defined its primary steps: recognition and splitting of stalled ribosomes into subunits, signaling of the heat shock response, ubiquitination of nascent chains, and extraction of polyubiquitinated nascent chain from the 60S subunit for degradation. I propose to dissect the mechanism of two unresolved steps of the RQC pathway and define its endogenous clients. Aim1 will investigate an essential step in signaling the heat shock response: the addition of C-terminal Alanine and Threonine (CAT) tails to stalled nascent chains. I will reconstitute this unusual mRNA-independent polypeptide elongation reaction in vitro to identify the minimum required components, define the energy source, and order the primary steps of this process. Aim2 will reconstitute nascent chain extraction in vitro to determine how a stalled polypeptide is delivered from the ribosome to the proteasome. Aim3 will identify the physiological clients of the RQC by trapping in vivo intermediates and identifying associated mRNAs or partially synthesized nascent chains.

Many sexually reproducing microorganisms are self-fertile, but in-breeding and out-breeding strategies have different outcomes on genetic diversity. Recent work from the Martin lab showed that self-fertile fission yeast S. pombe cells exhibit an exploratory polarity patch at their periphery, which allows them to preferentially pair with non-sister cells. However, the mechanism of this preference and its prevalence in nature are unknown. Here, I will study whether out-breeding is a general feature of fission yeast cell mating and dissect the molecular mechanism and genetic basis of this phenomenon. I will first develop a zygote FACS sorting method, which I will use together with high-resolution live-cell microscopy to study how pheromone concentration, cell size and transcriptional timing influence exploratory polarization and mate choice. I will then investigate whether out-breeding is a general feature amongst the recently sequenced collection of natural variants. If I detect pre-zygotic barriers amongst these strains, I will use pedigree analysis to probe for possible causative mutations. Finally, I will combine FACS sorting with experimental evolution to isolate strains favoring in- or out-breeding and uncover the genetic basis of the mating preference via comparative genomics/transcriptomics. Along the evolutionary experiment, I will also follow the exploratory polarization patterns by live-cell microscopy to relate the molecular mechanism of mating preference alteration with genetic changes. Finally, I intend to integrate the identified mutations into wild type lab strain to reconstruct the altered Cdc42 dynamic patterns and breeding preference.

To understand how cellular machineries work, we typically rely on reconstituted systems that often do not represent the complexity existing in vivo. We lack innovative methods to describe protein-protein interactions at high-resolution, specially the very transient ones, in their physiological environment. Chromatin is a good example of a system whose complexity cannot be fully reconstituted in vitro. Indeed chromatin is regulated by hundreds of chromatin remodelling enzymes and hundreds of possible combinations of histone post-translational modifications (PTMs) and variants. This complexity cannot be fully reconstituted in vitro.
We propose a novel combination of synthetic biology, mass spectrometry and high-resolution imaging to define the molecular details of how proteins function on chromatin in their physiological environment at high resolution. The challenge is to use photochemical traps installed by genetic code expansion in histones of living cells to “freeze” interactions of proteins bound to chromatin, especially the very transient ones that could be disrupted or missed by conventional purification or bulk chemical cross-linking. The trapped protein-chromatin complexes will be analysed by cryo-electron microscopy. By mass spectrometry we will map the interactions between remodelers and histones in vivo and we will quantitatively describe all chromatin PTMs associated to specific remodelers. This way, we will be able to analyse the spatio-temporal activity of chromatin-bound complexes at high-resolution at specific time points or upon specific stimuli.

Adult mammalian tissues are composed of heterogeneous cell types generated during development. To maintain homeostatic function, the identity of each of those cell types remains stable over time, and tissue renewal is normally sustained by dedicated stem cells. After injury, however, differentiated cells in many tissues can exit their quiescent state, acquire stem cell features and cross developmental identity boundaries to participate in the repair process. In the adult mammalian brain, injury-induced cellular plasticity (i.e. the acquisition of a new identity) remains poorly understood.
Here I plan to identify which brain cells exit their normally quiescent state upon stroke and study the molecular regulation of this process in situ. To this end I propose a novel strategy combining unbiased genetic fate mapping of stroke-generated cells with single-cell and spatially-resolved gene expression analyses. Specifically, stroke-generated cells will be marked with a genetic tag, identified using single-cell RNA-seq, and their contribution to repair traced over time. Next, we will use spatial transcriptomics to identify molecular triggers of cell plasticity, as well as their cellular source within their native environment. Finally, the identified molecules will be functionally tested for their capacity to enhance endogenous recovery after stroke by increasing cellular plasticity. This study will shed light on the cellular and molecular mechanisms governing the self-repair capacity of the brain.

Amino acid and peptide radicals have been implicated in a wide variety of biochemical processes and physiological disorders, including Alzheimer’s disease, arteriosclerosis and aging. At a molecular level, they are associated with protein damage and enzyme function, with the glycyl radical enzyme (GRE) family as important yet mostly unknown contributor. Using the high reactivity of peptide backbone radicals, GREs enable strict and facultative anaerobic organisms to catalyze a diverse range of chemically difficult reactions, including anaerobic toluene metabolism, the conversion of choline to trimethylamine (TMA) via C–N bond cleavage or glycerol fermentation. In taking advantage of the reactivity of the protein-based radical intermediates, tools will be developed to characterize novel GREs within intact microbial communities. For the first time, the concept of activity-based protein profiling will be expanded to include an entirely new class of enzymes. Furthermore, assigning functional and interactive relationships is suspected allow for the development of specific small molecules inhibitors to improve host health thereby opening up new opportunities in the way medicinal chemistry approaches certain disease. An additional key obstacle in studying GREs is the posttranslational mechanism of activation, which involves separate activating enzymes (AEs). The reconstruction of activase activity can be problematic and therefore, unnatural amino acid mutagenesis using designed glycine radical precursors to install a radical in the enzyme active site in a separate chemical step will facilitate studying GREs.

Some animals have great regenerative capability to regrow whole bodies even from small fragments. This ability relies on their pluripotent adult stem cells, which are capable of self-renewal and differentiating into any missing body part. During regeneration, positional signals such as Wnt signaling are crucial for stem cell regulation and specification. In planarians, inhibition of Wnt signaling promotes formation of head-specific cell types. However, the molecular control of stem cell fate by Wnt signals is unclear. In part, this is due to the limitation of lineage tracing in the well-established planarian system. Here I propose to use the acoel, Hofstenia miamia, as an alternative system to dissect this process. Hofstenia is an early diverging bilaterian and evolutionarily distant to planarians, and yet possesses whole-body regenerative ability and similar patterning pathways. In particular, Hofstenia produces large amounts of accessible embryos that are injectable, making lineage tracing of stem cells possible. I will first identify conserved Wnt signaling components by generating spatial transcriptomes along the anterior-posterior axis. Next, I will test the role of Wnt signaling in stem cell fate specification by using RNAi of Wnt pathway genes together with single-cell RNA sequencing. These data will reveal distinct transcriptional landscapes and identify Wnt-responsive cells within stem cell populations. Finally, I will develop lineage tracing methods for live imaging of stem cell differentiation and behavior. Together, this study will provide a basic understanding of how stem cell fate is regulated by positional signals during regeneration.

Cellular signalling pathways usually involve cascade processes. For some of these processes, ensemble average analyses cannot answer biological questions originating from stochastic biomolecular interactions. Though single-molecule analyses are sometimes helpful but still the oligomeric forms of many membrane protein complexes remain controversial. Protein or polymer scaffold lipid discs have emerged as platforms for monodispersed proteins. It is difficult to manipulate them to study protein-protein stoichiometric interactions. To overcome this, and motivated from DNA origami approaches, I propose to use DNA scaffolds to form lipid nanodiscs (DSN). Using DSN I will develop a novel programmable platform to study membrane protein-protein interactions in an isolated environment. First, I will create DNA nanorings, functionalised along their inner circumference with hydrophobic groups to hold a lipid membrane. Computational approaches will be used to optimise the designs along with experimental characterisation. The functional nature of DSNs will be established with an embedded P450 oxidoreductase enzyme. To develop a programmable platform, embedded proteins will be tethered at the edges of DSN with DNA linkers. Using DNA strand-displacement methodology proteins will be released to diffuse freely and interact in the membrane. As a proof of concept, an experiment will be set up using two formyl peptide receptors. Interactions will be analysed using AFM, single-molecule spectroscopy etc. Such platform, if achieved, could prove revolutionary for single-molecule analysis of protein oligomerization for studying, e.g., signal pathways, interaction energetics, metabolism etc.

Traditionally, model systems in experimental and theoretical condensed matter physics have provided a simplified/stylized version of target problems enabling fundamental advances. Over the years, soft condensed matter physics has undertaken the goal of explaining basic properties of living matter phenomenologically. This led to tremendous insights into specific phenomena such as behaviour of individual biological macro-molecules or mechanochemistry of biological forms, but the fundamental goal of understanding what the exact boundary is between living and non-living active matter remains elusive. Based on recent advances in soft-matter physics, here we propose an alternative approach to this question via developing an experimental abiotic (non-biological origin) physical model system capable of exponential self-replication and evolution (Aim 1a). With this framework in place, we intend to write down the fundamental properties of self-replicating and evolving matter in conjunction with the physical environment, laid out in terms of laws of physics (Aim 2), linking genotypes and phenotypes (Aim 3). These microfluidic experiments will be compared to robust hydrodynamic Molecular Dynamics (MD) simulations and theoretical Stokesian fluid mechanics, where hydrodynamic interactions and thermal noise are accounted for (Aim 1b). Together, these techniques could help formulate a theory for the essential ingredients of directed evolution and reproduction subject to physical constraints. We thus aim to discover new robust, abiotic architectures capable of self-replication of physical matter and information (Aim 4) and further exploit these findings in exponential manufacturing.