Animals as diverse as mammals, birds and insects use odors to find mates, hosts and food sources. This is a difficult task, because natural odors occur in complex turbulent air plumes, and the relevant target odors intermingle with plumes of multiple background odors that stem from a variety of natural and anthropogenic sources (vegetation, exhaust fumes, etc.). It is essential for animals to be able to separate target from background odors. We hypothesize that animals may use temporal information from natural odor fluctuations to achieve odor-background segregation, as odorants from the same source fluctuate together in synchrony, while odorants from different sources do not. Behavioral studies have shown that insects can indeed use fast temporal stimulus cues. However, it is not yet known how the brain accomplishes the difficult task of odor-background segregation.
In this project we will investigate the neural mechanisms of odor-background segregation using the honey bee as model organism. Honey bees visit many different flower species for nectar and pollen. However, over a series of trips an individual forager bee only visits a single flower species on which it has found nectar previously, a phenomenon called floral constancy. To localize the target flowers in a natural environment bees use several cues, including odors. As the bee is challenged by multiple backgrounds in every flower patch, bees must be equipped with efficient mechanisms of odor-background segregation. We will use behavioral experiments to identify naturally evolved strategies of odor-background segregation (Brian H Smith, USA). Physiological recordings will inform us about neuronal processing and the receptor neurons’ and the brain’s different roles (Paul Szyszka, Germany). We will use computational models (Thomas Nowotny, UK) to formulate mechanistic explanations of the process of odor-background segregation and generate predictions to test in experiments. To overcome the limitations of computer simulations, we will finally transfer successful models to the embodied context of odor tracking robots (Ryohei Kanzaki, Japan). Understanding how small and fast fluctuations in relative concentrations of odor mixtures are used by animals to reliably identify odor sources will help us identify common principles of how animals’ brains represent and process their environment.

Plants are sessile organisms that must sense and respond to their environment to grow and survive. To facilitate the integration of diverse environmental stimuli including water, sunlight, and nutrients, plants have evolved a complex signaling system that utilizes small molecules called phytohormones. Among the various phytohormones, Gibberellic Acid (GA) has a number of effects on plant growth and development, such as seed germination, root and shoot elongation, flowering and fruit patterning. GA is widely used in agriculture at specific developmental stages, such as promoting fruit set, increasing the size of seedless grapes and improving the outlook of apples and citrus. Importantly, genetic perturbation of GA signaling played a critical role in so called 'first green revolution', which has given rise to the modern day high-yielding cultivars of maize, wheat, and rice. Although spatiotemporal distribution of GA must be tightly regulated, it is largely unknown how GAs are delivered through plant tissues and where they accumulate during plant growth and development. Our overarching aim is to dissect the mechanisms underlying active GA transport and uncover physiological roles of GA redistribution during plant development. Taking advantage of our unique fluorescently labeled GA surrogates, we have discovered a novel protein that transports GA in vitro and in vivo. To the best of our knowledge, this is the first bona fide GA transporter so far. We hypothesize that this newly discovered GA transporter plays major roles in GA transport during plant growth and development. We will approach our goals by combining unique expertise from each lab, including electrophysiology and mass spectrometry-based in vitro transport assays, X-ray crystallography, and developmental genetics and in vivo transport assays using fluorescently labeled GAs. Our proposed research is innovative because it is the first detailed investigation for an active GA transport mechanism. We expect to determine the spatiotemporal distribution of GA at the cellular level, as well as uncovering the structural and mechanistic insights into the GA transporters, which may be used to rationally intervene GA delivery in plants. We believe that controlling GA delivery through manipulation of GA transporters has the potential to generate a second green revolution to improve agricultural traits.

Morphogenesis sculpts 2D epithelial sheets into 3D structures. While much work has been devoted to understanding the mechanisms underlying morphogenetic processes, little is known about how they arose during the course of evolution and whether the structures that they produce underwent dynamic changes to gain new functions. To address this open question, we envision a new model system that synthesizes the powers of Drosophila genetics, evolutionary, developmental, and data-intensive computational biology. Our work focuses on the cephalic furrow as an epithelial structure that forms transitorily during Drosophila gastrulation and eventually reverts back to an unfolded state. This cephalic furrow arose recently in a derived lineage of the insect order Diptera, appears to perform a critical, yet unknown function during embryogenesis, but does not contribute to an organ. We will utilize cephalic furrow formation as model system to reconstruct the past and explore the future evolutionary possibilities of a complex epithelial structure. Our analysis is based on a triad of species within a well-defined phylogeny: two distantly related diperan species of the derived lineage that form the cephalic furrow (Drosophila melanogaster and Megaselia abdita) and a basal dipteran species that naturally lacks a cephalic furrow (Chironomus riparius). We first employ a comparative genomics approach to identify the previously unknown cephalic furrow effector genes to unveil the evolutionary history of genome rewiring that led to the emergence of the cephalic furrow. We then link the cephalic furrow genetic network to the control of cellular and tissue dynamics using in vivo quantitative imaging and in silico modeling approaches, thereby deciphering the function of the cephalic furrow in the context of morphogenetic movements during dipteran gastrulation. We use our model to predict a minimal genetic circuit that can reconstitute cephalic furrow formation in Chironomus riparius and ultimately, artificially evolve the cephalic furrow in Drosophila melanogaster by reprogramming its cells into distinct differentiation pathways, thereby systematically exploring its past and future evolution.

Ribosomes are the molecular machines that synthesize cellular proteins, and their proper function is essential for life. Ribosome assembly, maturation and translation involves hundreds of reactions, each with the potential for error. Although the ribosome has been studied for decades, the processes that governs the recognition and disposal of malfunctioning ribosomes and the immediate cellular consequences of damaged ribosomes remain poorly understood. The goal of my proposed project is thus to produce defective ribosomes and characterize in molecular detail how these damaged ribosomes are recognized and selectively degraded. First, I will produce green fluorescence protein (GFP)-labeled defective ribosomes to identify components of the degradation machinery using high-throughput microscopy-based cell screening. I will further develop a novel spatial-restricted pulse-chase purification method in living cells, which allows time-resolved isolation of defective ribosomes. Moreover, I will characterize purified damaged ribosomes by state-of-art mass-spectrometry to identify specific post-translational modifications and interaction partners. Using this molecular information, I will then specifically misregulate ribosome degradation to study the immediate and long-term effects on the cellular proteome and the resulting physiological consequences.

Overall, the proposal holds the promise to significantly enhance our understanding of the molecular mechanisms governing ribosome quality control and their role in maintaining proteostasis in eukaryotic cells.

My project aims to identify the neural circuits underlying parental behavior. I will first perform anterograde and trans-synaptic retrograde viral tracing from genetically specified neurons that have been shown to control parental behavior in both sexes. These experiments will reveal which areas these neurons target, from which neurons they receive input, and whether they fall into functionally different subgroups (Aim 1). Crucially, these experiments will also reveal structural sex differences in parenting circuits.

Parental behavior consists of discrete motor sequences, such as pup retrieval, grooming or nursing. I will artificially activate projections to identified downstream areas in behaving mice to uncover (a) which of these areas control parental behavior and (b) whether different target areas control discrete aspects of parental behavior (Aim 2).

While female mice are spontaneously maternal, males exhibit a drastic change in their behavior towards pups after mating: virgin males normally attack and kill pups but they become paternal after mating (post-mating switch). In the final phase of this project, I aim to uncover the neural circuit correlates of the post-mating switch (Aim 3). I will stimulate presynaptic areas while recording evoked postsynaptic responses in single parenting neurons in a brain slice preparation. Due to its sensitivity, this electrophysiological mapping approach is ideally suited to detect functional changes that occur without obvious structural changes.

Together, these results will advance our understanding of how parental behavior is controlled by neural circuits in the brain and how it can be influenced by social experience.

How does the brain encode sensory perceptions? Although we know approximately how sensory input is routed through different brain areas the “language” used by the brain to represent sensory experiences remains elusive. Brain cells are known to translate external sensory stimuli into electrical pulses called “spikes” to process sensory information. The patterns of spikes contain a code, the neural code, that is used for communication. Theoretically, if we can “crack” the neural code then we will be able to read and write sensory information to the brain. This project aims to combine approaches from cellular, systems, behavioural and computational neuroscience in order to demonstrate that the brain uses two distinct neural codes simultaneously to represent the sensation of touch. To do this, we will study different types of inhibitory cells in the brain that we hypothesize are responsible for carrying the two different codes. Using electrical recordings of individual cells, computer models of neural circuits, and optical recordings of electrical activity in the circuits of mice feeling objects with their whiskers, we will study how these two types of cells integrate information encoded in different ways, and examine how they affect activity in the brain. Using what we learn about these two types of inhibitory cells we will then develop and a novel, optical approach to bias the codes in a mouse’s brain as it performs sensory discrimination tasks with its whiskers. By shifting the mouse’s brain from one code to the other we hope to be able to generate illusory percepts in the mice—making them think they are feeling something with their whiskers that they are not actually feeling. If we can do this for both of the codes that we hypothesize are used by the brain, then we will have demonstrated for the first time that the brain uses two codes simultaneously.

In Drosophila, the medulla part of the optic lobes receives direct color and polarized light information from the R7 and R8 photoreceptors as well as indirect motion information from lamina neurons. 40,000 neurons of 70 different types are processing this information. Expression of the transcription factors that drive this complexity is precisely regulated, spatially and temporally. To understand the role of each transcription factor in determining the characteristics of each of these diverse neurons, I will use supervised learning techniques to develop an algorithm able to predict the phenotype of a differentiated neuron given its “transcription factor code”.
I will first describe the phenotype and profile the transcriptome of most neuronal types in the medulla. To increase the depth of my analysis, I will exploit cutting-edge techniques that allow characterization of the expression profiles of single cells. I will thus create an exhaustive map including detailed phenotypic and molecular characters and transcription factor expression of each of these neuronal types. I will use this map as input for a machine-learning algorithm. I will test the model both in vivo and ex vivo by expressing transcription factor combinations in naïve neuronal precursors and by comparing the phenotype of their progeny with the prediction of the model.
I will use this model to determine transcription factor regulatory networks, study them in Drosophila and in other animal systems to understand the evolution of visual processing centers. The model will also help me program the differentiation of desired neurons from neuronal precursors, contributing on the long term, to regenerative medicine.

Colorectal cancer (CRC) develops during a multi-step process from small lesions of the intestinal epithelium. While genetic mutations in the canonical Wnt signalling pathway are considered to be the initial step of cancer formation, the interaction of the tumour with its microenvironment plays a crucial role in tumour progression. Tumour-reactive immune cells (ICs) such as cytotoxic T lymphocytes (CTLs) are part of the tumour microenvironment and have been shown to be associated with better prognosis. However, cancer cells can escape IC-mediated elimination for example by preventing CTL activation through stimulation of inhibitory receptors on T cells. This process of cancer immunoediting is still poorly understood in many cancer types such as CRC. I therefore aim to study the interaction between cancer cells and tumour-infiltrating ICs to understand how IC anti-tumour responses are repressed. To identify the subtypes of tumour-reactive ICs found in human CRC tissue, I will first perform immunophenotyping. Using different functional assays, I will then assess whether IC heterogeneity is reflected by differential immunocompetency. Subsequently, I am to develop a co-culture system of CRC organoids and tumour-reactive ICs to investigate their interaction under near native conditions, since suitable model systems to study this process in vitro do not exist. Exploiting this new method, I will eventually test different strategies modulating IC activity to reinstate anti-tumour responses against the CRC cells. My work will provide an invaluable tool to investigate IC anti-tumour responses and will give new insights into the role of IC heterogeneity in tumour biology.

Structural characterization of biomacromolecules is essential for understanding cellular processes, investigation of disease mechanisms, and drug development. There is a great demand for characterization methods that can provide low-cost and high-throughput structural information. I propose to develop DNA PUPPETEERED CALIPERS (DPC), which is a biophysical tool for measuring long-range distance restraints of biomacromolecules. DPC employs microbeads that are tethered to a surface via single DNA strands; these strands act as nanoscopic calipers that bind to specific sites on the target macromolecules. A novel REFLECTANCE INTERFERENCE CONTRAST CENTRIFUGAL FORCE MICROSCOPE (RIC-CFM) will enable measurement of a very large number of calipers simultaneously, each with near-angstrom precision. Another key innovation is RECONFIGURABLE LOOPING - a technique that will be used to reversibly (de)activate specific parts of the caliper, which is essential for accurate calibration of the device. This approach does not require large analyte concentrations or elaborate sample preparation. Such a highly sensitive, precise, and massively parallel detection method is unprecedented, and will, for the first time, allow rapid, inexpensive, and high-throughput determination of biomacromolecular structural features. Moreover, DPC will enable the study of molecules under alternating mechanical forces or changing molecular environments (e.g., for the study of protein folding/unfolding/misfolding; or for the detection of allosteric binding events). I anticipate that DPC will become a powerful tool that can revolutionize research in structural biology, bionanotechnology, and biomedicine.

Gene expression is precisely controlled in time and space during the development of metazoan organisms. While numerous studies have established how spatial information is integrated by gene regulatory regions known as enhancers, the mechanisms enabling temporal coordination of gene expression across a field of cells remain vastly unknown. Using Drosophila melanogaster as a model system, my general goal is to understand the developmental consequences of subtle alterations in the timing of gene activation. For example, I have recently shown that pausing of RNA polymerase II at promoters sets a transcriptional synchrony that is required for successful gastrulation. With this project, I propose to discover additional mechanisms involved in the regulation of timing by introducing targeted mutations into various core promoter elements and precisely quantifying the resulting expression temporal dynamics in vivo with single cell sensitivity. We also propose to characterize the mechanisms of transcription memory, whereby transcription activation is inherited through multiple divisions.

Using insights acquired from study of these mechanisms, we will address the important question of the functional role of temporal coordination of gene expression for cell fate specification during development.

The second aim will investigate the existence of, and requirements for, temporal coordination in gene expression in a complex but stereotyped morphogenetic process, cardiogenesis.

Major upstream cardiogenic factors will be examined to document and alter their transcriptional temporal dynamics and address their potential priming through transcription memory.

The discovery of intron-derived RNAs and cytoplasmic intron-retaining transcripts (CIRTs) hints that intronic RNAs, previously regarded as “junk”, play an important role in cellular processes. In order to unravel the dynamics of intron-derived RNA synthesis, transport and function in vivo a novel RNA visualization tool is needed. This is due to the fact that current RNA imaging methods, based either on the fusion of a fluorescent protein (FP) to an RNA-binding protein (RBP) or on fluorescence hybridization, are laborious and limited due to the low signal-to-noise ratio, the need to re-design RNA-binding proteins for each new target RNA, and the inability to detect endogenous RNAs. Thus I propose to develop an innovative, highly sensitive and easy-to-use RNA imaging tool by utilizing the powerful prokaryotic adaptive immunity system CRISPR/Cas and the most modern fluorescent labeling techniques. I will then use the resulting CRIPSR/Cas based RNA visualization tool to investigate CIRTs. To date, neither the mechanisms by which CIRTs are synthesized and processed, nor their functions are known. Thus I will take on a multi-disciplinary approach: apply state-of-the-art CRISPR/Cas RNA imaging and combine it with gene editing technologies and the most advanced tools in biochemistry and structural biology to investigate CIRTs. Hence this interdisciplinary project will set a new milestone towards the development of an easy-to-use endogenous RNA imaging tool in vivo and provide important insights into the localization, interaction, structure, and function of CIRTs by correlating the live cell imaging and structural and functional studies.

Protein organization in cells is classically studied at disparate scales, either through their association in complexes, or by their localization in compartments. We propose to investigate a scale in between, corresponding to micron-scale protein assemblies. While several such assemblies have been characterized recently, as illustrated by P-bodies or protein gels, basic principles related to their formation in vivo remain unknown. Here we propose to design a minimal system that consists of a pair of proteins with the potential to form such large assemblies in vivo. Using this minimal self-assembling system as a model, we will dissect the relationship between its molecular properties (e.g., geometry of the interacting proteins or affinity between them) and its macroscopic properties (e.g., size, regularity, or kinetics of assembly). Aim 1 will be the design of self-assembling protein pairs. We will use homomers as basic building blocks, because the interaction between homomers can result in the formation of infinite and regular structures. We have in fact validated one such design in vivo, and we will expand the range of designs to cover a spectrum of molecular properties. Aim 2 will consist in dissecting the kinetics of self-assembly and disassembly by confocal fluorescence microscopy, enabled by fusing the basic building blocks of the minimal system to fluorescent proteins. Finally, in Aim 3 we will seek to obtain structural information of the assemblies formed by in vivo STEM-tomography. Akin to the first synthetic gene networks, this approach might help single out design principles of otherwise highly complex biological processes.

OCD is a disorder defined by intrusive recurrent thoughts and repetitive compulsive actions. Recent studies in two OCD mouse models have yield contradictory results [6,7], most likely because these studies perturbed the entire corticostriatal circuitry instead of specific cell types. It is therefore important to understand the neural circuits mediating OCD with an appreciation for the heterogenetity of the corticostriatal circuitry. Striatum neurons are composed of anatomically and functionally heterogeneous subpopulations with different cortical inputs. I hypothesize that inhibitory or excitatory signals for certain OCD behaviors are transmitted by different components of the corticostriatal circuitry. In my proposed work, I first plan to dissect the circuitry coding of specific OCD behaviors using different mouse transgenic lines and optogenetic approaches. Cre-dependent, light-activated opsins (ChR2 or NpHR) will be delivered to several GENSAT BAC-Cre driver lines with expression in different cell populations of the corticostriatal pathway. Optogenetic manipulation of these specific corticostriatal pathways will be used to uncover the causation of OCD behaviors. Furthermore, synaptic plasticity of striatal neurons will be examined in genetically identified corticostriatal pathways. Finally, taking advantage of TRAP translational profiling method, I seek to find the molecular mechanism of OCD in different striatum and corticostriatal cell types. For this purpose, I will profile the identified cell types in the two existing mouse models of OCD before and after drug treatment and identify key molecules contributing to OCD behaviors and their amelioration.

The spontaneous generation of living organisms without descents from similar organisms was decisively put to rest with the experiments of Pasteur during the 19th century. As a consequence, the logic of self-reproduction, one of the most fundamental features of biological systems, is very difficult to break down. An approach to this problem consists of synthesizing cell analogs in the laboratory from its basic natural molecular components.
The chemical synthesis of living entities is a standing challenge not achieved yet. Various approaches have been taken to construct synthetic reduced cells in the laboratory, a goal that became experimentally imaginable over the past two decades. The construction of protocells, which explores scenarios of the origin of life, has been the original motivation. With the emergence of the synthetic biology era, bottom-up engineering approaches to synthetic cells are now conceivable. The modular design emerges as the most robust method to construct a minimal cell from natural molecular components. Although significant advances have been made for each piece making this complex puzzle, the integration of the three fundamental parts, information-metabolism-self-organization, into cell-sized liposomes capable of sustained reproduction has failed so far. Our inability to connect these three elements is also a major limitation. New experimental platforms and methods should be taken to accelerate the cell-free synthesis of complex biochemical systems.
Our scientific collaboration, composed of four team members, will address the problem of biological self-reproduction by using an engineering versus evolution approach to cell analogs. While “information-less” protocells will be generated by driving Miller-Urey experiments, a prototype of minimal cell will be constructed by integrating the three indispensable molecular parts making living cells: information, metabolism and self-organization (compartment). Unique experimental platforms developed by the team members will be used to achieve major advances in the synthesis of minimal living systems in the theoretical context pioneered by von Neumann in the 50s. The societal impact of our work includes a deeper comprehension of the emergence of self-reproduction with and without genetic information and the development of powerful platforms for the construction of living entities in vitro.

The development of the retina occurs via the production of over 60 cell types from a pool of multipotent retinal progenitor cells (RPCs). The photoreceptors and horizontal cells (HCs) are part of this complex circuitry, and they are first cells to establish synaptic connection during retina formation. Their connections with bipolar cells form the fundamental unit of the center-surround organization, which is shared among sensory systems. In the chick embryo, HCs come in three distinctive subtypes: H1, H2 and H3. Recent study showed that retinal progenitor cells were producing restricted subsets of horizontal cells and cones only. How specific RPCs make any specific type of neuron is currently not understood. We plan to use these recent observations concerning the genesis of cones and HCs subtypes as an entry point to this question. We propose to investigate the mechanisms of the development and specification of cones and the HC subtypes, made by specific types of RPCs, those that make a pair of H1s, a pair of H3s, or a cone and an H2 cell. To address these questions, we will use expression profiling, electroporation and viral infection in the chick along with morphological and immunohistochemical studies. Specification of each type of HC, which are interneurons, may shed light on the process of diversification of other types of interneurons, which are the most diverse category of neurons in the CNS. In addition, the fact that the HC likely arose early in evolution, may predict that its specification mechanisms were the forerunner of many other types of specification mechanisms.

While many one-to-one plant-microbe interactions have been studied in detail in lab experiments, nature exposes plants to large communities of microbes that are also interacting amongst themselves. Many of these microbes evade or tolerate the plant immune system and colonize leaves, posing a risk to the plant. Because maintaining an immune system is costly, successful plants streamline their defense capabilities. In Arabidopsis thaliana, many putative pathogen resistance genes, as well as pattern recognition receptors that recognize microbial molecules, are uncharacterized. These immune gene families are extremely variable, with great genetic variation maintained even within single field sites. The functional consequences of these immune gene repertoires in wild plants are essentially unknown. Characterization of the plant microbiome using marker genes like 16S rDNA has helped to address these concerns, but most single locus studies bias against the identification of entire domains of microbes and lack the sensitivity to distinguish closely-related pathogens and mutualists. This research explores the evolutionary genetics of plant-microbe interactions in A.thaliana in a wild setting. I will leverage high-throughput sequencing and classification methods to genotype and quantify both eukaryotic and prokaryotic microbes on a sub-species scale, and associate them with plant loci. I will survey wild plants, sow experimental crosses, and sow plants mutated for specific immune genes identified from this work and the literature. These experiments will allow me to address how host genetic diversity affects microbial associations and fitness in nature.

Linking circuits to behavior in mammals is a major challenge of neuroscience. In the mouse, we know which visual circuits control the pupil size or the optokinetic head control, two simple reflexes that require little visual processing. Here, we propose to dissect the visual pathway underlying a more complex innate behavior: the avoidance of looming objects. Mice rapidly escape or freeze when a shadow approaches overhead, which is essential to survive predators. How the visual system can extract such visual feature and trigger motor actions in milliseconds is not established. However, we do know that a type of ganglion cell, named the PV-5, responds specifically to looming stimuli. We think that this retinal channel might mediate the behavior. Given the rapid processing required, we hypothesize that this "looming-sensitive" retinal signal is processed via dedicated small brain nuclei from the accessory optical system, and does not recruit the main pathway going to the visual cortex involved in image formation.
We propose to test this hypothesis with an innovative experimental approach. We will use whole brain ultrasound imaging, a technique that I developed, on a head-fixed mouse to detect all regions specifically activated by looming stimuli. Using the same technique on behaving mice, we will discriminate which of these regions are specifically involved in escape or freezing. Using transynaptic retrograde tracing, we will then identify which ganglion cells project to these nuclei and verify if they are indeed PV-5 cells. Finally using optogenetics, we will specifically activate the PV-5 cells to see on which brain regions it acts and how it affects the mouse behavior.

We propose to apply cell-free synthetic biology to study fundamental aspects of transcriptional regulation, gene regulatory network evolution, and network robustness. Cell-free synthetic biology emerged recently as an in vitro alternative for network engineering. Cell-free synthetic biology implements biological systems in a coupled transcription – translation reaction and therefore is a well-defined environment that is easy to control and interrogate. Since genetic networks can be implemented in vitro as linear dsDNA templates, as opposed to plasmids, it also circumvents time consuming cloning and transformation steps, enabling rapid prototyping of genetic systems. We have recently implemented cell-free systems on a microfluidic microchemostat array, which performs transcription-translation reactions at steady state for 30+hours, allowing us to implement complex genetic networks in vitro. Here we propose to apply this novel concept to study fundamental principles of genetic networks. We will interrogate two different types of oscillators (phase and relaxation) and determine how cis- and trans-changes to the network impact network function and robustness. Specifically, we plan to map the precise cis-binding energy landscape of these networks by characterizing a large number of network variants with modified transcription factor binding sites. To implement trans-element changes we will employ synthetic Zn-finger transcriptional regulators with defined specificity landscapes to determine how changes to the trans-element influence network performance and robustness. Finally, we will also attempt to rationally evolve network function through a promiscuous transcription factor intermediate. To determine how robust these networks are to external perturbations, we will change reaction temperatures, and independently modulate transcription and translation rates. Finally, we will characterize a subset of our networks on the single cell level in vivo to evaluate network robustness in a regime were stochasticity dominates. Gene regulatory networks are complex systems that are difficult to quantitatively characterize in vivo. The in vitro cell-free synthetic biology approach proposed here has the potential to contribute significantly to the mechanistic understanding of complex biological systems.

Callyspongiolide, a unique polyketide-derived macrolide extracted from a Callyspongia species of marine sponge, has recently been shown to possess remarkable antiproliferative properties against cancer cells. Although various cytotoxic substances have been isolated from sponges belonging to the Callyspongia genus, this represents the first macrolide obtained from such species. As it belongs to the family of macrolides, its cytotoxicity could be related to antimitotic properties; however the actual mechanism of action is still unknown. This project aims to determine the mechanism of action of this compound and to develop the first enantioselective total synthesis of Callyspongiolide. A 23-step synthetic pathway has been proposed, starting from low cost and easily accessible starting materials, which should provide a sufficient supply of material to be employed for biological assessment of this new and potentially powerful marine natural product. The synthetic pathway has been designed to allow control over the molecule’s substituents and over the configuration of all chiral stereocenters facilitating analog synthesis.

Aging is one of the most prominent biological processes whose molecular and cellular mechanisms are poorly understood. Several studies demonstrate that aging is a progressive process involving a gradual dynamics of the genome, transcriptome and epigenome, and defects in organelle homeostasis. The current stem cell hypothesis of aging proposes that living organisms are as old as their tissue-specific adult stem cells. The adult mammalian brain contains pools of adult neural stem cells (aNSCs) that are crucial for learning and memory. The pool size and the functionality of aNSCs decrease with age. Although adult neurogenesis plays a central role during aging, the global aged-induced molecular dynamics and key factors that regulate aNSC aging are largely unknown. Here, I propose an integrative research approach that aims at answering basic biological questions regarding aging regulation in mammalians brain. My proposed study includes (1) performing a systematic, genome-wide profiling of aNSCs during brain aging, (2) identifying novel key players that regulate mammalian brain aging in vivo, and (3) modeling human aging using a 3D culture that is derived from human embryonic stem cells. This work will provide considerable information about molecular landscapes during aging of aNSCs, and will explore several key factors that regulate brain aging in mice and humans. Overall, the findings of this research will help to broaden our understanding of the mechanisms that promote or prevent cellular aging, and hopefully, will further provide potential implications for regenerative medicine to help rejuvenate brain cells.