A fundamental question in the study of social insects is how division of labor, the major organization principle of the colony social structure, emerges out of the behavior and interactions of many simple individuals. This self-organized phenomenon is central to the efficiency, stability and adaptivity of ant colonies. While a large body of knowledge has been accumulated with regard to the determinants of division of labor, and several conceptual models that account for various of its aspects have been suggested, the inherent difficulties in performing controlled, repeatable experiments have prevented the validation of these theoretical ideas with empirical measurements. Here I propose to make use of a unique model system, colonies of the clonal raider ant C. biroi, which combines the ability to conduct controlled experiments, with precise manipulation of group size and composition, with a new setup that enables the simultaneous tracking of individual ants in dozens of colonies. Using this system, I intend to establish a framework for a systematic study of division of labor. I will design and perform experiments in order to characterize dynamical and statistical properties of division of labor, with emphasis on the effect of group size and heterogeneity, with the intention to constrain, fit and select between possible theoretical models of division of labor, and link them to plausible biological mechanisms. On the long term, I wish to use this experimental-theoretical framework in order to understand and characterize the dynamical processes underlying the emergence of division of labor, with emphasis on the symmetry breaking that occurs when a social group is formed.

The elucidation of the three-dimensional structure of biomolecules has transformed our understanding of biological mechanisms and enabled the rational design of pharmaceutical drugs. Cryo-electron microscopy (cryo-EM) plays an increasingly important role for the structural investigation of biomolecules. Despite technical advances and several successful results, atomic and near-atomic details remain difficult to reach. In parallel, our ability to control the spatial arrangement of molecules at the nanometer scale has dramatically increased. The laboratory of Prof. Peng Yin has successfully established a collection of methods to construct complex and diverse DNA nanostructures, which provides new opportunities to push the limits of structural biology. The proposed research project aims at leveraging DNA nanostructures to foster high-resolution structure determination by cryo-EM especially for small biological objects, difficult-to-crystalize macromolecules and complexes. To achieve this goal, we plan to engineer programmable DNA frameworks with user-defined and functionalized cavities to uniformly anchor and direct the orientation of guest biomolecules. The framework will guide particles detection, alignment and classification to provide strong signal-to-noise ratios necessary for single-particle reconstruction at high resolution. As proof of concept, we will attempt to solve the structure of the SWR1 chromatin-remodeling machinery to shed light on the mechanism of histone exchange involved in transcription regulation. In addition, implications for crystal-free X-ray crystallography are expected from this work that may originate preliminary developments.

DNA replication is essential for an organism to reproduce and grow. The outstanding biological question that will be addressed in this research project relates to structural dynamics of the DNA replisome. By extending our structural knowledge of DNA replication using cryo-EM, we would be able to identify protein-protein interactions potentially suitable for the design of novel antibiotics.

Recent technical developments in single particle cryo-EM have enabled high-resolution reconstructions of an increasing number of macromolecular complexes. However, structures of flexible complexes are still of limited resolution, partly due to unintentional averaging of particle projections in various conformations. It is possible to reduce the number of conformations by optimizing sample preparation conditions. Nevertheless, reconstructions are often contaminated by structural heterogeneity, a problem out of reach for current algorithms. The project will address this limitation by developing novel image processing algorithms. Extracting information from specific parts of complexes on an image by image basis will increase the quality and information content on each region, improving the whole data-set and allowing high-resolution reconstructions of flexible protein complexes, not currently accessible by any technique.

This project combines addressing the important biological question of DNA replication, with the development of new methods to enable imaging of continuous molecular flexibility in cryo-EM. As was illustrated with gamma-secretase in the Scheres group, working on a sample outside current analysis limitations drives the development of methods, while providing insight into outstanding biological questions.

One of the fundamental questions in cancer biology is what makes primary cancer cells metastasize specific organs. Albeit metastatic prostate cancer (mPCa) can be found in lymph nodes, lungs and brain, more than 90% of all mPCa will be found in the bone. Metastasis is an elaborate process that requires multiple genetic alterations; hence the molecular framework responsible for organ determination is poorly understood. Traditionally, metastatic potential of cancer cells is measured using limited in-vitro models, such as trans-well migration assays or using in-vivo mouse models that do not fully reflect the human metastatic process and are inefficient for large scale systematic screening. The Garraway lab has excelled in using genome scale functional screening techniques, using both gain and loss-of-function approaches to systematically dissect drug resistance mechanisms in solid tumors. I plan to exploit these tools and knowledge in order to establish a screening platform that will be used to explore the molecular mechanisms behind mPCa bone tropism. This array will be based on an in-vitro, 3D microfluidics culture system that emulates major steps of the metastatic process, such as extravasation, migration and colonization, in a most pertinent way. I will use this system to methodically overexpress a library of thousands of human cDNA’s in primary prostate cancer cells, followed by functional classification of genes, that when overexpressed will promote mPCa bone metastasis. Additionally, I plan to delve into the role of the androgen receptor (AR), a regulator of transcription that is central in the growth and survival of mPCa, in regulating mPCa bone metastasis.

Building a functional centrosome
Centrosomes, conserved organelles in eukaryotic cells, are essential for fundamental cellular functions such as microtubule nucleation and accurate cell division. Regulated centrosome biogenesis is critical for normal animal development and its dysfunction results in developmental disorders such as Seckel syndrome, dwarfism and microcephaly. Centrosomes consist of centrioles surrounded by a protein network of peri-centriolar-material (PCM). In order for centrosomes to be functional as a microtubule-organizing center, it must first successfully recruit PCM around centrioles. The complexity of PCM assembly, recruitment to centrosomes and their regulation remains a mystery. Thus, we apply our interdisciplinary collaborative expertise combining genetics, biochemistry and structural biology to address a fundamental question in cell biology of how PCM is successfully assembled in order to build a functional centrosome needed for the viability of an organism. First we will identify the minimum centrosomal components required for the functional assembly of a centrosome. We will then study regulators involved in the functional assembly of centrosomes in-vivo in Drosophila to identify molecular switches that trigger PCM assembly and co-ordinate spatiotemporal PCM recruitment at the whole organism level. To understand the structural basis for the functional assembly of a centrosome at the atomic resolution level, our collaborating laboratory will apply its expertise in biophysics and X-ray crystallography to determine the crystal structure of proteins that mediate centrosome biogenesis. Based on this structural information, we will then develop mutants to dissect functional centrosome assembly in Drosophila and human cells. Collectively, the proposed interdisciplinary collaborative studies will provide new and fundamental mechanistic insights into the assembly of a functional centrosome in a normally functioning cell.

Most of the mechanisms used by the bacterial pathogen Mycobacterium tuberculosis to survive inside its human host were characterized using mutagenesis as a starting point. Nevertheless, the genetic manipulation of the bacterium is labor-intensive because of the high illegitimate recombination rate and the slow growth of the bacterium rendering multiple steps for mutant screening time-consuming. In addition, mutagenesis is not adapted for the study of noncoding RNAs (ncRNA) though they constitute major regulators of gene expression. As an alternative to mutagenesis, I would like to develop an RNA-targeting strategy to control the expression of genes and ncRNAs in mycobacteria. To this end, I propose to improve an already established RNA-targeting strategy called External Guide Sequence (EGS). This technique consists in the expression of short RNAs (or EGS) that pair with target RNAs and give birth to a structure mimicking an immature transfer RNA. This structure is then recognized by the endogenous ribonuclease RNAseP that cleaves the target RNA. As a proof of concept, I will set up the silencing of heterologous and endogenous genes in M. tuberculosis. I will also try to improve EGS strategy by the design of EGS targeting the start codon and/or the ribosome-binding site of messenger RNAs to facilitate the gene silencing at a large scale. In addition, I would also try to develop a multiple gene silencing assay using EGS. Finally, I would like to widen this technology by its rerouting for noncoding RNA degradation. I hope to make this genetic tool universal in terms of targeting both genes and ncRNAs and applicable in all bacteria.

Intriguingly, somatic cells can be directly reprogrammed back to naive pluripotency by defined, exogenously expressed transcription and growth factors. Induced pluripotent cells can initiate differentiation into a variety of somatic cell types, providing a powerful platform for understanding basic aspects of embryonic development that harbors substantial promise for human therapeutics for diverse diseases. During reprogramming, a fully differentiated regulatory landscape is converted to a permissive landscape capable of directed differentiation toward a multitude of cell types. During directed differentiation, this permissive landscape is narrowed, resulting in a fully differentiated cells expressing a fraction of potential genes and gene regulatory modules. However, a high-resolution picture of these epigenetic transformations is effectively entirely missing. A mapping of the regulatory changes caused during reprogramming and directed differentiation would provide the first regulatory “movie” of cells executing these programs, fundamentally changing our understanding of how biological state is set and how it evolves. We will combine highly efficient programming and reprogramming systems developed in the Hanna lab with cutting edge epigenetic analysis methods developed in the Greenleaf lab to 1) map chromatin dynamics at base-pair resolution during near-deterministic somatic cell reprogramming using a sensitive assay of transposase accessible chromatin (ATAC-seq). These data will allow a temporal understanding of the chromatin, TF binding, and nucleosome positioning changes that occur during the reprogramming process, providing causal information regarding the regulatory regions and protein factors that drive phenotypic changes. We will then 2) identify regulators that drive the human naive and primed pluripotent cell differentiation competence towards authentic primordial germ cell (PGC) lineage, using a similar workflow. Finally, we will 3) reveal regulatory variability in human pluripotent cell populations expanded in a variety of newly developed growth conditions by probing the epigenome at the single-cell level. Our analysis of controlled developmental systems will decipher the dynamics and interdependence between different layers of chromatin regulation, leading to basic understanding of epigenesis with immediate application toward therapeutic ends.

Tendons are specialized tissues that connect bone to muscle, transmitting the force generated by these structures to allow for body movement. Tendon injuries due to acute trauma overuse or age related degeneration account for 30-50% of all sports related injuries and nearly half of all job related illness in the US. Studies have shown that tendons initiate a regenerative response but rarely regain their original biomechanical properties. Despite how common tendon injuries are, we still have limited knowledge of the molecular pathways regulating tendon cell maintenance and injury response. To address this, we will employ a robust method to study the cell proliferation dynamics of adult tendon tissue using the TetO-H2B-GFP reporter mice. The TetO-H2B-GFP transgenic mice have been used in several systems to monitor cell division history, including hematopoietic stem cells and muscle stem cells systems (Foudi et al., 2009, Chakkalakal et al., 2012). Next, we will use pulse-chase labeling of these transgenic mice to identify label retaining cells residing in the tendon and determine if they exhibit stem cell activity in an injury model. Finally, we will isolate the H2B-GFP label-retaining stem cells and perform RNAseq to identify unique molecular signatures of tendon stem cell populations. This work will impact our understanding of tendon cell behaviors during maintenance and injury conditions, and aims to discover new gene pathways unique to the label retaining stem cell populations. These discoveries could ultimately provide new therapeutic targets for the treatment of tendon injuries.

Insect-transmitted parasites endanger our health and our food supply and are the causal agents of e.g. malaria and yellow fever and numerous plant diseases in economically important crops. Surprisingly, only a few laboratories worldwide investigate which characteristics at the parasite molecular level are required for insect transmission and represent key drivers of parasite epidemics. We hypothesize that evolution of the triangular host-parasite-insect relationship is a battle between genes rather than individual organisms.
Aster yellows phytoplasmas (AYPs) represent parasites that excel at controlling their plant hosts, dramatically modifying their development and thereby not only affecting the plant-AYP interaction, but also the interactions between plants and AYP vector leafhopper populations, for example by increasing plant attractiveness to the leafhoppers. When studying such an extended phenotype that even changes the development of other species within its own ecological web, it becomes evident how developmental outcomes are intertwined with ecological ones. Therefore, we selected this triangular host-parasite-insect system as a paradigm to unravel how mechanistic aspects of gene function are linked to the ecological niche of the organism and the environment.
AYP express vir genes, encoding effectors, which products have a dramatic impact on plant architecture and hence, crop yield, but which facilitate AYP spread by their vector insects. The relative AYP fitness increase that can be achieved by those different modifications of plant development can only be determined by understanding their impact on the population dynamics and migratory behavior of the insect vectors, which in its turn allow for the expansion and spread of the AYPs.
Unraveling these complex interactions requires an interdisciplinary approach and multi-scale modeling framework, in which we can zoom out from the molecular up to the ecological level. We will compare and contrast a leafhopper migration-driven context found in Wisconsin (USA) with a residential-population driven context found in Poland (Europe), allowing us to separate the relative importance of the plant and the insect dynamics. These distinct ecological settings also allow for testing hypotheses regarding how the ecological embedding is determining which effector genes are selected for, both regarding type and severity.
This study will result in a better understanding of general principles of any triangular host-parasite-vector interaction web in their full multi-level context. Moreover, it will set up a modeling framework that can be straightforwardly applied to a broader class of disease studies, including human diseases such as malaria.

Human influenza viruses are transmitted by inhalation of virus-laden aerosol, whereas avian viruses are disseminated by the fecal-oral route. Thus, in order to become transmissible between humans, an avian virus must adapt to a different mode of transmission. It is well established that the hemagglutinins in the envelope of avian viruses recognize differently glycosylated receptors on host cells. However, we propose that host adaptation also requires changes in the structure, fluidity, and lipid selection of the viral envelope glycoproteins hemagglutinin and neuraminidase, the proton channel, and the matrix protein.
There are several requirements on the envelope’s biophysical properties for optimal transmissibility: The membrane must be fluid at body temperature (42 °C in birds versus 33 °C in the human respiratory tract) to allow virus budding, but it should solidify to protect virions against damage during transmission between organisms. After internalization by a new host the membrane must liquefy again to allow infection. The phase state of the membrane depends on temperature, but lipid compositions determine at which temperature phase changes occur. In turn, this will have an effect on the structure and organization of the viral envelope proteins and their interaction with the viral membrane, particularly regarding their ability to laterally interact, cluster and adopt certain conformations. Hitherto, these aspects have been widely neglected when studying the adaptation of influenza virus to new hosts.
We will take a multi-pronged approach to address this problem. Avian and human viruses will be grown at different temperatures and in different types of host cells. The lipidomes of these viruses will be determined. The phase and mechanical properties of the viral envelopes under changing environmental conditions will be examined. Structural changes of the viral envelope proteins will be determined at high resolution and the reasons for their altered behaviors will be sought by determining differing interactions with specific lipids in each case. Hypotheses about the molecular reasons for envelope adaptation to different host conditions will be formulated based on these results and ultimately tested for their relevance in viral envelope lipid selection and virus morphology by generation of recombinant viruses lacking lipid binding sites.

Prions are protein-based epigenetic inheritance units with the unique ability to switch between native and prion forms. This switch results in profound alterations in function. Under normal conditions, this switch may reduce the fitness of the organism. However, under acute stress, the organism may survive by trying untapped and hidden phenotypes revealed by the prion state. Intriguingly, many RNA binding proteins are predicted to have prion domains and prion switching of such proteins can dramatically alter cellular networks. I propose to experimentally characterize all yeast RNA binding prions using well-established prion assays. Furthermore, using stringent and quantitative RNA-protein interaction assays together with prion assays, I will characterize the functionality of RNA-binding proteins in their prion form and determine which RNAs they bind in the two different states. Differential RNA binding of prions will be a proof-of-principle for the concept of beneficial gains-of-phenotypes under stress conditions.
Moreover, several neurodegenerative disease pathologies have been associated with mutations in the prion-like domains of RNA binding proteins. The mutations are thought to alter RNA homeostasis upon prion-like folding of the protein. I will test one such prion candidate RNA binding protein, the eIF4G translation initiation factor. Two point mutations in eIF4G are linked to parkinsonism. I will test if these mutations increase the prionogenicity of eIF4G and/or cause differential binding of RNAs.
My work will provide valuable insights into the prion biology of RNA binding proteins and how mutations in them can alter cellular physiology in disease.

Modularity of gene regulation has evolved in response to organisms adapting to varying environments, and enables independent variation of functional systems as well as synchronized responses to single stimuli. I propose to study the changes in modular gene regulation involved in natural adaptation by testing (I) the long-standing hypothesis that suppression of recombination can create linked groups of adaptive alleles in differentially adapting populations, and (II) a novel hypothesis that linked groups of adaptive alleles can give rise to modular regulation. By adapting recently developed genetic methods to use in stickleback fish, I will not only test these hypotheses in an empirical setting for the first time, but also produce a wealth of novel information on the effects of adaptive divergence on recombination and gene regulation, as well as further advance the stickleback as the premier model organism for studying natural adaptation with molecular resolution. With this project I will switch from analyzing organ development in the mouse to exploring genome-wide effects of adaptation on recombination and gene regulation in fish. Learning the methods and skills required to carry out this project in the high-quality environment provided by my host laboratory I will significantly expand my expertise and gain the tools necessary for a future as an independent researcher.

Many microbes produce toxins that kill members of other species. Intriguingly, however, some microbes specifically target members of their own species - termed fratricide. Such behavior seems obviously deleterious; unchecked, it can have detrimental effects on the fitness of individuals and their populations, even leading to self-inflicted extinctions of clonal populations. Surprisingly, however, growing evidence suggests that fratricide is widespread. Under what circumstances can fratricide emerge as the product of evolution? Under what conditions should this trait be expressed, and how should it be regulated?
In this proposal, we aim to address these questions using advanced biophysical techniques and theoretical modeling.
We will use Streptococcus pneumoniae as a model system. Previous biochemical studies indicate that, in S. pneumoniae, fratricide is regulated by a complex intracellular regulatory network and coordinated by cell-to-cell signaling. We aim to:
1) Characterize the dynamics and regulation determining the fates of individual cells during fratricide.
Using advanced biophysical techniques, we will characterize the real-time dynamics of the pertinent regulatory processes, cell fates, and multicellular interactions with single-cell resolution.
2) Elucidate how fratricide can emerge as a stable product of evolution. We will perform competition experiments between the wild-type strain and various mutants under a wide range of spatio-temporal settings and analyse them using quantitative models.
Bridging molecular, cellular and multicellular scales, our proposed research will produce an integrated understanding of the regulatory mechanisms and evolutionary origins of fratricide. The conceptual and quantitative framework obtained from this work will be largely system-independent, applicable to various types of competition among conspecifics.

Aging is a process fundamental to all organisms. Exhaustion of stem cells and their niche is a hallmark of aging, as it limits cell turnover and thus tissue homeostasis. In humans, aging-related dysfunctions of stem cells are linked to many diseases such as neurodegeneration and cancer. The testis of Drosophila melanogaster offers an excellent model to study aging of stem cells, with well characterized signaling pathways implicated in their maintenance and where phenomena like de-differentiation were discovered. Much attention has been paid to the decline of stem cell number and of self-renewal factors, but there is a gap in our knowledge of other (i.e., non-gonadal) extrinsic signals and of mechanisms other than de-differentiation that replenish the stem cell population as a function of time. In this project, I will determine if de-differentiation of non-stem cells contributes to the maintenance of the germline stem cell population. I will attempt to identify the roles of the anti-oxidant activity pathway Nrf2 and of other potential non-autonomous signals in aging, focusing on the hypothesis that tissues other than the niche can contribute to the maintenance of stem cells and prevent their loss during aging.

Recent advances in sequencing technologies have greatly expanded our knowledge about mRNA transcription rates, abundance, translational activity and half-lives. By contrast, a mechanistic understanding of the cytosolic mRNA fate is still scarce: what are the cis determinants (i.e. mRNA sequence, chemical modifications) and trans factors (mRNA binding proteins, small RNAs) that signal for translation, repression or decay? How are these signals read out and implemented? One puzzling aspect of mRNA biology is that upon stress, mRNAs become sequestered into granular assemblies, so-called P-bodies (PBs). PB formation is conserved from yeast to human, and they fulfill pivotal mRNA storage tasks in e.g. neurons and oocytes. Still, they are poorly characterized in terms of composition, assembly dynamics and most importantly their precise function is unknown.
In order to better understand the life of an mRNA and the function of PBs, I propose to
a) perform mass-spectrometric analyses of the composition of mRNA-particles (mRNPs) destined for translation, repression and decay to identify key factors specific for each pathway
b) to reconstitute PB formation in vitro from yeast lysate, a unrivaled opportunity to manipulate and study their assembly and function
c) to establish a system to visualize and quantitate mRNA-protein-interactions in real time, by microscopy.
These studies will help us to decipher the regulatory code of mRNA turnover and the mysterious function of granular assemblies like PBs, and to better comprehend the many diseases associated with these fundamental pathways. Further, it might allow us to develop novel tools to manipulate the activity state of an mRNA.

Avian feather patterning has long been studied as a model of two-dimensional periodic pattern formation. Each feather emerges from the site where embryonic dermal cells aggregate. Previous studies have suggested that the dermal condensation at regular intervals is based on the spatial patterns of interacting morphogens. However my recent finding implied that the existence of cell-cell interactions between two types of dermal cells: one is the dermal cells of outer boundary of feather buds and the other is those of interior of buds. Time-lapse observation showed a unique behavior of dermal cells of outer boundary. At the beginning of feather patterning, the dermal cells was uniformly distributed throughout the region beneath epidermis. As skin develop, the cells actively moved to avoid the center of feather buds and localized in the outer boundary, suggesting segregating mechanisms between two types of dermal cells. This looks like a meshwork in the skin. The patterning process of mesh structures seems to be directly involved in the size of buds and distance between each buds. Thus, in this proposal, I aim to uncover the molecular basis of the pattern formation. To this end, I will use reconstitution assay in which dissociated dermal cells can regenerate complete feather buds, allowing the analysis of cell interaction and easy gene-transfer into dermal cells. We have candidate genes that regulate the spacing feather buds, so I will also examine whether these genes are involved in the interaction of dermal cells by gene suppression. This study will provide new insight into developmental biology.

The heart is the first organ to acquire its function during embryonic development and has a vital role in pumping nutrients/oxygen. In the early vertebrate embryo, the heart is a single primitive contractile cardiac tube. In mammals, it originates from a horseshoe-shaped cardiac crescent continuous across the midline that merges at the midline to form a tube. While in recent years significant progress has been made in our understanding of the gene regulatory logic that controls early heart development, approaches toward a comprehensive framework of the cell behaviors underlying the formation of the primitive heart tube from the cardiac crescent are still missing. In this project, we propose to combine genetic approaches to label in vivo the individual cardiac progenitor cells, state-of-the-art microscopy techniques (i.e. 2-photon and light sheet microscopy) to live-image the morphogenesis of the entire heart tube at cellular resolution in mouse and computational methods to extract quantitative meaningful morphogenetic parameters. Through this approach, we aim to elucidate the complex 4D tissue movements and associated cell dynamics- such as migration, division and changes in cell shape- that occur during heart tube morphogenesis. We aim to subsequently use these findings to analyze the putative cellular defects in embryos mutant for transcriptional regulators that show cardiac abnormalities. While much of the cardiac defects observed have been described by looking at the terminal phenotype, our goal is to understand how these defects arise from seeing directly the dynamics of the collective behavior of the cells that form the heart.

Structural genome variation and DNA methylation enable plants as sessile organisms to adapt to changing environments. Our current understanding of the interaction between structural variation and methylation, the underlying mechanisms and genome-wide extent, is limited by the lack of true reference genomes. In this project I will extend our knowledge by exploiting the Arabidopsis thaliana 1,001-genomes and methylomes project as an unprecedented resource created in the host lab. By utilizing groundbreaking single-molecule real time sequencing (PacBio) together with Bionano Irys optical DNA mapping, I propose to create true de novo reference genomes for 50 selected A. thaliana ecotypes. I will then characterize effects of DNA methylation on genome stability in methylation deficient mutants. Subsequently, I propose to generate A. thaliana populations from differentially methylated lines, and a mutant, and generate Bionano optical genome and methylome maps for F1 and inbred F2 progeny. From these large data sets, I will bioinformatically infer the genome and population-wide extent of SVs and DNA methylation to understand their potential associations. I will further expand on their causal relationship after selfing of heterozygous F1 progeny, and identify e.g. whether there is a bias towards the homologous chromosome containing the variation. I propose to use the created datasets to also test the hypothesis that changes in DNA methylation can trigger structural genome variation other than through retrotransposition. Results of this project will benefit our understanding of the evolutionary drive and consequences behind different shapes in the (epi)genome.

A well-recognized problem in rational drug design is inability to successfully predict the potency of the drug even for enzymes where the structure is known with atomic resolution. One of the missing pieces in the puzzle is the dynamical nature of the water hydrating the protein surface. This hydration water has properties significantly distinct from the bulk and mediates most of the biomolecular interfacial interactions, while its dynamics is intimately linked to the protein dynamics. The complexity of the problem becomes even clearer if one considers that the native environment where the protein functions is the heterogeneous and crowded environment of the living cell. Intracellular environment is known to alter the water dynamics due to high extent of macromolecular crowding. Therefore, if one wants to have a realistic picture of the drug-enzyme interaction in situ, it is essential to take into account the dynamical properties of both the solvating water and the protein itself in its native environment.
Recently a sensitive in-vitro method to probe hydration dynamics with (sub-) nanometer resolution based on Overhauser Dynamic Nuclear Polarization (ODNP) was developed in the laboratory of Prof. Songi Han (UCSB). The main goal of this proposal is to develop ODNP into a method that will be applicable to in-vivo studies in living cells. When developed this method will allow to monitor changes in hydration dynamics of the biomacromolecules that are induced by the heterogeneous in-cell environment and to compare them with the ones induced by the mimics commonly employed in the in-vitro studies such as cell extracts and solutions artificially crowded with high MW polymers.

Persistence in Mycobacterium tuberculosis is a phenomenon of cells where a fraction is resistant to a killing assault as a consequence of a transient gene expression or protein modifications. I believe that the persistence phenomenon is being unmasked at the presence of bactericidal drugs or host adaptive immunity and it is the single most important impediment to tuberculosis control. While several genes were previously proposed as contributing to the emergence of persistence, the complete regulatory network is still unknown and it is clearly not attributed to one 'persister gene'. My goal is to define the genetic network causing the persistence phenotype in Mycobacterium tuberculosis. I plan to tackle this problem by exploring the roles of the transcription initiation sigma factors in persistence in the presence of bactericidal drugs. This will be done by a combination of precise null deletions of the twelve nonessential sigma factors together with ChIP-seq and RNA-seq analysis. In addition to the in vitro assays, I will be also testing my hypothesis in vivo, by examining the effect of sigma factor mutant strains as well as mutants of the genes transcribed by each sigma factor. All these mutants will be made in the background of the sigma factor deletion strain (double mutants) and tested in an in vivo mouse model for Mycobacterium tuberculosis cells that are unable to persist.
By combining the in vitro high throughput screens with the in vivo experiments, I expect to define the genetic network causing persistence in Mycobacterium tuberculosis.