Over the last few years there has been an enormous development in biological research and the birth of the fields of genomics and proteomics. One common requirement of these disciplines is the need for sensors that allow identification of specific DNA sequences or protein interactions. This proposal is focused on the development of new fluorescent tools and sensors for studying biological processes. First, using rational and combinatorial methods, new fluorescent GFP-like mini-proteins will be designed that can be incorporated at the DNA level into any recombinant protein. Second, fluorescent switchable sequence-specific DNA-binding peptides and protein complexes will be designed for the study of the basic mechanisms of DNA recognition. Finally, their application as probes for specific DNA sequences and gene expression control will be tested. Support from HFSP is fundamental for the development of these projects, since they close a gap between the purely chemical research carried out in our department and new biological-focused research in the frontier between chemistry and biology. The experience accumulated in the last few years in our department in the field of fundamental bioorganic chemistry gives us the opportunity to aim to more ambitious objectives, which can have practical implications in biological research.

DESIGNING A QUANTITATIVE MODEL OF THE CELL I propose to develop a quantitative model that captures main determinants of the molecular biology of the cell and its transitions between different states, for example during cell growth. This will lead to a more detailed understanding of how actions and interactions of proteins are controlled and how they determine the overall state of the cell. For these projects, I will join Prof. Edward Marcotte’s lab since it offers excellent facilities for both computational and wet-lab experiments in that field. First, I will explore a specific subset of yeast proteins in detail, e.g. the proteins of the mating pathway. This will reveal the possible dimensions of my analyses. Second, I will design a quantitative model of the cell that revolves around a series of transitions between discrete cell states, where these “states” are defined by gene expression profiles. I will identify the “basis set” of transcriptional patterns that best explain the majority of existing yeast expression data for each cellular state. The basis set will be used in a neural network that predicts expression profiles, e.g. for gene knockouts. It may even be possible to describe the cellular behaviour in a simple hidden Markov model which emits gene expression patterns for particular cellular states. Next, I will use the basis set of expression profiles to extract subnetworks from a probabilistic network of gene functional relationships and examine their properties. Finally, in order to better understand the robustness of these networks, I will analyse how gene duplicates are distributed across the network, e.g. how they conserve their position.

The selective expression of var genes forms the basis of immune escape and pathogenesis of the agent of severe malaria, Plasmodium falciparum. In order to study epigenetic mechanisms involved in the regulation of var gene expression, yeast orthologues involved in nucleosomal promoter repression will be disrupted in the parasite. Altered var gene expression will be assayed by measuring mRNA and protein levels and performing functional studies. Complementing this approach, two var gene promoters will be analysed. Wild type and mutated var gene promoters upstream from a resistance marker will be stably integrated into wild type and knockout parasites established in the first part. By enforcing expression through drug selection, elements involved in promoter activation or repression will be determined under different chromatin constraints. To identify cis-element binding factors, a pull-down assay of an episomally maintained plasmid, containing a var gene promoter positioned upstream of a resistance marker, will be performed. Bound proteins will be separated by denaturing gel-electrophoresis and subsequently analysed by mass-spectrometry. My previous research activities focussed on mosquito vector innate immune responses towards Plasmodium. The fellowship would enable me to extend my expertise towards the understanding of P. falciparum survival strategies in the human host. The profound training in parasite and vector biology would be optimal for my future scientific career in the field, enabling me to take novel and broad research paths to understand parasite development and transmission.

The brain detects noxious stimulation of the skin, viscera and other internal structures via different subsets of primary nociceptive sensory neurons. These subsets possibly engage distinct circuitry all the way up to the brain, explaining the ability to distinguish the bodily sites at which noxious stimuli are detected. The objective of this proposal is to get new molecular markers for such subsets and especially for viscera nociceptors that project to the gut and other internal organs and that form the basis of interoception, the brain’s perception of the body’s internal states. The ultimate goal is to use them, in conjunction with already identified markers for nociceptors innervating other targets, to compare their sensory maps, so as to illuminate the logic of brain decoding in the understudied visceral pain. In order to do that we will dissect the function, by gene targeting, of MrgB4/B5 members of the GPCR family of Mrgs, which is believed to be involved in nociception. Since the MrgD member is specifically expressed in cutaneous afferents, it is implied that the other Mrgs might be characteristic for nociceptor subsets innervating different organs, including viscera. Additionally, we will conduct a screen that will be based on the comparison of the gene expression profile of single cells from cutaneous nociceptors (marked by MrgD) and nociceptor afferents enriched by other markers for visceral targets. The outlined research will introduce me to the problems, methods and logic of the new field of neurobiology, giving me the chance to devise novel and creative ways, in which I can bring my previous training in mouse molecular genetics to bear on solving these problems.

A fundamental question in vertebrate development is how regional identity is specified in the developing embryo. For instance, the developing hindbrain is transiently subdivided into seven contiguous segments, termed rhombomeres, a highly conserved process in vertebrate development. The regional domains defined by the boundaries of each rhombomere form the scaffold upon which the subsequent specialization of neural function in the brain is built. Zebrafish provide a powerful model to genetically dissect the molecular mechanisms involved in this process. Each rhombomere has a unique identity based on segment-specific expression of Hox genes Hox gene function is crucial for rhombomeric identity. Recent work in Drosophila has shown that the zinc-finger proteins Teashirt, Disco, and Disco-related behave genetically as Hox cofactors. We have cloned zebrafish homologs of Teashirt, and have found that some of these are expressed in Hox-like patterns during zebrafish development, as would be predicted of a Hox cofactor. I propose to determine whether Teashirt genes are required for Hox gene function in the developing hindbrain of zebrafish. I will identify Tsh mutants in zebrafish and study their effects on known Hox-dependent processes. Second, I will investigate whether these Teashirt proteins interact physically with Hox proteins or other components of the Hox DNA binding complex. Lastly, I will clone zebrafish disco homologs and study their expression and function. Ultimately, this research will help our understanding of how neural patterning is established and maintained in vertebrates, an important first step in understanding higher neural function.

Colour patterns are involved in many aspects of the lives of animals ranging from mate recognition and predator avoidance to thermoregulation. As pigmentation patterns are major functional end products in animals, their development must be tightly regulated. The biosynthesis of the most common pigment in animals, melanin, is well known. However, the ways by which pigmentation patterns are formed and evolved are poorly understood. Because pigmentation patterns are rapidly evolving traits in the Drosophila genus, they offer an ideal framework to study how modifications in gene regulation lead to phenotypic evolution. This proposal aims to investigate a key locus for melanin pattern evolution in Drosophilids, the yellow gene and its cis-regulatory elements. The wings of Drosophila biarmipes males display a melanin spot, which is important for sexual behaviour and selection. Yellow protein expression in the pupal wing correlates to this spot. In Drosophila melanogaster, Yellow protein is absent in the corresponding area and no spot is present. This is due to functional differences in the upstream regulatory region of yellow. Intriguingly, the transcription factors are present for making the spot in D. melanogaster. I propose to localize functional differences in cis-regulatory elements of the yellow gene of different Drosophila species and to identify the transcription factors that recognize sites that have changed.

The phenomenon that all members of a plant species exhibit resistance to the pathogens that infect other plant species is known as ‘non-host’ resistance. In Arabidopsis the mutation LRA5+-1 reduces non-host resistance to some bacterial and fungal phytopathogens. While there is no macroscopic phenotype associated with the mutation, transcriptomic data show that LRA5+-1 plants lack basal expression of defence transcripts, indicating that a central node in defence signalling is affected. LRA5+-1 has been mapped to a ~100 kb interval on chromosome II. Complementation and sequencing studies are in progress, and the cloning of LRA5+-1 is expected shortly. I will use biochemical approaches to elucidate the role of LRA5+-1 in non-host resistance. LRA5+-1 is a dominant gain-of-function mutation. Therefore both the mutant and the wild-type proteins will be analysed. To this end I will generate transgenic lines of Arabidopsis expressing epitope-tagged versions of LRA5+-1 and LRA5 (LRA5s). This will allow me to study the dynamics of the proteins in response to plant defence signalling molecules and pathogen challenge, and to identify proteins associated with LRA5s by coimmunoprecipitation. The molecular and biochemical approaches chosen to study LRAs are not only likely to unravel some of the mechanisms underlying non-host resistance they will also furnish me with firsthand knowledge of new techniques that will be of great value to me when I come to lead a research group of my own one day.

The proposed research aims at the design of hybrid structures composed of functional molecular building blocks and nanometer scale systems. Combining my previous background in molecular model systems and the host lab expertise in nanoscience will allow me to expand my scientific skills and scope. We suggest developing a versatile and robust architecture for studying fundamental biological and chemical processes. Understanding the fundamental processes and phenomena utilized by nature for efficiently carrying out catalysis, assembly, locomotion, and other vital mechanisms requires tools capable of differentiating the ensemble behavior and the underlying individual molecular properties. Thus, we will progress towards an experimental platform where the ‘sensing’ (i.e. observable response), and the studied motif can be tailored in a modular manner. Such capabilities are expected to be attainable by the combination of recent advances in nanodevices, chemical modification of surfaces, and biosynthesis in Prof. Lieber’s group and implementation of my previous research experience. The development of molecular functionalized nanowire hybrids that integrate the molecular-level complexity with an efficient transudation and enhancement of the information produced as a result of specific molecular interactions and response may provide a highly desired bridge between the molecular level physical environment and a well-resolved readout signal. Recent progress achieved at Prof. Lieber’s group demonstrated that the selective detection at single molecule level (that is, stochastic sensing) is possible with nanowire or nanotube field- effect transistors.

Antiandrogen (antagonist) therapy is the standard therapy for prostate cancer. But many men eventually fail and die of a relapse of the hormone-refractory, androgen-independent prostate cancer (AIPC). Understanding the mechanisms of AIPC is the key towards developing new therapies. Androgen receptor (AR) regulates gene expression in the presence of androgens (agonists) or antogonists, recruiting coactivator or corepressor complexes, respectively, to promoters. The host lab found that N-CoR corepressors are required for the inhibitory action of antagonists. The antagonism actions of currently used antagonists depend on N-CoR, or they function as agonists in the case of AIPC. So to identify N-CoR-independent compounds/ligands is the key to understand and prevent AIPC. I will identify antagonists in N-CoR-deficient cells, and use them to elucidate mechanisms of alternative coregulator assembly. The N-CoR-deficient prostate cancer cells will be employed in a screen of compounds libraries. Compounds will be scored in the high throughput, reporter-based screening as antagonists in those cells. Positives will be evaluated for their ability to repress AR target genes in AIPC cells, to recruit alternative corepressors and/or to release coactivators on promoters of AR target genes. The ability of compounds will be ultimately tested using cell culture and in vivo xenograft models of AIPC. This fellowship will provide me a thorough training in endocrinology/biochemistry, which is a perfect complement to my PhD study in genetics. Also it is a unique opportunity to conduct independent research at a top-ranking lab and will serve as a transition of my career to an independent scientist.

Glycosyltransferases, the enzymes that are responsible for the synthesis of the vast majority of oligosaccharides in the biosphere, are an attractive alternative to chemical approaches for the synthesis of complex oligosaccharides. My proposed research is to use directed evolution methodologies in order to engineer glycosyltransferases for the enzymatic synthesis of novel oligosaccharides. More specifically, I plan to evolve bacterial glycosyltransferases, to utilize readily synthesized and inexpensive glycosyl fluorides as donor sugars for transfer to a range of different glycosyl acceptors. In addition I propose to change the selectivity of the glycosyltransferases to accommodate different sugar acceptors and open the possibility of enzymatically synthesizing novel oligosaccharides. The engineered enzymes will shed light on the mechanisms of glycosyltransferases and the oligosaccharide products may be of value in the search for new therapeutic agents. The fellowship experience will give me the opportunity to conduct research in the large and important field of carbohydrate processing enzymes, which will be different from my previous research experience where I never dealt with carbohydrates or glycosyltransferases. I will be exposed, during the fellowship period, to a variety of ideas and techniques that are unique to the glycobiology world and this will enhance my professional growth, eventually as an independent investigator conducting research in this important field.