The recently identified rhomboid intramembrane proteases possess the unusual characteristic of cleaving substrates within the hydrophobic environment of the lipid bilayer. Although present throughout evolution, little is known about the biological role(s) of rhomboids outside of Drosophila, where they cleave EGF receptor ligands. In particular, the biology of the mammalian rhomboids remains elusive as few substrates for these exist to date. Identification of substrates is clearly essential in order to understand the biological role of these proteases. Here I will exploit my prior experience in proteomics to identify novel rhomboid substrates. Importantly, the physiological validity of the novel substrates will be dissected using model organism approaches existing in the Freeman laboratory. This fellowship differs from my current research experience in several ways. I will be moving into a completely new research area based on rhomboid proteases. I have no prior experience in this field. Critically, this fellowship will enable me to train in Drosophila and mouse model organism work for the first time. This experience will provide me with a solid experimental niche applicable to many biological problems as an independent researcher. Working under the mentorship of Dr Freeman in the Laboratory of Molecular Biology in Cambridge will provide me with the necessary training, mentorship skills and publication record to achieve my goal of establishing my own research group in 2-4 years.

The function of neuronal circuits relies on the formation of specific synaptic connections among different classes of neurons. While much is known on axon guidance, topographic mapping, synapse formation, and plasticity of excitatory principal neurons, GABAergic circuitry development is an area that has not been studied. For instance, little is known concerning the generic and specific mechanisms that promote arborization of immature GABAergic interneurons (GIs): what chemotrophic factors may influence these activities and how? Here, I propose to take advantage of our already established BAC transgenic mice that labelled at synaptic resolution GIs with GFP, to study the role of “guidance cues” (i.e. Slits, ephrins, semaphorins, netrins and GPCR) in guiding and organizing GIs axons. Using a primary culture of GIs I will first test the physiological effect of these molecules in shaping GIs axons. In addition, I will over-expressed, mutated or down regulated these molecular cues both in vitro and in vivo and test their effects on the GIs axonal targeting and patterning. I expect to find molecules that will be important for general axonal branching pattern as well as molecule that will be key regulators of cell type recognition. Finally, using calcium imaging I intend to study the signaling pathway induced relevant guidance cues in GIs and correlate with branching pattern and axonal directionality both in vitro and in vivo. This project will bring together multiple aspect of neuroscience form the cell biology to axon guidance, cell signaling and network organization. The HFSP CDA program will help me to established this new research area in my home country.

Most research into sensory-perceptual processes focuses on a single sensory modality, but our every-day experiences are filled with information accumulated from multiple modalities simultaneously. Thus a fundamental question that remains largely unanswered is how we combine information across modalities in constructing our perceptual experiences. The purpose of the present project is to understand how information collected through one modality shapes or tunes the ongoing percept in another modality. We will use psychophysical methods to study the perceptual experience of human subjects as they simultaneously feel a tactile stimulus and view an image of the stimulus. In a typical sequential sensory discrimination task, subjects are required to compare two consecutive sensory stimuli. We will initially focus on a frequency discrimination task - either between two visual flickers or two vibrotactile stimuli. Separate experiments will measure subjects’ performance (discrimination thresholds and response time ) and adaptation for isolated visual and tactile presentations and for combined visuo-tactile presentation (while the vibrotactile stimulus and the visual flicker are completely congruent or when they exhibit different degrees of spatial or temporal discrepancies). After a long exposure to one modality, we will test the degree of adaptation present in the same versus alternative modality. These experiments will help us understand how the brain fuses multiple sensory modalities to generate a seamless experience of the world. It will also provide insights about how input in one modality can affect the basic mechanisms of stimulus processing in the other.

De novo design of functional proteins is emerging as a powerful tool to improve or totally alter their original functionality. Such studies shine light on the basic principles that govern protein folding, protein interactions with proteins or other molecules, and may facilitate the development of devices for biotechnology applications. However, the progress towards these goals is limited due to the fact that most of the currently used synthetic scaffolds do not possess surface area that is large enough to coordinate complex and massive biological ligands. I suggest here the design, synthesis and evaluation of artificial Leucine Rich Repeats (LRR) proteins as universal recognition entities for the preparation of receptors and biomimetic catalysts. The LRR motifs possess extended ligand binding surfaces and play central role in a wide scope of biomolecular recognition events in cells. Their analogs will be prepared by chemical methods that allow introducing sequence diversity from natural as well as non-natural amino acids. Efficient binders of proteins and nucleic acids ligands, and catalysts for reactions that induce conformational changes in proteins, will be identified in dynamic combinatorial libraries of such proteins, a method by which the host is being selected and amplified by its guest at the expense of competing structures. I anticipate that the modular synthesis and screening approaches will allow the development of LRR binders for large variety of biomolecules, which will be utilized for parallel detection in vitro and within cells.

Multisensory encoding of the spatial location of stimuli is a challenging issue in the sensorimotor processing field. At the last stage of sensorimotor processing, the frontal cortex is a key region to study such mechanisms. Of interest to us, the frontal eye field (FEF) represents both visual and auditory stimuli in an eye-centered reference frame (Russo and Bruce 1994).This is not surprising as this region is implicated in the generation of saccadic eyes movement. However, stimulations of the FEF not only produce eye movements but also head movements (Tu and Keating 2000). Consequently, multisensory reference frames have to be re-evaluated under natural conditions, where both eyes and head contribute to gaze shifts. The aim of the project is to assess frames of reference for the representation of visual and auditory signals in FEF neurons in head-free monkeys. Dr. Crawford's lab is the only lab in the world performing head-free, 3-D recordings in the monkey. He has developed specific techniques and analysis tools for describing reference frames under this natural configuration (Martinez-Trujillo et al 2004). My own expertise is that of multisensory reference frames on head-restrained animals (Avillac et al 2005). Consequently, this collaboration will provide me totally new skills on head-free and 3D recordings. It will offer me the opportunity to give considerable advances in the field of multisensory space representation, in which these techniques have never been used before. References Avillac et al 2005 Nat Neurosci. 8:941-949 Martinez-Trujillo et al 2004 Neuron 44:1057-1066 Russo and Bruce 1994 J Neurophysiol 71:1250-1253 Tu and Keating 2000 J Neurophysiol 84: 1103-1106

Transcription is the first step in the pathway of eukaryotic gene expression. Transcriptional regulation underlies cellular differentiation, development, oncogenesis, signaling and other fundamental processes. The aims of the proposed research concern the structural characterization of RNA polymerase II transcription initiation complexes. Structures will be determined by electron microscopy and image processing, electron crystallography, and X-ray crystallography. Comparisons of structures containing various components will reveal possible conformational changes underlying the transcription mechanism. Therefore structural characterization of such complexes will not only reveal information about interactions between components but also provide new insight into the mechanism of transcription initiation. In this way, “static” structural information may be combined to elucidate the mechanism of a dynamic process. The ultimate goal is the structure determination of a complete 48-protein RNA polymerase II transcription initiation complex, and thus elucidation of the mechanism of transcription initiation and its control. This experience will introduce me to a different model organism (yeast) that will allow me to explore novel molecular engineering tools. This fellowship will broaden my horizons in the research of regulation of gene expression since I will change from the pre-mRNA splicing field to the transcription field. Finally, this fellowship differs from my previous research experience in the structural study of a macromolecular complex as I will have the opportunity to combine electron microscopy with X-ray crystallography