Schizophrenia is a devastating mental disorder and impairment in cognitive processes, such as working memory, might represent the core features of the disease. The Merzenich group is elaborating a brain plasticity-based training strategy designed to prevent the schizophrenia onset in prodromal and at-risk individuals, and to ameliorate its symptoms in patients. The training strategy is designed to re-strengthen working and associative memory. In preliminary studies with less complete training tools (both using about half the repertoire that is now available) conducted at UCSF and Yale, it was observed positive impacts of training on almost all measures. This project intends to evaluate how changes in the disease symptoms are followed by functional changes in the brain. Biochemical markers affected by disease progression and treatment will be searched. In animal models it will be evaluated the capacity to increase neural functions by training. Possible biomarkers correlation to those changes will also be studied. These objectives will be assessed by conducting elaborated blood analyses and imaging studies before vs after training. In animal models the focus will be the development of working memory and cortical and cortico-thalamic feedback, as these systems are profoundly dysfunctional in schizophrenic. Biochemistry evaluation of tissue samples from these animals will be conducted as well. By using this striking training induced approach this project may extend our understanding of brain plasticity capacities and its use in mental disorder treatment.

The goal of this project is to develop a computational method to compensate space variant aberration (SVA) encountered in 3D widefield fluorescent microscopy due to refractive index (RI) variations within the specimen. We will attempt to remove this aberration via a space-variant convolution (SVC) modeling, and then a space-variant deconvolution (SVD) operation. - The first step is to obtain the 3D refractive index map of the specimen by using differential interference contrast microscopy. Then the actual florescent imaging is performed. Next, the SVC model, which will express the image in terms of the object, will be built from the RI map. The convolving function, which changes spatially, indeed captures the effect of the SVA caused by the local RI variations. Finally, the object will be recovered from the image by performing an SVD. - To represent the image formation as an SVC, we will express each image voxel as a weighted integral over the object. We will call the weighting function the voxel response function (VRF). Next, each VRF will be expressed as a product of the traditional point spread function (PSF) and a local aberration function (LAF). Then we will find a formula for fast computation of all LAFs from the RI map. This part is the key in making the whole project computationally tractable. - We will formulate the SVD problem as an optimization task, where the required object is a minimizer of a cost function consisting of a data fidelity term and a regularization term. The data fidelity term consist of the SVC operation over the candidate object and a comparison of result with the measured image. We will construct a multigrid algorithm for solving this optimization problem.

The specific aim of this proposed research is to understand the mechanism by which Polycomb group proteins repress gene expression. This goal will be achieved by using combination of structural and biochemical methods. My research will focus on PcG proteins of PRC1. I will study the structures and functions of the PcG proteins of PRC1 and its functional complexes with nucleosome(s) and the transcription machinery. X-ray crystallography will be used to determine the three dimensional structures of individual PcG proteins of PRC1. In addition, the core PRC1 complex, also in its functional state with nucleosome(s), will be investigated with hybrid methods by x-ray crystallography and electron microscopy. Biochemical studies based on the structural information will be performed, to elucidate the mechanism of PRC1 in chromatin remodeling. I will also try to determine the interactions and structures of PRC1 and the components of general transcription machinery. Biochemical methods will be used to map the interactions between the components of the general transcription machinery with the PRC1. I will determine the structure(s) of these assemblies to understand the mechanism of PRC1 silencing by direct interaction with the basal transcription machinery. This combined structural and biochemical approach is critical to understand the way PRC1 functions in gene regulation and how the deregulation of PcG expression causes cancer.

Most physiological and behavioural processes in mammals are governed by a complex intrinsic circadian clock. The mammalian circadian clock is composed of a central pacemaker in the brain that coordinates subsidiary oscillators in most peripheral tissues. The intracellular clock mechanism involves interacting positive and negative transcriptional feedback loops that drive rhythmic expression of key clock components. Experimental evidence suggests a strong link between metabolism and circadian rhythms. Circadian clocks respond to feeding cycles and control the level of many cellular metabolic enzymes and nutrients. These observations point out a feed back-loop between the circadian clock and the cellular metabolic status. However, the molecular mechanisms that govern the interplay between metabolism and circadian oscillators are not yet clear. Recently it was demonstrated that, at least in vitro, the cellular redox state and NAD(P)+/NAD(P)H levels regulate the activity of the clock components. Here we propose to further study the interplay between cellular redox state and circadian clocks in living cells. We intend to develop a novel method that follows NAD(P)+/NAD(P)H levels in living cells and animals. Such a method will allow us to study the cross talk between cellular NAD(P)+/NAD(P)H levels and circadian oscillators. This project is aimed at understanding fundamental aspects of the molecular mechanisms within the mammalian circadian clock in connection with cellular metabolic state, in particular the involvement of cellular NAD(P)+/NAD(P)H levels in the phase entrainment of peripheral circadian oscillators.

Polyadenylation is a fundamental step in the formation of almost every eukaryotic mRNA. The 3’ poly(A) tail plays key roles in the mRNA life cycle, including nuclear export, turnover, and translational activity. Despite more than 40 years of studies on mRNA polyadenylation, the mechansism of poly(A) tail length control is still poorly understood. We have recently characterized an evolutionarily conserved nuclear poly(A) binding protein (PAB2) in the fission yeast Schizosaccharomyces pombe. Significantly, mRNAs from pab2-null are hyperadenylated, indicating a key role for PAB2 in polyadenylation control in vivo. Our objective is therefore to understand how PAB2 determines and regulates poly(A) tail length control. To achieve this objective, the proposed research is designed to test the hypothesis in which PAB2 is recruited by the RNA polymerase II transcriptional machinery and transferred to 3’ ends of pre-mRNAs to regulate polyadenylation. We also intend to determine the functional role of a posttranslational modification where specific arginine residues of PAB2 are methylated. Given the critical role of fission yeast PAB2 in poly(A) tail length regulation, our rationale is that a detailed understanding of PAB2 function will provide novel and significant insights into the mechanism of polyadenylation control in eukaryotes. The HFSP long-term fellowship was instrumental in the establishment of my current research program and my return to Canada yet, I now need further support to continue this important work. An HFSP CDA award would give me the financial and scientific independence, support, and tools that I need to make this work possible.

The interaction between several cell types inside a neuronal circuit enables an organism to perceive information from its surroundings and produce an appropriate behaviour. To understand this processing of sensory information, the contribution of each cell type to the logic of neuronal circuitry must be defined. The Drosophila visual system, with its relatively simple anatomy and its amenability to genetic manipulation of neuronal activity is ideal for such studies. The aim of the proposed work is to decipher how the neurons of the fly medulla, the neuropil of the optic lobe specialised in colour vision, process colour information coming from the sensory end of the circuit, the photoreceptors. Using two-photon imaging of genetically encoded sensors of neuronal activity, I propose to develop a system in which the activity of the different subtypes of medulla neurons is precisely monitored when light of a specific wavelength is applied. In parallel, I will validate techniques to artificially depolarise or hyperpolarise genetically targeted neurons that will serve to map the next level of connectivity in this circuit. This work will enable me to build a functional map of the colour vision system and will lead to an understanding of how colour is represented in the brain.

I propose to study olfactory signaling for the fellowship period. My primary goal will be to combine physiology and molecular biology to study the recognition of odorants by receptor proteins, ad to study the downstream signaling steps. Because the standard approach of expressing and studying cloned odorant receptors in heterologous expression systems has so far met with limited success, I propose to approach the problem in the opposite direction, namely, studying these receptor proteins in primary olfactory receptor neurons with a panel of odorants by using electrophysiology (suction-pipette recording) and then characterizing the molecular structures of the same receptors by using molecular biology (single-cell polymerase chain reaction). Although an analogous approach has been undertaken previously by others using Ca-imaging to probe receptor responses to odorants, this assay is not as detailed in characterizing receptor-odorant interactions as electrophysiological recording, especially the suction-pipette recording method that I propose to use, which has a resolution capable of examining the response of a receptor cell to the binding of a single odorant molecule. A correlation between the properties of the odorant receptor proteins and their structures will be an important step toward understanding the olfactory code. In addition, I propose to study two questions not previously addressed by others. One is constitutive activity of the odorant receptors, and the other is adaptation mechanisms of the olfactory receptor cell.

The integrity of stratified epithelial sheets in skin is dependent on intercellular adhesion, which is in part mediated by adherens junctions. These junctions are dynamic, allowing for tissue remodeling during morphogenesis and tissue repair following injury. Wound healing is an orchestrated process that involves movement of epidermal cells to close the gap, inflammatory responses to prevent infection at the injured site, and hyperproliferation to replenish lost cells. Targeted loss of the adherens junction protein alpha-catenin in skin results in enhanced inflammatory, hyperproliferative, and migratory behaviors, changes in epithelial cell morphology, and development of squamous cell carcinoma in situ. The molecular roots of these phenotypes appear to be intrinsic to the epidermal keratinocyte. I have developed an approach to dissect the mechanisms underlying the complex phenotypes arising from alpha-catenin loss in skin. I devised a series of RNAi screens in wild type and alpha-catenin mutant keratinocytes to assay how the genes that are differentially expressed in these cells contribute to each phenotype. I then propose functional analyses of my identified factors to address the phenotypic consequence of their loss in skin, the nature of their interaction with alpha-catenin, and their function in wound repair and squamous cell carcinoma tumor development and progression.