Genomic instability is a hallmark of cancer cells. It has been recently reported that the DNA damage checkpoint is constitutively activated in human precancerous lesions. Interestingly, precancerous cells also express common fragile sites, which are regions of the genome that frequently break as a consequence of a replication stress. The nature of the replication stress that accounts for this genomic instability is not known. The aim of this research project is to characterize the genomic instability occurring spontaneously in precancerous lesions. My work will consist in the characterization of these replication defects which are source of checkpoint activation and the identification of their genomic localization. We will first determine the nature of the DNA replication stress and its consequences on genomic instability. We will focus on common fragile sites, which are unstable in precancerous lesions. We will also focus on the rDNA array, which is to date the only known region of the human genome containing replication-impeding sequences. This work will notably be done by analyzing the replication dynamics of individual chromosomes by DNA molecular combing. Then, we will search for and extend our analysis to other replication pause sites over the genome. Finally, we will check whether the replication fork arrest at natural pause sites is a regulated process, in human cells at a precancerous stage. Indeed, I am convinced that a better understanding of cancer-associated cell cycle defects will permit the development of more efficient therapies against cancer.

Multiple sources of sensory information can be used to define the spatial locations of targets for arm reaching movements. Reaching toward an object in space requires a transformation of visual, auditory, and proprioceptive signals into a motor plan suitable to drive the arm. To carry out these sensorimotor transformations and specify the desired displacement, the brain must integrate sensory signals related to the target and reaching hand positions. Whereas the fundamental relevance of multisensory processing is becoming increasingly clear, the neural mechanisms underlying these cross-modal interactions and the spatial-aspects of sensorimotor transformations, primarily the frames of reference component, are not well understood. Here we will examine reach performance of patients with parietal damage to determine whether planning of reach movements to visual and auditory targets use a common cortical network to transform these signals into motor commands. The outcome of this process is fundamental to perception, cognitive processing, and the control of action. This is an essential step in establishing collaboration with leading authorities, Drs. Yves Rossetti & Laure Pisella, in multimodal sensorimotor integration and human motor control and the achievement of my principal career goal, which is to operate a human motor control laboratory in Canada and institute a research program that is highly interdisciplinary in nature - combining neuropsychological patient investigations, innovative modern techniques to measure or evoke functional activity in the human brain, and traditional cognitive and psychophysical methodologies.

I am interested in determining the molecular role of RecQ type helicases at the replication fork. During replication, the DNA polymerase encounters obstacles such as DNA lesions and areas of strong secondary structure, which cause the replication fork to stall. The cellular response to stalling consists of triggering a repair response and activation of the cell cycle checkpoint. The absence of either process facilitates genomic instability. The maintenance of stalled replication forks depends on the presence of RecQ helicases. RecQ proteins are intricately connected to the cell cycle checkpoint through binding the single-strand binding protein RPA (the yeast RFA homologue). RPA is also an essential subunit for the S-phase checkpoint kinase and is part of the DNA synthesis machinery required for replication restart. Specific goals include: 1. Determine the importance of the phosphorylation through Mec1/ATR kinase on either RPA or RecQ helicases. Does modification enhance function of the helicase at the fork? 2. Determine the structure of Sgs1-RFA1 complex, and that of the WRN helicase bound to DNA. The structure of these proteins is an important finding currently lacking in the field. Together with the biochemical approach I plan to carry out, these studies will provide much needed insights into structure and function of RecQ helicases at the DNA replication fork and in their role in safeguarding genomic integrity.

The possibility of cell regeneration in the human brain was thought impossible, until the discovery of two small niches of neurogenic stem cells in the human brain that continue to generate new neurons throughout life. Stem cells in the subventricular zone normally migrate towards the olfactory bulb and become olfactory neurons, but multiple studies in rodents have shown that these cells have the potential to migrate towards areas of CNS pathology.

My recent research emphasis has shifted to the analysis of transcriptional control of gene expression by nearby regulatory elements and to a recognition of the role of long-range interactions and three-dimensional organization of chromatin within the nucleus. Insulators are now known as a major endogenous mechanism for epigenetic control of gene expression. Although many recent studies have been devoted to the identification and characterization of the CTCF-dependent insulators, there is still little information and mechanistic understanding of insulators distinct from CTCF. Arylsulfatase insulator (ArsI) is distinct from the CTCF family of insulators and is known to work in various animals from humans to plants. Unlike CTCF-dependent insulators, the ArsI sequence does not have CTCF-binding sites and can work independent of nuclear matrix. ArsI insulator is also found well conserved in sea urchins and there were over 100 putative sites for this fragment in the genome, implying the significance of this DNA fragment. Using biochemical and computational approaches, I would like to identify the proteins that interact with ArsI and reveal the mechanisms of ArsI regulation in vivo. I also expect the study on ArsI mechanisms may lead us to find novel mechanisms of gene regulations, chromatin modifications that are conserved in all organisms.

The main goal of this study is to understand how taste information is represented by primary receptor cells, and how it is transformed in subsequent brain areas. Previous studies in other sensory modalities showed that the sensory stimuli are represented differently in higher brain areas than in receptor neurons. It is likely that analogous transformations of sensory information may also occur in the gustatory system. We use Drosophila as a model for investigating gustatory coding. The molecular organization of the gustatory system in flies is remarkably similar to vertebrates. However, the fly brain is simpler and more genetically tractable than the vertebrate brain, yet can generate complex chemosensory behaviors. This project has 4 aims: 1) To develop a method for separating the taste responses of gustatory receptor neurons (GRNs) and to determine their molecular receptive fields. 2) To identify the second-order gustatory neurons in the subesophageal ganglion (SOG). 3) To determine the molecular receptive fields of SOG neurons and the connectivity between GRNs and SOG neurons. 4) To determine the spatial organization of taste responses in SOG neurons and its importance for gustatory coding. This project will increase our knowledge of how taste stimuli are encoded in the brain and is likely to contribute to our general understanding of neural coding and sensory transformation. Finally, this work should generate new strategies for combining systems neurophysiology with functional imaging and emerging genetic tools, with applications in the investigation of other complex neural systems.

Recent studies highlight the importance of nano structure morphology, shape, and order in the biological context. It was demonstrated that aligned nanostructures can serve as signaling factors for cell guidance, enhance nerve regeneration, and affect the extra cellular matrix (ECM) production of human ligament fibroblast. By utilization of bottom-up nanomaterial capabilities and assembly methods we will form new synthetic model systems for the study of biological interactions at nanoscale interfaces. We will focus on Matrix metalloproteinases (MMPs) that are a class of metalloproteins involved in tissue organization, degradation of the ECM, including collagens, elastins, and gelatin. MMPs are normally regulated by hormones, growth factors, and cytokines. Substantial knowledge has been gained regarding the biochemical factors that are involved in the regulation of MMP activity. However, until recently the ability to systematically study the morphological contributions at the nanoscale to protein function has been limited. This is highly desirable for MMPs in particular as their activity milieu is composed of nanoscale fibrous structures. Among the unique properties of these model systems is the introduction of nanostructured morphologies as structural cues in addition to the chemical and physical parameters. The relationships between the activity, scale, structure, and physical parameters will be studied in a systematic manner in order to develop molecular level understanding nanoscale effects on biological systems. These capabilities will be eventually applied to study living cells-nanostructure hybrids.

Schizophrenia is a mental disorder that affects ~1% of the world’s population and features social and cognitive dysfunction as well as psychosis. Today, schizophrenia is believed to result from the dysfunction of specific neuronal networks in the brain. However, very little is known about the neuronal functional deficits that give rise to the cognitive and psychotic symptoms. Previous studies have shown that gamma-band oscillations are aberrant in schizophrenia patients. This pattern, thought to result from the repetitive activity of specific inhibitory neurons in the cortex, may be related to the cognitive symptoms and serves as a clue to the neuronal mechanisms of the disease. However, the links between activity of specific neuronal populations and schizophrenia have not been directly tested. The proposed project is aimed at examining the involvement of cortical inhibitory interneurons in the pathogenesis of schizophrenia. The project will be based on optical control of the excitability of the parvalbumin-expressing interneurons in the cortex. I will use channelrhodopsin and NpHR to control the rate of activity and the temporal pattern of spike generation in these cells. I will use electrophysiology and fast optical imaging of voltage-sensitive dyes to evaluate the effect of the activity of parvalbumin-expressing cells on network dynamics and their contribution to gamma oscillations in the isolated slice preparation. Then, using an optic-fiber neural interface I will apply similar light-stimulation protocols to the brains of live, behaving animals and directly study the links between the activity of inhibitory neuron networks, gamma oscillations and behavior.

The random generation of diversity is the feature of immune system, which is essential to counteract numerous pathogens. However, it also allows immune system to attack self tissues and produce autoimmune diseases. Therefore self tolerance, the inability to make an immune response to self, is crucial to avoid autoimmune disease. For T lymphocytes, potentially auto-reactive cells are eliminated during their differentiation in thymus through encountering the autoantigens which are ectopically expressed in thumus. The AIRE (autoimmune regulator) is a key molecule to promote transcription of many ectopic genes in thymus and a critical element for self tolerance. Though it has structural features of a transcription factor, it is difficult to be reconciled with classical site-specific transcriptional factors, because it impacts on extensive transcriptions. To reveal the precise mechanism for transcriptional regulation by aire, we propose to identify the aire associating proteins by the yeast two-hybrid screening method with Ras Recruitment System. In addition, to elucidate the DNA-binding specificity of aire, we will perform high throughput sequencing-by-synthesis approach, ‘picotiter plate pyrosequencing’, following immunoprecipitating the Aire-DNA complex with anti-Aire antibody. The binding sites on genomic DNA will be examined by analyzing the obtained sequences through the database of mouse genome sequences. All these findings will contribute to reveal the molecular mechanisms for transcriptional regulation by AIRE and profound understanding of self tolerance, including the organ-specificities of autoimmune diseases.

Microtubules (MTs) are cytoskeletal polymers that exhibit highly dynamic behavior at one end, referred to as the MT plus end. Defined by their localization at MT plus ends, plus-end-tracking proteins (+TIPs) regulate MT dynamics and link MTs to cellular structures such as the cell cortex and kinetochores. EB1 protein emerges as a particularly significant +TIP due to its presence in many of the MT plus end interactions, however, the exact mechanisms of its binding to MT ends and its role in regulating MT dynamics are largely unknown. To address these questions controlled in vitro studies with purified components are needed. This research project employs novel in vitro MT growth/shrinkage assay with two-color total internal reflection fluorescence single-molecule imaging technology to: (i) investigate the binding mechanism of EB1 to MT plus end and determine whether EB1 associates with a newly polymerized lattice and remains at a fixed position or moves with the growing end by simultaneous single-molecule-imaging of EB1 and a growing MT (ii) determine the role of EB1 in MT dynamics by varying EB1 concentrations and recording binding events, as well as accompanying MT plus-end processes (iii) investigate the interactions of EB1 with other MT plus-end proteins (e.g. CLIP-170, XMAP215 and MCAK) to determine the effects of such interactions on binding of EB1 to MT plus end and on MT dynamics. The proposed research will provide an essential understanding of key behaviors and functions of EB1, which is known to have a role in important processes such as directed cell migration, regulation of cell growth and polarization, and cell division.

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.