Behavioral studies have shown that in addition to three-dimensional (3D) shape, two dimensional (2D) surface reflectance information in faces is important for face recognition. The neural representation of these two types of information, however, is not well understood. It is unclear weather shape and surface reflectance retain unique representations, or integrate into a single representation in the human brain, and if so along which time course. The goal of this project is to investigate the neural correlates of 3D shape versus 2D reflectance independently using neuroimaging (fMRI) and electrophysiological (EEG) methods. I will first determine the general spatial and temporal characteristics of neural responses to faces that vary only in 3D shape or in 2D reflectance. I will then “decode” neural response patterns to shape versus to reflectance using a pattern-based classification approach. The results of this project should provide insight into the respective role of 3D shape versus 2D reflectance, both at behavioral and neural level. My previous research experience is in human psychophysics and computational modeling. This project will allow me to master all aspects of neuroimaging and electrophysiological methods, and implement them in conjunction with behavioral measures and computational models. If successful, these converging techniques can be applied as a powerful combination in my future work on other high-level cognitive functions. The host laboratory will provide me with an excellent opportunity to learn neuroimaging and electrophysiological techniques and will allow me to shift my research focus to a neural domain.

The olfactory system is a great example of neuronal diversity and specificity of both neuron identity and connectivity. Sensory neurons expressing the same olfactory receptor (OR) make connections with the same target glomerulus in the antennal lobe in the brain. This creates the challenging task of correlating OR expression with targeting specificity of the neuron. I am interested in the genetic program that governs neuronal diversification and links it to a neuron’s connectivity, and ultimately allows the construction of functional neuronal networks. I will use the relatively simple and genetically tractable olfactory system of Drosophila melanogaster to identify and characterize the molecular components of this genetic program. First, I will continue to analyze a few of the most interesting mutants I have previously discovered in a genetic screen that have defects in either neuron specification or neuronal connectivity. Second, I will analyze candidate groups of molecules using RNAi gene targeting to identify the proneural and neuron-specific transcription factors involved in neuronal diversification, and the targeting receptors and ligands required for connection specificity. Finally, I will characterize the role of FGF receptor Breathless in the specification and/or connectivity of one specific ORN class. This research is likely to gain new insights in aspects of neuronal diversification and connectivity in all kinds of complex nervous sytems. The CDA will help me to establish myself as an independent researcher, and to build a research group of substantial size and impact and competitive financial support.

The soil bacterium Burkholderia pseudomallei (B.ps) is a major health risk in tropical Australia and SE-Asia as it causes the serious and emerging disease melioidosis in humans and animals. Its natural habitat is unknown which is a major limitation for public health education programs.
We have developed a sensitive and specific molecular assay to detect B.ps in soil and performed a pilot study collecting 800 soil samples in rural Darwin, Australia to study the habitat of B.ps. An association between B.ps and grass-rich areas was found and an increase of B.ps load at these sites in the wet season when the root mass largely increases. B.ps was also detected in soil closely associated with roots of Tully grass (Brachiaria humidicola cv Tully). This suggests the rhizosphere of some grasses to be a potential reservoir of B.ps.
By a newly set up collaboration with Dr. Michael Schmid (Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Department Microbe-Plant Interactions), we aim to study the colonization behaviour of B.ps in the rhizosphere of selected grasses from rural Darwin using optical techniques such as fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). During my visit of Dr. Michael Schmid’s laboratory in June 2009, I will learn and apply FISH as well as CLSM.
This interdisciplinary project combines our previous findings based on real-time PCR based methods with the latest imaging technology and will largely contribute to the elucidation of the reservoir and microhabitat of B.ps. This will be important to inform effective public health measures to contain the spread of this potentially deadly bacterium.

The vascular system is essential for animal survival as it transports nutrients and oxygen throughout the body. Although several recent breakthroughs have enhanced our understanding of the genetic control of vasculogenesis and angiogenesis, the molecular mechanisms of vascular development still remain largely unknown. In general, forward genetic screen is one of the most powerful tools to reveal new clues of vascular development, and zebrafish is the best suited animal for such genetics screen among the animals with a closed circulatory system. Moreover, the transparency of zebrafish embryos and larvae enables one to observe the entire image of three-dimensionally expanded vascular system and to perform in vivo live imaging of blood vessel development. The accessibility of zebrafish allows various manipulations such as generation of chimeric fishes, pharmacological analysis. Therefore, we use zebrafish to reveal mechanisms of vascular development. Previous forward genetic screening identified the mutant with vascular defect, mts (Jin et al., 2007.) Through the analysis of this mutants, we propose to investigate the mechanisms of vascular development. Moreover, we will observe the individual steps of blood vessel development in detail, paying particular attention to individual cell behavior, cytoskeletal dynamics, and the process of endothelial cells polarization by in vivo time-lapse imaging. With the establishment of conditional knock down technology, we will attempt to reveal the mechanism of these steps. The analysis described above should give us further insights into blood vessel development.

CD4+CD25+ regulatory T cells (Tregs) have been shown to dominantly suppress a wide range of immune responses and constitute a major mechanism of peripheral tolerance. The goal of this proposal is to understand the T cell receptor (TCR) affinity and signaling requirements for Treg development in the thymus and in the periphery. The link between TCR affinity and conventional thymocyte selection is well established, and recent work in the host lab has revealed that small changes in ligand affinity dramatically affect activation and subcellular localization of Ras/MAP kinase signaling intermediates. In contrast, data regarding the affinity requirements for Treg selection have yielded conflicting results, and a direct comparison of the relative contributions of TCR affinity and peptide concentration is lacking. Using a MHC II restricted TCR transgenic mouse model I will define and compare the parameters for conventional CD4+ T cell and Treg development in response to a panel of ligands with varying affinity for TCR. In addition, I will examine the signaling pathways initiated by TCR engagement on Tregs. My previous research focused on understanding the cellular mechanisms responsible for disease development in an unexpected mouse model of autoimmunity. In the proposed research, I will build on these skills while expanding my knowledge of T cell development and signal transduction using microscopy and molecular immunology techniques. This work will provide me with the technical and conceptual tools to address both basic immunology and immune pathology questions.

Protein aggregation is a common feature of neurodegenerative diseases such as Huntington, Parkinson and Alzheimer´s disease. I will use C. elegans, which is an established model for human degenerative diseases to study cellular and tissue specific responses to protein aggregation. The aim of the research described in this proposal is to elucidate whether expression of polyQ in the cytosol results in a competition for molecular chaperones and thereby affecting the functionality of proteins targeted to the organelles. Using fluorescent imaging techniques, the specific translocation of model proteins into the ER and other organelles will be monitored upon induced polyQ aggregation in the cytosol. I propose to determine the threshold of polyQ for interference with the trafficking process and the functionality of the protein and its dependence on longevity and the capacity of the PQC system. For that, the protein trafficking process will be analyzed in various mutant backgrounds resulting in a shortened or expanded lifespan and a deletion or overexpression of a particular chaperone or chaperone network, respectively. As a next step, I aim (i) to compare the simultaneous intracellular trafficking to different organelles using a dual labeling approach and (ii) to study the impact of aggregation on protein trafficking in different tissues such as neuronal and muscle cells. Finally, based on the obtained data, a genetic approach using RNAi is planned to identify novel modifiers of the interrelation between protein aggregation and intracellular signaling/ trafficking.

The vibrissal system is an active sensing system, whereby sensorimotor processing is mediated by anatomically closed feedback loops that emanate from the brainstem through loops that close at the level of the neocortex. The goal of proposed research project is to understand the functional ramifications of the confluence of sensory and motor signals in the deep layers of vibrissa primary sensory (S1) cortex. Combining methods that enable photo-activation of Channelrhodopsin-2 positive layer 5b neurons, extracellular recordings, intrinsic optical imaging, behavioral paradigms, and anatomical tracing techniques, my goal is to map function onto anatomical sensorimotor loops of the vibrissa system during different behavioral states (anesthesia and wakefulness) and modes of sensory activation (active and passive). In particular, I will be studying corticotrigeminal gating and modulation of evoked activity in the lower-most sensorimotor loop that encompasses the brainstem trigeminal complex and facial nucleus motoneurons. A series of experiments are proposed. Initial experiments in anesthetized mice will measure activity throughout the trigeminal pathway evoked by photo-activation of ChR2-positive L5b pyramidal cells. Second, modulatory effects of trigeminal activity due to L5b pyramid photo-activation will be studied during whisking behavior in anesthetized and behaving mice. Control experiments will differentiate the modulatory effects of L5b pyramids on ongoing trigeminal activity during active versus passive touch. Experiments in chronically implanted behaving mice will be conducted in order to test behavioral relevance of the modulatory effects of L5b activation.

I propose to elucidate the in vivo functions of neuroligin, a postsynaptic adhesion molecule at the molecular and organismal levels. Several in vitro studies have suggested that the neuroligins (along with neurexins) act as the main organizers of excitatory and inhibitory synaptic functions. In addition, genetic studies have implicated both of these proteins in autism. However, although these findings provide some initial insight into autism biology, it is not yet known whether neuroligins really regulate the excitatory-to-inhibitory (E/I) ratio, and how imbalances of this ratio may be related to autism. I will employ three separate biological approaches for assessing the molecular and cognitive functions of neuroligins. 1) Cell biology: I will examine whether interactions of neuroligin-1/2 with various inhibitory scaffolds are responsible for controlling the E/I ratio. 2) Mouse genetics: based on results from (1), I will generate knockin mice in which neuroligin-1/2 cannot interact with scaffolds and use electrophysiology and behavioral assays to examine the neurological consequences. 3) Biochemistry: To date, the neurexins are the only known adhesion partners of the neuroligins. Accordingly, I will attempt to identify novel adhesion partners of the neuroligins. These three approaches will be combined to shed new light on the question of how neuroligins regulate the E/I ratio, and may lead to the establishment of a new animal model for autism. Furthermore, since my past research mainly focused on molecular/cell biology in cultured neurons, this work will give me important exposure to new techniques, such as mouse genetics, electrophysiology and behavioral assays.

Alpha herpesviruses are a subfamily of the Herpesviridae that follow a common pathway of infection. They establish a primary infection at the mucosal or epithelial surfaces and then leave that site by invading the peripheral nervous system where they establish a reactivatable latent infection. Interestingly, comparison of microarray analyses of the cellular response of different cells types from different mammalians to infection with two different alpha-herpesviruses (PseudoRabies Virus and Herpes Simplex Virus-1) had revealed only a few common host genes that are over-expressed during viral infection. These few genes have no other common feature and their roles in the virus reproductive cycle or in the host response to the infection were not identified. We will use a set of short interfering RNA (siRNA) to silence these genes during the infection process in the cellular level. Genes, that their inhibition will be found to interfere with viral infection, will be tested further in animal models. Microscopic, genetic and biochemical assays will be used to corroborate the results and identify the role of these genes in viral propagation

We would like to study dynamics of the body movement and the activity of the underlying motor neurons that control this movement, as the worm Caenorhabditis elegans as it crawls on a rigid surface. In the first stage of the proposed project, we will excite the motor neurons one or several at a time, while simultaneously measuring the calcium activity in the other motor neurons of an immobilized worm. The results of these measurements will reveal the connectivity of the motor neurons. In the second stage of the proposed project, we will measure calcium activity and develop the capability of photo-exciting single motor neurons in a freely moving animal. My previous experience in the field of optics finds useful applications in developing the tools for this purpose. Through statistical analysis of the excitation patterns of the neurons and the changes in the body movement of the worms on photo-excitation of motor neurons, we will try to understand the dynamics of the neural circuit that controls worm locomotion.

How stem cell proliferation is controlled to obtain a tissue of correct cellularity (homeostasis) is an unsolved problem in biology. Failure to identify the molecular mechanism of organ size determination by traditional methods may be due to the fact that environmental cues control the process. Indeed, physical paradigms are emerging in the control of cellular function. We propose to elucidate the role of physical, mechanical forces as signal of “cell density” in the control of cell cycle progression of adult intestinal stem cells (ISCs) in Drosophila melanogaster midgut. The midgut epithelium is homeostatic. Old differentiated cells turn-over weekly, while the gut is replenished by division of self-renewing ISC stem cells. ISCs divide while adhering to both neighbouring midgut epithelial cells and the extracellular matrix. In this position, we expect ISCs to sense and respond to mechanical forces. Forces are expected to arise from mitotic division of ISCS, cell mass and volume increase of daughter cells, and from constant loss of old differentiated cells. Therefore, when the rate of cell growth in part of the intestine is too high, increased mechanical pressure (force per area) might restrict further ISC proliferation. This mechanical feedback on the stem cell cycle is an intriguing model for tissue homeostasis, because it is inherently caused by non-uniform growth. With previous tools it has not been technically possible to address the physiological role of mechanical forces in vivo. We propose to load force on the ISC by selective genetic manipulation of the midgut tissue. We will quantify the consequence for the stem cell proliferation rate.

A series of behavioral and modeling studies is proposed with the goal of improving our understanding of 1) how normal-hearing humans localize speech sources in realistic complex environments consisting of other speech sources, and 2) how this ability relates to the ability to use spatial attention to enhance understanding of the speech of one of multiple talkers.Compared to previous studies performed in simpler environments, the characteristics of the complex environment explored here will include: multiple distractor sources, presence of reverberation, and uncertainty about the number and spatial distribution of the distractors. The results will be important for bridging the gap between our understanding of the basic parameters that influence performance in simple laboratory experiments and behavior in complex every-day situations. Also, as current approaches for improving performance in listeners with hearing loss fail when the listening environment becomes more complex, these experiments will serve as a basis for future research on the effect of hearing impairment on the everyday auditory tasks crucial for successful communication. The proposed research will enhance Kopco’s professional development by 1) allowing him to learn new experimental techniques and use state-of-the-art equipment and facilities for studying spatial hearing in real and virtual environments, 2) enabling him to interact with one of the world-wide leading centers of hearing research to combine his modeling skills with the spatial hearing expertise of the host’s group to create an interdisciplinary research program, and 3) providing the basis for a long-term collaboration between the laboratories.

Cells live in a dynamic environment and are confronted with a large variety of signals. These signals can be molecules such as nutrients, physical parameters such as temperature, DNA damage, light levels etc. Cells process these signals and respond by synthesizing the appropriate type and number of proteins at the right time for an optimal response. This processing is achieved through networks of interacting transcription factors, called transcription networks. Although such biological systems can consist of thousands of interacting proteins and perform sophisticated computations, they exhibit inherent simplicity: it appears that recurring ‘motifs’ of interaction patterns occur serve as the network’s fundamental building blocks, not unlike the intentionally designed modular networks in computer chips. Unknown, however, is why and how biological network motifs evolved and what environmental conditions promote and sustain their ‘designed’ traits. Here we propose experiments to quantitatively study the dynamic evolution of network motifs. We will direct the evolution of an important network motif, the feed-forward loop, in E.coli bacteria grown in controlled, microfluidic structures and probed by fluorescence spectroscopy. These environments either favor or disfavor the presence of the motif and are theoretically predicted to respectively create or destroy the motif. We will learn, for the first time, how network motifs evolve and adapt to the environment. Studying the evolution of network motifs can identify the conditions that lead to molecular design and allow us to understand biological design across organisms.

Fungi are a major player in soil functioning. However, their community structure and diversity are still not fully understood. This project will contribute to a theoretical framework that links soil physics, fungal biology, mathematical biology and statistics in order to assess fungal community dynamics and diversity in heterogeneous soils. It will take advantage of the recent advances in computer tomography and microscopy allowing us to reach a new level of understanding of the mechanisms governing fungal behavior at microscopic scales, and for the first time to examine species interactions in a 3D environment. The main goal of the proposed study is to determine how soil microscopic heterogeneity affects fungal species interactions in soils with contrasting structural compositions. This will be achieved through X-TEK HMX micro-tomography scanning of undisturbed soil samples followed by characterization of soil pore structures and geometries. Pore geometry information will then be combined with modeling multi-species fungal interactions in 3D soil environment, generating theoretical evaluations of the pore volume characteristics that have a significant impact on community evolution under different land use and management regimes. My long-term research goal is to apply my expertise in soil physics and spatial statistics to develop a better understanding of the mechanisms governing micro-scale activities and spatial patterns of soil biota. This project will allow me to learn state-of-the-art measurement and modeling techniques and to establish collaborations with microbiologists and biological modelers, both tasks crucial for further progress of my research.

Social
and emotional development are particularly influenced by early-life
experience, a critical period in neural development when neurogenesis
is maximally robust. Although experimental approaches in animals and
humans are well-established to probe the biology of genetic determinants,
the mechanistic influence of environmental stimuli and behavioral experiences
have been more difficult to elucidate. Together with recent data suggesting
that epigenetic modifications function as a critical nexus between genetic
alleles and environmental variables, we reasoned that experience-dependent
epigenetic plasticity of neural stem cells could exert a powerful influence
on early neural development. In the current proposal, we plan to utilize
the well-characterized in vitro clonal neurosphere assay to examine
differential CpG methylation in neural stem cells isolated from mice
exposed to prenatal stress versus normal gestation. Our approach will
utilize both an unbiased technique (Restriction Landmark Genomic Scanning),
as well as focus on specific genes (GR, Reelin, BDNF, FGF-2) previously
shown to have developmental plasticity in their patterns of CpG methylation.
Further, using clonal expansion of individual neurospheres, we have
the unique opportunity to infer the in vivo origin of the observed
epigenetic changes, by comparing
the stem cells themselves with their progressively differentiated progeny. Through behavioral studies, neural
stem cell culture, and molecular biology, this interdisciplinary proposal
will advance my long-term career goal of understanding the mechanisms
by which early-life experience influences both normal brain function
and the risk of psychopathology.

Prefrontal cortex (PFC) contributes to a diverse set of cognitive functions, including valuation, navigation, inference and concept learning, but the underlying mechanisms are poorly understood. It is believed that these cognitive capabilities arise from the functional organization of neural circuitry within the PFC. GABAergic interneurons show great diversity in their connectivity, morphology, biochemistry and intrinsic physiology . This phenotypic diversity provides the variety and fine structure of inhibitory interactions which confer balance, complexity, and flexibility to cortical circuits . In order to gain insights into how different classes of interneurons affect network activity and animal behaviour we plan to label two distinct classes of interneurons (Pv and CR positive interneurons) with rhodopsins (ChR2 and NpHR). In this way we aim to both manipulate their activity and also identify specific classes of neurons with recording electrodes in behaving animals during odor discrimination tasks. This multi-disciplinary approach will begin to offer us mechanistic explanations in terms of the underlying neural circuitry. about how prefrontal cortex performs cognitive functions

We propose to investigate the neural basis of motor intention using a combination of transcranial magnetic stimulation (TMS) and magnetoencephalography (MEG). Specifically, we test the direction of influence of activity between the dorsolateral prefrontal cortex and the pre-supplementary motor area. Both of these areas have been implicated in a previous study on motor intention (Lau et al Science 2004), but the direction of interaction between them is unclear.
Potential impact to the research field This issue of directionality is important to the research field, as most current models of control of actions assume that the prefrontal cortex sends top-down signals to posterior areas, and yet the empirical status of this assumption is not completely clear. By manipulating the activity in one area, and monitoring the oscillatory signal in another connected area, we hope to convincingly resolve this issue of the directionality of influence between two areas. From a metholodological standpoint, we hope that this could also make an impact on the research field as this strategy of investigating connectivity by combining TMS and MEG is relatively novel, and has great potential.
Professional growth In pursuing this project, I hope to acquire the skills of conducting advanced data analysis for MEG, and address the technical issues involved in combining it with TMS. These skills will be essential for my future research. I also hope that my visit to the Donders center can help me to establish long-term strategic collaborations with researchers there, who have expertise that would greatly compliment my research interests.