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.

Alzheimer’s disease (AD) is one of the most rampant epidemics of the modern world. As populations grow older, the number of patients affected by AD reaches massive proportions. Despite the alarming statistics, we have a poor understanding of the pathophysiological mechanisms underlying it. The septo-hippocampal brain system is intrinsically associated with AD. Septal cholinergic neurons and rhythmic network activity degenerate and the cholinergic theory of AD pathophysiology has long been accepted as the main mechanism behind this disease. Some evidence suggests that the glutamatergic neurons of the septum are also affected by AD. However, little is known about the role of septal glutamatergic inputs to the hippocampus and their effect on hippocampal function. The aim of my research project is to investigate the glutamatergic projection from the medial septum to the hippocampus and its effect on the modulation of rhythmic electrical activity in this network. The experiments I propose will characterize the glutamatergic pathway in the septo-hippocampal system using a mouse brain slice preparation. Anatomical and biophysical knowledge from my initial results will be applied to experiments that involve a combination of cutting-edge research methods from different fields of neuroscience such as electrophysiology, two-photon uncaging of glutamate, dynamic clamp and brain modeling. This combined approach will yield crucial data to the understanding of the septo-hippocampal system and how glutamatergic septal neurons contribute to oscillatory activity in the hippocampus. My results may also contribute towards developing novel therapeutic targets for the treatment of AD.

The IkB kinase (IKK) complex is a central component of the NF-kB pathway that controls a variety of biological processes including inflammation, immunity, cell survival and tumorigenesis. The IKK complex composed of three subunits, IKKa, IKKb and IKKg/NEMO is primarily dedicated to phosphorylation-dependent degradation of IkB proteins, thereby resulting in activation of NF-kB. However, phenotypic analysis of IKKa knockout mice indicates that IKKa also has important functions that are NF-kB-independent. Moreover, preliminary results in our laboratory suggest a crosstalk between IKKa and TGFb signaling without the involvement of NF-kB. However, the molecular details and the full physiological significance of this crosstalk in skin and prostate cancer are not fully clear. I plan to investigate the relationship between IKKa and TGFb using three independent model systems. First, I will examine the genetic interaction between IKKa and TGFb signaling in the Drosophila wing system. Then, I will investigate the mechanistic details of IKKa-TGFb crosstalk in mammalian cell culture systems including primary keratinocytes and prostate epithelial cells. Finally, I will examine whether the regulation of IKKa activity by the TGFb pathway plays an important role in prostate cancer development and progression using a suitable mouse model. Since both IKKa and TGFb signaling pathway are known to enhance metastatic progression, it is likely that they work together in metastatogenesis and improved understanding of the molecular details underlying their interaction is likely to lead to new therapeutic strategies for prevention of metastatic disease, the major cause of cancer-related death.

Most phenotypic traits are the result of the complex interplay between genotype and environment. Much progress has been made in identifying sequence variation. However it remains unclear, how this genetic variation translates into phenotypic variation. More than 60 years ago, mechanisms that buffer genetic and environmental variation had been proposed theoretically (canalization). Canalization allows accumulation of cryptic genetic variation, an important source for the evolvability of novel traits. Recent work identified Hsp90 as molecular buffering mechanism that allows robust wild-type phenotypes. The molecular bases by which Hsp90 stabilizes phenotypes remains nonetheless unknown. I propose a quantitative genetics approach using natural populations of the plant Arabidopsis thaliana to determine Hsp90-buffered genetic variants and to dissect the molecular bases of trait robustness. Once Hsp90-responsive alleles are identified, I will address if and how selection acts upon these Hsp90-responsive genes. Based on recent studies, I hypothesize that other highly connected cellular machines such as the small RNA pathway and chromatin remodeling may play a similar role. I will therefore test central genes in the small RNA pathway for their ability to buffer genetic and environmental perturbation. In summary, I want to identify and dissect molecular mechanisms that allow organisms to express robust phenotypes and why in some cases genetic variation is no longer buffered and gives rise to phenotypic diversity. My results will improve our understanding of how genetic and phenotypic variation evolves.

One major determinant of cell fate are patterns of chromatinization of genetic loci that are established during differentiation and that limit the possible outcomes of general signaling events to the activation of lineage-specific genes. We recently showed that an epigenetic switch controlled by subunit exchange within a neural, ATP-dependent SWI/SNF-like nBAF chromatin remodeling complex is deterministic for the transition from proliferating stem/progenitor cells to post-mitotic neurons. Despite their essential role in neural development, very little is known on the importance of chromatin-based epigenetic mechanisms in normal and leukemic hemopoiesis. The main objectives of this research program are to define the roles and regulatory mechanisms of a novel family of hemopoietic SWI/SNF-like hBAF ATP-dependent chromatin remodeling complexes in the developing hemopoietic system and in leukemia. In aim 1, we will use state-of-the-art proteomics technologies to define the subunit composition and post-translational modifications of hBAF complexes in normal hemopoietic and leukemic stem cells. In aim 2, we will use molecular genetics approaches to address the potential roles of the stem cell-specific BAF45a and BAF53a subunits of these complexes (and eventually other novel hBAF subunits identified in Aim #1) in regulating the self-renewal/proliferation, survival and differentiation of normal hemopoietic and leukemic stem cells. Together, these approaches will provide fundamental insights concerning the role of chromatin-based epigenetic mechanisms in normal and leukemic hemopoiesis and help build my career as an independent investigator.

In living cells almost all transfers of mass (e.g. chemical reactions), information or energy involve protein-protein interactions (PPIs). For this reason, understanding PPIs is key to comprehending all biological processes. Despite the importance of PPIs, the mechanisms by which they are created during the course of evolution are still poorly understood. An estimated 1,600 to 16,000 PPIs have been re-wired between human and chimpanzee. This high turnover of PPIs implies a high evolvability, i.e., that the number of mutations needed to create a new PPI is low. I would like to investigate this issue and quantify in molecular terms how easy it is to create a new PPI. To this end I will use in vivo directed evolution to engineer new PPIs, and dissect and rationalize the sets of mutations found to trigger new interactions. This will allow me to estimate several parameters that describe the evolvability of PPIs, and determine how these parameters depend on certain factors such as protein family, sequence similarity to a known binder, or functional constraints. This project will provide an estimate of the basal “stickiness” (i.e., the intrinsic propensity to interact weakly and non-specifically) of proteins for each other, and the rate at which the strength and specificity of this basal stickiness can be improved by introducing mutations. This knowledge is fundamental to a better understanding of the principles of formation and evolution of PPIs, and for finding and designing drugs that modulate them. In addition, this project will provide a novel strategy to engineer PPIs in vivo, which has implications to protein design as well as nanotechnology.