Not only are animals able to record the time of day when some behaviourally relevant event has happened, but also they take advantage of this knowledge in their subsequent behaviour. The main goal of the project is to understand how flies are aware of time; and the basic idea is making use of learning and memory to uncover this ability. An initial focused approach consists of evaluating whether memory processes are modulated by the circadian clock. Training in Dr. Dubnau’s laboratory, at Cold Spring Harbor Laboratory (CSHL) is vital for the success of this project since it will provide access to the memory paradigm, provide hands-on experience to manipulate and interpret the behavioral data, and whenever possible, answer several questions regarding a potential circadian modulation of memory, with emphasis on possible mechanisms. In doing so, new issues will certainly take shape which might end up requiring modifications in the original setup to be able to study time awareness at home. The request for a Short-Term Fellowship is based on the fact that it would enable me to spend some time overseas, which in itself will open new venues in my career: not only it will be instrumental in successfully carrying out my post-doctoral project, but also it will allow the setup of this new behavioral paradigm in my home institution; besides, my lab would benefit immensely from the opportunity to spread the research in circadian modulation of memory and, hopefully, time awareness. Training at a high profile institution such as CSHL will most likely impact my career as a young independent researcher. Moreover, it will give a powerful know-how to bring back home and share with others.

Replicative DNA helicases are enzymes that unwind DNA during DNA replication. MCM2-7 is a hexameric protein complex present in all eukaryotes that is composed of six nonidentical, but highly related AAA+ ATPase proteins. There is a wide agreement that the MCM2-7 complex functions as a replicative DNA helicase in replication. So far, it is still a mystery how MCM2-7 complex unwinds DNA on chromosome. Most well-studied replicative DNA helicases function by encircling and translocating along single-stranded DNA (ssDNA). However, various lines of indirect evidence implicate that the MCM2-7 complex may encircle and translocate along double-stranded DNA (dsDNA). In this proposal, we expect to determine whether the MCM2-7 complex translocates along ssDNA or dsDNA during the DNA unwinding. To distinguish these two fundamentally different mechanisms, we will use ChIP to examine the effects of specific DNA lesions on MCM2-7 translocation. Meanwhile, by performing DNA footprinting on a helicase stalled at a specific location, we will independently investigate the way in which MCM2-7 binds to DNA. The proposed study will not only answer one of the biggest open questions in the replication field, but also be instrumental to understand the roles of the many proteins that are required to recruit and activate MCM2-7 during replication. Moreover, the knowledge gained from this study will also provide key insight into the architecture of the replisome.

Calcineurin (Cn) is a serine/threonine protein phosphatase that plays pivotal roles in many physiological processes. Most prominently, calcium-activated Cn dephosphorylates multiple residues within the regulatory domain of the nuclear factors of activated T cells (NFATs). Effective phosphate removal by Cn requires its docking to NFAT. There are still many open questions regarding the biological and structural-related aspects of this mechanism such as what is the mechanisms by which phosphorylated and dephosphorylated NFAT interact with Cn? and how dephosphorylation exposes a nuclear localization sequence?
This plan proposes to structurally characterize the Cn-NFAT association using NMR techniques. This determination of three-dimensional structure for the NFAT regulatory domain before and after the dephosphorylation process will have important implications for understanding the T-cell activation and fundamental signaling pathway mechanisms.

Homologous recombination (HR), albeit being an important mechanism for the repair of DNA double-strand breaks, and thus the maintenance of genome stability, has to be tightly regulated, since unscheduled or excessive HR can lead to gross chromosomal rearrangements characteristic of cancer cells. In yeast, the Srs2 helicase is known to suppress aberrant recombination events, but functional homologues in higher eukaryotes have remained elusive for a long time. Recently, the Boulton lab has identified SPAR1 in a C. elegans screen as a functionally analogous protein to Srs2. SPAR1 is conserved from C. elegans to humans and exhibits many of the genetic and biochemical hallmarks of yeast Srs2.
The aim of this project is to investigate the mechanisms of how HR is restrained in higher eukaryotes. To do so, a two-pronged approach will be employed: The first approach comprises the detailed biochemical characterisation of SPAR1. To this end, I will design an in vitro assay to elucidate a potential role of SPAR1 in synthesis-dependent strand annealing (SDSA). In addition, I will set out to purify and characterise SPAR1 containing complexes from human cells. Novel interacting partners of SPAR1 will be characterised for their role in regulating recombination and their influence on SPAR1 activity. In a parallel approach, I will perform a GFP reporter-based genetic screen in C. elegans to identify spar-1 like genes, which will be subsequently tested for their anti-recombinogenic function in a human cell culture system.
Integration of the results from both approaches can be expected to add considerably to our understanding of how and by which factors homologous recombination is regulated.

Toll-like receptors (TLR) signaling cascades recognize pathogenic components and initiate the innate immune response, which is largely mediated through gene expression changes. Variation in TLR signaling underlies differences in immune homeostasis and susceptibility to infectious and autoimmune diseases. It is unclear, however, how genetic variation between individuals leads to inflammatory diversity and whether variation in gene expression mediates the phenotypic differences. Here, I propose to elucidate the regulatory mechanisms underlying the influence of genetic polymorphism on gene expression variability in inflammatory responses. I will develop new algorithms for simultaneous mapping of multiple gene expression traits and for direct identification of their underlying regulatory mechanisms. The scalable algorithm I will design will be applicable for complex mammalian systems, will provide concrete mechanistic hypotheses, and will be made available to the biomedical community. I will apply this algorithm to genome-wide gene expression and phenotype measurements in dendritic cells from segregating mouse populations exposed to different combinations of TLR ligands. The resulting hypotheses will be then tested in lab, identifying the molecular mechanisms that underlie heritable variation in innate immunity and inflammation. I hope that my work will help to understand individual variation in immune disorders and will constitute an important step toward personalized medicine.

In this project, we will investigate the biological function of the dengue virus helicase. Dengue virus is an important human pathogen that poses a serious health problem around the world. Using reverse genetics, infectious clones, and in vitro assays, we are investigating the interaction of the helicase with the viral RNA replication machinery. Here, we propose to initiate a collaboration to investigate mechanistic aspects of the unwinding activity of this viral helicase. I will learn a technology, developed in the host laboratory, to analyze the RNA unwinding process by single molecule methods. To this end, I will be trained to use optical tweezers. Then, we will design appropriate RNA substrates to determine thermodynamic and kinetic parameters of the unwinding process. The combination of the virology studies that I am carrying on in my home laboratory, together with the mechanistic studies that I propose in this project, will help us to define the biological role of the dengue virus helicase. Importantly, this short term fellowship will be the starting point of a new international collaboration that will allow me, as a young investigator, to have access to new technology and to learn novel biophysical approaches to address a relevant biological question.

Hepatitis C virus (HCV) affects 120 million individuals world-wide and causes serious liver disease in up to 20% of infected patients. To date, the molecular events leading to HCV infection and to HCV-induced liver pathology remain largely elusive.
HCV enters the liver via sinusoidal blood vessels and infects hepatocytes from the basolateral side. HCV entry into hepatocytes requires the tight junction protein claudin-1. In the healthy liver, claudin-1 mediates intimate contacts of adjacent hepatocytes and forms the physical barrier between basolateral and apical compartments. It is unknown whether and how HCV gains access to tight junctions. Since claudin-1 is essential for hepatocyte function, we hypothesize that claudin-1 engagement by HCV contributes to liver pathology. Therefore, I will first investigate how HCV uses claudin-1 for entry and second how this affects the integrity of polarized primary hepatocyte cultures.
To visualize how HCV engages tight junctions, I will establish labeling techniques for HCV and apply single particle tracking methods. Live-cell imaging of HCV and its host receptors will provide a detailed spatio-temporal view of HCV entry. Moreover, I will clarify which cellular trafficking pathways are exploited by HCV using inhibitors and gene silencing.
Engagement of the cell-cell adhesion molecule claudin-1 likely alters liver integrity. I will investigate tight-junction function of HCV-infected primary polarized hepatocytes using permeability assays and electrophysiology. This together with the above imaging techniques will help understand how HCV exploits cellular adhesion molecules for entry and what impact this has on liver pathology.

Dendritic cells (DCs) are the most efficient antigen presenting cells of the immune system, due to their capacity to present peptides derived from endocytosed exogenous antigens on MHC class I molecules. This process is called cross-presentation, resulting in the activation of specific CD8+ T cells (cross-priming).
My proposed work will elucidate functional and molecular mechanisms for efficient cross-priming of viral antigens in vivo and in vitro.
I will establish new experimental systems to assess cross-priming efficiency of viral antigens delivered by autophagic or apoptotic cells used as exogenous antigen sources.
In particular, I will characterize the mechanisms through which autophagic cells internalized by DCs elicit more efficient cross-priming compared to apoptotic cells.
I will evaluate whether antigen processing within the autophagic cell prior to phagocytosis by DCs is required for efficient cross-priming.
By using knock out mice, I will investigate whether specific signaling pathways involving TLR-7, TLR-9 and RIG-I are engaged upon autophagic cell internalization by DCs.
Finally, I propose siRNA screening approaches targeting autophagic cells to identify key molecules potentially responsible for their efficient antigen cross-presentation by DCs.
My proposed work will establish more physiological conditions to characterize the cross-presentation pathway in vivo. Not only this project will give a substantial contribution in the field of immunology, but it will also have important implications for immunotherapeutic approaches.

The brain represents sensory information by complex patterns of electrical activity in groups of neurons. Deciphering this neuronal population code is one of the fundamental challenges in neuroscience. To study this, the neural network of the retina is superbly suited. It combines good experimental accessibility with a well-defined role as the first stage of visual processing and displays intriguing multi-neuronal activity patterns, such as synchronization and other forms of correlated activity. I therefore intend to study how multi-neuronal activity patterns in the retina contribute to representing the information that the brain receives from the eye. In particular, I will ask which types of visual stimuli are best represented by different kinds of multi-neuronal activity patterns. I will begin with synchronous activity of neuron pairs and subsequently include larger groups of neurons and non-synchronous activity patterns. The features of the identified stimuli will provide insight into the role of these patterns for information transmission. My approach will be to design experiments that actively search for suitable stimuli while monitoring the neuronal activity with multi-electrode arrays. An essential ingredient will be the development of closed-loop experiments. Based on automated analysis of the recorded data, this will allow implementation of appropriate search algorithms for systematic adjustments of the applied visual stimuli during the experiment. For this multi-disciplinary endeavor, the support and recognition provided by the CDA will function as an ideal framework to assemble a strong international research team from a diversity of scientific backgrounds.

Epidermis is a multi-layered epithelium composed of hair follicles (HF), interfollicular epidermis (IFE), and sebaceous glands (SG). Its patterning is initiated during embryogenesis by signals coming from the underlying dermis, and is later maintained in adult skin by different populations of stem cells residing in HF bulge, IFE and SG. Patterning changes according to the body region. Several signalling pathways have been implicated in HF spacing and IFE specification in back skin, but the mechanisms responsible for pattern variations remain unknown. Mouse tail represents a good model to study this question because of its highly stereotyped arrangement of HF into triplets, facilitating hair counting, and its different populations of interfollicular epidermal cells, identifiable using markers. The aim of this proposal is to identify the factors regulating HF and IFE patterning using mouse tail as an experimental model. I will analyze interactions between different signalling pathways for their ability to modify HF and IFE patterning. To this end, I will implant beads soaked in molecules that regulate signalling pathways in wild-type and transgenic mouse tail epidermis both in vitro and in vivo. I will test the role of the dermis in controlling tail patterning by changing epidermis orientation relative to dermis, before and after establishment of patterning. Finally, I will investigate the role of the identified pathways in adult tails during induction of ectopic HF. These experiments will enlighten mechanisms regulating embryonic and adult skin patterning and contribute in understanding both development and stem cell regulation in skin.

While animals may be able to attribute knowledge, ignorance and motivation to other individuals (a human ability termed 'Theory of Mind'), direct evidence for such attributions by non-humans has proved very difficult to attain. In order to overcome the difficulty, inherent to non-verbal methods, of revealing what animals expect other individuals to do, I will develop and use a behavioural paradigm to enable obtaining such data. I will first train hand-raised Western scrub-jays (Aphelocoma californica), which readily cache food items under laboratory conditions, to perform an 'expectation test'. Storers will be rewarded when they perform specific behaviours according to their predictions regarding the location in which an observer jay is likely to look for the cached food. Then, I will use this test to reveal whether jays can attribute a false-belief to a conspecific which has observed them caching in one spot, but not subsequently re-caching the item in another spot, while remembering the correct present location themselves. Extending this paradigm to other ecologically-relevant scenarios, I will test whether jays keep track of the knowledge-state of many individuals simultaneously, whether they attribute knowledge based on auditory information to others and whether they act to deceitfully decrease the knowledge of potential cache pilferers. This study may provide a novel and much needed paradigm for the field of animal cognition, advancing our understanding of whether and how animals represent the minds of other individuals in their social environment.

Deregulation of the epidermal growth factor receptor (EGFR) has been identified as a common component of multiple cancer types including head and neck, ovarian, gastric, breast, and colorectal cancers. Over the past two decades, we have learned much about how signals are conveyed from EGF receptor at the membrane to its targets in the cytoplasm or nucleus. However, most traditional methods, have adopted a reductionist approach, focusing on one or a few molecules of interest. Thus, while the traditional methods have yielded abundant information about individual proteins in EGFR signalling, they have been less successful at providing an integrated understanding of biological systems. Here, we will develop a technology that enables the high-throughput, system-wide, and quantitative characterization of an intracellular signalling network, under different perturbation conditions and as a function of time. Our analysis of the mechanics of the EGFR signalling network will provide more thorough characterization as well as opportunities for the development of targeted novel cancer therapeutics. Further, dissecting the EGFR network at systems level should serve as a benchmark to evaluate future efforts in data-driven model discovery. Ultimately, it is our hope that this combination of high-throughput data collection and probabilistic modeling will provide a general route to the rational design of combination therapies.

The mammalian hippocampus is crucially involved in spatial navigation. In this context, hippocampal excitatory principal cells are referred to as place cells, if they distinctly enhance their firing frequency when the animal enters a specific region in the environment, the place field. Hippocampal interneurons are also known to show place-modulated firing activity, although less pronounced and influenced by additional contextual factors. Yet, the dynamic interplay of principal cell and interneuron firing and the topography of neuronal activity patterns underlying behavioral states are little understood. In this fellowship I therefore propose to identify the principal rules of local hippocampal ensemble activity in freely exploring rodents. The major aims are to (1) determine the proportion of place cells to non-spatially selective neurons, (2) characterize the fine-scale topography of place-specific firing in both principal cells and interneurons, and (3) analyze the dynamic interplay between principal cells and interneurons in hippocampal ensembles. To do so I will follow a progression of concerted approaches: Two-photon microscopic imaging of hippocampal neurons in vivo will first be established in anesthetized animals. Transgenic mice expressing GFP in interneurons will then be used to differentiate activity patterns in principal cells and interneurons, and ensemble activity of hippocampal neurons will fiber-optically be monitored in exploring rodents. Extensive data analysis will finally describe the local topography of principal cell and interneuron activity and unravel the balance between local excitation and inhibition in freely behaving animals.

Animals and their organs become patterned as
they grow. Several signaling pathways have been shown to regulate patterning
(e.g. Wingless/Wnt, Hedgehog, Fibroblast Growth Factor, Notch pathways) or
growth (e.g. Hippo and Insulin Signaling pathways) aspects of development. One
of the most intriguing and challenging questions in developmental biology today
is how these processes are coordinated in time and space. Decapentaplegic
(Dpp), a Drosophila member of the transforming growth factor-ß
(TGF-ß) family,
serves as a paradigm to answer this question because it has the ability to
regulate both patterning and growth.

My first aim
focuses on the logic of transcriptional regulation by Smad proteins, the
nuclear effectors of the TGF-ß pathways. The Drosophila Smad proteins, Mad and Medea, lead to activation of some target genes while
inhibiting others. The putative cofactors required for Dpp-dependent activation
have not been identified thus far. Here, I will test
the hypothesis that T-box containing proteins, Dorsocross 1, 2, and 3, fulfil
this function.

In the second
part, I aim to monitor Dpp signaling dynamics in vivo. Fusing Dpp
responsive elements to cDNA constructs that encode highly unstable versions of
GFP or RFP will allow us to detect small and transient fluctuations in Dpp
response. These experiments aspire to achieve a detailed spatio-temporal
analysis of the dynamics of Dpp signaling in growing tissues. Such in vivo
studies will allow putting forward or excluding certain models of how Dpp
exerts its multiple functions.

Engineered biomaterial systems can be used to answer mechanistic and applied questions of stem cell biology such as how to control stem cell fate decisions, generate populations of differentiated cells, or optimize the regenerative capacity of a biomaterial. Cardiac stem cells (CSCs) have recently been identified and hold significant promise. In vitro they are clonogenic and multipotent, able differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells; in vivo they can modify cardiac healing post-injury. The microenvironment (niche) surrounding CSCs in vivo is thought to provide extrinsic signals that influence CSC fate (quiescence, self-renewal, differentiation, apoptosis, motility). Cell behavior is modulated by a spatio-temporally integrated set of biophysical parameters, but rigorous study of the role extrinsic factors play in CSC fate has not been undertaken. I hypothesize that biomaterial systems with spatio-temporal control over display of matrix, cell, and soluble cues will enable systematic investigation of mechanisms underlying CSC mechanobiology. I propose a HFSP Short Term Fellowship to initiate a collaboration with Garry Duffy (RCSI, Ireland) that integrates our expertise in biomaterial systems and cardiac stem cell biology to quantify the influence of cell-cell and cell-matrix interactions on early CSC fate decisions. We will generate critical pilot data and start preparing joint extramural grant applications. CSC niche analogs will enable mechanistic study of extrinsic regulation of physiological and pathological CSC processes and will prove critical for developing CSC-biomaterial composites to induce cardiac regeneration.

Until recent times our knowledge of the microbes living within the
human body was limited to the small proportion that could be cultured.
However, within the last few years advances in sequencing technologies
have facilitated the first comprehensive surveys of the human
microbiota. These surveys have revealed not only an extraordinary level
of microbial diversity, but also suggest that there has been
significant co-evolution of host and microbiota. The goal of this
project is to carry out large-scale mapping of the host-microbe
interactions that are driving this co-evolution. To achieve this I will
use microarrays to measure host (human) gene expression and use
barcoded pyrosequencing of a portion of the 16S rRNA gene to measure
microbial abundance. These measurements will be carried out in multiple
biopsies of both healthy individuals and those with inflammatory bowel
disease (IBD). Host gene expression profiles will be used to define
disease state as well as identify features of microbial community
structure associated with patterns of host response, and then
comparisons between samples will be carried out to determine the genes
and microbes associated with health and disease. The results will be
used to shed light on the etiology of IBD and more generally on the
nature of host-microbe interactions.

Telomeres are critical for genome maintenance, as they prevent the natural chromosome ends from being recognized as damaged DNA. While telomere length decreases during the replication of normal cells, immortal cancer cells acquire the pathways that keep telomere length abnormally. Understanding the dynamics and differences of telomere regulation in normal and tumor cells is essential for comprehension of chromosome inheritance mechanism and the successful combating of cancer. Human telomeres associate with a number of factors that can influence chromosome integrity, such as the telomeric core complex and proteins involved in damage checkpoints, repair and recombination. Since cohesin, a ring-like complex that holds sister chromatids, is involved in checkpoints, repair and recombination pathways, it is tempting to hypothesize that cohesin acts in telomere maintenance in interaction with these pathways. To deepen our knowledge about telomere dynamics in both normal and cancer cells, I propose two specific aims: AIM 1 will focus on the novel role of cohesin in telomere maintenance, and AIM 2 is designated to explore telomere dynamics in cancer cells. I will examine the cell cycle dependent localization of cohesin at telomeres and the telomeric effects of cohesin suppression in AIM 1. Since SMC1, a cohesin subunit, is phosphorylated by damage checkpoint kinase, I will also investigate the importance of the phosphorylation in telomere dynamics. In AIM 2 I will study the dynamic binding of proteins and the replication pattern at telomeres in different types of immortal cells, with the goal to utilize the results to develop targeting strategies for different cancer cell types.

During sky compass navigation migratory monarch butterflies integrate the time of day with
skylight cues to generate a correct flight vector. How this integrative
processing is achieved in the brain will be addressed in the proposed project.
Electrophysiological, anatomical and molecular tools will be used to address three aims. First, the anatomy
of the monarch butterfly brain and the organization of regions relevant for
navigation will be studied. Differences in gene transcription levels in compass
related neuropils will be examined between migratory and non migratory monarch
butterflies by microarray analysis. Second, intracellular recordings will be
developed to characterize neurons involved in the processing of compass cues,
especially polarized light and spectral gradients. Recordings will compare both
butterfly forms and focus on brain regions characterized in aim 1. Third,
influences of clock-gene expressing neurons on the compass system will be
identified to find the neural correlates of time compensation. As such cells
have been found in the central complex, and this brain region contains a map of
compass directions in the locust, it is a potential interface between the
compass and the clock system. To test this, activity of clock neurons as well
as their connection to compass cells will be characterized. RNAi-mediated gene
silencing will be used to disrupt the internal clock and identify effects on
the activity of compass and clock cells.
The development of an electrophysiological approach to the monarch butterfly will
combine this method with molecular biology and significantly advance our
understanding of time-compensated compass navigation.

Gangliosides are glyco-sphingolipids present on most vertebrate tissues, but are over expressed in several tumor cells and are fundamental for tumor biology. Their own self and glycolipidic nature makes them poorly immunogenic. The findings in healthy individuals and cancer patients of circulating anti- ganglioside antibodies (Abs) indicate that specific B cells have not been clonally deleted. Some anti-ganglioside Abs have proved to be involved in B and T lymphocyte idiotypic networks and could mediate the activation of cells able to regulate the immune response against these self antigens. Many anti-ganglioside Abs are of germline origin and encoded by genes preferentially used by B-1 cells, which suggest the involvement of B-1 cells in the immunity against gangliosides. The active mechanisms controlling the immune response against non-protein antigens like gangliosides are still unknown, but B-1 cells and natural Abs could be involved. This fellowship would allow me to acquire experience in a laboratory with high expertise and cutting-edge technology, nonexistent at the moment anywhere in my home country, to purify different B-1 cell populations and study their phenotype and cytokine secretion in the presence of gangliosides and anti-ganglioside Abs. This study could prove the existence of natural anti-ganglioside Ab networks and define the role of B-1 cells in the regulation of the immune response against glycolipidic antigens. These results could be useful not only in the development of active specific anti-tumor glycovaccines, but also in the elucidation of glycolipid responses and the function of B cell subsets.

This project aims at addressing an important question in evolutionary developmental biology: Which genes show a different expression pattern between species and what is the underlying mechanism? There is strong support that highly conserved non-protein-coding elements (CNEs) are often associated with transcriptional regulation of crucial developmental genes. I will conduct a systematic computational search for genomic regions that are highly conserved in many species but lost in another species. Since strong conservation implies function, the underlying hypothesis is that loss of such a CNE leads to changes in the expression pattern of a target gene. This hypothesis will be experimentally tested by (i) verifying a cis-regulatory role for the CNE and (ii) comparing the expression pattern of a gene having the CNE to a gene lacking the CNE. For example, the experiments might reveal that (i) a CNE is a brain-specific transcriptional enhancer and that (ii) the target gene in a species having this CNE is expressed in brain while the target gene in another species lacking this CNE is not expressed in brain. This computational and experimental hybrid approach allows to associate CNE loss with divergence in gene expression. The goal of this project is to provide insight into the evolution of CNEs, to contribute to our understanding of gene expression regulation by conserved regions and to associate diverged gene expression with lost conserved elements.