The lipid sphingosine-1-phosphate (S1P) plays critical roles in mammalian biology. It is essential for both immune cell
circulation and embryonic blood vessel development, and has been implicated in inflammation and the angiogenesis that accompanies tumor growth. Despite the importance of this signaling lipid, the regulation of S1P production and distribution is still poorly understood.
I plan to ask two related questions about S1P biology.
First, I will ask how the S1P gradient between tissues and circulation is maintained. How is circulatory S1P kept out of tissues? I will focus initially on the contribution of Lipid Phosphate Phosphatase 3 (LPP3) in destroying circulatory S1P as it exits blood vessels with transudated plasma.
Second, I will investigate the significance of keeping tissue S1P low (beyond enabling immune cell circulation): I will test the hypothesis that S1P is an inflammatory stimulus in vivo. I plan to focus on inflammation of the central nervous system because the S1P analog FTY720 has shown promise in Phase II clinical trials for multiple sclerosis. I will ask whether FTY720 is effective because it stops recruitment of T cells to the spinal cord by halting lymphocyte circulation, or because it acts directly on the innate immune system.
Taken together these projects will contribute to our understanding of S1P regulation and its role in pathology, and more generally to our understanding of inflammation, which increasingly recognized to be a factor in a large number of diseases.

Life is dependent on proper functioning of the cellular machinery, which is dependent on the ability of proteins to fold into their functional states. When proteins fail to fold properly “misfolding” or maintain their native folded state, they adopt unstable conformations which undergo self-assembly to form highly ordered aggregates in and outside the cells, depending on the protein. Protein misfolding and aggregation play a central role in the pathogenesis of several incurable systemic and neurodegenerative diseases that affect the population today. Therefore, detailed mechanistic understanding of protein aggregation and amyloid formation in and outside the cell is essential for i) developing effective therapeutic strategies; ii) improving our understanding of the fundamental chemical and physical roles that govern protein folding and self-assembly; iii) translating this knowledge to new advances in biology, medicine, biotechnology and engineering. Towards achieving these goals, we will take advantage of recent advances in chemistry to develop new chemical tools and experimental that would allow us to monitor, manipulate and study these processes in real time in and outside the cell. The knowledge to be gained from the proposed studies will have significant impact on our understanding of the molecular basis of amyloid diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Type II diabetes, and contribute to advances in biotechnology, where protein aggregation remains a major challenge in the development of protein therapeutics.

Freezing avoidance in Antarctic fishes is enabled by the presence of antifreeze glycoproteins (AFGPs) in most of their body fluids, but how they deal with circulating ice remains unresolved. We have shown that AFGPs are synthesized in the exocrine pancreas and the anterior stomach and secreted into the gut, where they serve in defence against imbibed ice. We have also identified high levels of AFGPs in the blood, where they defend against ice crystal growth in the circulation. In this investigation we will combine biology with synthetic organic chemistry to address how AFGPs get from the gut into the blood and how fishes remove circulating ice.

To determine how AFGPs traffic from the gut to the blood, we will synthesise fluorescently-labelled AFGPs and use confocal microscopy to visualize the pathway of uptake following oral intubation. Mass spectrometry of blood samples obtained after the introduction of isotopically-labelled AFGPs will be undertaken to confirm translocation from the gut to the circulation.

We postulate that removal of ice from the circulation occurs in the spleen via a receptor-dependent interaction involving ice-bound antifreeze. Recently, we have identified a second antifreeze protein, termed antifreeze potentiating protein (AFPP), which also adsorbs to ice. This new observation leads us to hypothesize that the adsorption of both types of antifreeze is required for the recognition and removal of circulating ice.

To identify the AFGP receptor and the cell involved, we will prepare dual function orthogonally-labelled AFGP probes containing a UV-activatable cross-linker that will covalently bind to the putative receptor on photoactivation. An azide chemical reporter group will also be incorporated into the AFGP probe to enable the use of “click chemistry” to attach a biotin-conjugate to the cross-linked putative AFGP receptor. The receptor will be identified following streptavidin affinity chromatography of cell membrane extracts prepared following the culture of spleen cells in the presence of excess probe. Such probes, in conjunction with routine cytological analysis, will also enable us to identify the cell type involved. Following complete characterization of AFPP we will attempt synthesis and similar modifications to identify its receptor. Taken together with knowledge of the pathway of AFGPs to the blood, we will finally have a complete picture of the mechanism of organismal freezing avoidance.

The activity of nerve cells in behaving animals correlates with sensory processing, decisions and motor control. Such correlations are necessary, but not sufficient, to link specific aspects of brain activity to behaviour. In order to obtain causal evidence it is critical to manipulate identified neuronal circuits in the brain during behaviour. This demanding goal is the objective of this research program, where as a team we will develop, test and apply genetic technology for highly specific control of neuronal networks in the mammalian brain which will be quantitatively related to behavioural report of sensory perception. Edward Callaway of the Salk Institute in La Jolla, California will develop genetic tools for specifically controlling the activity of well-defined populations of cells in the rodent brain. Carl Petersen of the Brain Mind Institute in Lausanne, Switzerland and Mathew Diamond of the International School for Advanced Studies in Trieste, Italy will use these genetic tools to analyse the neuronal basis of perception through simultaneous measurement and manipulation of cortical activity during performance of tactile detection and discrimination tasks. Carlos Brody of Princeton University will carry out information theoretic analysis and neuronal network simulation necessary for quantitative evaluation of causal relationships between neuronal activity and behavioural report of perception. Together this team of investigators aims to provide pioneering evidence for different functional roles of the diverse types of neurons found in the neocortex. Furthermore, the rigorous testing of such genetic technology in vivo in the mammalian brain will provide an important step towards the many potential clinical applications for treating brain disorders.

Living cells must be highly organized, therefore many of their components are transported to particular places and anchored there. Disruptions in this transport and localization cause disease, particularly neuropathologies. Many copies of proteins are made from each working copy of a gene (messenger RNAs, mRNAs). Proteins are moved to their correct location or, more efficiently, made there from mRNAs that have been moved. Although the mechanisms that move proteins to their destinations have been intensively investigated, those that carry mRNAs have been much less studied. Indeed, RNAs have recently been discovered to participate in a growing number of new roles and unexpected qualities in cells.

The molecular “zip codes” hidden in RNAs that specify destinations have only been deciphered for a few RNAs. An important goal of this project is to create intelligent methods that can find these codes. Here we will address fundamental unanswered biological questions- are many or just a few key RNAs localized? How many different zip codes exist? Have these codes been conserved through evolution? Are transport codes and anchoring codes identical or separable? Does localization change on insult or in disease?

Through an international collaboration we now have a unique opportunity to answer these questions. In mammalian cells, genome-wide screening has provided us with a large set of mRNAs that localize to cell protrusions and axonal growth cones. In yeast, we have identified a set of mRNAs unexpectedly bound to membranes. These data offer an unrivaled opportunity to develop new computer programs for zip code identification, and to refine these methods by testing predictions with experiment. We will also investigate the evolutionary conservation of the zip codes from yeast to man. By combining the diverse expertise of a cell biologist, a yeast geneticist, and a computer scientist, we are in a unique position to uncover fundamental principles of mRNA localization. Our ultimate goal is to unravel a fundamental dictionary of RNA zip codes.

NOD-like receptors (NLRs) play important roles in innate immunity as intracellular sensors of microbial components and cell injury. Upon binding of ligands some NLRs associate with ASC and caspase-1, to form a multiprotein complex called inflammasome. The inflammasome activates caspase-1, which then triggers the release of pro-inflammatory cytokines as well as cell death. Although 16 putative NLRs have been identified in humans, the function and the ligands of most of them remain unknown. Infection of macrophages with Francisella tularensis, an intracellular pathogen, has been shown to activate the inflammasome in a type I IFN dependent manner. This process depends on the expression of the Francisella Pathogenicity Island genes, which are necessary for intracellular replication and which encode a putative type VI secretion system.
I hypothesize that new, specific host cytosolic sensors are involved in sensing F. tularensis ligands in the cytosol and that these ligands are released in a FPI-dependent manner. The goals of this work include the identification of new components of the host pathways involved in inflammasome activation in response to F. tularensis and the identification of new PAMPs (pathogen associated molecular patterns) that are involved in triggering innate immunity.
The study of the signaling pathways leading to inflammasome activation will contribute to the understanding of the role of the inflammasome in the host response to pathogens. In addition, the results might help to understand the role of the inflammasome in various autoinflammatory and rheumatic diseases as well as in the response to adjuvants, in order to develop new therapeutics and vaccines.

Axons of retinal ganglion neurons traverse molecularly distinct territories and choice points on their way to the optic tectum. The navigational behavior of their growth cones is governed by extracellular guidance factors that elicit chemotropic responses through local protein synthesis and degradation. Cyclic nucleotide signaling modulates these processes to allow response switching. Little is known about the orchestration of molecular mechanisms in axonal navigation, mainly due to a lack of intracellular real-time assays. In isolated neurons and the Xenopus brain, we wish to study how guidance cues are interpreted and integrated by the growth cone to produce the desired behavior. For this, we will apply a novel optical sensor paradigm that can offer a detailed quantitative view of the molecular navigation reactions in the growing tip, while minimizing possible deleterious effects of the introduction of sensor constructs. These approaches will be used to generate uniquely high-content information on the key reactions of local protein translation, folding, and proteasomal degradation. These powerful tools and highly interdisciplinary approaches will allow the dynamic dissection in vitro and in vivo of molecular events elicited by extracellular signals, and the molecular functional definition of their interplay at key choice points in the visual pathway where axons change their behavior in response to changes in guidance cues.

We propose to bring together an international and intercontinental team of biophysicists, cell biologists and neuroscientists to investigate the cellular and molecular mechanisms by which large proteinaceous aggregates can cross biological membranes. We propose three specific research aims. First, we will use state-of-the-art technology to understand the properties of protein aggregates that endow them with membrane-penetrating properties. The second aim seeks to determine the extent to which internalized aggregates can move between cells within a tissue and the role of endocytic, autophagic and cytoskeletal processes. Third, we propose to assess the extent to which protein aggregates can nucleate the aggregation of endogenous proteins within the brains of specially engineered mouse lines, and determine the role of motor-based axonal transport in this process. Together, this highly collaborative project seeks to understand the fundamental cellular and molecular basis for a newly discovered phenomenon that may play a critical role in the pathogenesis of diverse conformational diseases.

Tumor development is driven by the accumulation of genetic and epigenetic alterations. Though mutations induced by translocations have long been seen as critical to the development of hematological malignancies, only recently have technological advances allowed genome scale analysis of rearrangements in epithelial cancers. These are most often detected as copy number polymporphisms by low to moderate resolution comparative genomic hybridizations (CGH). Here, we propose the study of structural alterations in cancer genomes at single base resolution through the development of a new methodology. These studies use Array CGH datasets to identify regions containing structural rearrangements. We will recover these regions from tumor genomes using an Array-based genomic capture strategy developed in the Hannon laboratory. Using next generation sequencing, we will decipher the precise structure of genomic rearrangements, which lead to detection as copy number loss and gain. These studies will provide the first deeply detailed picture of chromosomal alterations in epithelial cancers, which may lead to the discovery of novel oncogenes and tumor suppressors, whose function is directly altered by structural rearrangement. Signatures of structural variation could become prognostic indicators and may also permit the development of extremely sensitive detection methods for tumor cell genomes for non-invasive monitoring of disease status. Finally, having a large collection of tumor-associated genomic breakpoints may lead to an understanding of the mechanisms underlying genomic instability in cancer.

Background & Hypotheses

The establishment and maintenance of specialized plasma membrane and cytoplasmic domains leading to growth at restricted regions of the cell cortex is fundamental to the form and function of many polarized cell types, ranging from yeasts to neurons. It results from the concerted action of cytoskeletal components and a variety of highly evolutionarily conserved ‘polarity factors’. Although substantial progress has been made in identifying factors and describing their individual modes of action, how their collective (‘systemic’) interactions mediate with precision the initiation, maintenance and termination of localized cell growth and convey plasticity and robustness to the polarized growth process remains, even for the simplest cells, poorly understood.

Objectives

Our objective is to carry out the most detailed quantitative study of the ‘systemic’ regulation of polarized cell growth to date, using cells of the fission yeast Schizosaccharomyces pombe. The fission yeast with its relative genomic simplicity, genetic tractability, uniform size and shape, and extremely well-characterized and comparatively simple polarization machinery, is ideal for this study.

Research plan

A) Which polarity factors and factor interactions are sufficient to generate localized areas of sustained cell growth: we will identify and quantify, among known fission yeast polarity factors of the ‘Tea1 cluster’ and the ‘Cdc42 cluster’, the ‘core’ of factors and factor interactions that suffice to give rise to localized areas of sustainable polarized growth.

B) How the localization, initiation and termination of polarized growth are regulated in space and in time: we will investigate how the ‘core’ factor network together with the cytoskeleton and the cell cycle machinery can define the position and timing of growth zone assembly.

Approaches

For every project and subproject, we will use a combination of genetics, quantitative microscopy and computer modelling and work iteratively between those approaches.

Significance of anticipated results

By this approach we hope to gain novel insights on how polarized cell growth is fine-tuned in fission yeast, and to significantly advance our fundamental understanding of general design principles of cell polarization relevant to other eukaryotic cells.

In bacteria, the rigid external cell wall (CW) and the intracellular actin-like (MreB) cytoskeleton are major determinants of cell shape. Synthesis and chemical composition of the CW, a three dimensional polymer network that is one of the most prominent targets for antibiotics, are well understood. However, despite decades of study, little is known about the complex CW ultrastructure and the molecular mechanisms that control CW morphogenesis in time and space. MreB homologues assemble into dynamic filamentous helical structures thought to control shape by serving as organizers for the movement and assembly of macromolecular machineries that effect CW biogenesis. However, the mechanistic details used by the MreB cytoskeleton to fulfill this role remain to be elucidated.

We will combine powerful genetics and biochemistry available in the model bacterium Bacillus subtilis with modern high-resolution light microscopic techniques to study the dynamics of the cytoskeleton and of CW assembly. Scanning probe microscopy and magnetic tweezers will be used to probe the ultrastructure and mechanical properties of the CW. Parameters measured by the different approaches will be combined to generate a mathematical model that quantitatively describes the features of B. subtilis morphogenesis.

Specific tasks will be:

1. To characterize the interactions between cytoskeleton and CW biogenesis. We will quantitatively characterize the distribution and dynamics of fluorescently-labeled cytoskeletal components, CW enzymes and adaptor proteins.

2. To analyze cell surface properties of B. subtilis strains. The pattern of CW polymers will be visualized using high-resolution light microscopy and Scanning Probe Microscopy (SPM). Results will be evaluated after genetic or chemical perturbations of the cytoskeleton or CW organization. Mechanical properties of the CW and the influence of polymer organization on CW rigidity will be measured using force spectroscopy (SPM and magnetic tweezers).

3. To build a mathematical model of B. subtilis morphogenesis. Initially a cluster/network representation of morphogenesis will be generated from previous studies and refined with data from our experiments. Quantitative parameters from our experiments will then be combined with the formal network background to generate a dynamic spatial model with predictive power that will be tested experimentally.

The control of cerebral vessel diameter is of fundamental importance in maintaining healthy brain function because it is critical to match cerebral blood flow to the metabolic demand of active neurons. The hypothesis that we will test is that astrocytes are essential communicators between blood vessels and neurons to regulate cerebral blood flow. Although there are numerous data indicating that astrocytes have critical roles in these processes there are still outstanding questions and critical gaps in our knowledge. A key shortcoming in previous studies is the difficulty to selectively activate neurons versus astrocytes. This grant will fund research to develop and apply the necessary tools for two photon uncaging and imaging in vivo. This team will apply novel two photon uncaging approaches in the cortex in vivo to address fundamental questions of how astrocytes signaling can alter cerebral blood vessels. A major question concerning the roles of astrocytes in the regulation of cerebral blood flow is the mechanism by which astrocytes are induced to cause either constrictions or dilations as a result of calcium transients. It is clear that astrocytes have the ability to induce either response in adjacent blood vessels. A recent study from MacVicar’s lab using a new two photon sensitive caged calcium (DMNPE-4) from Ellis-Davis’ lab has shown that the metabolic state of the surrounding tissue determines the polarity of the astrocyte influence on blood vessel diameter. This was determined in studies in brain slices in which oxygen levels and metabolic controls could be rigorously controlled and manipulated. The major pressing issue is to test these mechanisms described in brain slice studies using in vivo experiments. This collaborative effort will provide the expertise to do this. The Charpak lab has years of experience examining two photon signals in the olfactory bulb in vivo. The MacVicar and Charpak labs will combine efforts to examine astrocyte effects on blood vessel dynamics in the cortex and olfactory bulb in vivo. They will use new caged probes developed by the Ellis-Davis’ lab. New probes are necessary to provide the sensitivity that is required for in vivo photolysis and to provide new compounds to be uncaged for these investigations.

DNA in eukaryotic cells is packaged into chromatin, and access to DNA is mediated and regulated by the histone proteins that coat and package the DNA. A wealth of functional analysis has shown that reversible histone post-translational modifications are central regulators of all processes that act on the genome, including replication, transcription and repair. However, a structural basis for how specific combinations of post-translational modifications control these processes is currently lacking. By reconstituting designer nucleosomes and nucleosome arrays containing histones bearing site-specific post-translational modifications and combinations of such modifications we will study the effect of histone post-translational modifications on the structure and function of nucleosomes and the higher order structure of chromatin (the ‘30 nm’ fiber). Specific histone modifications will be incorporated using a novel genetic code expansion method that permits the incorporation of modified amino acids at preselected positions using bacterial expression. We will study the effect of these modifications on histone, nucleosome, and chromatin structure using an ensemble of biophysical techniques. State-of-the-art single molecule techniques including magnetic tweezers based force spectroscopy and single pair Fluorescence Resonance Energy Transfer will be employed to resolve the effects of histone modifications on DNA organization within the nucleosome. We will test and expand physical models for epigenetic changes on the structure and dynamics of the ‘30 nm’ fiber. In addition, we will identify and characterize new proteins or complexes that specifically interact with modified histones. Thus we will combine synthetic biological and biophysical approaches to create a molecular and biophysical understanding of how histone modifications control cellular function.

Background

Jasmonates regulate specific developmental processes and responses to biotic or abiotic stresses. The active hormone, Jasmonoyl-isoleucine (JA-Ile), is perceived by the F-box COI1. As a result, the JAZ repressors are targeted by SCFCOI1 for degradation, releasing transcription factors that in turn mediate the plant responses (Chini et al, Nature, 2007).

Biological Significance

JA-Ile-mediated plant responses are very abundant and diverse. However, the molecular basis for their specificity is completely unknown. This project aims to identify agonist and antagonist molecules of JA-Ile, which will be instrumental in other projects to study the molecular basis of specificity in JA-Ile-mediated responses.

Methods

We will carry out two screens exploiting the huge chemical library available at the Raikhel lab and two experimental frameworks routinely performed in our lab: the JA-Ile-dependent degradation of JAZ proteins and the hormone-dependent COI1/JAZ interaction in Y2H.

Prospects

The proposed Screens aim to identify two set of molecules:

A) JA-Ile Agonist, which could represent either activators of hormone biosynthesis or novel active forms of the plant hormone, possibly modulating specific responses (addressing the specificity of JA-Ile responses).

B) JA-Ile Antagonist molecules. Such compounds have not been identified yet, therefore, they will represent powerful tools for the pharmacological analysis and dissection of the signalling pathway.

In 2010, I will apply for a 5-year position. This project results will be crucial to set up large-scale genetic screen to identify new regulators of the JAZ-mediated pathway and therefore my future indipendent line.

The success of a cell's survival under different growth conditions depends on its ability to control the coordinated transcription of selected genes in response to external stimuli. In bacteria, transcriptional regulation of genes depends on the binding of proteins to DNA, but also on the physical configurations of the resulting mesoscopic protein/DNA complex, the nucleoid. Particular nucleoid-shaping transcription factors (NSTFs) have the task of modulating the nucleoid's dynamic structure in response to changes in environmental conditions. NSTFs may thus affect the expression of many genes - some of which code for the same NSTFs. The current transcription network view of regulation considers a graph of all genome interactions, but disregards the effects due to changes of the nucleoid. What role does the nucleoid's structure play in the hierarchy of events leading to the large scale transcriptional response to changing growth conditions? Can this be incorporated into a generalized network description? To address these questions, we will develop a predictive integrated account of transcription in bacteria, including the thermodynamic state of the nucleoid as dictated by the local and global interplay of NSTFs and supercoiling, and apply it to the study of the cell's global response to a nutritional shift. The interdisciplinary nature of the team will help us build a theoretical generalized network model, make quantifiable predictions, and test them experimentally. In order to obtain quantitative systematic measurements of nucleoid structure we will (i) study a library of specifically designed nucleoid structure-sensitive reporter gene constructs at different chromosomal loci, (ii) develop new optical/microfluidic technologies allowing for time-resolved single cell measurements in liquid medium, (iii) perform in situ nucleoid purification and subsequent biophysical characterization, and (iv) develop new modeling approaches to characterize the biophysics of the nucleoid as a "complex polymer". These results will allow, for the first time, a complete description of the mechanisms of cellular adaptation to specific environmental changes through transcription, integrating the interplay of global nucleoid organization/structure with the classical network model. This project lies at the frontier of biology and statistical/soft-matter physics.

70% of mankind’s current energy needs are met by burning fossil fuels. This is already problematic since oil and gas supplies are limited and because of the adverse environmental effects of rising levels of carbon dioxide in the atmosphere. Moreover this situation is set to get worse as current predictions estimate that our energy needs will double by 2050. Mankind is therefore facing a major challenge to find new sources of clean renewable fuels. Photosynthesis is a biological process able to use solar energy to produce such fuels. The essence of photosynthesis can be described in terms of the following three partial reactions: light harvesting, charge separation, and electron transfer to take electrons from water in order to reduce carbon dioxide to carbohydrate (a fuel).

Since solar energy at the earth’s surface is rather diffuse, any artificial system designed to convert it into a fuel will need to incorporate a light-harvesting or light-concentration step. The first question any engineer would ask is: what is the design tolerance of such a system? How much architectural flexibility is possible while still retaining both high efficiency light-harvesting and energy delivery to the ‘sink’, where that energy can be productively utilized? The project addresses this problem by interrogating natural light-harvesting systems with sophisticated spectroscopic and microscopic techniques, which can tell us how their supra-molecular, meso-scale architecture relates to their overall light-harvesting efficiency.

We use as model complexes purple bacteria, which are very simple photosynthetic organisms, for which the structure and function of individual native light-harvesting complexes have been identified. We will first investigate native membranes of purple bacteria by the combined use of time- and space-resolved spectroscopies, in order to correlate their local organization with their light-harvesting function. We will then use this information to begin to construct arrays of artificial light-harvesting complexes, based on synthetic analogues of natural pigments, on surfaces where we can control supra-molecular architecture. Such arrays can, in the long term, be used in devices for producing solar fuels.

How do we suppress inappropriate responses? One influential theory proposes that the anterior cingulate cortex works in conjunction with the lateral prefrontal cortex (PFC) to signal and suppress inappropriate actions. Despite a wealth of studies investigating the neuroelectric signatures of response conflict monitoring, almost nothing is known about how these different brain regions interact to form a cognitive control network. The proposed research combines existing theory with cutting-edge analysis techniques to test the novel hypothesis that oscillatory phase synchrony—a millisecond-resolution measure of transient functional interactions among brain regions—underlies the PFC’s ability to monitor and adjust inappropriate actions. The research comprises 3 Specific Aims. Aim 1: To examine how neural oscillatory synchrony links the medial and lateral PFC, and motor cortex, into a functional network. This will be done by measuring EEG while human participants engage in difficult reaction time (RT) tasks, and examining dynamic changes in phase synchrony before and after fast vs. slow RTs. Aim 2: To use transcranial magnetic stimulation (TMS) to understand how patterns of PFC oscillations and oscillatory coupling are altered by a TMS pulse. Aim 3: To test whether oscillations play a causal role in the cognitive control network by examining the effects of TMS during different oscillation cycles on response selection. In addition to combining different psychological and neurobiological theories in one unified framework, the proposed research has the potential to provide a novel neurobiological mechanism by which we can monitor our behavior and control inappropriate responses.

During mammalian embryogenesis, mesenchymal cells differentiate into stromal lymph node organizer cells (LNo). In the course of a continuous differentiation program, LNo specialize into stromal cells that specifically accommodating either T or B cell clustering, this process forms the characteristic cellular architecture of secondary lymphoid organs. Gene-targeted mice have provided insight into the hematopoietic cells interacting with stromal cells during their differentiation, and several of the molecules essential for this process have been elucidated. However, in vivo studies are intrinsically limited in their ability to allow for the simultaneous and tightly controlled manipulation of multiple cells and signals. In this project, we will develop artificial lymph node (ALN) environments in vitro that will allow us to study the complex cellular and molecular networks underlying LN organogenesis with unprecedented control. Using the ALN environments, we will unravel 1) Minimal cellular and molecular requirements for LN development; 2) Organization of stromal cells into distinct T and B cell specific areas and 3) A role for non-paracrine forces including mechanical stresses, oxygen tension, cell movement and extracellular matrix remodeling in development of LNs. To achieve this, 3D remoldable collagen scaffolds will be microfabricated that capture the anatomy of the developing LN microenvironment. The scaffolds will be seeded with appropriate human or mouse cells and connected to externally controlled fluidics to allow for direct modulation of the cellular (LTi, stroma, lymphocytes) and chemical (signalling molecules, inhibitors) microenvironments within the scaffold at any given time. Additionally, this approach will allow for direct modulation of physiological forces (oxygen, shear stress and wall tension). Seeded cells will be allowed to differentiate and organize while these parameters are varied. Stromal cells, LTi cells, lymphocytes and the extracellular matrix will be monitored in situ by real time 4D multicolour imaging and cells will be harvested from the scaffolds such that gene expression signatures between different conditions can be measured and compared and correlated with imaging results. From this data we will develop both physiological and genomic maps of LN formation and generate fully functional ALNs, providing a unique platform to study LN development and function in real time and allow visualization and modulation of biological and pathological processes.

Serotonin (5HT), like dopamine (DA), has long been implicated in the control of behavior, including decision-making and reinforcement learning (RL). Its relevance for behavior is illustrated by its implication in a variety of neuropsychiatric disorders ranging from affective disorders such as anxiety and depression to impulse control disorders. However, while the two neuromodulators are tightly related and have a similar degree of functional and clinical importance, compared to DA, we have a much less specific understanding about the mechanisms by which 5HT affects behavior. We address this issue by leveraging existing understanding of DA and a constellation of methods that has shed light on DA function: integrating functional magnetic resonance imaging (fMRI), psychopharmacology and neuronal recordings in behaving nonhuman and human primates, all guided by computational theory. We will test, and ultimately revise, a hypothesis that 5HT biases decision-making through an interaction with dopaminergic RL mechanisms.

Specifically, we will test the hypothesis that 5HT and DA have coordinated effects that serve to couple multiple axes that might otherwise be expected to have independent effects on decision making. We will manipulate different axes in behavior independently in a learned decision-making task, using functional neuroimaging in humans and neuronal recordings from DA and 5HT nuclei in monkeys to study correlations between neural activity and behavioral context. We will also assess the neuromodulators’ causal roles, by manipulating DA and 5HT in the same task and studying the effects on behavior and neural activity.

To maximize the comparability of the data, we will use experimental procedures as close to identical as practical between species, and use computational models in data analysis to bridge different levels of measurement. Experiments will be grounded in and quantitatively analyzed using current theoretical RL models, and will in turn inform the revision of these models.

Alpha-synuclein (AS) is the major constituent of Lewy bodies, a pathological hallmark of Parkinson’s disease (PD). Amyloid-like deposits of AS are generated by toxic oligomeric species of the protein, which are now considered to be a major cause of cell death in PD. The mechanisms by which the monomeric protein suffers structural rearrangements that lead to oligomerization are not yet known and are a subject of intense research in the field. The most appealing hypothesis is the one of oxidation-mediated oligomerization of AS, causing the formation of highly toxic early amyloidogenic species. Such intermediates appear to be scarcely populated, likely involving important structural rearrangements, which paves the way towards an effective therapeutic strategy against PD based on the stabilization of the native monomeric protein. The study of the early stages of fibrillogenesis requires the development and application of new methods. I propose tackling this challenge in a novel way, through exciting multidisciplinary research by scaling up the NMR techniques combined with methods of molecular dynamics simulations, together with the use of the more advanced single-molecule fluorescence techniques. The impact of metal-catalyzed oxidation in the structure of monomeric and oligomeric AS by means of low and high resolution structural techniques will be evaluated, shedding light into the role of oxidative damage as an effector in protein aggregation. The information obtained will be paramount in order to validate the use of pharmacological chaperones of AS as new therapeutic agents to treat PD.