Cell division consists in the duplication of both the genome and the cytoplasm into two genetically identical cells. Multicellular organisms rely on the constant generation of new cells to survive. During cell division, motor proteins organize microtubules in a bipolar structure, the spindle, which serves as scaffold to transmit the necessary forces for chomorome segregation. Although the spindle is apparently static, it is highly dynamical and there are constantly fluxes of molecules. There is no satisfactory quantitative understanding of how such a highly dynamical structure results from the self-organization of individual components and maintains its shape even under external perturbations. Moreover, there are no any direct measurements of the correlation between the dynamics of the spindle components and the forces involved in its maintenance, which are ultimately responsible of chromosome segregation. I propose to experimentally examine the relationship between global properties (force, shape and length) and local properties (motor and tubulin densities, kinetics and treadmilling velocities) in the spindle. I will develop an experimental set up to simultaneously probe force production and dynamics based on magnetic tweezers and fluorescence imaging. I will also develop a quantitative model based on contiuum coarse grained descriptions taking explicitly in account the coupling between local forces and motor kinetics. This fellowship will grant me the possibility to change my research direction from biophysics to experimental cell biology and consolidate my career towards having my own research group addressing cell biology problems in an interdisciplinary approach.

Advances in genetics are giving researchers unparalleled access to the complexities of the nervous system. Key to this advance is the ability to drive the expression or removal of select genes in discrete neuronal populations. It is now possible to tag cells, track their development and potentially modify their function at any time during this process. This is particularly beneficial in terms of an evolving, high dynamic environment such as the developing cerebral cortex. The aim of the present application is to relate the embryonic specification of GABAergic interneurons to the emergence of functionality in the early postnatal cortex. We propose that interneurons acquire their mature characteristics via a cascade of genetic programs regulated by a transcriptional code akin to that determined in the spinal cord. However our knowledge of this progression has been distorted by (1) the diversity of cortical interneurons (2) the spatial and temporal distances that interneurons have to travel from the point of proliferation to integration and function. To resolve these issues we will use inducible fate-mapping technologies to track and profile discrete temporal cohorts of interneuron. A multidisciplinary approach will be used to identify when and how interneurons begin to diverge in terms of acquiring their functional identity. It will focus on (1) changes in gene expression, notably transcription factors (2) alterations in responsiveness to environmental cues e.g. neurotransmitters (3) in vivo recruitment into emergent network activity. The resultant profile will provide novel insight into the function and conversely potential malfunction of this emergent complex network.

The purpose of this fellowship is to gain experience of functional connectivity analyses in a centre that specializes in this approach for aging research, pioneering work in this field having been conducted by Professor Cheryl Grady at the Rotman Research Institute (RRI). I aim to perform such analyses on two existing fMRI data sets of normal aging, in order to learn the specific techniques developed by Professor Grady and her colleagues.
Experiment 1, uses data collected by the host supervisor (Prof Grady) investigating auditory spatial and non-spatial working memory. 1-back working memory data is available for two subject groups (16 younger and 18 older adults). Differences in the patterns of functional connectivity between the groups, and by stimulus type will be assessed.
Data for Experiment 2 was collected as part of the GENIE study at St George’s University London, where I have been investigating neural correlates of cognitive aging. Fifteen older adults (aged >70 yrs) completed a verbal working memory n-back task (with 4 conditions). Changes in functional connectivity specifically with task difficulty will be assessed and combined with diffusion tensor imaging (DTI) to explore the white matter changes and their association with loss of connectivity.
The fellowship will allow me to establish collaboration with Professor Grady, one of the leading researchers in neurocognitive aging and also visit the RRI, a première aging research institute in the world, and learn from their expertise using this technique. I will be able to apply this analysis to other data sets on my return to the UK, and develop further research combining functional connectivity and DTI analyses.

The goal of this project is to investigate the changes in structure of the ventricular wall of the mouse heart during embryogenesis, in order to better understand the origins of the human cardiomyopathy, ventricular non-compaction. This recently recognised disease is characterised by abnormally prominent and extensive trabeculae, (finger-like projections from the muscular wall), and a relative thinning ("non-compaction") of the wall itself. Neither the aetiology of non-compaction nor its pre-symptomatic progression is understood, but the formation of trabeculae and compaction of the ventricular wall normally occur during embryonic development. The disease is likely therefore either to be developmental in origin, or result from untimely onset of embryonic genetic programme in adults. Familial isolated ventricular non-compaction (IVNC) patients, detected from childhood years, suffer from cardiac dysfunction with poor prognosis. As treatment options are limited, the utmost important challenge is to better understand the aetiology of the disease so that pre-symptomatic diagnostic tools might become available. This requires a better characterisation of ventricular wall remodelling during development and availability of animal models of non-compaction. I propose to (I) investigate ventricular trabeculation in the mouse embryo, using 3D modelling and morphometric analysis (II) identify genes regulating normal ventricular wall development through the use of transgenic mutants in which trabeculation can be induced or suppressed (III) attempt to construct a mouse model of non-compaction based on mutations known to be associated with IVNC.

T cells are involved in virtually all aspects of human disease including; viral, bacterial and anti-cancer immune responses as well as during autoimmunity and organ transplant rejection. We have developed multimeric forms of peptide-major histcompatibility complexes (pMHCs) which are currently being used to identify and grade T cells in the clinic. However, the ability of these multimers to stain T cells depends on the strength of the tripartite interaction that controls T cell activation between the T cell receptor (TCR), pMHC and T cell co-receptor. Most CD4+ T helper cells (Th cells), due to the combination of generally weak TCR/pMHC class II interactions and an extremely weak pMHC/CD4 co-receptor interaction, are beyond the limits of multimer staining. This represents a major bottleneck for the analysis of Th cells in human disease. Using our previously published methods of ‘intelligent design’, I aim to modulate the pMHCII/CD4 interaction to improve its affinity. In this way, it will be possible to develop pMHCII multimers with enhanced ability to identify Th cells. The HFSP short-term fellowship will allow me to visit Prof Lawrence Stern at UMASS medical school, a world leader in the field of molecular immunology. This collaboration is necessary for the success of this project because it will allow me to learn essential techniques and approaches and to forge new contacts with internationally renowned scientists. This proof-of-concept study will provide a novel resource that has far reaching implications for the initiation of new avenues of research concerning the role of Th cells in disease.

Maintenance of the ratio between the nuclear and cytoplasmic volumes, referred to as the nucleocytoplasmic ratio (N/C ratio), is a fundamental problem of cell biology. Its implications however are unknown. The fission yeast Schizosaccharomyces pombe shows a generally constant N/C value, and its simple geometry is a great advantage to addressing the question of N/C ratio homeostasis. We therefore propose to use S. pombe as a model organism to investigate the molecular mechanisms that control the N/C ratio, and to determine the physiological consequences of its maintenance and potential modifications. This project first relies on a detailed analysis of nuclear and cellular growth rates throughout mitosis and meiosis, and when cells exit these cycles. Second, a genome-wide reverse genetic screen will be performed in order to identify genes, whose products regulate the N/C ratio. Finally we will characterize the cellular processes that may depend on specific N/C values. Such an approach will shed light on this poorly understood property of eukaryotic cells.

The cytochromes P450 (P450s) form a superfamily of oxidative haemoproteins that catalyze a wide array of important oxidative reactions in Nature. The aims of this project are to structurally characterize and mechanistically investigate a number of members of the cytochrome P450 superfamily that accept their substrate bound to a carrier protein. Such P450s play important roles in bacterial biosynthesis, for example in the biosyntheses of biotin and vancomycin-type antibiotics. No P450-carrier protein complex has yet been structurally characterized, which prevents the rational redesign of vancomycin enzyme/substrate pairs, which possibly could lead to new antibiotics. To overcome this lack of knowledge, this project will initially characterize the complex formed between P450BioI, found in the biotin operon of Bacillus subtilis, and acyl carrier protein bound fatty acids. This complex is a stable one, and is able to be purified in an intact state to allow crystallographic studies to be performed. Crystallization and analysis of this complex will provide important information concerning P450-carrier protein interactions and how they are stabilized. This knowledge will then be applied to the vancomycin biosynthetic P450s OxyA, OxyB and OxyC, in order to improve the tightness of binding of these P450s to their substrate bound to peptidyl carrier protein, which will allow structural characterization of these important protein complexes. An investigation of the mechanism and substrate tolerance of these enzymes will then be performed, with the ultimate goal of allowing the development of new vancomycin analogues.

Dendritic cells (DCs) play a major role of immune sentinels. These cells present foreign antigens to activate immune responses but maintain tolerance at the steady state. Today, two major subclasses of DCs have been characterized by profound differences in their in vivo localization and phenotype. The aim of the project will be to decipher the distinct roles of CD8+DEC205+ and CD8-33D1+ DC subsets in the generation and homeostasis of a self tolerant immune system and to determine how their ablation impact on protection of mice against autoimmune diseases. DC biology was limited to date because, all experiments with DC subsets required ex vivo DC manipulation and non-specific methods of antigen delivery. To solve these problems we will use an established method to specifically target antigens to each DC subtype in vivo, and, in the first part of the project, we will generate knock-in mice allowing selective and timely inducible elimination of either DEC205+ or 33D1+ DCs. these cells will exhibit fluorescent tracers in intent to track their ablation after drug treatment as well as to study the ontogeny of these DCs subsets in situ. In the next part of the project, we will use these tools to study the role of DEC205+ and 33D1+ DCs in the differentiation of T-cells, the establishment of central and peripheral tolerance and in the development of autoimmune diseases like encephalomyelitis or diabetes. Our final goal being to understand the biology and behavior of the different families of DCs, this specific in vivo approach will probably profoundly modify our comprehension of the immune system, which has important therapeutic consequences.

Brain insulin signaling - crucial for memory formation – is impaired in Alzheimer’s disease. Brain levels of insulin and its receptors are lower in Alzheimer’s disease and I recently discovered that a toxic form of a protein found in Alzheimer’s brains removes insulin receptors from nerve cells, rendering those neurons insulin resistant. This sheds light on the emerging notion that Alzheimer is a novel brain-specific form of diabetes. The toxic protein, known to attack memory-forming synapses, is identified as Abeta oligomers. This proposal thus concerns a fundamental new connection between two fields, diabetes and Alzheimer’s disease, and its implications for therapeutics. I propose to establish the impact of Abeta oligomers on insulin signaling and aim to find ways to make brain insulin receptors resistant to the deleterious impact of Abeta oligomers. The possible beneficial effects of drugs used for treatment of type 2 diabetes will be determined. I will use in my studies both in vitro and in vivo experimental approaches comprising: (i) mature cultures of rat hippocampal neurons (ii) a transgenic mouse model for Alzheimer's disease (iii) stereotaxic injection of oligomers into the brains of adult rats and (iv) regenerated human neuronal cultures. It is well known that the transition from postdoc to independent investigator is hard, and it can be especially difficult for young scientists to establish a research group upon return to a developing country. Particularly in Brazil, science funding is in severe shortage and the CDA support would make a world of difference for the development of my independent scientific career.

In eukaryotes, DNA methylation results in gene silencing and is implicated in genome defence and epigenetic regulation of genes. In Arabidopsis, the DRM2 methyltransferase possesses de novo and maintenance DNA methylation activities through a poorly understood process termed RNA-directed DNA methylation (RdDM). Preliminary phylogenetic and genetic analyses suggest that DRM3, a previously uncharacterized protein, is a catalytically dead paralog of DRM2, conserved throughout the plant kingdom, and that might act analogously to its mammal Dnmt3L ortholog in promoting de novo DNA methyltransferase activity. The major goal of the proposed work is to better understand DRM2-mediated DNA methylation and DRM2 targeting, in particular through the elucidation of the role played by DRM3 in regulating DRM2. One important approach will be to analyze the genome-wide location/targeting of DRM2 and DRM3, which will allow us to test models about the generality of the DRM2-DRM3 relationship and characterize the sequences bound by DRM2. Furthermore, DRM3 activity will be biochemically characterized to address whether and how DRM3 activates DRM2 and/or mediates its targeting to specific loci. The dynamic regulation of both proteins will also be investigated, throughout development and upon various stresses. The results of this study will shed light on the process of de novo methylation in Arabidopsis and may generate general insights into the processes occuring in other eukaryotes as well.

A key question in plant biology is
how different morphologies in plant shape were produced during evolution.
Plant leaves have remarkable morphological diversity, thus presenting
an attractive opportunity to investigate this problem. Both dissected
and simple leaves initiate as entire primordia from the shoot apical
meristem (SAM) but post-initiation, leaf morphology diverges through
mechanisms that are largely unknown. The Arabidopsis thaliana
close relative Cardamine hirsuta displays compound leaves and
has emerged as a powerful model system for studying processes responsible
for evolutionary diversification of leaf morphology. Leaflet formation
in C.hirsuta requires the establishment of growth foci that form
after leaf initiation at the leaf margin in response to an auxin activity
maxima organized via the PIN1 auxin efflux facilitator. However, the
the pathways that direct species-specific deployment of auxin activity
maxima that support leaflet formation are unclear. I propose to address
this problem by investigating whether regulated auxin biosynthesis by
the YUCCA pathway might contribute to species-specific leaflet
formation .