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 .

The ability to move in response to a chemical gradient confers a significant evolutionary advantage to cells. However, the earliest cells during the origin of life were very primitive systems. The goal of this proposed research is to understand how such simple systems can use the energy stored in external chemical gradients to move in an anisotropic manner. To achieve this we will first develop an experimental set-up able to establish a reliable static concentration gradient. The motion of simple vesicles will then be observed and modeled in order to quantitatively understand their behavior. The second step of the project will be to encapsulate particles, including biological reactants, inside the model proto-cells to see if a polarity is induced in the cell, and how the cell motion is affected. The expected output of this research is to understand the role of physical mechanisms in the development of cell migration at an early stage of evolution. My background as an experimental physicist will help me to set up the devices to be used in the experiment. This fellowship will assist me as I want to shift the focus of my research towards biophysics. I am particularly motivated to undertake this project at the Bauer center because of the superior infrastructures and specialized experience of the researchers offered by the center. Indeed, I look forward to being trained in new biological techniques by Irene Chen and to collaborating with Michael Brenner on the modeling aspect of the project. The experience with which this fellowship will provide me will be an important step towards my goal of becoming a professor and researcher of biophysics in a university setting.

Cell viability and division are dependent on the controlled degradation of protein regulators. This degradation occurs via the action of ubiquitin ligases, which conjugate the 76 kDa ubiquitin moiety to exposed lysine residues on target proteins. Polyubiquitinated proteins are subsequently degraded via a protein complex called the proteasome. The events occurring between protein ubiquitination and degradation, including transport to the proteasome, are poorly understood. While the proteasome can bind some ubiquitinated proteins directly, the genes encoding these subunits, in contrast to the proteasome itself, are not essential. Therefore, the cell must have other mechanisms to recognize proteins’ polyubiquitin. The Dsk2, Rad23 and Ddi1 proteins of S. cerevisiae are proposed to function as soluble ubiquitin ‘receptors’ that bind to both the proteasome and to ubiquitinated proteins via distinct domains. RAD23 and DDI1 are up-regulated after DNA damage and mutations in these genes cause sensitivity to DNA damaging agents. I will exploit these properties in a comprehensive proteomic approach aimed at identifying targets of the Rad23 and Ddi1 ubiquitin receptors in yeast. I will then analyze receptor-target interactions to elucidate the mechanism by which receptors facilitate protein turnover. Towards the end of my fellowship, I will explore the interaction of conserved receptor-target pairs in mammalian cells. This project represents a change in research direction that will expose me to new technology and facilitate my development as an independent scientist.

A major function of the circadian clock is to precisely coordinate responses of an organism to daily environmental stimuli. The molecular mechanisms underlying the integration of environmental stimuli and the clock are poorly understood in all organisms. My objective is to understand how environmental signals are integrated into the circadian clock and the function of specific genes involved in this integration, using Arabidopsis as a model system. My proposal is designed to provide insight into the function of EARLY FLOWERING3, a gene that modulates the effects of light into the clock. The specific aims of this proposal are to: 1. Characterize interactions between ELF3 and known clock associated proteins in vivo. Biochemical and genetic interactions between specific clock proteins that are co-expressed and interact in vitro will be analyzed. 2. Identify novel proteins that interact with ELF3. Yeast two-hybrid assays and affinity purification techniques coupled with mass spectrometry will be used to identify novel ELF3 interacting proteins. Identified candidates will be analyzed by biochemical and genetic approaches in vivo. 3. Identify suppressors of elf3 mutant phenotypes. A suppressor screen will be performed to identify genes that genetically interact with ELF3. Relevance: The circadian clock allows for the proper coordination of daily human behaviors, such as sleep/wake cycles. Research to elucidate the complex molecular mechanisms underlying the circadian clock in plants will complement similar analyses in other systems, ultimately leading to a better understanding of the circadian clock in humans, allowing for the treatment of circadian disorders.

Progress in bioengineering, biotechnology and biomedicine is limited by our understanding of how genetic control systems work in living cells. The lack of methods for studying kinetics and gene regulation in single cells seriously impairs our prospects to gain deeper insight and to develop better quantitative models of such control systems. This project proposal is focused on transcription factors (TFs), proteins that regulate gene regulation in all kingdoms of life. The main objective is to help develop new experimental methods for analyzing intracellular transcription factor dynamics at high temporal resolution and spatial precision of multiple transcription factors simultaneously. The methods will be used to answer fundamental questions in bacterial physiology related to transcription factor mediated gene regulation and dynamics in individual living bacterial cells. It aims at understanding how bacterial TFs coordinate the expression of genes at the level of single cells. The experimental challenge of studying TF mediated gene regulation in individual bacteria is that it is a single molecule process where one or a few TF molecules bind one or a few specific binding sites on the bacterial chromosome. This proposal describes new state-of-the-art single molecule microscopy methods for studying the dynamics of multiple TF molecules in individual living cells. We will we gain an understanding of how transcription factors operate in living cells, how their copy number is maintained, and how TF activity at competing binding sites are controlled. Direct observation of TF dynamics will also be used to probe gene regulation at unprecedented time resolution.

Phosphoinositides are constituents of biological membranes that are centrally involved in signalling and serve as membrane anchors. Phospholipase C (PLC) is activated by G-protein-coupled receptors (GPCRs), many of which are targets for drugs treating neurological and psychiatric disorders. PLC cleaves phosphatidylinositol (4,5)-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG), which modulate cytosolic calcium, ion channels and plasma membrane transporters. Many ion channels are also modulated by PIP2 depletion. To gain a mechanistic understanding of this important cellular signalling pathway, I will use kinetic measurements and modelling. On a single-cell level, PIP2, IP3 and DAG will be measured following GPCR activation using PIP2-sensitive ion channels and Förster Resonance Energy Transfer (FRET). In particular, I will address source, rate and limiting factors of PIP2 regeneration following its depletion by PLC. I will also measure lifetimes of IP3 and DAG. These measurements will be important to understand temporal precision of PLC signalling, the questions of refractory period and local confinement and the effects of prolonged stimulation. A kinetic model of phosphoinositide signalling will be determined from the measurements. Since many factors operate simultaneously and feedback loops icrease complexity, only modelling will allow us to estimate whether our current understanding of PIP2 signalling is correct and sufficient. The model will provide insight into the functioning of this signalling cascade and inform new experiments.

The main idea of my proposed visit in the lab of Prof. Millar is to start and establish collaboration between his group and my group in order to include the light input into the clock models. I will use data from Prof. Millar’s and Dr. Halliday’s lab to develop an integrated, dynamic model of the clock and its phytochrome light receptor. I intend to start with a combination of the clock model developed in Edinburgh and the model for the phyB dynamic developed by myself in Freiburg. In an iterative cycle between theory and experiment we plan to extend this starting model.
Furthermore, my efforts to understand the PIF3-PHYB interaction fits very well into the research activities of the ROBuST project coordinated by Dr. Karen Haliday at the University of Edinburgh. This project aims to understand the principles and molecular mechanisms that underlie temperature buffering or sensitivity of the circadian clock. Both I and the ROBuST project are modeling the interactions between phytochromes and their interacting proteins. One important component not investigated in the ROBuST framework so far but by myself (thesis S. Sonntag 2008) is the light induced degradation of transcription factors like PIF3 via the phyB pathway. Dr. Halliday and I have agreed to collaborate on this effort. My visit in Edinburgh will link the activities in Edinburgh and Freiburg to understand light dependent mechanisms of plant development. No clock model for any organism currently has an explicit and detailed photoreceptor mechanism, so this work would be a first. It would also provide a link between two areas of plant science.

The plasma membrane of eukaryotic cells imposes a semi permeable barrier essential for the integrity of the cell. Unlike small hydrophobic substances that can diffuse across the plasma membrane, macromolecules rely on the endocytic system to enter cells. Most of the internal-ized cargo is degraded along the endocytic pathway or recycled to the cell surface. However, a fraction of it escapes degradation and gains access to the cytosol as evidenced by the penetration of encapsulated viruses, the success of DNA and RNA transfections, or the cross-presentation of protein antigens requiring proteasomal digestion. The mechanisms by which endocytosed macromolecules can gain access to the cytosol remain unexplored, and constitute the focus of my proposed studies. I will study the mechanism of egress of endocytosed proteins to the cytosol in vitro and in vivo. To this end, I will set-up cell-based assays to detect the presence of endocytosed molecules in the cytosol of living cells. In parallel, I will use experimental animal models to identify cell types that efficiently deliver exogenous macromolecules to the cytosol, to evaluate its regulation by signaling pathways that may affect it, and to compare the effi-ciency of this process following different endocytic routes in vivo. Finally, to identify genes involved in the molecular machinery involved in the egress of endocytosed proteins to the cytosol, I will optimize the in vitro assays for large-scale RNAi screens. This work will help elucidating the molecular mechanism of egress of endocytosed proteins to the cytosol and will contribute to the formulation of novel routes of drug delivery into cells.

MicroRNAs (miRNAs), the cellular cousins of siRNAs, are post-transcriptional regulators of gene expression. In the past, much progress has been made to understand their biogenesis. Now, the biochemical diversity of small RNA-containing complexes and the biological context of miRNA-mediated regulation are important questions for the field. Drosophila miR-277 is an exceptional tool to address both of these: First, this miRNA is predominantly loaded into Ago2, the siRNA acceptor protein, which is unable to silence miRNA-type reporter genes. Since no siRNA-type targets for miR-277 exist in Drosophila, a study of miR-277 function will also lead us to novel activities of the Ago2-complex. One hypothesis is that miR-277 participates – via Ago2 - in regulation of AU-rich elements. Second, miR-277 is a non-essential gene and mutant flies are morphologically normal, thus allowing the study of miRNA function in adult physiology without major developmental defects. We will determine miR-277 target genes and the affected cellular pathways by transcriptome and proteome analysis. A combination of these results, the mRNA-to-protein ratio-changes, allows dissection of the miR-277 dependent silencing mechanisms on a genome-wide scale. Finally, we will determine what regulates miR-277 transcription and combine this with the our validated miR-277 functions to construct the miR-277 dependent control circuits. According to miRNA target predictions, mitochondrial metabolism is controlled in part by miR-277. This is consistent with our preliminary results and we hope to strengthen the concept that miRNAs contribute to the robustness of an organism towards changes in its environment.

Innate immune response is facilitated by microglia in the CNS. The microglia is not only maintains the brain's plasticity, they also protect it, as well as aid the brain to functionally recover from injuries. However, a dysfunction of microglia may promote neurodegeneration and eventually, a retraction of neuronal synapses leading to cognitive deficits. The focus of this proposal is on microglial cells and how their diversity, specifically with regard to phagocytosis and restorative or neuroprotective properties, can help us understand the development of Alzheimer’s disease(AD). By the identification of specific stages of microglial cell activation with neuroprotective or neurotoxic properties, we aim to unveil mechanisms in AD pathology that lead to impaired brain function. Three major objectives of the project are as follows: 1)Characterization of regional microglial activation markers during aging and the development of AD-like pathology. 2)Analysis of microglia effects on neurodegeneration in models of AD to identify microglia markers and. 3)Functional Effects of AD-associated microglia on neuronal survival and plasticity. In order to achieve this, we will use a carefully chosen series of unique experimental models of AD based on existing technology of advanced live imaging and molecular cell intervention. By characterizing the different types of microglial activation during the development of Alzheimer’s like pathology in an animal model, we aim to define future novel molecular targets in restorative therapeutic approaches to neurodegerative diseases