HIV-1 and HIV-2 are closely related viruses that share up to 60% of their genetic sequence and both can cause severe immunodeficiency disease. Nonetheless, most people with HIV-2 infection will die of other cause and the virus remains relatively confined to West Africa whereas 90% of HIV-1 infected people rapidly develop AIDS and the virus has spread throughout the world. Preliminary studies suggest that HIV-2 is not attenuated compared to HIV-1, but that the immune system is more efficient in controlling the replication of HIV-2. Whilst a considerate amount of knowledge about HIV-1 infection has accumulated over the last two decades, much less is known about HIV-2 infection and in particular the reason for its reduced pathogenicity. We hope that gaining a better appreciation of the immune control of HIV-2 infection will help in our understanding of the requirements for protective immunity in HIV infection. My previous research was based on primary human deficiencies with little inference of medecine, whereas this project is aimed at understanding of the cellular immune response to HIV-1 and HIV-2 viruses, working on infected patients with their consent, in close collaboration with clinicians and epidemiologists. We think that medical knowledge can improve our comprehension of immune responses in general and the development of efficient cytotoxic responses in particular.

B-cells are the antibody-producing cells of the immune system. Their development is a complex, stepwise process. While random B-cell receptor gene re-arrangements ensure a diversified antibody repertoire, B-cells recognising self-antigens must be deleted during their development to prevent autoimmunity. This process is initiated by the interaction of the B-cell receptor (BCR) on immature B-cells with stromal cells in the bone marrow, where the recognition of self-antigen transmits a signal that induces a change of specificity (receptor editing) or the death of the B-cell. We aim to investigate how the BCR works in concert with adhesion molecules and other co-receptors during this process. Unique real-time visual systems in combination with the use of cell lines and animal models will allow to relate the dynamic re-organisation of the BCR/adhesion molecules during B-cell/stromal cell interactions to cellular responses on a single-cell level. These studies will lead to a more detailed understanding of the processes involved in B-cell development and in the prevention of autoimmunity. While I will benefit from my expertise in B-cell development, I will shift my focus to the mechanisms of B-cell mediated autoimmunity. Further, I will learn a variety of new techniques. As I am fascinated in the processes involved in conveying an extracellular signal or environmental clue into a cellular response, this work will broaden my insight and expertise in a field where I hope to establish myself as an independent research scientist in the future.

I am interested in genetic factors that mediate cellular growth and body size control. The cyclin-dependent protein kinase complex CycD/Cdk4 regulates growth through a previously unknown mechanism. In a genetic screen, I identified two proteins: mRpL7/L12 and Hif-1 prolyl hydroxylase (Hph), which function, in a common pathway, downstream CycD/Cdk4. mRpL7/L12 is a mitochondrial ribosomal protein. Hph regulates the cellular response to low oxygen (hypoxia). I showed that Hph, through hydroxylation of yet unidentified proteins, has a novel function in growth control. The screen was successful, but not designed to saturate the genome. Thus I propose to continue the screen, on a larger basis, to identify more components of this pathway. In particular, I will be focusing on new, growth-specific, hydroxylation targets of Hph. CycD/Cdk4 stimulate the metabolism, however, our understanding how the metabolism and cellular growth are linked is still limited. In a parallel approach, I propose biochemical assays and two-dimensional gels to better understand the effect of CycD/Cdk4 and Hph on the metabolism. A combination of these assays will allow a rapid characterization of the mutants isolated in the genetic screen, and will give a global view of CycD/Cdk4’s function on the metabolism. The University of Zurich offered me an ‘Oberassistenz’ position, which allows me to perform independent research. This position does not include salary and funding. Next year, I will be applying for a ‘Forderungsprofessur’ (assistant prof.) of the Swiss National Science Foundation. Therefore the CDA would allow me to carefully prepare such a position, and to gain expertise in guiding lab personal.

The tumour suppressor protein p53 induces cell cycle arrest or apoptosis of potentially malignant cells in response to oncogenic stress, preventing the malignant transformation. p53 is mutated in around 50% of human cancers. The mechanism and regulation of p53-induced apoptosis are of great importance, but are still poorly understood. Recently, the protein ASPP2 was shown to specifically stimulate the apoptotic response of p53. The research aim is to elucidate the molecular mechanism and regulation of the p53 – ASPP2 interaction. We will use an interdisciplinary approach combining protein biochemistry and biophysical techniques such as NMR, fluorimetry and calorimetry. First, we will study the kinetics and thermodynamics of the p53 – ASPP2 interaction and use NMR to study the dynamics of these proteins upon binding each other. To elucidate its mechanism of action, we will study the effect of ASPP2 on the DNA-binding of p53 core domain, and how it stimulates the transcriptional activity of p53 despite binding it in its DNA binding site. We will then study the thermodynamics, kinetics, and NMR dynamics of ASPP2 interactions with the apoptosis-related proteins Bcl-2 and NF-kB and explore the possible role of these proteins in ASPP2 regulation at the molecular level. Being a young scientist at the beginning of my independent career, the project is of vital importance for my professional growth. My research interests are the molecular mechanism and dynamics of interactions between macromolecules, and how to restore such interactions when impaired. The project will be the first application of my experimental system for a biological pathway that is crucial for the cell.

I will study the importance of Transforming Growth Factor beta (TGFb) signalling for the regulation of embryonic cell proliferation. A mutation in a receptor for drosophila Activins, the least probably least studied TGFb ligands, specifically impairs cell proliferation. I will therefore genetically analyse the importance of different Activin signalling components for the control of cell division. As excessively proliferating tumour cells present alterations in cellular organisation, I will study trafficking and organisation of the Activin signalling machinery and determine its importance for the regulation of cell proliferation. I will analyse the organisation of zebrafish Bone Morphogentic Protein signalling to determine the importance of cellular architecture for vertebrate TGFb signalling in epithelial and mesenchymal tissues. Finally, I will use drosophila and zebrafish to study the function of SARA, a TGFb signalling adapter that associates with the central spindle during mitosis, suggesting a potential link between TGFb signalling and the mitotic apparatus. By studying proliferation control, I want to tackle a major aspect of development which I have not considered previously. Training in cell biology and drosophila genetics will considerably widen my methodological playfield. The experience of this fellowship will therefore be both different from and complementary to my previous research. Beyond the period of the fellowship, the ability to take advantage of both drosophila and zebrafish, two major model organisms, and the acquired competence in cell biology will allow me to work in the exciting new field at the interface between cell and developmental biology.

Vesicle trafficking of receptors has been recognised as an integral part of signalling in animals. In plants, BRI1-receptor mediated signalling is one of the best understood to date. However, nothing is known about the interplay between signal perception and regulation of BRI1 transport. I propose to investigate BRI1 trafficking and its importance for signal transduction. BRI1-GFP is detected in root cells, well-suited for cell biological manipulations. Many non-lethal brassinosteroid (BR) mutants are available and facile Arabidopsis transformation protocols will allow me to screen for PCR-mutagenised BRI1 trafficking mutants in defined genetic backgrounds. This will be done in-planta, with proteins that are stably expressed to endogenous levels. The combination of genetic, molecular biological and cell biological tools will give new insights into brassinosteroid signalling and possibly establish Arabidopsis as an attractive model for investigation of receptor cell biology. I acquired good skills in molecular genetics and cell biology. However, I never undertook mutant screens and only occasionally utilised live-imaging with GFP-fusion proteins. Nonetheless, I have precise ideas of the advantages and limitations of using fluorescent proteins. At the Salk I will find all the necessary equipment and I will be in a group with large experience in genetic screens and BR biology. I will be able to build on my skills, but also to explore new imaging and cloning techniques and to establish new mutagenesis methods in Arabidopsis. In the future I hope to exploit this new mutagenesis approach and to further dissect the interplay of endosomal trafficking and signal transduction.

MicroRNAs are evolutionarily conserved non-coding RNAs of ~22 nucleotides (nt) that have emerged in recent years as regulators of gene expression and development. I propose to study the role of miRNAs during vertebrate morphogenesis using zebrafish as a model system. I will first take a combined biocomputing and cloning approach to identify miRNAs and their targets. Using gain- and loss-of-function approaches, I will then perform a screen to identify miRNAs that orchestrate early development and organogenesis. Finally, I will focus on a small subset of miRNAs to determine how their targets control development. During my previous research I addressed how the Wingless morphogen gradient is established and how it regulates tissue growth, using Drosophila as a model system. This project provided new insights into the regulation of signaling pathways during development and gave me a thorough training in Drosophila developmental genetics and signaling. My proposed postdoctoral project shifts my focus to the study of RNA regulation during zebrafish development. Hence, I will change both my area of research and the model system of choice. My proposed project will be a new, independent line of research in Dr. Schier's laboratory and employ a novel combination of molecular biology, in vivo imaging, phenotypic characterization and biocomputing approaches. The expertise in vertebrate developmental genetics and zebrafish biology in Dr. Schier’s laboratory will provide an excellent training and research environment. A HFSP fellowship would allow me to broaden my scientific training and establish an independent line of research that will form the basis of my future laboratory.

1) The nuclear envelope (NE) serves to compartmentalize the nucleus and cytoplasm of higher eukaryotic cells. In multicellular organisms the stable NE is dynamically rearranged during cell division. Since the precise molecular mechanisms of NE reformation at the end of mitosis are still unknown, identification of novel proteins involved in this process may provide us with valuable information on how it takes place. Caenorhabditis elegans is a powerful multicellular experimental system to study such phenomena. By entire Caenorhabditis genomic RNA-mediated interference (RNAi) screen several novel genes with possible function in NE assembly have been identified. I plan to determine, which of these new candidate genes have real function in NE assembly, and then characterize the selected genes using C. elegans embryos as in vivo and Xenopus egg extract as in vitro system. Combination of the results got with the above-mentioned experimental systems might be of great potential in describing the molecular mechanism of NE assembly. 2) In the proposed research work I intend to use new in vivo (C. elegans) and in vitro (Xenopus egg extract) experimental systems, which differ from the system (Drosophila) that I have used until now. Furthermore, I also plan to use many modern biochemical techniques and approaches, which are new for me. 3) Experiences reached with the proposed research work as well as with the mental training in the outstanding laboratory of Prof. Mattaj, together with my previous research experiences, will broaden my intellectual horizon and help me to be prepared for new challenges and to solve the critical scientific questions in the future.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one pathway of the ubiquitin (Ub) system, which was extensively studied by the Varshavsky laboratory over more than two decades. I propose to carry out experiments whose chief subject are the Ub ligases (E3 enzymes) of the UBR family that mediate the N-end rule pathway in both the yeast Saccharomyces cerevisiae and the mouse. The aims are to identify new physiological substrates of this pathway and to dissect its regulation in yeast. The understanding thus obtained will be relevant not only to this pathway but also to the Ub system at large. My graduate research comprised an extensive study of metabolic pathways using biochemical approaches. In my postdoctoral work, I intend to complement this experience by learning, in the Varshavsky lab, the techniques and approaches of molecular genetics as applied to the N-end rule pathway, a universally present proteolytic system. Working with yeast and mammalian cell cultures is also new to me. The proposed studies will, therefore, allow me to broaden my knowledge of eukaryotic biochemistry and genetics. In sum, the proposed research fundamentally differs from my graduate research experience. Carrying out the biochemical and genetic dissection of the N-end rule pathway, and studying the targeting of protein substrates to the proteasome will expose me to a broad range of methods and concepts that I did not encounter, thus far, in my graduate research, which was focused on protein physical chemistry and enzymology. The proposed projects are therefore crucial to my further development as a scientist.

Recent studies reveal that mammalian stem cells (SCs) are more versatile in choosing fates than previously appreciated. This property holds tremendous promise, particularly for less controversial adult SCs, in tissue regeneration. Challenges that remain unsolved include the relation between adults and their embryonic counterparts, and the mechanisms regulating renewal and lineage determination. Skin epithelium provides an excellent model for investigating these problems, since it can be maintained and propagated in culture. My research focuses on isolating and characterizing SCs, enabling me to explore how self-renewal is achieved and to identify mechanisms involved in regulating lineages. After isolating multipotent epithelial SCs and their committed progeny from mouse skin, I will compare their programs of mRNA expression, placing emphasis on known markers of hematopoietic and other SCs, including cell cycle and chromatin structure genes that might provide clues to the slow-cycling properties of SCs. Real time PCR, subsequent in situ and immunofluorescence will reveal which markers are expressed in the skin niche, and how these patterns change with commitment. The most interesting of these genes will be explored by RNAi in vitro and in vivo first and eventually by transgenic technology. My prior training has focused on cell-cell communication in a tissue in Drosophila. Through my post-doc, I hope to understand the complex mechanisms controlling SC self renewal and fate decisions in skin, and similarity between adult and embryonic SCs. This training will be important for my career goals as a professor of Cell and Developmental biology focusing on SC research.

SERA5 is emerging as a key protein in the growth of the blood-stage cycle of the human malaria parasite P. falciparum, but its precise biological function remains unknown. Current evidence implicates a role for SERA5 most likely in the rupture or invason of infected red blood cells. In this project I aim to determine the function of SERA5 and in doing so establish a powerful new genetic technology of broad applicability to the malaria field. I propose to delete the gene encoding SERA5 using a novel conditional knockout approach, which will provide insight into its function and its relationship to downstream cellular pathways and processes. Furthermore, I plan to determine the localization of SERA5 and its downstream substrates, which will help unravel its role in the rupture/invasion pathway. Ultimately, understanding invasion will aid in the discovery of new ways to therapeutically intervene and prevent malaria. To achieve the goals of this proposal, I will study the key molecular pathways and interactions that a protease must make in the process of malarial rupture/invasion. My prior work involved the development of chemical probes and inhibitors to understand the overall function of a protease, but not the molecular details. To accomplish these new goals I will need to learn the combined use of a host of genetic and molecular biology tools with cell biology and proteomics analysis. This fellowship will allow me to add powerful genetics techniques to my skill set as well as a deeper understanding of malaria biology.

In mammals, dosage compensation of X-linked genes between sexes is achieved by X-chromosome inactivation in female cells. The Xist gene, which encodes a non-coding RNA is located on the X-chromosome and is required and sufficient for X-inactivation. At the onset of X-inactivation Xist expression is upregulated on the future inactive X-chromosome (Xi), and the RNA associates with chromatin to coat the entire chromosome in cis. The spread of Xist RNA along the chromosome correlates closely with silencing of the inactive X-chromosome. Although the mechanisms by which Xist RNA orchestrates X-inactivation are largely unknown, it has been demonstrated that the chromatin of the Xi accumulates epigenetic modifications upon transition through the stages of X-inactivation. Xist RNA could act as a scaffold that directs chromatin-modifying activities in a defined spatio-temporal manner to mediate the ordered series of epigenetic modifications required for the progressive hetero-chromatinization of the Xi. These large scale and uniform changes of histone modifications on the Xi provide a powerful system to study facultative heterochromatin formation. Here, I propose to study different stages of the X-inactivation process, including the mechanisms of choice, enactment and maintenance of X-inactivation. These studies will help to understand how Xist RNA spreads along the Xi, and how Xist RNA mediates silencing, elucidating the mechanism of X-inactivation and obtaining a more complete picture of the interplay between RNA and chromatin modifiers and hence provide insight into the epigenetic regulation of gene expression in mammalian cells.