Gene expression regulation occurs through a series of tightly regulated steps. To ensure a fast response to external stimuli, cells use many post-transcriptional regulation checkpoints that affect the metabolism of mRNA, from its transcription to its degradation. The spatiotemporal aspects of these levels of regulation are limited so far by the fact that we cannot follow single mRNA particles (mRNPs) over timescales comparable to their lifetimes. Here I propose to specifically label mRNA molecules with quantum dots, which are bright and photostable fluorescent dyes. Using this method, I will track single mRNPs over their lifetime, with high resolution. By using the expression of endogenous beta-actin gene as a model, I will first investigate the kinetics of mRNA transcription. I will measure transcription rates and assess their variability over time in a cell population. This will provide fundamental insight into transcription noise and its contribution to gene expression variability. I will then track single mRNPs as they travel through the cytoplasm, are translated and finally get degraded. By simultaneously imaging mRNPs, the relevant organelles (P-bodies where mRNA decay likely occurs) and protein synthesis, I will analyze the pathways of mRNPs in the cytoplasm and provide quantitative kinetic information for each phase of the ‘mRNA cycle’. I will then be able to quantitatively assess the effects of various factors. I will study how localizing sequences on the mRNA and binding proteins direct the dynamics of mRNPs. I will then investigate the pathways and kinetics of decay and translation inhibition mediated by small interfering RNAs or micro RNAs.

Telomerase, by preserving telomere integrity, determines the proliferative capacity of cells. Consistent with this role, telomerase is upregulated in highly proliferating cells such as cancer cells and embryonic stem cells. Beyond telomere maintenance, telomerase has also been implicated in transcriptional regulation, DNA repair, protection against apoptosis and proliferation of adult stem cells. Interestingly, telomerase undergoes complex alternative splicing, resulting in at least 14 different transcript variants. Telomerase positive embryonic as well as cancerous tissues almost always co-express one or several telomerase splice variants at high levels. Because alternative splicing of telomerase is observed in distant species such as rice, human and chicken, it is suggestive that this presents an evolutionary conserved process of telomerase expression. All these observations invite the question of whether telomerase isoforms, rather than full-length telomerase, account for the observed non-telomere-synthesizing functions of telomerase. No study has addressed this possibility so far, not least because the means for detection and identification of the protein isoforms were not available. Using innovative methods to examine the currently understudied role of telomerase alternative splicing, this project has the potential to uncover novel functions of telomerase. Furthermore, it will shed light on post-transcriptional regulation of telomerase in human development and contribute to our understanding of how we age or suffer from diseases such as cancer.

Polarity is a vital property of cells. In development, cell polarisation is tightly coordinated with cell cycle progression to generate cell diversity, but the molecular mechanisms remain largely unknown. The family of Dual-specificity tYrosine phosphorylation-Regulated Kinases (DYRK) may function at the interface between cell polarisation and proliferation: DYRKs mediate the survival of tumour cells, and are also essential for cell polarisation in multiple organisms. Interestingly, the human DYRK1A gene is localised in the Down’s syndrome critical chromosomal region and its overexpression contributes to Down’s syndrome features. I propose to use the powerful experimental tools of the fission yeast model system to study the regulation and function of this kinase family. Fission yeast cells are rod-shaped cells that grow by extension at cell ends and divide by medial fission. The DYRK pom1p localises to cell ends, and is required for correct cell morphology, for the medial positioning of the division site and for cell cycle control. Using complementary approaches of chemical genetics, live cell imaging and biochemistry, we will 1) investigate the molecular mechanisms of pom1p localisation to cell ends, 2) dissect how pom1p regulates cell cycle progression and 3) identify direct pom1p substrates. I expect the results of this work to provide an integrated insight into the role of a DYRK within a single cell and establish novel molecular links between cell polarisation and cell proliferation. The HFSP CDA will be essential to establish myself as an independent researcher, as it will provide both international recognition and financial support to develop my research programme.

B cells constitute an essential part of the adaptive immune system by producing antibodies, which neutralize toxins and infected or malignant cells. B cells circulate in peripheral blood and concentrate in secondary lymphoid organs, where they migrate and search for antigens. Upon antigen recognition the cells rapidly stop and lose their ameboid shape, quickly spreading and then slowly contracting over the surface of antigen presenting cells. Concomitantly with this response, B cells completely reorganize their cell surface receptors giving rise to the immunological synapse (IS), which is a central step in B cell activation. Thus, understanding B cell migration and IS formation at the molecular level is essential to comprehend the adaptive immune response, of which very little is currently known. Here, we propose to identify novel genes that are critical for the regulation of B cell morphology. We will address this with a genome wide RNAi screen. The key candidates will be subsequently functionally characterized by cutting edge imaging techniques, such as TIRF and confocal microscopy, both in vitro and in vivo (intravital two-photon imaging). Antigen stimulation will be assessed in a highly controlled manner using synthetic lipid bilayers which, together with the imaging techniques, are readily available in the host laboratory. Furthermore, our studies will be boosted by the use of genetics to create B cells deficient in key candidates. Understanding the basic mechanisms of B cell migration and membrane antigen recognition is of great importance, not only for understanding the protective immunity of our bodies, but also for designing better antibodies for therapeutic use.

The host laboratory has shown the Notch1 gene to be mutated in over 50% of T-cell acute lymphoblastic leukemia (T-ALL) patients, and that inhibition of Notch signaling in established tumor cells impairs their proliferation and can induce apoptosis. Despite these results, it remains to be determined the role Notch plays in tumor initiation. Although mouse models of Notch signaling-induced T-ALL were first established over a decade ago, the cellular target and molecular mechanisms underlying oncogenic transformation in this disease have yet to be defined. We propose to identify which cellular subsets are susceptible to Notch-induced transformation. In defining the relevant cellular context(s) for transformation, we will be able to investigate critical downstream molecular events. We will employ genome scale approaches to profile these molecular events initially, with subsequent validation by traditional methods. Our mouse studies will make use of genetic conditional alleles such that we can exert exquisite control over Notch signaling. We will complement these studies with similar experiments employing primary human hematopoietic progenitor cells in a NOD/SCID xenograft model. Taken together, these studies will significantly advance our understanding of the initial steps required for T-ALL induction and lend critical insight into the biology of leukemia stem cells.

Autism is a common neurodevelopmental disorder characterized by abnormalities in social, communicative, and behavioral functioning. Although the etiology of autism remains largely unknown, cytogenetic and genetic studies have suggested that Autism may be influenced by genomic imprinting of 15q11-13, through copy number gains of 15q11-13 occurring in around 1-3% of autism cases. This proposal will focus on the homologous pairing of 15q11-13 in human neuronal cells, previously observed by the host supervisor Dr. Janine M. LaSalle. In addition, Dr. LaSalle observed that homologous pairing of 15q11-13 was deficient in RTT, AS, and autism brain. To model 15q11-13 maternal duplication in a neuronal cell line, I will develop a systematic in vitro approach for studying the epigenetic consequences of cytogenetic disruptions to 15q11-13 using monochromosomal hybrids containing individual human chromosomes 15 of defined parental origin with and without targeted deletions of regulatory sequences. Modified paternal or maternal copy of human chromosome 15 will be transferred into SH-SY5Y cells by microcell fusion. Then, FISH analysis will be performed to compare how the homologous alleles of 15q11-13 are organized in human neuronal cells with and without modified human chromosome 15 copy number or sequence changes. Thus, the short-term fellowship from HFSP will facilitate an important collaboration with Dr. LaSalle who has developed novel methods for measuring and quantiating FISH signals in neuronal nuclei. Moreover, this collaborated research will undoubtedly shed much additional light on the molecular mechanisms of Autism and RTT and provide an important research tool for the field.

Recent studies based on McDonald-Kreitman (MK) tests have indicated that adaptations occur much more frequently during evolution than previously thought. However, MK-based approaches can give substantially misleading results under realistic demographic scenarios such as recent population expansions. In addition, these approaches can only be used to infer the rate of adaptation, but not its strength. Spatial correlations in neutral polymorphism and functional divergence along the genome in principle contain information necessary to infer the properties of the adaptive process in much finer detail. This is because if adaptations are really that common and, in addition, if some of them provide substantial fitness advantage, the resulting recurrent selective sweeps should leave very specific signatures in the spatial distribution of polymorphisms along the genome. These data are only now becoming available and very few methods for harnessing it exist so far. Here, we propose to develop a coalescent simulation of the genome-wide patterns of polymorphism that incorporates recurrent selective sweeps, genetic recombination, genetic drift and common demographic scenarios to quantitatively investigate the relationships between various evolutionary parameters and the spatial statistics of selective sweeps. We will use this simulation to evaluate the predictive power of current methods and to design new methods for inferring characteristics of the adaptive process under realistic evolutionary and demographic scenarios. By applying this method to various sources of newly available population genetics data we hope to help elucidate the parameters of adaptation on a genome-wide scale.

Embodiment theories suggest a reciprocal relationship between bodily expression and the way in which emotions are processed. In line with this notion a number of studies have shown, that depressive states resonate with the muscular-sceletal system. For example various studies evidence that currently depressed patients show specific gait characteristics (e.g., reduced gait velocity, stride length, increased standing phase and gait cycle duration). In the proposed research we plan to give currently depressed individuals online feedback on their gait characteristics. Acquisition of motion data will be done at the BioMotionLab at Queen’s University, a facility with ample experience in motion-capture technology. We will be able to analyze a patient’s gait in real-time and then to give them visual feedback, indicating the appearance of their gait. We conjecture that giving currently depressed individuals feedback about the way they walk and to assist them to walk in a more healthy style could de-escalate pathological configurations between body, cognition and emotion. After the feedback has been given to the participants an emotional stroop test with negative words will be administered to measure the effects of gait feedback on cognitive processing of emotional information. Additionally, effects of feedback on mood ratings and automatic negative thoughts will be assessed. The stay at the BioMotionLab will enable me do learn a very innovative approach to analyze gait patterns and to give feedback on gait characteristics. This might help me to build up my own BioMotionLab in the future where I might be able to conduct extensive research on embodiment in clinical psychology.

Clathrin-mediated endocytosis is the process by which cells capture molecules from the extracellular environment and package them into vesicles in the cytosol. The molecular mechanism of endocytosis is highly conserved among eukaryotes, and involves more than 60 endocytic proteins. I propose to examine with the model organism Saccharomyces cerevisiae the mechanism by which actin-dependant plasma membrane invagination is achieved. One protein module, the WASP/WIP/myosin module, seems to play a central role in this process, by regulating and organizing the actin cytoskeleton at the plasma membrane to generate forces. I propose to use an interdisciplinary approach that will combine cell biology, yeast genetics and actin biochemistry to: 1/ establish the mechanism by which the proteins of the module are successively recruited at the membrane 2/ measure how activities of these proteins are coordinated to trigger actin polymerization and 3/ understand the key role of type I myosins in driving membrane invagination. The ultimate goal of this project is to reconstitute membrane invagination in vitro, which will involve progressively assembling proteins from this module on artificial liposomes and determining how they induce membrane deformation, in the absence or in the presence of actin. This project will represent a change in research directions for me, from the field of actin biophysics and single molecule imaging, to cell biology and live-cell imaging. In my future professional career, I hope to be able to combine these approaches simultaneously to address complex biological problems.

Centrosomes are the primary microtubule organizing centers of animal cells and consist of a pair of centrioles surrounded by pericentriolar-material. Centrosomes are crucial for many fundamental processes, including cell polarity, nuclear positioning, spindle assembly, and cell migration. Thus, centrosome number must strictly be regulated both in somatic cells and in the germline. In proliferating cells, a single centrosome duplicates to give rise to two units that are inherited by daughter cells. During gametogenesis one of the parental centrosomes must be eliminated, so that only one centrosome is inherited by the zygote. In most metazoans centrioles are eliminated in the female germline, while they are maintained during spermatogenesis and are thus contributed strictly paternally to the zygote. The molecular basis underlying the differential inheritance of centrioles in the female and the male germline is not understood. My goal is to take advantage of the unique tools developed in C.elegans to study centrosome biogenesis and investigate the mechanisms regulating centriole inheritance in the germline. I propose to pursue three complementary approaches to dissect this process: 1.Analyze the mode, timing and candidate factors affecting centriole elimination during oogenesis 2. Characterize sas-1, a mutant affecting centriole maintenance during spermatogenesis 3.Conduct a functional genomic screen to identify novel genes inducing centriole elimination in the female germline.

Understanding the effect of spatial confinement on protein folding is directly relevant to biological processes such as folding in a chaperonin cavity or in a ribosome tunnel. In addition, macromolecular "crowding" effects in the cellular environment can significantly alter folding from what is observed in vitro. In certain limits, the protein behavior in such a crowded environment can be equivalently modeled as protein localization in an effective confining boundary. Although several previous studies have provided important insights into this problem, a quantitative understanding of the shift in folding / unfolding thermodynamics and kinetics as a function of the confinement characteristics such as dimensionality and size of the cavity and its interactions with the protein molecules and under different crowding conditions is lacking. Our goal in this project is twofold, (i) elucidate the effect of confinement on protein folding transition states and folding rates as a function of confinement size and interactions between the protein atoms and the confining walls and (ii) projection of the dynamics of our multi-dimensional systems onto suitable low-dimensional reaction coordinates (RCs) in order to understand better the underlying mechanism. To accomplish these goals, we plan to utilize a coarse-grained description of proteins which has already been found to be useful to describe several aspects of protein folding mechanisms. The successful outcome of this research will contribute towards our understanding of protein folding, association and aggregation under “in vivo” like conditions.

The synapse is a specialized cell-cell contact site between neurons or neurons and muscles, and precision of synapse formation is critical in the formation of a neural circuit. While previous studies have identified the molecules that are sufficient for synaptogenesis, little is known about molecular mechanisms that inhibit synaptogenesis with non-target cells. To understand how neurons inhibit synaptogenesis between axons and non-target cells, I will utilize the DA9 neuron in C. elegans, in which the presynaptic terminal is restricted to the specific domain in the axon by the local activity of Wnt and its receptor Fz. As this signaling does not appear to belong to any known Wnt signaling cascade, I will identify the downstream components through characterizing mutants that have a similar phenotype to wnt mutants in terms of localization of synaptic markers. Concurrently, I will try to elucidate how Wnt signaling inhibits synaptogenesis, based on two hypotheses: 1. Wnt inhibits synapse formation 2. Wnt stimulates synapse elimination. Stage specific expression or inactivation of Wnt will reveal which step Wnt actually regulates. I also plan on examining the relationship between Wnt signaling and SCF complex (E3 ubiquitin ligase) in synapse elimination, as SCF complex is required for synapse elimination in other neurons. As Wnt signaling is essential in normal human development, and its deregulation causes various diseases including neurological disorder, understanding the molecular mechanisms of inhibition of synaptogenesis by the novel Wnt pathway will contribute to therapeutic treatment of these diseases, as well as in understanding the development of the human brain.

Sec4p is a Rab GTPase required for transport of secretory vesicles from the Golgi to the plasma membrane in yeast. Sec4p localizes on the secretory vesicle and controls vesicle transport, vesicle tethering and vesicle fusion with plasma membrane. In each case, activation of Sec4p by its guanine nucleotide exchange factor Sec2p is necessary for the function. Ypt32p, another Rab that regulates the export of cargo from Golgi to the plasma membrane, is required to recruit Sec2p to the secretory vesicle. Thus, Sec2p links two different Rabs and constitute a Rab-GEF cascade. However, the regulation mechanism of these Rabs in the transport pathway is unclear. The host laboratory has shown that Sec2p also bind to Sec15p, a component of the exocyst complex implicated in vesicle tethering at the plasma membrane. Since Ypt32p and Sec15p bind to Sec2p in a competitive manner, the mechanism that triggers the change in Sec2p to switch from binding Ypt32p to binding Sec15p would be important in regulating the Rab-GEF cascade. Moreover, Sec2p localization relies on the production of phosphatidylinositol 4-phosphate (PI4P) in the Golgi by the phosphatidylinositol 4-kinase Pik1p. In this proposal, I will focus on the regulatory mechanism of Sec2p in the exocytic pathway. First, I plan to establish the roles of Ypt32p and Sec15p in Sec2p localization by identification of their binding sites in Sec2p. Second, I will examine the domain in Sec2p that binds PI4P and other phosphoinositides. Finally, I will test the effects of altering the subcellular phosphoinositide distribution on Sec2p localization and functions, and examine the role of Pik1p on Sec2p localization to the secretory vesicle.

Fanconi Anemia (FA) is a cancer susceptibility syndrome caused by mutations in genes encoding factors involved in replication-dependent removal of interstrand DNA crosslinks. Most FA members identified to date form a nuclear complex that ubiquitinates the proteins FANCD2 and FANCI in response to DNA damage, leading to formation of DNA repair foci. FA is thought to coordinate three different pathways: homologous recombination, nucleotide excision repair and mutagenic translesion synthesis (TLS) to remove crosslinks, but its mode of action is unclear. This research will study the FA-controlled repair process by focusing on identification and characterization of new members of the pathway. In order to understand how they promote mutagenic DNA synthesis, I will investigate if FA proteins recruit TLS enzymes directly, or otherwise I will identify the factors that connect them. For this, I will use both a proteomic approach to unravel protein complexes associated with TLS polymerases, and perform whole-genome RNAi screens to identify genes required for FA-dependent mutagenesis. Also, to study how the repair process is activated, I plan to uncover novel proteins recruited by ubiquitinated FANCD2-FANCI complexes, by purifying FANCD2 under conditions that stabilize the modified form. To assay if the candidates found by all these approaches are involved in the FA pathway, I will investigate if they exhibit the genetic and biochemical hallmarks of this pathway. This research is expected to reveal insights into the mechanism by which FA steers the repair of DNA crosslinks, and how deregulation of this essential genome maintenance mechanism leads to cellular transformation.

The transfer of mechanical energy via hollow structures is of great interest in many areas of engineering. Tubes as waveguides are matters of investigation since they are subject to many constraints, one being external acoustic radiation via the tube wall. In human hearing aids for example, acoustic radiation from tube walls causes feedback. Acoustic insects like bush-crickets detect sound with a system of paired ear drums on each foreleg, just below the knee. An air-filled tube, the acoustic trachea, delivers sound from the acoustic spiracle on the side of the body down the foreleg to the internal face of the eardrums. The wall structure of this tracheal tube is flexible and elastic, allowing sharp curvatures (produced by leg articulation) along its length. This ‘air tube’ is thus confined inside a fluid-filled chitin cylinder. The acoustic trachea of insects and human hearing aids tubing have remarkably analogous functions the former is however effective at much smaller length scales, opening up the opportunity of further miniaturisation of acoustic wave guides. This project aims at elucidating the mechanisms by which ears endowed with wave guides function to generate high sensitivity and directional accuracy. The role of accessory structures backing a conventional eardrum has been largely neglected thus far. Using mathematical modelling, experimental and theoretical data will be exploited to understand the modalities of sound propagation these auditory systems. The principles of sound propagation in biological wave guides may find direct applications for the development of novel detection technologies, notably in hearing aids and smart miniature pressure sensors.