Alzheimer’s disease (AD) is one of the most rampant epidemics of the modern world. As populations grow older, the number of patients affected by AD reaches massive proportions. Despite the alarming statistics, we have a poor understanding of the pathophysiological mechanisms underlying it. The septo-hippocampal brain system is intrinsically associated with AD. Septal cholinergic neurons and rhythmic network activity degenerate and the cholinergic theory of AD pathophysiology has long been accepted as the main mechanism behind this disease. Some evidence suggests that the glutamatergic neurons of the septum are also affected by AD. However, little is known about the role of septal glutamatergic inputs to the hippocampus and their effect on hippocampal function. The aim of my research project is to investigate the glutamatergic projection from the medial septum to the hippocampus and its effect on the modulation of rhythmic electrical activity in this network. The experiments I propose will characterize the glutamatergic pathway in the septo-hippocampal system using a mouse brain slice preparation. Anatomical and biophysical knowledge from my initial results will be applied to experiments that involve a combination of cutting-edge research methods from different fields of neuroscience such as electrophysiology, two-photon uncaging of glutamate, dynamic clamp and brain modeling. This combined approach will yield crucial data to the understanding of the septo-hippocampal system and how glutamatergic septal neurons contribute to oscillatory activity in the hippocampus. My results may also contribute towards developing novel therapeutic targets for the treatment of AD.

The IkB kinase (IKK) complex is a central component of the NF-kB pathway that controls a variety of biological processes including inflammation, immunity, cell survival and tumorigenesis. The IKK complex composed of three subunits, IKKa, IKKb and IKKg/NEMO is primarily dedicated to phosphorylation-dependent degradation of IkB proteins, thereby resulting in activation of NF-kB. However, phenotypic analysis of IKKa knockout mice indicates that IKKa also has important functions that are NF-kB-independent. Moreover, preliminary results in our laboratory suggest a crosstalk between IKKa and TGFb signaling without the involvement of NF-kB. However, the molecular details and the full physiological significance of this crosstalk in skin and prostate cancer are not fully clear. I plan to investigate the relationship between IKKa and TGFb using three independent model systems. First, I will examine the genetic interaction between IKKa and TGFb signaling in the Drosophila wing system. Then, I will investigate the mechanistic details of IKKa-TGFb crosstalk in mammalian cell culture systems including primary keratinocytes and prostate epithelial cells. Finally, I will examine whether the regulation of IKKa activity by the TGFb pathway plays an important role in prostate cancer development and progression using a suitable mouse model. Since both IKKa and TGFb signaling pathway are known to enhance metastatic progression, it is likely that they work together in metastatogenesis and improved understanding of the molecular details underlying their interaction is likely to lead to new therapeutic strategies for prevention of metastatic disease, the major cause of cancer-related death.

Most phenotypic traits are the result of the complex interplay between genotype and environment. Much progress has been made in identifying sequence variation. However it remains unclear, how this genetic variation translates into phenotypic variation. More than 60 years ago, mechanisms that buffer genetic and environmental variation had been proposed theoretically (canalization). Canalization allows accumulation of cryptic genetic variation, an important source for the evolvability of novel traits. Recent work identified Hsp90 as molecular buffering mechanism that allows robust wild-type phenotypes. The molecular bases by which Hsp90 stabilizes phenotypes remains nonetheless unknown. I propose a quantitative genetics approach using natural populations of the plant Arabidopsis thaliana to determine Hsp90-buffered genetic variants and to dissect the molecular bases of trait robustness. Once Hsp90-responsive alleles are identified, I will address if and how selection acts upon these Hsp90-responsive genes. Based on recent studies, I hypothesize that other highly connected cellular machines such as the small RNA pathway and chromatin remodeling may play a similar role. I will therefore test central genes in the small RNA pathway for their ability to buffer genetic and environmental perturbation. In summary, I want to identify and dissect molecular mechanisms that allow organisms to express robust phenotypes and why in some cases genetic variation is no longer buffered and gives rise to phenotypic diversity. My results will improve our understanding of how genetic and phenotypic variation evolves.

One major determinant of cell fate are patterns of chromatinization of genetic loci that are established during differentiation and that limit the possible outcomes of general signaling events to the activation of lineage-specific genes. We recently showed that an epigenetic switch controlled by subunit exchange within a neural, ATP-dependent SWI/SNF-like nBAF chromatin remodeling complex is deterministic for the transition from proliferating stem/progenitor cells to post-mitotic neurons. Despite their essential role in neural development, very little is known on the importance of chromatin-based epigenetic mechanisms in normal and leukemic hemopoiesis. The main objectives of this research program are to define the roles and regulatory mechanisms of a novel family of hemopoietic SWI/SNF-like hBAF ATP-dependent chromatin remodeling complexes in the developing hemopoietic system and in leukemia. In aim 1, we will use state-of-the-art proteomics technologies to define the subunit composition and post-translational modifications of hBAF complexes in normal hemopoietic and leukemic stem cells. In aim 2, we will use molecular genetics approaches to address the potential roles of the stem cell-specific BAF45a and BAF53a subunits of these complexes (and eventually other novel hBAF subunits identified in Aim #1) in regulating the self-renewal/proliferation, survival and differentiation of normal hemopoietic and leukemic stem cells. Together, these approaches will provide fundamental insights concerning the role of chromatin-based epigenetic mechanisms in normal and leukemic hemopoiesis and help build my career as an independent investigator.

In living cells almost all transfers of mass (e.g. chemical reactions), information or energy involve protein-protein interactions (PPIs). For this reason, understanding PPIs is key to comprehending all biological processes. Despite the importance of PPIs, the mechanisms by which they are created during the course of evolution are still poorly understood. An estimated 1,600 to 16,000 PPIs have been re-wired between human and chimpanzee. This high turnover of PPIs implies a high evolvability, i.e., that the number of mutations needed to create a new PPI is low. I would like to investigate this issue and quantify in molecular terms how easy it is to create a new PPI. To this end I will use in vivo directed evolution to engineer new PPIs, and dissect and rationalize the sets of mutations found to trigger new interactions. This will allow me to estimate several parameters that describe the evolvability of PPIs, and determine how these parameters depend on certain factors such as protein family, sequence similarity to a known binder, or functional constraints. This project will provide an estimate of the basal “stickiness” (i.e., the intrinsic propensity to interact weakly and non-specifically) of proteins for each other, and the rate at which the strength and specificity of this basal stickiness can be improved by introducing mutations. This knowledge is fundamental to a better understanding of the principles of formation and evolution of PPIs, and for finding and designing drugs that modulate them. In addition, this project will provide a novel strategy to engineer PPIs in vivo, which has implications to protein design as well as nanotechnology.

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.