Both microRNAs (miRNAs) and small interfering RNAs (siRNAs), distinct classes of 21–23 nt guides for the RNA Induced Silencing Complex (RISC), silence the expression of target mRNAs. In Drosophila, miRNAs and siRNAs are sorted according to their structures into different Argonaute (Ago) protein effector complexes. siRNAs are loaded into Ago2-RISC, which cleaves target mRNAs with near perfect complementarity to the RNA guide miRNAs are loaded into Ago1-RISC and regulate target RNAs only partially complementary to the miRNA guide. In animals, most miRNAs silence such targets by accelerating decay of the mRNA target or repressing its translation. Recent data suggest that the Dcr-2/R2D2 heterodimer sorts siRNAs into Ago2 complexes, but the proteins that load miRNAs into Ago1 remain to be identified. The evolutionary homolog of Drosophila Ago1 in humans is called Ago2. Thus, identifying the Ago1 loading machinery in flies should help explain how human RISC is assembled. Here, I propose experiments to identify the fly miRNA loading and sorting machinery and to investigate the molecular basis for its recruiting miRNAs but not siRNAs to Ago1-RISC. I will use biochemical and molecular methods in cultured Drosophila cells and cell extracts to identify miRNA-binding proteins and test their ability to load miRNAs into Ago1. Using genomic methods, I will test the prediction that Ago2-RISC is highly specific, regulating only highly complementary target RNAs, whereas Ago1-RISC is promiscuous, capable of regulating many, partially complementary mRNAs. Finally, I will test if small RNAs in cultured human cells are also differentially sorted among the four human Ago proteins.

Recent advances in algorithms and hardware mean that membrane protein simulations are rapidly approaching physiological timescales (> microsecond), enabling fully converged protein folding simulations. However, these studies have exposed striking differences between various simulation methods, some of which contradict currently established partitioning theory. Unfortunately, there is no suitable experimental dataset to compare to. This means parameters and/or partitioning theory cannot be verified and adjusted, making lipid-bilayer simulations unreliable at long timescales. I propose to address this problem by experimental measurement of the secondary structure and membrane partitioning properties of a simple peptide test-system as a function of temperature. The synthetic WALP peptide is ideal since it serves as a model trans-membrane peptide and can also be efficiently folded into membranes in the computer. The structure of the peptide embedded in the lipid bilayer of vesicles will be explored using both using circular dichroism and Fourier transform infrared spectroscopy. The orientation of the peptide with respect to the membrane normal can be deduced from measurements in oriented bilayers on glass plates. Proton deuterium exchange measurements in combination with mass spectrometry will be used as a control and also provide an efficient way to determine if the peptide is not inserted, or inserted in a unfolded form. After completion of the project simulations will be performed under conditions identical to the experiment. This will allow for very detailed calibration of lipid parameters and simulation protocols on the microsecond timescale.

Parasitism and disease are powerful determinants of host ecology and evolution, but the relative importance of different modes of parasite-mediated selection is unclear. Like all cases of Darwinian selection, parasite-mediated selection leaves behind distinct patterns of genetic diversity. By analyzing these patterns, signatures of positive, negative, or balancing selection can be identified for specific host alleles, such as those involved in immunity to parasites. While genome-wide surveys of selection exist for a few species, fewer studies have explored the mode of selection acting on innate immune genes in naturally occurring vertebrate host-parasite systems. In this project I will analyze the mode of natural selection on the Toll-like receptor 1 (TLR1) gene in wild rodents. TLR1 has a cell surface receptor domain known to be involved in pathogen recognition in vertebrates, so is a direct target of pathogen-mediated selection and an obvious candidate for studies of parasite-mediated selection. The study populations are found in Northern and Southern Sweden, and are exposed to numerous pathogens, including Borrelia sp., the causative agent of Lyme disease. The host institution provides the unique opportunity to study a naturally occurring vertebrate host-pathogen interaction, and possesses the technical and intellectual expertise that will aid the project’s success. This fellowship will give me with the opportunity to expand my current research on host-parasite coevolution, and acquire new molecular and bioinformatics skills, while nurturing new collaborations.

The hand is a unique product of evolution. Many features of the central and peripheral control of the human hand are poorly understood, especially as most hand and finger movements are complex and use many muscles. It is not known how this activation of multiple muscles is coordinated by the brain. Recent studies suggest that descending pathways from the brain might diverge to supply the motoneurons innervating different muscles with common input and thereby underpin the coordinated activation of the participating muscles (Hockensmith et al 2005; McIsaac & Fuglevand 2008). Such common input is indicated by the presence of short-term synchronization in the firing times of motoneurons supplying different muscles. The proposed research will examine whether short-term synchronization is involved in the cortical coordination of complex finger movements that require the activation of multiple muscles simultaneously, and then especially the synchronization between motor units of intrinsic and extrinsic hand muscles. This will give insight into how the brain coordinates simultaneously the multiple muscles that allow human manual dexterity.
Furthermore, it will allow me to work with a distinguished scientist in a leading laboratory, to learn new techniques, and thus broaden my scientific horizon. This will benefit my long-term scientific career.

In recent years it was shown that a significant variability exists in the behaviour of genetically identical cells. This is due to the random nature of the chemical reactions inside the cell. Such ‘molecular noise’ is often studied in the context of gene expression. However, the impact of molecular noise on systems based on protein-protein interactions, such as signalling pathways, has remained unexplored. We propose to study the effect of noise on a well-studied signalling pathway: the High Osmolarity Glycerol (HOG) response in the budding yeast S. cerevisae. When S. cerevisae senses high external osmolarity it responds by accumulating glycerol in the cell to relieve the pressure difference across the membrane. The HOG pathway consists of at least two osmosensing receptors that input to a mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade outputs to Hog1, a transcription factor that translocates to the nucleus upon activation. We will study the variability in timing and magnitude of the HOG response in single cells by monitoring the nuclear accumulation of a Hog1-YFP fusion protein, using microfluidic techniques to deliver controlled osmotic shocks. In addition, we will systematically change the concentration of components of the HOG pathway by putting them under inducible control. In this way, we hope to clarify the role of ‘extrinsic noise’, due to fluctuations in the expression of the different HOG components, and ‘intrinsic noise’, due to the random dynamics within the HOG pathway. Since elements of the HOG response, such as the MAPK cascade, are evolutionary conserved from yeast to mammals our results will likely be widely applicable.

The human brain adapts rapidly to drugs of abuse like cocaine. Humans can become either tolerant of, or sensitive to cocaine depending on the pattern of exposure, and these neuroadaptive responses contribute to drug dependence and addiction. Presently, the molecular events underlying drug tolerance and sensitisation are poorly understood. The aim of this project is to examine changes in neurochemistry in the honey bee brain associated with cocaine tolerance and sensitisation. This project will examine mechanistic similarities between honey bee and human brains, and also determine the utility of the honey bee as a simple model system with which to explore brain adaptation to cocaine. I will treat free-flying bees with controlled doses of cocaine, and use behavioural assays to measure the onset of tolerance and sensitisation. Then I will use high pressure liquid chromatography to compare the levels of biogenic amines in different regions of the brains of cocaine tolerant and sensitive bees compared to controls. This work will reveal which biogenic amine systems, and which brain regions, respond most strongly to cocaine in honey bees. The project will be a significant and synergistic development of my research interests. I have an established interest in dopamine as a modulator of natural behaviour. Through this collaboration I will learn the neurobiology of drugs of abuse, techniques for non-invasive pharmacololgical treatment of free-flying bees, and how to measure levels of the biogenic amines in dissected regions of individual bee brains. These techniques and new knowledge will enhance my research capabilities, and further my career as a comparative neurobiologist.

Genomic instability is a hallmark of cancer cells. It has been recently reported that the DNA damage checkpoint is constitutively activated in human precancerous lesions. Interestingly, precancerous cells also express common fragile sites, which are regions of the genome that frequently break as a consequence of a replication stress. The nature of the replication stress that accounts for this genomic instability is not known. The aim of this research project is to characterize the genomic instability occurring spontaneously in precancerous lesions. My work will consist in the characterization of these replication defects which are source of checkpoint activation and the identification of their genomic localization. We will first determine the nature of the DNA replication stress and its consequences on genomic instability. We will focus on common fragile sites, which are unstable in precancerous lesions. We will also focus on the rDNA array, which is to date the only known region of the human genome containing replication-impeding sequences. This work will notably be done by analyzing the replication dynamics of individual chromosomes by DNA molecular combing. Then, we will search for and extend our analysis to other replication pause sites over the genome. Finally, we will check whether the replication fork arrest at natural pause sites is a regulated process, in human cells at a precancerous stage. Indeed, I am convinced that a better understanding of cancer-associated cell cycle defects will permit the development of more efficient therapies against cancer.

Multiple sources of sensory information can be used to define the spatial locations of targets for arm reaching movements. Reaching toward an object in space requires a transformation of visual, auditory, and proprioceptive signals into a motor plan suitable to drive the arm. To carry out these sensorimotor transformations and specify the desired displacement, the brain must integrate sensory signals related to the target and reaching hand positions. Whereas the fundamental relevance of multisensory processing is becoming increasingly clear, the neural mechanisms underlying these cross-modal interactions and the spatial-aspects of sensorimotor transformations, primarily the frames of reference component, are not well understood. Here we will examine reach performance of patients with parietal damage to determine whether planning of reach movements to visual and auditory targets use a common cortical network to transform these signals into motor commands. The outcome of this process is fundamental to perception, cognitive processing, and the control of action. This is an essential step in establishing collaboration with leading authorities, Drs. Yves Rossetti & Laure Pisella, in multimodal sensorimotor integration and human motor control and the achievement of my principal career goal, which is to operate a human motor control laboratory in Canada and institute a research program that is highly interdisciplinary in nature - combining neuropsychological patient investigations, innovative modern techniques to measure or evoke functional activity in the human brain, and traditional cognitive and psychophysical methodologies.

I am interested in determining the molecular role of RecQ type helicases at the replication fork. During replication, the DNA polymerase encounters obstacles such as DNA lesions and areas of strong secondary structure, which cause the replication fork to stall. The cellular response to stalling consists of triggering a repair response and activation of the cell cycle checkpoint. The absence of either process facilitates genomic instability. The maintenance of stalled replication forks depends on the presence of RecQ helicases. RecQ proteins are intricately connected to the cell cycle checkpoint through binding the single-strand binding protein RPA (the yeast RFA homologue). RPA is also an essential subunit for the S-phase checkpoint kinase and is part of the DNA synthesis machinery required for replication restart. Specific goals include: 1. Determine the importance of the phosphorylation through Mec1/ATR kinase on either RPA or RecQ helicases. Does modification enhance function of the helicase at the fork? 2. Determine the structure of Sgs1-RFA1 complex, and that of the WRN helicase bound to DNA. The structure of these proteins is an important finding currently lacking in the field. Together with the biochemical approach I plan to carry out, these studies will provide much needed insights into structure and function of RecQ helicases at the DNA replication fork and in their role in safeguarding genomic integrity.

The possibility of cell regeneration in the human brain was thought impossible, until the discovery of two small niches of neurogenic stem cells in the human brain that continue to generate new neurons throughout life. Stem cells in the subventricular zone normally migrate towards the olfactory bulb and become olfactory neurons, but multiple studies in rodents have shown that these cells have the potential to migrate towards areas of CNS pathology.

My recent research emphasis has shifted to the analysis of transcriptional control of gene expression by nearby regulatory elements and to a recognition of the role of long-range interactions and three-dimensional organization of chromatin within the nucleus. Insulators are now known as a major endogenous mechanism for epigenetic control of gene expression. Although many recent studies have been devoted to the identification and characterization of the CTCF-dependent insulators, there is still little information and mechanistic understanding of insulators distinct from CTCF. Arylsulfatase insulator (ArsI) is distinct from the CTCF family of insulators and is known to work in various animals from humans to plants. Unlike CTCF-dependent insulators, the ArsI sequence does not have CTCF-binding sites and can work independent of nuclear matrix. ArsI insulator is also found well conserved in sea urchins and there were over 100 putative sites for this fragment in the genome, implying the significance of this DNA fragment. Using biochemical and computational approaches, I would like to identify the proteins that interact with ArsI and reveal the mechanisms of ArsI regulation in vivo. I also expect the study on ArsI mechanisms may lead us to find novel mechanisms of gene regulations, chromatin modifications that are conserved in all organisms.

The main goal of this study is to understand how taste information is represented by primary receptor cells, and how it is transformed in subsequent brain areas. Previous studies in other sensory modalities showed that the sensory stimuli are represented differently in higher brain areas than in receptor neurons. It is likely that analogous transformations of sensory information may also occur in the gustatory system. We use Drosophila as a model for investigating gustatory coding. The molecular organization of the gustatory system in flies is remarkably similar to vertebrates. However, the fly brain is simpler and more genetically tractable than the vertebrate brain, yet can generate complex chemosensory behaviors. This project has 4 aims: 1) To develop a method for separating the taste responses of gustatory receptor neurons (GRNs) and to determine their molecular receptive fields. 2) To identify the second-order gustatory neurons in the subesophageal ganglion (SOG). 3) To determine the molecular receptive fields of SOG neurons and the connectivity between GRNs and SOG neurons. 4) To determine the spatial organization of taste responses in SOG neurons and its importance for gustatory coding. This project will increase our knowledge of how taste stimuli are encoded in the brain and is likely to contribute to our general understanding of neural coding and sensory transformation. Finally, this work should generate new strategies for combining systems neurophysiology with functional imaging and emerging genetic tools, with applications in the investigation of other complex neural systems.

Recent studies highlight the importance of nano structure morphology, shape, and order in the biological context. It was demonstrated that aligned nanostructures can serve as signaling factors for cell guidance, enhance nerve regeneration, and affect the extra cellular matrix (ECM) production of human ligament fibroblast. By utilization of bottom-up nanomaterial capabilities and assembly methods we will form new synthetic model systems for the study of biological interactions at nanoscale interfaces. We will focus on Matrix metalloproteinases (MMPs) that are a class of metalloproteins involved in tissue organization, degradation of the ECM, including collagens, elastins, and gelatin. MMPs are normally regulated by hormones, growth factors, and cytokines. Substantial knowledge has been gained regarding the biochemical factors that are involved in the regulation of MMP activity. However, until recently the ability to systematically study the morphological contributions at the nanoscale to protein function has been limited. This is highly desirable for MMPs in particular as their activity milieu is composed of nanoscale fibrous structures. Among the unique properties of these model systems is the introduction of nanostructured morphologies as structural cues in addition to the chemical and physical parameters. The relationships between the activity, scale, structure, and physical parameters will be studied in a systematic manner in order to develop molecular level understanding nanoscale effects on biological systems. These capabilities will be eventually applied to study living cells-nanostructure hybrids.

Schizophrenia is a mental disorder that affects ~1% of the world’s population and features social and cognitive dysfunction as well as psychosis. Today, schizophrenia is believed to result from the dysfunction of specific neuronal networks in the brain. However, very little is known about the neuronal functional deficits that give rise to the cognitive and psychotic symptoms. Previous studies have shown that gamma-band oscillations are aberrant in schizophrenia patients. This pattern, thought to result from the repetitive activity of specific inhibitory neurons in the cortex, may be related to the cognitive symptoms and serves as a clue to the neuronal mechanisms of the disease. However, the links between activity of specific neuronal populations and schizophrenia have not been directly tested. The proposed project is aimed at examining the involvement of cortical inhibitory interneurons in the pathogenesis of schizophrenia. The project will be based on optical control of the excitability of the parvalbumin-expressing interneurons in the cortex. I will use channelrhodopsin and NpHR to control the rate of activity and the temporal pattern of spike generation in these cells. I will use electrophysiology and fast optical imaging of voltage-sensitive dyes to evaluate the effect of the activity of parvalbumin-expressing cells on network dynamics and their contribution to gamma oscillations in the isolated slice preparation. Then, using an optic-fiber neural interface I will apply similar light-stimulation protocols to the brains of live, behaving animals and directly study the links between the activity of inhibitory neuron networks, gamma oscillations and behavior.

The random generation of diversity is the feature of immune system, which is essential to counteract numerous pathogens. However, it also allows immune system to attack self tissues and produce autoimmune diseases. Therefore self tolerance, the inability to make an immune response to self, is crucial to avoid autoimmune disease. For T lymphocytes, potentially auto-reactive cells are eliminated during their differentiation in thymus through encountering the autoantigens which are ectopically expressed in thumus. The AIRE (autoimmune regulator) is a key molecule to promote transcription of many ectopic genes in thymus and a critical element for self tolerance. Though it has structural features of a transcription factor, it is difficult to be reconciled with classical site-specific transcriptional factors, because it impacts on extensive transcriptions. To reveal the precise mechanism for transcriptional regulation by aire, we propose to identify the aire associating proteins by the yeast two-hybrid screening method with Ras Recruitment System. In addition, to elucidate the DNA-binding specificity of aire, we will perform high throughput sequencing-by-synthesis approach, ‘picotiter plate pyrosequencing’, following immunoprecipitating the Aire-DNA complex with anti-Aire antibody. The binding sites on genomic DNA will be examined by analyzing the obtained sequences through the database of mouse genome sequences. All these findings will contribute to reveal the molecular mechanisms for transcriptional regulation by AIRE and profound understanding of self tolerance, including the organ-specificities of autoimmune diseases.

Microtubules (MTs) are cytoskeletal polymers that exhibit highly dynamic behavior at one end, referred to as the MT plus end. Defined by their localization at MT plus ends, plus-end-tracking proteins (+TIPs) regulate MT dynamics and link MTs to cellular structures such as the cell cortex and kinetochores. EB1 protein emerges as a particularly significant +TIP due to its presence in many of the MT plus end interactions, however, the exact mechanisms of its binding to MT ends and its role in regulating MT dynamics are largely unknown. To address these questions controlled in vitro studies with purified components are needed. This research project employs novel in vitro MT growth/shrinkage assay with two-color total internal reflection fluorescence single-molecule imaging technology to: (i) investigate the binding mechanism of EB1 to MT plus end and determine whether EB1 associates with a newly polymerized lattice and remains at a fixed position or moves with the growing end by simultaneous single-molecule-imaging of EB1 and a growing MT (ii) determine the role of EB1 in MT dynamics by varying EB1 concentrations and recording binding events, as well as accompanying MT plus-end processes (iii) investigate the interactions of EB1 with other MT plus-end proteins (e.g. CLIP-170, XMAP215 and MCAK) to determine the effects of such interactions on binding of EB1 to MT plus end and on MT dynamics. The proposed research will provide an essential understanding of key behaviors and functions of EB1, which is known to have a role in important processes such as directed cell migration, regulation of cell growth and polarization, and cell division.