Abstract Jonathon Howard (Invited, Max Planck Institute, Germany) Motor proteins and the Cytoskeleton Our laboratory is interested in the biochemical and biophysical basis of cell shape. The shape of a cell is determined primarily by its cytoskeleton, which serves as a scaffold to support the plasma membrane, and as a network of tracks along which motor proteins transport subcellular structures. Our research is therefore focused on the mechanics of the cytoskeleton, with a particular emphasis on microtubules and microtubule-based motors. On one hand, we are interested in the mechanisms by which these proteins work: i.e. how do kinesins and dyneins convert chemical energy derived from the hydrolysis of ATP into mechanical work used to move along or to depolymerize microtubules? And on the other hand, we are interested in the roles that microtubules and their motors play in cell morphology and motility. For example, how do the dynamic properties of microtubules drive spindle and chromosome movements in mitosis, how does dynein drive axonemal motility? To address these questions, we are combining molecular biology techniques with image processing, modeling, mechanical measurements, single-molecule recordings and electron microscopy. Kazuhiko Kinosita (Invited, Waseda University, Japan) Single-Molecule Physiology of Protein Machines A single molecule of protein (or RNA) enzyme acts as a machine which carries out a unique function in cellular activities. To elucidate the mechanisms of various molecular machines, we need to observe closely the behaviors of individual molecules, because these machines, unlike man-made machines, operate stochastically and thus cannot be synchronized with each other. By attaching a tag that is huge compared to the size of a molecular machine, or a small tag such as a single fluorophore, we have been able to image the individual behaviors in real time under an optical microscope. A huge tag helped visualize the gait of a two-foot linear motor myosin V, which appears to 'walk' using a lever action and Brownian motion. Stepping rotation of the central subunit in a single molecule of F1-ATPase has also been videotaped, showing that this rotary motor, driven by ATP hydrolysis, runs at an amazingly high speed with an energy-conversion efficiency that may approach nearly 100%. This motor, when forced to rotate in the reverse direction, becomes a generator that synthesizes ATP from ADP and phosphate. The mechanism of the two-way energy conversion, from chemical energy of ATP hydrolysis to mechanical work of rotation and from mechanical rotation to chemical synthesis, can now be discussed in detail. In the lecture, I will present our experimental results on myosin V and F1- ATPase. Phase behaviour of two lipids related to programmed cell death: Using deuterium nuclear magnetic resonance spectroscopy, we studied the phase behaviour of increased amounts of ceramide on a sphingomyelin-ceramide mixture as a function of temperature. Ceramide is associated with early steps in the signalling of apoptosis, and sphingomyelin, a major constituent of cell membranes, is the precursor to ceramide. Moreover, Ceramide has been proposed as a therapeutic agent in cancer treatment. We were able to generate three main results: adding ceramide to sphingomyelin 1) broadens the gel-liquid crystalline phase transition, 2)increases the starting temperature of the phase transition, and 3) increases the order of ceramide chains in the liquid crystalline phase. During the transition, the gel component has a lower sphingomyelin-ceramide ratio than the liquid crystalline component. Hence, high local ceramide concentrations in cell membranes may allow ceramide to take part in a physical signalling mechanism. It is very likely that the physical state of the membrane may be shifted much closer to the gel phase when there is a high ceramide concentration. It is hypothesized that downstream signalling components are able to recognize such regions of the membrane. Labeling the N and C termini of collagen prior to pulling Collagen, a triple helical protein, is found in various forms and tissues in the human body and, in degenerate form, can be associated with disease. In order increase our understanding of collagen, we would like to pull on collagen in order measure the molecule's stiffness. Our plan is to label the amino and carboxyl termini differently and pull on these ends with differently labeled beads in an optical tweezers instrument. The labeling strategy includes the addition of ATP-g-S to N-terminal tyrosines and biotin to C-terminal cysteines on collagen III. We will use amino beads linked to Sulfo-SMCC cross-linker to form a covalent link to thiols on modified tyrosines and Streptavidin coated beads to bind to biotin labels on modified cysteines. In order to determine the labeling process does not alter the triple helical structure of collagen III (i.e remains in native vs. denatured state), we are investigating the use of native gel or circular dichroism method! Model of intracellular calcium oscillations due to negative feedback We present a mathematical model for calcium oscillations and fast adaptation in the cilia of olfactory sensory neurons. Stoichiometric network analysis is used for analysing the kinetic equations and finding the oscillatory regime. The underlying mechanism is based on direct negative feedback and does not require any autocatalysis such as calcium-induced calcium release. Results of the model using physiological parameter values agree quantitatively with experiment, both with respect to oscillations and to fast adaptation. The bifurcation diagram of the model is calculated to make predictions regarding the occurence of oscillations. Metagenomic applications for SCODA DNA extraction and purification It is estimated that less than 1% of all microorganisms observed in nature can be cultured in the laboratory. This leaves researchers unable to study more than 99% of microorganisms in some environments - microorganisms that sometimes have unique and potentially very useful abilities such as waste degradation, or synthesis of compounds that could find use as drugs or antibiotics. Metagenomics, the genomic reconstruction of unculturable microorganisms, is a powerful new tool for accessing the untapped resources of biodiversity in environmental samples. We present a novel method for sample preparation capable of directly extracting intact, high molecular weight DNA directly from environmental samples. This ability to extract and purify high MW DNA from difficult samples such as soil attempts to provide a breakthrough in representative metagenomic library construction for metagenomics that may enable the discovery of many future drugs and antibiotics. Towards Engineering Artificial Polyprotein-based Molecular Springs. Polyprotein of GB1 is an ideal elastomeric protein springs under biological settings and underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. In addition, they are also potential building blocks for the bottom-up construction of functional nanomechanical devices as well as materials of superb mechanical properties. To mimic the naturally occurring elastomeric proteins, we combine protein engineering and single molecule atomic force microscopy techniques to demonstrate that it is feasible to use non-mechanical proteins to engineer artificial polyprotein-based molecular springs. We engineered a polyprotein made of eight identical tandem repeats of a non-mechanical protein GB1, the B1 immunoglobulin-binding domain of protein G from streptococcal. Although GB1 is not evolved for mechanical functions and does not have known mechanical function, single molecule AFM measurements revealed that polyprotein of GB1 exhibits significant mechanical stability, which is comparable to that of naturally occurring elastomeric proteins, and does not show mechanical fatigue after hundreds of repeated stretching-relaxation cycles. The fast folding kinetics (a folding rate constant of 700 s-1) enables the polyprotein GB1 to recover its mechanical stability quickly. These fine mechanical features make polyprotein of GB1 an ideal elastomeric protein. This study opens up the possibilities to utilize non-mechanical proteins to engineer artificial elastomeric proteins with tailored nanomechanical properties for nanomechanical application and in material sciences. Mechanical model for coordinated, processive transport of dimeric molecular motors Single-molecule experiments have been used to observe the motion of the motor domains (heads) and the cargo of processive, dimeric molecular motors such as kinesin and myosin-V, within a single step of their hand-over-hand transport. We use information from existing experimental data about a motor's average trajectory, stepping behavior and fluctuations to theoretically model the mechanisms of force generation underlying the observed motion. We use a minimal, mechanistic model in which most parameters are constrained by experimental observations. By varying parameters that correspond to open questions about the transport mechanism, we predict qualitative performance features that can be tested experimentally. In particular, we numerically simulate two coupled particles that are each exposed to a one-dimesional, time-dependent free energy landscape that switches stochastically among several discrete states. By tuning features of the free energy landscape, we explore the roles of diffusion, deterministic conformational change (power stroke), and cooperative motion of the two binding domains. We use a conformational feedback scheme to model the role of internal strain in the coordinated motion of motor domains. Model predictions are obtained through Langevin dynamics simulations as well as analytical solutions to the system's Fokker-Planck equations. We will use this model as the basis for experimental realizations of artificial molecular motors in which mechanically coupled beads are exposed to a time-dependent potential profile created with a scanning optical trap. This experimental setup will provide a powerful tool for studying the physics of motors that operate in an environment dominated by thermal fluctuations. Optical tweezers in biophysics Optical tweezers is applied in biophysics for its proper force range and sub-nanometer resolution. The studies of biopolymers and molecular motors using optical tweezers lead to the concept of "single molecule experiment". In this introductory talk, several classical experiments from the past ten years are presented to show the classical application of optical tweezers in biophysics. Using FRAP to Determine Binding Kinetics of Cell Surface Proteins Protein interactions between cells are important for cell adhesion and the kinetics of these interactions are pivotal determinants of global cell responses. Using total internal reflection microscopy (TIRF) or confocal scanning laser microscopy (CSLM) and fluorescently tagged membrane proteins, we show how an extension of the commonly used technique of fluorescence recovery after photobleaching (FRAP) can be used to determine the on- and off-rates of interacting proteins on cell membranes. We will describe several experimental systems where this protocol can be used, including in vitro determination of protein binding kinetics. Lastly, we discuss several shortcomings of the method. Using an STM to image biological specimen. A single molecule protein sensor to investigate heterogeneity of protein populations Heterogeneity of protein populations with respect to conformation and folding is an important aspect of investigations in protein function. Organic nanopores, which are capable of astonishing resolution in single molecule detection and identification , can be used to study heterogeneity between individual proteins. It has been shown that electrophoresis of a DNA hairpin molecule with an 8-9 base pair stem into the vestibule of an ¦Á-hemolysin organic nanopore results in a unique current blockade transitioning between three distinct current states ("toggling"), the frequency of which depends on the penultimate base pair and the base pair terminus of the DNA hairpin. By exploiting this current signature it may be possible to resolve the structure-related hydrodynamic parameters of a protein by attaching the protein to the DNA hairpin such that the toggling pattern of the probe molecule is altered in a manner that is characteristic to the bound protein. We present progress on the development of a single molecule protein sensor based on a PEG-DNA tether attached to a 9 base pair DNA hairpin, and measurement of the kinetics of interaction between the construct and the ¦Á-hemolysin nanopore, with and without the presence of protein. Mathematical Model of Cell Polarization Based on Inhibitory Crosstalk of Rho GTPases. Cdc42, Rac, and Rho are small GTPases known to regulate cell polarization and motility, by affecting actin nucleation, uncapping, depolymerization, and acto-myosin contractility. Studies of crosstalk in these three proteins have led to a number of proposals for their interaction. At the same time, observations of the spatio-temporal dynamics of Rho-family proteins give evidence of spatial polarization and mutual exclusion between Cdc42/Rac and Rho. We formulate a mathematical model to account for such observations, based on the known underlying biology of these proteins. We investigate which of the crosstalk schemes proposed in the literature is consistent with observed dynamics, and derive a simple module that can correctly describe these dynamics (assuming crosstalk is mediated via Rho GEFs). We show that cooperativity is an essential ingredient in the interactions of the proteins, and that fast spatial segregation is related to bistability of the underlying ODE's. The fast diffusion of the inactive forms of these proteins is essential for stabilizing the transition fronts in the PDE formulation of the model, leading to robust spatial polarization, rather than travelling waves. Towards DNA uptake by Haemophilus influenzae Analyzing the robustness of synchrony in GnRH neurons when a common pool of GnRH hormone is considered Gonadotropin Releasing Hormone (GnRH) secreted by GnRH neurons plays key roles in the onset of puberty and the regulation of hormone secretion in the pituitary. GnRH neurons are intrinsically capable of generating pulsatile and episodic neurosecretion of this hormone, but the underlying mechanism for GnRH-pulse generator still remains obscure. The discovery of GnRH receptors allowing GnRH to exert autocrine regulation on its own release, led Krsmanovic et al. (2003) to propose a mechanism underlying this effect. A mathematical model describing the proposed mechanism has been developed by Khadra et. al. (2006). The model was further extended to study synchrony in GnRH neurons by incorporating the idea of a common pool of GnRH hormone. In this talk, we shall analyze several aspects of this mathematical model, particularly robustness. We shall show that coupling of a heterogeneous family of GnRH neurons will not significantly alter the general behaviour of the pulse generator. Indeed, we shall show that no more than 50% of these coupled neurons must be active participants in the process to generate pulsatility. The effects of averaging in the parameter-values, as well as the volume of the extracellular medium will be also discussed. In addition, several model predictions explaining the type of behaviour observed experimentally upon the injection of GnRH-agonist will be stated. These results will further demonstrate the properties of synchrony observed and the reliability of the model proposed. Single Collagen Molecule Manipulation Using Magnetic Tweezers Collagen is a biologically relevant protein whose structure is that of a triple helix. In the past, only bulk measurements have indirectly measured the persistence length and thermodynamic stability of collagen. In this study, we attempt to directly determine the extension of a single molecule of collagen as a function of twist and extensional force using a magnetic tweezers apparatus. These data will be used to determine the rigidity of collagen, characterize the stability of the triple helix, and contribute to the phase diagram for low forces (0.1 – 1 pN) and twist. Details of the magnetic tweezers apparatus, as well as, methods of analysis are presented. Conductivity Measurement of Single DNA Molecules using Conductive-Atomic Force Microscopy (c-AFM) From a nanoelectronics perspective, DNA possesses ideal structure and molecular recognition properties, which make DNA excellent candidate for molecular electronics. However, to understand the mechanism of electron transport along DNA is an essential step for the development of DNA-based molecular electronics. That is why intense interests on this topic have been shown in biophysics society. New insights to this issue have been brought recently by series of direct conductivity measurements through DNA molecules. In this talk, we would give some review of previous electrical experiments done on DNA molecules, different techniques as well as difficulties for single molecule measurements. Then we would talk about our specific work on DNA molecule with c-AFM, which shows short DNA acts more like semiconductor with a wide gap. In this aspect, DNA molecules are not good candidate for the original proposal as one dimensional molecule wire. However, it does not exclude that DNA molecules still have appealing future in molecule electronics. A Three-Dimensional Model of Cellular Electrical Activity We present a three-dimensional model of cellular electrical activity. This model takes into account the three-dimensional geometry of biological tissue as well as ionic concentration dynamics, both of which are neglected in conventional models of electrophysiology. We use both asymptotic and analytic methods to study the system of equations. We find in particular that the model possesses multiple temporal and spatial scales. This has important consequences for the development of an efficient numerical scheme. This modeling methodology is applied to cardiac physiology. Numerical simulations with this model is used to explore the characteristics of a recently observed anomalous mode of cardiac action potential propagation: cardiac propagation without gap junctions. A Novel Method and Technology for Electrophoretic Concentration of Low Abundance or High Molecular Weight Nucleic Acids A Novel Method and Technology for Electrophoretic Concentration of Low Abundance or High Molecular Weight Nucleic Acids We have developed a unique, cost-effective electrophoretic technology and subsequent prototype instrument that enables highly efficient oncentration of nucleic acids from contaminated or dilute samples, with no need for centrifugation, filtration, or fluid flow. This technology uses two-dimensional nonlinear electrophoresis to recover DNA or RNA from zeptomolar concentrations, and to recover fragments up to 1Mb in length without shearing. Nucleic acids are electrophoretically injected into a concentration medium from a liquid or gel-based sample up to 5 mL in volume. Time varying electric fields are applied to the concentration medium such that the highly nonlinear electrophoretic response of nucleic acids causes them to move towards a common focus location. Since this method acts only on molecules with highly nonlinear electrophoretic behaviour, it preferentially concentrates nucleic acids over other organic and inorganic molecules. The focusing fields completely pr! event dispersion, allowing injection from multiple samples into the same recovery volume. Subdomain Unfolding of T4 Lysozyme Detected by Single Molecule Atomic Force Microscopy Single molecule atomic force microscopy has evolved into a powerful tool in studying protein's folding and unfolding dynamics at the single molecule level. Most of the single molecule AFM studies focused on small proteins that comprise only one domain and fold via an all-or-none process. To extend single molecule AFM technique to more complex proteins, we employ T4 lysozyme as a model system. Although T4 lysozyme is a small protein, it comprises two subdomains, one is an amino-terminal alpha/beta lobe and the other one is a pure alpha-helix carboxy-terminal lobe. Chemical denaturation studies showed that the chemical unfolding of T4-lysozyme is a two-state process. By flanking T4-lysozyme with protein GB1, we were able to use single molecule AFM to stretch T4-lysozyme and investigate its mechanical unfolding behaviours. In contrast to the simple two-state behavior observed in ensemble chemical unfolding studies, we discovered that T4-lysozyme can unfold following multiple distinct pathways. In addition to the all-or-none unfolding pathway, we captured two distinct stepwise unfolding pathways of T4-lysozyme. The sizes of the unfolding intermediate states observed during these stepwise unfolding pathways correlate well with the two subdomains of T4-lysozyme. These findings reveal the dynamic and heterogeneous picture of the unfolding reaction for T4 lysozyme. Development of solid-state nanopore-based biosensors (poster to be co-presented with Dhruti Trivedi) Applications of genomics to health care require genotyping technologies with higher sensitivity, improved selectivity, faster response times and lower cost. The necessity for new sensing technologies to meet these demands is driving extensive research efforts in many areas of science and engineering. In particular nanopore-based single-molecule detection schemes are emerging as leading candidates for high throughput DNA sequence analysis. This presentation will report work perform in our group, towards direct
detection of DNA, employing solid state nanopores. Within the past year
we have developed the ability to reliably produce synthetic pores at the
2¨C10 nm scale, opening up exciting new possibilities for expansion of
the work carried out on proteinaceous alpha-Hemolysin pores. The fabrication
process of nanometer-scale pores in freestanding silicon nitride membranes
using a transmission electron microscope (TEM) will be described. I-V
curves used to characterize the nanopore impedance and size will be presented;
potential rectification behaviour will be discussed. Methods for reducing
the significantly higher (¡Á100) background noise level in synthetic pore
compare to their organic counterparts will be examined. The low signal
to noise ratio is attributed to the relatively high capacitance of the
Si/SiNx structure and due to the presence of charge carriers. Finally,
preliminary DNA translocation results will be presented along with a range
of chemical surface modification strategies to improve on the performance
of solid state nanopore-based biosensors. Probing biolgical systems with time varying signals Electrical engineers routinely use frequency response measurements to predict the function and input/output relationships of electrical circuits; we are interested in finding out how much information can be gathered from biochemical systems by making analogous measurements. Here we will describe initial work done in developing instrumentation for delivering time varying signals to cell populations as well some preliminary data and modeling results. Development of solid-state nanopore-based biosensors (poster to be co-presented with Vincent Tabard-Cossa) Applications of genomics to health care require genotyping technologies with higher sensitivity, improved selectivity, faster response times and lower cost. The necessity for new sensing technologies to meet these demands is driving extensive research efforts in many areas of science and engineering. In particular nanopore-based single-molecule detection schemes are emerging as leading candidates for high throughput DNA sequence analysis. This presentation will report work perform in our group, towards direct detection of DNA, employing solid state nanopores. Within the past year we have developed the ability to reliably produce synthetic pores at the 2¨C10 nm scale, opening up exciting new possibilities for expansion of the work carried out on proteinaceous alpha-Hemolysin pores. The fabrication process of nanometer-scale pores in freestanding silicon nitride membranes using a transmission electron microscope (TEM) will be described. I-V curves used to characterize the nanopore impedance and size will be presented; potential rectification behaviour will be discussed. Methods for reducing the significantly higher (¡Á100) background noise level in synthetic pore compare to their organic counterparts will be examined. The low signal to noise ratio is attributed to the relatively high capacitance of the Si/SiNx structure and due to the presence of charge carriers. Finally, preliminary DNA translocation results will be presented along with a range of chemical surface modification strategies to improve on the performance of solid state nanopore-based biosensors. Electronic detection of functional bio-molecules with Nanopores Electronic bio-molecule detection using nanopores is emerging as a method for highly sensitive detection and characterization of bio-molecules. Nanopores allow single molecule detection with very high resolution as the pore size approaches molecular dimensions. By analyzing changes in ionic current through the nanopore we get insight on the size and concentration of molecules present in or passing through the pore. We are developing methods for electronic detection of proteins in solution and for DNA analysis. Protein detection has a very wide range of applications, starting from detection of specific ligands to the implementation of an electronic form of ELISA. We successfully obtained antigen recognition by attaching antibodies to arrays of nanopores. Upon exposure to the antigen the nanopore conductance is highly affected as the antibody changes conformation. In particular we will be presenting experiments involving reversible adsorption of Horse Radish Peroxidase (HRP) to gold-coated nanopores. Upon application of an electric field through the nanopore this charged protein moves close to the pore and due to its high affinity to gold it is adsorbed. The attachment of proteins to the pore walls causes a change in the conductivity and surface charge of the nanopore. In the experiments the effect on pH is investigated as the affinity of proteins to gold changes depending on the conditions. We will also be presenting an electrostatic model describing the effect of increasing amounts of charged molecules inside a nanopore. Furthermore we are investigating detection of methylated groups on single DNA strands using organic nanopores. Once this procedure is optimized it could give great insight on the DNA methylation patterns and their relation to cancer and in general to the cell cycle. Single-Molecule Conductivity Measurements of DNA Constructs for Biosensor Applications The idea of using DNA as a molecular wire in an electrical circuit is technologically attractive, as it represents a "biological" solution to the problem of making nanoscale electrical connections. This concept would allow the construction of deoxyribosensors (DNA constructs that specifically bind certain analytes) to monitor the safety of food products. Current-voltage information on single or small clusters of molecules can be obtained by conductive atomic force microscopy (cAFM) while the surface morphology is also examined. We have initially investigated the electrical properties of octanedithiol attached to a gold substrate and to a gold nanoparticle (Au-monolayer-Au) via the Au-S bonds. Current-voltage (I-V) curves were observed as integral multiples of the fundamental curve, indicating the quantized electron transport behaviour. Molecular contract detected b y optical tweezers I want to use optical tweezers technology to detect the contraction of a single strand DNA, which has multiple aptamer repeat units, upon binding with its ligand. Simulations of Aging and the Plastic Deformation of Polymer Glasses Roughly, glass is not an equilibrium state of matter. It forms when a liquid is quenched such that the liquid degrees of freedom are frozen out before the material can "find" the crystalline state. Some materials such as polymers and polydisperse mixtures only ever form glasses in the solid state. Ageing is the process by which the material very slowly explores its configurational space and approaches equilibrium. This has dramatic consequences on the dynamic and mechanical properties of the glass. We explore this phenomenon with molecular dynamics simulation of a model ball and spring polymer system. Distinct domains of HCN channels regulate voltage-independent, cAMP-dependent effects on open probability and deactivation. BACKGROUND: HCN2 and HCN4 are the primary isoforms of the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel which regulates heartbeat pacemaking. We are investigating how the mouse HCN2 and HCN4 isoforms respond differently to cyclic AMP (cAMP), and the structural determinants of these differences. Binding of cAMP is well known to upregulate HCN channel activity, and consequently heart rate, both by shifting half-maximal (V1/2) voltages to less hyperpolarized values and by increasing the speed of channel activation. We are studying both these effects plus two additional effects, a cAMP-induced increase in the maximal voltage-activated open probability ("Pmax enhancement", defined as Gmax/Gmax, basal), and a cAMP-induced increase in the time constant of deactivation (tdeact), two phenomena not previously quantified in detail. METHODS: Recombinant chimeric derivatives of mouse HCN4, containing selected domain substitutions from mouse HCN2, are expressed in Xenopus laevis oocytes and tested for hyperpolarization-activation (V1/2 and kinetics) using patch-clamp of excised inside-out patches. We measure cAMP-induced Pmax enhancement using a maximally activating voltage in both the absence and presence of cAMP. tdeact values are determined using a maximally activating voltage followed by steps to various depolarized voltages, and fitting tail currents with a monoexponential function. RESULTS: The presence of the transmembrane domain of HCN4 in chimeric channels correlates with both slower activation and with a pronounced cAMP-induced increase in tdeact, implicating this region in channel kinetics, even though HCN2 and HCN4 show similar basal V1/2 and cAMP-induced V1/2 shift. The C-terminal domain influences cAMP-dependent increases in Pmax, suggesting this region is involved in an inhibitory mechanism that is relieved upon cAMP binding; notably, this inhibition is far less prominent in HCN4 than HCN2. CONCLUSIONS: We expect cAMP positively regulates HCN channels in the heart in at least four distinct ways: By shifting V1/2 to less hyperpolarized values, increasing the speed of activation, slowing deactivation, and increasing current amplitude. The latter three effects are manifested differently in mouse HCN2 and HCN4 isoforms. We are now working to pinpoint the particular residues involved in both a voltage-independent cAMP-induced increase in maximal open probability and a cAMP-induced slowing of channel deactivation. Coarse-grained DNA Modelling with MD Current research lies in modelling DNA with coarse-grained Gay-Bern-like potentials in molecular dynamics simulations. Zuckermann, Martin J.
Participants NMR/MRI I am interested in modeling of cellular processes (e.g. cell division, polarity establishment and maintenance, motility) with an emphasis on the role of biopolymers and the cytoskeleton. Processes that involve spontaneous pattern formation and geometry sensing are of particular interest. My research is in computational biology. Specifically, i am interested in analyzing and developing dynamical models for genetic regulatory networks. My lab develops and applies bioinformatic techniques to decipher the regulatory logic of gene expression. We are also interested in solving dynamical systems that model the temporal behaviour of biochemical networks. Current work is focussed on chromatin regulation and signalling cascades in Drosophila. Physical properties of cyanobacteria Single-molecule biophysics: My current research interests centre on predicting mechanical properties from molecular structure. This broad topic includes studying protein folding / unfolding at the single-molecule level, determining free energy surfaces from nonequilibrium experiments, and the development of optical trapping strategies to manipulate biomaterials. I am also interested in the mechanisms of novel molecular motors. I am interested in relationships between structure and dynamics in soft condensed matter. Our group studies lipid vesicles, gels and polymers. Modeling of cellular physiological processes. I have had some experience in the following areas, although my modeling experience thus far is limited: *Cardiac muscle - measurement of spatial distributions of Ca2+ related proteins involved in Excitation-Contraction Coupling; *Hair Cell - electro/chemical/mechanical transduction of sound induced vibrations in the cochlea; *E. Coli cell division machinery - min protein oscillations; *CNS regeneration following injury - quatification of axon outgrowth behaviour in response to Olfactory Epithelium derived treatments *simple reaction diffusion systems I am enrolled as a Computer Science/Microbiology and Immunology combined major at UBC. My current research, in Dr. Marziali's Lab, is focused on investigating new research techniques to describe the intracellular signaling mechanisms that occur in yeast cells. I am interested in what effects an oscillating signal at different fixed frequencies might have on the metabolic pathways of yeast cells. Specifically I am subjecting yeast cells to a time varying temperature signal and then I am trying to understand how this affects the biochemical pathways of the cells. The approach I take in analyzing the data is derived from electrical circuit analysis and is aimed at revealing any non-linearities the response might contain. I am developing the hardware and software for controlling the setup and also the software for acquisition and analysis of the data. Signalling to actin: modules that regulate cell motility (coauthor: Dr. Adriana T Dawes) Gradient sensing, polarization, and chemotaxis of motile cells involves the actin cytoskeleton, and regulatory modules, including the phosphoinositides (PIs), and small GTPases (Rho proteins). Here we model their individual components in the context of a 1D dynamic model for protrusive cell motility, (parameter values derived from in vitro and in vivo studies). In response to a spatially graded stimulus, the model produces stable amplified internal profiles of regulatory components, and initiates persistent motility (consistent with experimental observations). By connecting the modules, we find that Rho GTPases work as a spatial switch, and that PIs filter noise, and define the front vs. back. Relatively fast PI diffusion also leads to selection of a unique pattern of Rho distribution from a collection of possible patterns. We use the model to explore importance of specific hypothesized interactions, to explore mutant phenotypes, and to study the role of actin polymerization in the maintenance of the PI asymmetry. Phase behaviour of two lipids related to programmed cell death: Using deuterium nuclear magnetic resonance spectroscopy, we studied the phase behaviour of increased amounts of ceramide on a sphingomyelin-ceramide mixture as a function of temperature. Ceramide is associated with early steps in the signalling of apoptosis, and sphingomyelin, a major constituent of cell membranes, is the precursor to ceramide. Moreover, Ceramide has been proposed as a therapeutic agent in cancer treatment. We were able to generate three main results: adding ceramide to sphingomyelin 1) broadens the gel-liquid crystalline phase transition, 2)increases the starting temperature of the phase transition, and 3) increases the order of ceramide chains in the liquid crystalline phase. During the transition, the gel component has a lower sphingomyelin-ceramide ratio than the liquid crystalline component. Hence, high local ceramide concentrations in cell membranes may allow ceramide to take part in a physical signalling mechanism. It is very likely that the physical state of the membrane may be shifted much closer to the gel phase when there is a high ceramide concentration. It is hypothesized that downstream signalling components are able to recognize such regions of the membrane. single molecule biophysics I am interested in using single molecule atomic force microscopy to probe the folding and unfolding dynamics of proteins and understand molecular determinants of the mechanical properties of proteins. Development of scanning probe techniques and study of complex systems. The traditional applications of control theory have involved the use of feedback to control experimental instruments. However, the concepts in control theory have found applications in complex systems. On the other hand, approaches in other areas suggest ways to refine our understanding of control theory. It is interesting to compare different approaches. This practice will help us investigate a more fundamental A 3-D Model To Study Interplay Of Cell Signaling, Cell Adhesion And Chemotaxis In Multicellular Systems. My research involves the use of micropipette aspiration techniques to investigate the mechanical strength of lipid bilayer vesicles. Studies target the elasticity, viscosity and dynamic rupture of membranes under a wide range of timescales (loading rates). Application of theoretical statistical mechanics models allows for quantitative characterization of lipid-cholesterol interactions as well as the impact of antimicrobial peptides on membrane strength. Phase coexistence in membranes Interests: properties of lipid membranes containing unusual lipids or sterols, especially membranes with complex phase behaviour major technique used: solid state deuterium NMR The need for low cost DNA sequence detection in clinical applications is driving development of low-cost, sensitive technologies. We have recently developed a nanopore-based sensor for direct detection of mutations in a DNA sequence purely by electronic means, and without need for labels. The sensor employs an array of alpha-hemolysin nanopores, and uses electric fields to perform force spectroscopy on hundreds of DNA molecules in parallel. The analyte DNA molecule is hybridized to a single-stranded DNA probe molecule of defined sequence then forced to dissociate, as the probe is pulled through the pore. The relationship between dissociation time and force is strongly dependent on the dissociation energy, which is determined by the target sequence; we have demonstrated the ability to distinguish single base substitutions. I will begin by describing the original incarnation of the sensor, which uses one nanopore to achieve single-molecule detection. The apparatus, methods for data processing, and results obtained will be discussed. I will then describe a more recent development, where we use arrays of several hundred pores operated in parallel, yielding detailed information on the kinetics of hundreds of molecule dissociations in a single measurement. I will conclude with a discussion of how this method could lead to a chip-based rapid genotyping tool. Research interests include single-molecule biophysics, membrane transport, motors, optical techniques |