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DNA-Mediated Charge Transport for Long-Range Sensing and Protein Detection
Get PDFThe structural core of DNA, a continuous stack of aromatic heterocycles—the base pairs—that extends down the helical axis, gives rise to the fascinating electronic properties of this molecule that is so critical for life. This π-stacked structure facilitates a unique form of charge conduction, termed DNA-mediated charge transport (DNA CT). Experiments with diverse platforms, in solution, on surfaces, and with single molecules, collectively provide a broad and consistent perspective on the essential characteristics of this chemistry. Notably, DNA CT can proceed over long molecular distances, but is remarkably sensitive to perturbations in base pair stacking. These characteristics suggest that DNA CT may be used for long-range sensing both in nature and in nanoelectronic applications. Here, measurements of DNA CT with surface and single molecule platforms are used to (i) determine how ground state DNA CT varies over regimes of increasing distance and (ii) apply this chemistry to the electrical detection of DNA-binding proteins.
First, the design and fabrication of multiplexed, DNA-modified electrodes on silicon chips is reported. These lithographically patterned chips with 16 individually addressable gold electrodes allow for the measurement of DNA CT with four different types of DNA, side by side on the same surface, with four-fold redundancy. Discrimination of DNA with a single base mismatch and detection of sequence-specific restriction enzyme activity are both achieved with these chips. Scaling of these devices to microelectrode dimensions is also demonstrated. Importantly, these chips show greater reproducibility and consistency than commercially available rod electrodes. This greater signal quality, combined with the capacity to examine different samples side by side, opens the door for more complex applications of this platform.
The fully developed, multiplexed chips are first used to compare DNA CT over short and long distance regimes. DNA is evaluated in this context because the efficacy of a long-range sensor, in either nature or nanoelectronics, is determined largely by its capacity to facilitate CT in a manner that is minimally affected by the CT distance. DNA CT over 34 nm in 100-mer monolayers is found to yield electrochemical signals that are comparable in size to shorter 17-mer DNA. Signal attenuation from a single base-pair mismatch in the 100-mer is also comparable to that for 17-mers, and confirms that CT in these 100-mer films is DNA-mediated. Efficient cleavage by a restriction enzyme indicates that the 100-mer DNA adopts a native, upright conformation. The alkanethiol linker used to anchor the DNA to the electrode is found to limit the electron-transfer rate for both DNA lengths. Thus the impact of increasing the CT distance on DNA CT is too small to be resolved by this platform, even over 34 nm. These measurements put DNA among the longest and most conductive molecular wires reported to date.
Next, DNA CT with multiplexed chips is extended to the electrochemical detection of methyltransferases, proteins that are attractive targets because of their prominent role in the initial stages of many types of cancer. Electrochemical detection of binding and activity by these proteins is achieved by two different methods. First, DNA-binding and base-flipping by these proteins disrupts the DNA π-stack and may be used for direct “signal OFF” detection. Using this method, the concentration- and cofactor- dependence of SssI methyltransferase, the bacterial analog of human methyltransferases, are examined. Second, methylation-conferred protection of DNA against cutting by a restriction enzyme may be used for “signal ON” detection of methyltransferase activity. With this approach, the use of both unmethylated and hemimethylated DNA substrates is demonstrated for the sensitive detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Importantly, the electrochemical format of these assays requires minimal equipment, is low cost, and may be easily applied to high throughput studies, making it an accessible option for a variety of research and clinical settings.
Alongside work with this surface, electrochemical platform, a single molecule, carbon nanotube-DNA (CNT-DNA) platform is also used to evaluate DNA CT over increasing distances and to detect protein binding. CNT-DNA devices consist of a single molecule of DNA that is made to bridge a gap cut in a CNT covalently, such that current flow through the device is DNA-mediated. Upon introduction of DNA bridges of varying length (15-mer, 60-mer, and 100-mer), the device resistance is minimally affected, echoing the result of long distance electrochemistry experiments. These devices are also used to detect SssI methyltransferase binding by the direct “signal OFF” method used with multiplexed chips; DNA-binding and base-flipping disrupts DNA CT and shuts off current flow through the device. CNT-DNA devices are used to electronically measure the sequence-specific, cofactor-dependent, and reversible binding of SssI. DNA methylation catalyzed by SssI is also detected based on its alteration of the protein-binding affinity of the device. This detection approach, which relies on DNA as both a recognition element and electrical transducer, represents a unique strategy for the specific, single molecule detection of protein binding and activity.</p
Dynamics of Cell–Matrix Mechanical Interactions in Three Dimensions
Get PDFThe forces cells apply to their surroundings control biological processes such as growth, adhesion, development, and migration. In the past 20 years, a number of experimental techniques have been developed to measure such cell tractions. These approaches have primarily measured the tractions applied by cells to synthetic two-dimensional substrates, which do not mimic in vivo conditions for most cell types. Many cell types live in a fibrous three-dimensional (3D) matrix environment. While studying cell behavior in such 3D matrices will provide valuable insights for the mechanobiology and tissue engineering communities, no experimental approaches have yet measured cell tractions in a fibrous 3D matrix.
This thesis describes the development and application of an experimental technique for quantifying cellular forces in a natural 3D matrix. Cells and their surrounding matrix are imaged in three dimensions with high speed confocal microscopy. The cell-induced matrix displacements are computed from the 3D image volumes using digital volume correlation. The strain tensor in the 3D matrix is computed by differentiating the displacements, and the stress tensor is computed by applying a constitutive law. Finally, tractions applied by the cells to the matrix are computed directly from the stress tensor.
The 3D traction measurement approach is used to investigate how cells mechanically interact with the matrix in biologically relevant processes such as division and invasion. During division, a single mother cell undergoes a drastic morphological change to split into two daughter cells. In a 3D matrix, dividing cells apply tensile force to the matrix through thin, persistent extensions that in turn direct the orientation and location of the daughter cells. Cell invasion into a 3D matrix is the first step required for cell migration in three dimensions. During invasion, cells initially apply minimal tractions to the matrix as they extend thin protrusions into the matrix fiber network. The invading cells anchor themselves to the matrix using these protrusions, and subsequently pull on the matrix to propel themselves forward.
Lastly, this thesis describes a constitutive model for the 3D fibrous matrix that uses a finite element (FE) approach. The FE model simulates the fibrous microstructure of the matrix and matches the cell-induced matrix displacements observed experimentally using digital volume correlation. The model is applied to predict how cells mechanically sense one another in a 3D matrix. It is found that cell-induced matrix displacements localize along linear paths. These linear paths propagate over a long range through the fibrous matrix, and provide a mechanism for cell-cell signaling and mechanosensing. The FE model developed here has the potential to reveal the effects of matrix density, inhomogeneity, and anisotropy in signaling cell behavior through mechanotransduction.</p
On Erasure Coding for Distributed Storage and Streaming Communications
Get PDFThe work presented in this thesis revolves around erasure correction coding, as applied to distributed data storage and real-time streaming communications.
First, we examine the problem of allocating a given storage budget over a set of nodes for maximum reliability.
The objective is to find an allocation of the budget that maximizes the probability of successful recovery by a data collector accessing a random subset of the nodes.
This optimization problem is challenging in general because of its combinatorial nature, despite its simple formulation.
We study several variations of the problem, assuming different allocation models and access models, and determine the optimal allocation and the optimal symmetric allocation (in which all nonempty nodes store the same amount of data) for a variety of cases.
Although the optimal allocation can have nonintuitive structure and can be difficult to find in general, our results suggest that, as a simple heuristic, reliable storage can be achieved by spreading the budget maximally over all nodes when the budget is large, and spreading it minimally over a few nodes when it is small.
Coding would therefore be beneficial in the former case, while uncoded replication would suffice in the latter case.
Second, we study how distributed storage allocations affect the recovery delay in a mobile setting.
Specifically, two recovery delay optimization problems are considered for a network of mobile storage nodes:
the maximization of the probability of successful recovery by a given deadline, and the minimization of the expected recovery delay.
We show that the first problem is closely related to the earlier allocation problem, and solve the second problem completely for the case of symmetric allocations.
It turns out that the optimal allocations for the two problems can be quite different.
In a simulation study, we evaluated the performance of a simple data dissemination and storage protocol for mobile delay-tolerant networks, and observed that the choice of allocation can have a significant impact on the recovery delay under a variety of scenarios.
Third, we consider a real-time streaming system where messages created at regular time intervals at a source are encoded for transmission to a receiver over a packet erasure link;
the receiver must subsequently decode each message within a given delay from its creation time.
For erasure models containing a limited number of erasures per coding window, per sliding window, and containing erasure bursts whose maximum length is sufficiently short or long, we show that a time-invariant intrasession code asymptotically achieves the maximum message size among all codes that allow decoding under all admissible erasure patterns.
For the bursty erasure model, we also show that diagonally interleaved codes derived from specific systematic block codes are asymptotically optimal over all codes in certain cases.
We also study an i.i.d. erasure model in which each transmitted packet is erased independently with the same probability;
the objective is to maximize the decoding probability for a given message size.
We derive an upper bound on the decoding probability for any time-invariant code, and show that the gap between this bound and the performance of a family of time-invariant intrasession codes is small when the message size and packet erasure probability are small.
In a simulation study, these codes performed well against a family of random time-invariant convolutional codes under a number of scenarios.
Finally, we consider the joint problems of routing and caching for named data networking.
We propose a backpressure-based policy that employs virtual interest packets to make routing and caching decisions.
In a packet-level simulation, the proposed policy outperformed a basic protocol that combines shortest-path routing with least-recently-used (LRU) cache replacement.</p
Studies of Zγ Production and Constraints on Anomalous Triple Gauge Couplings in pp Collisions at √s = 7 TeV
Get PDFIn this thesis, we test the electroweak sector of the Standard Model of particle physics through the measurements of the cross section of the simultaneous production of the neutral weak boson Z and photon γ, and the limits on the anomalous Zγγ and ZZγ triple gauge couplings h3 and h4 with the Z decaying to leptons (electrons and muons). We analyze events collected in proton-proton collisions at center of mass energy of sqrt(s) = 7 TeV corresponding to an integrated luminosity of 5.0 inverse femtobarn. The analyzed events were recorded by the Compact Muon Solenoid detector at the Large Hadron Collider in 2011.
The production cross section has been measured for hard photons with transverse momentum greater than 15 GeV that are separated from the the final state leptons in the eta-phi plane by Delta R greater than 0.7, whose sum of the transverse energy of hadrons over the transverse energy of the photon in a cone around the photon with Delta R less than 0.3 is less than 0.5, and with the invariant mass of the dilepton system greater than 50 GeV. The measured cross section value is 5.33 +/- 0.08 (stat.) +/- 0.25 (syst.) +/- 0.12 (lumi.) picobarn. This is compatible with the Standard Model prediction that includes next-to-leading-order QCD contributions: 5.45 +/- 0.27 picobarn.
The measured 95 % confidence-level upper limits on the absolute values of the anomalous couplings h3 and h4 are 0.01 and 8.8E-5 for the Zγγ interactions, and, 8.6E-3 and 8.0E-5 for the ZZγ interactions. These values are also compatible with the Standard Model where they vanish in the tree-level approximation. They extend the sensitivity of the 2012 results from the ATLAS collaboration based on 1.02 inverse femtobarn of data by a factor of 2.4 to 3.1.</p
A Dynamical Systems Analysis of Vortex Pinch-Off
Get PDFVortex rings constitute the main structure in the wakes of a wide class of swimming and flying animals, as well as in cardiac flows and in the jets generated by some moss and fungi. However, there is a physical limit, determined by an energy maximization principle called the Kelvin-Benjamin principle, to the size that axisymmetric vortex rings can achieve. The existence of this limit is known to lead to the separation of a growing vortex ring from the shear layer feeding it, a process known as `vortex pinch-off', and characterized by the dimensionless vortex formation number. The goal of this thesis is to improve our understanding of vortex pinch-off as it relates to biological propulsion, and to provide future researchers with tools to assist in identifying and predicting pinch-off in biological flows.
To this end, we introduce a method for identifying pinch-off in starting jets using the Lagrangian coherent structures in the flow, and apply this criterion to an experimentally generated starting jet. Since most naturally occurring vortex rings are not circular, we extend the definition of the vortex formation number to include non-axisymmetric vortex rings, and find that the formation number for moderately non-axisymmetric vortices is similar to that of circular vortex rings. This suggests that naturally occurring vortex rings may be modeled as axisymmetric vortex rings. Therefore, we consider the perturbation response of the Norbury family of axisymmetric vortex rings. This family is chosen to model vortex rings of increasing thickness and circulation, and their response to prolate shape perturbations is simulated using contour dynamics. Finally, the response of more realistic models for vortex rings, constructed from experimental data using nested contours, to perturbations which resemble those encountered by forming vortices more closely, is simulated using contour dynamics. In both families of models, a change in response analogous to pinch-off is found as members of the family with progressively thicker cores are considered. We posit that this analogy may be exploited to understand and predict pinch-off in complex biological flows, where current methods are not applicable in practice, and criteria based on the properties of vortex rings alone are necessary.</p
Single Cell Proteomics Microchip to Profile Immune Function, with Applications in Stem Cell Biology, Translational Disease Mechanism Study and Clinical Therapeutics Monitoring
Get PDFIn response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.
To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.
In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs.
Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.</p
Towards Conditional RNAi : Shape and Sequence Transduction with Small Conditional RNAs
Get PDFRNA interference (RNAi) is a powerful biological pathway allowing for sequence-specific knockdown of any gene of interest. While RNAi is a proven tool for probing gene function in biological circuits, it is limited by being constitutively ON and executes the logical operation: silence gene Y. To provide greater control over post-transcriptional gene silencing, we propose engineering a biological logic gate to implement “conditional RNAi.” Such a logic gate would silence gene Y only upon the expression of gene X, a completely unrelated gene, executing the logic: if gene X is transcribed, silence independent gene Y. Silencing of gene Y could be confined to a specific time and/or tissue by appropriately selecting gene X.
To implement the logic of conditional RNAi, we present the design and experimental validation of three nucleic acid self-assembly mechanisms which detect a sub-sequence of mRNA X and produce a Dicer substrate specific to gene Y. We introduce small conditional RNAs (scRNAs) to execute the signal transduction under isothermal conditions. scRNAs are small RNAs which change conformation, leading to both shape and sequence signal transduction, in response to hybridization to an input nucleic acid target. While all three conditional RNAi mechanisms execute the same logical operation, they explore various design alternatives for nucleic acid self-assembly pathways, including the use of duplex and monomer scRNAs, stable versus metastable reactants, multiple methods of nucleation, and 3-way and 4-way branch migration.
We demonstrate the isothermal execution of the conditional RNAi mechanisms in a test tube with recombinant Dicer. These mechanisms execute the logic: if mRNA X is detected, produce a Dicer substrate targeting independent mRNA Y. Only the final Dicer substrate, not the scRNA reactants or intermediates, is efficiently processed by Dicer. Additional work in human whole-cell extracts and a model tissue-culture system delves into both the promise and challenge of implementing conditional RNAi in vivo.</p
Palladium-Catalyzed Asymmetric Allylic Alkylation: Insights, Application Toward Cyclopentanoid and Cycloheptanoid Molecules, and the Total Synthesis of Several Daucane Sesquiterpenes
Get PDFThe asymmetric construction of quaternary stereocenters is a topic of great interest in the organic chemistry community given their prevalence in natural products and biologically active molecules. Over the last decade, the Stoltz group has pursued the synthesis of this challenging motif via a palladium-catalyzed allylic alkylation using chiral phosphinooxazoline (PHOX) ligands. Recent results indicate that the alkylation of lactams and imides consistently proceeds with enantioselectivities substantially higher than any other substrate class previously examined in this system. This observation prompted exploration of the characteristics that distinguish these molecules as superior alkylation substrates, resulting in newfound insights and marked improvements in the allylic alkylation of carbocyclic compounds.
General routes to cyclopentanoid and cycloheptanoid core structures have been developed that incorporate the palladium-catalyzed allylic alkylation as a key transformation. The unique reactivity of α-quaternary vinylogous esters upon addition of hydride or organometallic reagents enables divergent access to γ-quaternary acylcyclopentenes or cycloheptenones through respective ring contraction or carbonyl transposition pathways. Derivatization of the resulting molecules provides a series of mono-, bi-, and tricyclic systems that can serve as valuable intermediates for the total synthesis of complex natural products.
The allylic alkylation and ring contraction methodology has been employed to prepare variably functionalized bicyclo[5.3.0]decane molecules and enables the enantioselective total syntheses of daucene, daucenal, epoxydaucenal B, and 14-p-anisoyloxydauc-4,8-diene. This route overcomes the challenge of accessing β-substituted acylcyclopentenes by employing a siloxyenone to effect the Grignard addition and ring opening in a single step. Subsequent ring-closing metathesis and aldol reactions form the hydroazulene core of these targets. Derivatization of a key enone intermediate allows access to either the daucane sesquiterpene or sphenobolane diterpene carbon skeletons, as well as other oxygenated scaffolds.</p
GPU-Accelerated Fourier-Continuation Solvers and Physically Exact Computational Boundary Conditions for Wave Scattering Problems
Get PDFMany important engineering problems, ranging from antenna design to seismic imaging, require the numerical solution of problems of time-domain propagation and scattering of acoustic, electromagnetic, elastic waves, etc. These problems present several key difficulties, including numerical dispersion, the need for computational boundary conditions, and the extensive computational cost that arises from the extremely large number of unknowns that are often required for adequate spatial resolution of the underlying three-dimensional space. In this thesis a new class of numerical methods is developed. Based on the recently introduced Fourier continuation (FC) methodology (which eliminates the Gibbs phenomenon and thus facilitates accurate Fourier expansion of nonperiodic functions), these new methods enable fast spectral solution of wave propagation problems in the time domain. In particular, unlike finite difference or finite element approaches, these methods are very nearly dispersionless---a highly desirable property indeed, which guarantees that fixed numbers of points per wavelength suffice to solve problems of arbitrarily large extent. This thesis further puts forth the mathematical and algorithmic elements necessary to produce highly scalable implementations of these algorithms in challenging parallel computing environments---such as those arising in GPU architectures---while preserving their useful properties regarding convergence and dispersion.
Additionally, this thesis develops a fast method for evaluation of computational boundary conditions which is based on Kirchhoff's integral formula in conjunction with the FC methodology and an accelerated equivalent source integration method introduced recently for solution of integral equation problems. The combination of these ideas gives rise to a physically exact radiating boundary condition that is nonlocal but fast. The only known alternatives that provide all three of these features are only applicable to a highly restrictive class of domains such as spheres or cylinders, whereas the Kirchhoff-based approach considered here only requires a bounded domain with nonvanishing thickness. As is the case with the FC scattering solvers mentioned above, the boundary-conditions algorithm is modified into a formulation that admits efficient implementation in GPU and other parallel infrastructures.
Finally, this thesis illustrates the character of the newly developed algorithms, in both GPU and parallel CPU infrastructures, with a variety of numerical examples. In particular, it is shown that the GPU implementations result in thirty- to fiftyfold speedups over the corresponding single CPU implementations. An extension of the boundary-condition algorithm, further, is demonstrated, which enables for propagation of time-domain solutions over arbitrarily large spans of empty space at essentially null computational cost. Finally, a hybridization of the FC and boundary condition algorithm is presented, which is also part of this thesis work, and which provides an interface of the newly developed algorithms with legacy finite-element representations of geometries and engineering structures. Thus, combining spectral and classical PDE solvers and propagation methods with novel GPU and parallel CPU implementations, this thesis demonstrates a computational capability that enables solution, in novel computational architectures, of some of the most challenging problems in the broad field of computational wave propagation and scattering.</p
Tools for Assessing Mitochondrial Dynamics in Mouse Tissues and Neurodegenerative Models
Get PDFMitochondria are dynamic organelles that undergo membrane fusion and fission and transport. The dynamic properties of mitochondria are important for regulating mitochondrial function. Defects in mitochondrial dynamics are linked to neurodegenerative diseases and affect the development of many tissues. To investigate the role of mitochondrial dynamics in diseases, versatile tools are needed to explore the physiology of these dynamic organelles in multiple tissues. Current tools for monitoring mitochondrial dynamics have been limited to studies in cell culture, which may be inadequate model systems for exploring the network of tissues. Here, we have generated mouse models for monitoring mitochondrial dynamics in a broad spectrum of tissues and cell types. The photoactivatable mitochondria (PhAMfloxed) line enables Cre-inducible expression of a mitochondrial targeted photoconvertible protein, Dendra2 (mito-Dendra2). In the PhAMexcised line, mito-Dendra2 is ubiquitously expressed to facilitate broad analysis of mitochondria at various developmental processes.
We have utilized these models to study mitochondrial dynamics in the nigrostriatal circuit of Parkinson’s disease (PD) and in the development of skeletal muscles. Increasing evidences implicate aberrant regulation of mitochondrial fusion and fission in models of PD. To assess the function of mitochondrial dynamics in the nigrostriatal circuit, we utilized transgenic techniques to abrogate mitochondrial fusion. We show that deletion of the Mfn2 leads to the degeneration of dopaminergic neurons and Parkinson’s-like features in mice. To elucidate the dynamic properties of mitochondria during muscle development, we established a platform for examining mitochondrial compartmentalization in skeletal muscles. This model system may yield clues to the role of mitochondrial dynamics in mitochondrial myopathies.</p