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Investigation of Receptors for the Modulation of Neuronal Growth by Chondroitin Sulfate
Get PDFA major obstacle to neural regeneration after injury in the central nervous system (CNS) is the environment encountered by injured axons. This environment is inhibitory due to proteins expressed by the CNS myelin as well as molecules present in the glial scar. Experimental results have implicated chondroitin sulfate proteoglycans (CSPGs) as major inhibitors of axonal regeneration after CNS injury, but until recently, the mechanisms of this inhibition were not well understood. Furthermore, the complex nature of the chondroitin sulfate (CS) chains made it difficult to study their contribution to CSPG function. This thesis describes a specific carbohydrate epitope, CS-E, that is primarily responsible for the inhibition of CNS axonal regrowth in the presence of CSPGs. We show that removal or blocking of the CS-E motif via genetic elimination of the enzyme responsible for generating CS-E or a monoclonal antibody that binds specifically to the CS-E motif significantly reduces the inhibitory activity of CSPGs on axon growth. Furthermore, we show that CS-E functions as a protein recognition element to engage receptors, including the transmembrane protein tyrosine phosphatase PTPσ, which had been previously established to be a receptor for CSPGs. Finally, we show that the protein tyrosine kinase receptor EphA4 is a novel receptor for the CS-E motif, and as with PTPσ, neurons deficient in EphA4 exhibit reduced inhibition by CS-E. Our results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides directs important physiological processes, and establish the importance of the chemical structure of CS chains in modulating the activity of CSPGs in vivo. The identification of receptors that mediate the inhibitory effect of CS-E advances our understanding of the mechanisms of axon regeneration following injury to the CNS when CS-E expression is upregulated. These findings provide us with the opportunity to develop therapies for the recovery of axonal outgrowth after damage to the nervous system, which in conjunction with blocking approaches targeting the CS motif, can provide a powerful strategy for allowing recovery after injury to the CNS
First-Principle Studies of the Initiation Mechanism of Energetic Materials
Get PDFIt is important to understand the initiation mechanism of energetic materials to improve and engineer them. In this thesis first-principle calculation is used to study the initiation of several explosives and propellants.
The second chapter is focused on a new energetic material, silicon pentaerythritol tetranitrate (Si-PETN), DFT calculations have identified the novel rearrangement that explains the very dramatic increase in sensitivity observed experimentally. The critical difference is that Si-PETN allows a favorable five-coordinate transition state in which the new Si−O and C−O bonds form simultaneously, leading to a transition state barrier of 33 kcal/mol (it is 80 kcal/mol for PETN) and much lower than the normal O−NO2 bond fission observed in other energetic materials (40 kcal/mol). In addition this new mechanism is very exothermic (45 kcal/mol) leading to a large net energy release at the very early stages of Si-PETN decomposition.
The third chapter is about nitrogen-rich compounds, which has high heat of formation and releases the energy by decomposing into stable N2 molecules. Two families of compounds, azobistetrazoles and azobistriazoles, were studied. Based on the calculated mechanisms, for azobistetrazoles with four N atoms in the five-member ring, a clearly-defined N=N fragment can always be found in the ring, and its decomposition starts with ring-opening to free one end of N=N followed by N2 dissociation and heat generation. This barrier is around 28-35 kcal/mol, which is low enough to dominate the sensitivity of material. For azobistriazoles, only 1,1’-azobis-1,2,3-triazole has a N=N fragment in the original 5-member ring and similar ring-opening - N2 dissociation pathway is favored. For the remaining compounds, an additional isomerization is necessary to release N2, which gives the barrier around 55~60 kcal/mol, making these compound less sensitive.
The fourth chapter shifts focus to hypergolic propellants. DFT calculations with B3LYP functional was applied to study the hypergolic reaction between N,N,N',N'-tetramethylethylenediamine (TMEDA), N,N,N',N'-Tetramethylmethylenediamine (TMMDA) and HNO3. Bond energies in TMEDA and TMMDA were calculated and compared with their alkane analogues to demonstrate that the lone-pair electrons on N atoms plays the role of activating adjacent chemical bonds. Two key factors relating to the ignition delay were calculated at atomistic level. The first factor is the exothermicity of the formation of the dinitrate salt of TMEDA and TMMDA. Because of the shorter distance between basic amines in TMMDA, it is more difficult to protonate both amines for the stronger electrostatic repulsion, resulting in the smaller heat of dinitrate salt formation by 6.3kcal/mol. The second factor is the reaction rate of TMEDA and TMMDA reacting with NO2 to the step that releases enough heat and more reactive species to propagate reaction. In TMEDA, the formation of the intermediate with C-C double bond and the low bond energy of C-C single bond provide a route with low barrier to oxidize C. Both factors can contribute to the shorter ignition delay of TMEDA.
The fifth chapter is about the other pair of hypergolic propellant, monomethylhydrazine (MMH) with oxidizers NO2/N2O4. Experimentally several IR-active species were identified in the early reactions, including HONO, monomethylhydrazinium nitrite (MMH•HONO), methyl diazene (CH3N=NH), methyl nitrate (CH3ONO2), methyl nitrite (CH3ONO), nitromethane (CH3NO2), methyl azide (CH3N3), H2O, N2O and NO. In order to elucidate the mechanisms by which these observed products are formed, we carried out quantum mechanics calculations (CCSD(T)/6-31G**//M06-2X/6-311G**++) for the possible reaction pathways. Based on these studies, we proposed that the oxidation of MMH in an atmosphere of NO2 occurs via two mechanisms: (1) sequential H-abstraction and HONO formation, and (2) reaction of MMH with asymmetric ONONO2, leading to formation of methyl nitrate. These mechanisms successfully explain all intermediates observed experimentally. We concluded that the formation of asymmetric ONONO2 is assisted by an aerosol formed by HONO and MMH that provides a large surface area for ONONO2 to condense, leading to the generation of methyl nitrate. Thus we proposed that the overall pre-ignition process involves both gas-phase and aerosol-phase reactions.
The sixth chapter is about another pair of hypergolic propellant, unsymmetrical dimethylhydrazine (UDMH) with oxidizers NO2/N2O4. We carried out the same level of quantum mechanics calculations as MMH to study this pair. We proposed that the oxidation of UDMH in an atmosphere of NO2 occurs via two mechanisms, similar with MMH: (1) sequential H-abstraction and HONO formation in gas phase, which has no more than 20 kcal/mol barrier and leads to the production of (CH3)2NNO and HONO. (2)UDMH reacts with asymmetric ONONO2 in aerosol phase, leading to formation of CH3N3 and then CH3ONO2, with a 26.8 kcal/mol enthalpic barrier, which is 10 kcal/mol higher than the corresponding reaction barrier for MMH. Thus we predicted the low production rate of CH3ONO2 for UDMH/NO2 pair. Experimental evidences support our mechanisms for both MMH and UDMH reacting with NO2.</p
Octopamine Neurons Mediate Flight-Induced Modulation of Visual Processing in Drosophila melanogaster
Get PDFActivity-dependent modulation of sensory systems has been documented in many organisms, and is likely to be essential for appropriate processing of information during different behavioral states. However, the mechanisms underlying these phenomena, and often their functional consequences, remain poorly characterized. I investigated the role of octopamine neurons in the flight-dependent modulation observed in visual interneurons in the fruit fly Drosophila melanogaster. The vertical system (VS) cells exhibit a boost in their response to visual motion during flight compared to quiescence. Pharmacological application of octopamine evokes responses in quiescent flies that mimic those observed during flight, and octopamine neurons that project to the optic lobes increase in activity during flight. Using genetic tools to manipulate the activity of octopamine neurons, I find that they are both necessary and sufficient for the flight-induced visual boost. This work provides the first evidence that endogenous release of octopamine is involved in state-dependent modulation of visual interneurons in flies. Further, I investigated the role of a single pair of octopamine neurons that project to the optic lobes, and found no evidence that chemical synaptic transmission via these neurons is necessary for the flight boost. However, I found some evidence that activation of these neurons may contribute to the flight boost. Wind stimuli alone are sufficient to generate transient increases in the VS cell response to motion vision, but result in no increase in baseline membrane potential. These results suggest that the flight boost originates not from a central command signal during flight, but from mechanosensory stimuli relayed via the octopamine system. Lastly, in an attempt to understand the functional consequences of the flight boost observed in visual interneurons, we measured the effect of inactivating octopamine neurons in freely flying flies. We found that flies whose octopamine neurons we silenced accelerate less than wild-type flies, consistent with the hypothesis that the flight boost we observe in VS cells is indicative of a gain control mechanism mediated by octopamine neurons. Together, this work serves as the basis for a mechanistic and functional understanding of octopaminergic modulation of vision in flying flies
Beyond the Blur: Construction and Characterization of the First Autonomous AO System and an AO Survev of Magnetar Proper Motions
Get PDFAdaptive optics (AO) corrects distortions created by atmospheric turbulence and delivers diffraction-limited images on ground-based telescopes. The vastly improved spatial resolution and sensitivity has been utilized for studying everything from the magnetic fields of sunspots upto the internal dynamics of high-redshift galaxies. This thesis about AO science from small and large telescopes is divided into two parts: Robo-AO and magnetar kinematics.
In the first part, I discuss the construction and performance of the world’s first fully autonomous visible light AO system, Robo-AO, at the Palomar 60-inch telescope. Robo-AO operates extremely efficiently with an overhead < 50s, typically observing about 22 targets every hour. We have performed large AO programs observing a total of over 7,500 targets since May 2012. In the visible band, the images have a Strehl ratio of about 10% and achieve a contrast of upto 6 magnitudes at a separation of 1′′. The full-width at half maximum achieved is 110–130 milli-arcsecond. I describe how Robo-AO is used to constrain the evolutionary models of low-mass pre-main-sequence stars by measuring resolved spectral energy distributions of stellar multiples in the visible band, more than doubling the current sample. I conclude this part with a discussion of possible future improvements to the Robo-AO system.
In the second part, I describe a study of magnetar kinematics using high-resolution near-infrared (NIR) AO imaging from the 10-meter Keck II telescope. Measuring the proper motions of five magnetars with a precision of upto 0.7 milli-arcsecond/yr, we have more than tripled the previously known sample of magnetar proper motions and proved that magnetar kinematics are equivalent to those of radio pulsars. We conclusively showed that SGR 1900+14 and SGR 1806-20 were ejected from the stellar clusters with which they were traditionally associated. The inferred kinematic ages of these two magnetars are 6±1.8 kyr and 650±300 yr respectively. These ages are a factor of three to four times greater than their respective characteristic ages. The calculated braking index is close to unity as compared to three for the vacuum dipole model and 2.5-2.8 as measured for young pulsars. I conclude this section by describing a search for NIR counterparts of new magnetars and a future promise of polarimetric investigation of a magnetars’ NIR emission mechanism.</p
Where Tori Fear to Trend: Hypermassive Neutron Star Remnants and Absolute Event Horizons or Topics in Computational General Relativity
Get PDFComputational general relativity is a field of study which has reached maturity only within the last decade. This thesis details several studies that elucidate phenomena related to the coalescence of compact object binaries. Chapters 2 and 3 recounts work towards developing new analytical tools for visualizing and reasoning about dynamics in strongly curved spacetimes. In both studies, the results employ analogies with the classical theory of electricity and magnitism, first (Ch. 2) in the post-Newtonian approximation to general relativity and then (Ch. 3) in full general relativity though in the absence of matter sources. In Chapter 4, we examine the topological structure of absolute event horizons during binary black hole merger simulations conducted with the SpEC code. Chapter 6 reports on the progress of the SpEC code in simulating the coalescence of neutron star-neutron star binaries, while Chapter 7 tests the effects of various numerical gauge conditions on the robustness of black hole formation from stellar collapse in SpEC. In Chapter 5, we examine the nature of pseudospectral expansions of non-smooth functions motivated by the need to simulate the stellar surface in Chapters 6 and 7. In Chapter 8, we study how thermal effects in the nuclear equation of state effect the equilibria and stability of hypermassive neutron stars. Chapter 9 presents supplements to the work in Chapter 8, including an examination of the stability question raised in Chapter 8 in greater mathematical detail
Virtuosity
No full text[Introduction] The moments before beauty are often the most chaotic. Legions of chattering spectators pick their way through the rows of seats, flipping fervently through the program, exclaiming praise and criticism upon the conductor, the repertoire, the concertmaster, and the soloist
Point of Care Molecular Diagnostics for Humanity
Get PDFDiagnostics of disease at POC (point of care) has been declared one of the Grand Challenge by the Bill and Melina Gates Foundation (BMGF). Infectious diseases constitute a major cause of disease burden and cause more than half a billion Disability-Adjusted Life Years (DALYs) and millions of deaths each year. They have an especially large effect on children under 5 years of age. We have analyzed data from the GBD 2010 (Global Burden of Disease) project to emphasize the damage caused by infectious diseases, and highlight the opportunity of using diagnostic tools to rapidly identify and treat diseases. To motivate the work of this thesis, we quantify the expected impact of appropriate diagnostic technologies.
We have also analyzed the requirements that a diagnostic tool should meet to generate the maximal global impact. We present various existing TPPs (Target Product Profiles) from different organizations and suggest some additions to these existing TPPs. We explain the particular molecular pathology technologies which have the potential to allow deployment of functional products in the developing world for point-of-care pathogen detection, especially in low-resource settings.
We perform a detailed analysis on existing polymerase chain reaction (PCR) systems and describe the problems caused with thermal performance and optical interrogation. We list the requirements that disposable cartridges for such instruments should meet and suggest a metal base design with polymer top. After detailed FEA simulations, we demonstrate that the thermal response can be modeled using a one-dimensional (1D) lumped element system. We show improvements in thermal response due to using a metal base and the effect of fluid height. We also performed thermal-structural simulations to quantify the stresses on the adhesive bonds of metal/polymer cartridges.
Next, we explain fabrication of these cartridges. We show methods to dispense adhesive using a robot and a custom made jig to spread the adhesive during curing. The cartridge was tested with different PCR reagents and we obtained reaction efficiencies approaching those of the commercial real time PCR machines. Our fabrication technique is useful to join dissimilar materials and is production friendly. By developing custom software, we observed the cartridge performance in a continuous manner. We could see the thermal response of cartridges by continuous fluorescence monitoring, and used reflective aluminum which increase light collection efficiency.
We then present a simple and robust new way for thermal cycling. Robust thermal cycling has been a major challenge conducting PCR, especially in point of care situations. Here, we suggest a contact cooling approach, in which the cartridge rests on a thin metal plate with an integrated thin heater constructed from flexible printed circuit board (PCB) material. We use a solenoid to move a metal plate to cool down the sample cartridge during cycling. The metal plate then rests on a larger heat sink to disperse the shuttled heat. Our design is dust and water proof and was verified on a bench-top prototype.
A novel optical design for fluorescence detection during qPCR is also described. We suggest a lateral illumination waveguide geometry with prism coupling that eliminates lenses and is integrated into an injection molded cartridge. The light is homogenized using a light guide, and we quantify the sources of scattered stray light from the chamber edge by performing ray tracing simulations to optimize the precise geometry. The design is tolerant to misalignments and enables easy coupling of LED light into the chamber. As the light collection efficiency is high, the size of the chamber can be very small. We tested real PCR reactions using this concept and observed a rapid integration time, enabling very fast reading.
Sample preparation has been another challenge for all point-of-care (POC) lab-on-chip devices for many years. Here, we propose a new design which is robust, fast, flexible and simple, and uses a sliding seal to move the collected sample between various reservoir chambers. The sample moves on a slider sandwiched between seals that shuttles a DNA binding membrane between different reactions. Thus, size and volumes of reagents can be increased without increasing dead volumes. This design is easily automated, and positive displacement of fluids can work with many reagents without worrying about their characteristics such as foaming. The speed of the sample preparation protocols is high and complex protocols can be ported on this design concept, which we tested on real clinical samples and obtained impressive results. We designed and injection molded devices to test and verify this concept.
Finally, we focus on instrumentation and software required to allow our technology to be used at the POC. We describe our embedded electronics and describe the powerful micro-controller and various high performance ICs that are used to construct a fully functional for sample to answer instrument. We developed various versions of software. The developer software allows us to control our system and bench top setup. Our end user product includes a tablet and cell phone software interface. Software was developed for a windows 8 tablet, windows 8 phone and an Android based devices.
To conclude, we very briefly describe the POC systems that are under development: A portable qPCR system with a separate cartridge design, and a universal sample to answer system that performs qPCR, sample preparation and sample to answer protocols in one box depending on the cartridge.
As per best of our knowledge the cost of this technology is much lower than any other option in its class. The sample to answer instrument is expected to cost less than 5. The performance is not compromised. We hope that this work can help bring a transformative change in the practice of pathology especially in the developing world.</p
Integrated Microsystems for Wireless Sensing Applications
Get PDFPersonal health monitoring is being considered the future of a sustainable health care system. Biosensing platforms are a very important component of this system. Real-time and accurate sensing is essential for the success of personal health care model. Currently, there are many efforts going on to make these sensors practical and more useful for such measurements. Implantable sensors are considered the most widely applicable and most reliable sensors for such accurate health monitoring applications. However, macroscopic (cm scale) size has proved to be a limiting factor for successful use of these systems for long time and in large numbers. This work is focused to resolve the issues related with miniaturizing these devices to a microscopic (mm scale) size scale which can minimize many practical difficulties associated with their larger counterparts currently.
To accomplish this goal of miniaturization while retaining or even improving on the necessary capabilities for such sensing platforms, an integrated approach is presented which focuses on system-level miniaturization using standard fabrication procedures. First, it is shown that a completely integrated and wireless system is the best solution to achieve desired miniaturization without sacrificing the functionality of the system. Hence, design and implementation of the different components comprising the complete system needs to be done according to the requirements of the overall integrated system. This leads to the need of on-chip functional sensors, integrated wireless power supply, integrated wireless communication and integrated control system for realization of such system. In this work, different options for implementation of each of these subsystems are compared and an optimal solution is presented for each subsystem. For such complex systems, it is imperative to use a standard fabrication process which can provide the required functionality for all subsystems at smallest possible size scale. Complementary Metal Oxide Semiconductor (CMOS) process is the most appropriate of the technologies in this regard and has enabled incredible miniaturization of the computing industry. It also provides options for designing different subsystems on the same platform in a monolithic process with very high yield. This choice then leads to actual designs of subsystems in the CMOS technology using different possible methods. Careful comparison of these subsystems provides insights into different design adjustments that are made until the desired functions are achieved at the desired size scale. Integration of all these compatible subsystems in the same platform is shown to provide the smallest possible sensing platform to date.
The completely wireless system can measure a host of different important analyte and can transmit the data to an external device which can use it for appropriate purpose. Results on measurements in phosphate buffer solution, blood serum and whole blood along with wireless communication in real biological tissues are provided. Specific examples of glucose and DNA sensors are presented and the use for many other relevant applications is also proposed. Finally, insights into animal model studies and future directions of the research are discussed. </p
Bright-Field and Fluorescence Chip-Scale Microscopy for Biological Imaging
Get PDFOptical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.
Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.
Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.
Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.
Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.</p
Crystals that Count! Physical Principles and Experimental Investigations of DNA Tile Self-Assembly
Get PDFAlgorithmic DNA tiles systems are fascinating. From a theoretical perspective, they can result in simple systems that assemble themselves into beautiful, complex structures through fundamental interactions and logical rules. As an experimental technique, they provide a promising method for programmably assembling complex, precise crystals that can grow to considerable size while retaining nanoscale resolution. In the journey from theoretical abstractions to experimental demonstrations, however, lie numerous challenges and complications.
In this thesis, to examine these challenges, we consider the physical principles behind DNA tile self-assembly. We survey recent progress in experimental algorithmic self-assembly, and explain the simple physical models behind this progress. Using direct observation of individual tile attachments and detachments with an atomic force microscope, we test some of the fundamental assumptions of the widely-used kinetic Tile Assembly Model, obtaining results that fit the model to within error. We then depart from the simplest form of that model, examining the effects of DNA sticky end sequence energetics on tile system behavior. We develop theoretical models, sequence assignment algorithms, and a software package, StickyDesign, for sticky end sequence design.
As a demonstration of a specific tile system, we design a binary counting ribbon that can accurately count from a programmable starting value and stop growing after overflowing, resulting in a single system that can construct ribbons of precise and programmable length. In the process of designing the system, we explain numerous considerations that provide insight into more general tile system design, particularly with regards to tile concentrations, facet nucleation, the construction of finite assemblies, and design beyond the abstract Tile Assembly Model.
Finally, we present our crystals that count: experimental results with our binary counting system that represent a significant improvement in the accuracy of experimental algorithmic self-assembly, including crystals that count perfectly with 5 bits from 0 to 31. We show some preliminary experimental results on the construction of our capping system to stop growth after counters overflow, and offer some speculation on potential future directions of the field.</p