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Planar Reflection of Gaseous Detonation
Get PDFPipes containing flammable gaseous mixtures may be subjected to internal detonation. When the detonation normally impinges on a closed end, a reflected shock wave is created to bring the flow back to rest. This study built on the work of Karnesky (2010) and examined deformation of thin-walled stainless steel tubes subjected to internal reflected gaseous detonations. A ripple pattern was observed in the tube wall for certain fill pressures, and a criterion was developed that predicted when the ripple pattern would form. A two-dimensional finite element analysis was performed using Johnson-Cook material properties; the pressure loading created by reflected gaseous detonations was accounted for with a previously developed pressure model. The residual plastic strain between experiments and computations was in good agreement.
During the examination of detonation-driven deformation, discrepancies were discovered in our understanding of reflected gaseous detonation behavior. Previous models did not accurately describe the nature of the reflected shock wave, which motivated further experiments in a detonation tube with optical access. Pressure sensors and schlieren images were used to examine reflected shock behavior, and it was determined that the discrepancies were related to the reaction zone thickness extant behind the detonation front. During these experiments reflected shock bifurcation did not appear to occur, but the unfocused visualization system made certainty impossible. This prompted construction of a focused schlieren system that investigated possible shock wave-boundary layer interaction, and heat-flux gauges analyzed the boundary layer behind the detonation front. Using these data with an analytical boundary layer solution, it was determined that the strong thermal boundary layer present behind the detonation front inhibits the development of reflected shock wave bifurcation.</p
Experimental Apparatus for Measuring Ultralow Thermal Conductivity Structures
No full textUltralow thermal conductivity materials play an important role in many
applications such as space exploration and thermal insulation. One of the primary
challenges in studying heat conduction in these materials is performing basic
thermal conductivity measurements, because even a small amount of steady heating
results in enormous temperature increases. While methods do exist to measure the
thermal conductivities of macroscopic materials, these techniques are difficult to
apply to the microscopic samples that are the only samples available for some
materials.
In this thesis, we describe an optical, non-contact experimental system for
measuring ultralow thermal conductivities. The system we designed and built is a
modified pump-and-probe system. With this system, we will be able to measure a
sample as small as tens of microns without having physical contact between the
tester and the sample. Different from a traditional pump-and-probe method, we
used a pump laser with a very low repetition rate of a few hundred Hz, which can
efficiently reduce the steady heating, and at the same time the transient heating is
still enough to give a measurement result.
In the following chapters, we will first discuss the background of this thesis,
including a brief introduction to the manufacture of the nano-lattice and the
principles of the pump-and-probe method. Then we will go through some
calculations done during the design process of the apparatus. Finally, we will
present the test results from the experiments and compare them against the
simulation.</p
Synthetic Regulation of Eukaryotic Gene Expression by Noncoding RNA
Get PDFSynthetic biological systems promise to combine the spectacular diversity of biological functionality with engineering principles to design new life to address many pressing needs. As these engineered systems advance in sophistication, there is ever-greater need for customizable, situation-specific expression of desired genes. However, existing gene control platforms are generally not modular, or do not display performance requirements required for robust phenotypic responses to input signals. This work expands the capabilities of eukaryotic gene control in two important directions.
For development of greater modularity, we extend the use of synthetic self-cleaving ribozyme switches to detect changes in input protein levels and convey that information into programmed gene expression in eukaryotic cells. We demonstrate both up- and down-regulation of levels of an output transgene by more than 4-fold in response to rising input protein levels, with maximal output gene expression approaching the highest levels observed in yeast. In vitro experiments demonstrate protein-dependent ribozyme activity modulation. We further demonstrate the platform in mammalian cells. Our switch devices do not depend on special input protein activity, and can be tailored to respond to any input protein to which a suitable RNA aptamer can be developed. This platform can potentially be employed to regulate the expression of any transgene or any endogenous gene by 3’ UTR replacement, allowing for more complex cell state-specific reprogramming.
We also address an important concern with ribozyme switches, and riboswitch performance in general, their dynamic range. While riboswitches have generally allowed for versatile and modular regulation, so far their dynamic ranges of output gene modulation have been modest, generally at most 10-fold. We address this shortcoming by developing a modular genetic amplifier for near-digital control of eukaryotic gene expression. We combine ribozyme switch-mediated regulation of a synthetic TF with TF-mediated regulation of an output gene. The amplifier platform allows for as much as 20-fold regulation of output gene expression in response to input signal, with maximal expression approaching the highest levels observed in yeast, yet being tunable to intermediate and lower expression levels. EC50 values are more than 4 times lower than in previously best-performing non-amplifier ribozyme switches. The system design retains the modular-input architecture of the ribozyme switch platform, and the near-digital dynamic ranges of TF-based gene control.
Together, these developments suggest great potential for the wide applicability of these platforms for better-performing eukaryotic gene regulation, and more sophisticated, customizable reprogramming of cellular activity.</p
Chemical Probes to Study Fucosylated Glycans
Get PDFFucosylated glycans have many critical biological roles, from leukocyte adhesion to host-microbe interactions. However, a molecular level understanding of these sugars has been lacking, in part due to the chemical and structural diversity of glycans that make them challenging to study. In order to gain a deeper understanding of fucosylated glycans, we have explored the use of chemical probes to study these structures. In Chapters 1 and 2, we apply a metabolic labeling technique for the investigation of fucosylated glycans in neurons, where they have been implicated in learning and memory processes. However, the molecular mechanisms by which these sugars influence neuronal processes are not well understood, and only a handful of fucosylated glycoproteins have been identified. In order to facilitate our understanding of these processes, we exploit non-natural fucose analogs to identify the fucose proteome in rat cortical neurons, identifying proteins involved in cell adhesion, neuronal signaling, and synaptic transmission. Moreover, we track fucosylated glycoproteins in hippocampal neurons, and show that fucosylated glycoproteins localize to the Golgi, axons, and dendrites, and are enriched in synapses. In Chapter 4, we report a new chemoenzymatic strategy for the sensitive detection of the Fucα(1-2)Gal epitope, which has been implicated in tumorigenesis as a potential biomarker of cancer progression. We demonstrate that the approach is highly selective for the Fucα(1-2)Gal motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of the motif on live cells, discriminating malignant from normal cells. These approaches represent new potential applications and strategies for the investigation of fucosylated glycans, and expand the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease
Innovations in Imaging System Design: Gigapixel, Chip-Scale and MultiFunctional Microscopy
Get PDFMicroscopy imaging is of fundamental importance in diverse disciplines of science and technology. In a typical microscopy imaging platform, the light path can be generalized to the following steps: photons leave the light source, interact with the sample, and finally are detected by the image sensor. Based on such a light path, this thesis presents several new microscopy imaging techniques from three aspects: illumination design, sample manipulation, and imager modification.
The first design strategy involves the active control of the illumination sources. Based on this strategy, we demonstrate a simple and cost-effective imaging method, termed Non-interferometric Aperture-synthesizing Microscopy (NAM), for breaking the spatial-bandwidth product barrier of a conventional microscope. We show that the NAM method is capable of providing two orders of magnitude higher throughput for most existing bright-field microscopes without involving any mechanical scanning. Based on NAM, we report the implementation of a 1.6 gigapixel microscope with a maximum numerical aperture of 0.5, a field-of-view of 120 mm2, and a resolution-invariant imaging depth of 0.3 mm. This platform is fast (acquisition time of ~ 3 minutes), free from chromatic aberration, capable for phase imaging, and, most importantly, compatible with most existing microscopes. High quality color images of histology slides were acquired by using such a platform for demonstration. The proposed NAM method provides a robust way to transform the problem of high-throughput microscopy from one that is tied to physical limitations of the optics to one that is computationally solvable. The active control of illumination sources can also be adapted for chip-scale microscopy imaging. To this end, we present a lensless microscopy solution termed ePetri-dish. This ePetri-dish platform can automatically perform high resolution (~ 0.66 micron) microscopy imaging over a large field-of-view (6 mm × 4 mm). This new approach is fully capable of working with cell cultures or any samples in which cells/bacteria may be contiguously connected, and thus, it can significantly improve Petri-dish-based cell/bacteria culture experiments. With this approach providing a low-cost and disposable microscopy solution, we can start to transit Petri-dish-based experiments from the traditionally labor-intensive process to an automated and streamlined process.
The second strategy in design considerations is to manipulate the sample. We present a fully on-chip, lensless, sub-pixel resolving optofluidic microscope (SROFM). This device utilizes microfluidic flow to deliver specimens directly across an image sensor to generate a sequence of low-resolution projection images, where resolution is limited by the sensor’s pixel size. This image sequence is then processed to reconstruct a single high-resolution image, where features beyond the Nyquist rate of the LR images are resolved. We demonstrate the device’s capabilities by imaging microspheres, protist Euglena gracilis, and Entamoeba invadens cysts with sub-cellular resolution.
The third accessing point in design considerations is the image sensor. Imager modification is an emerging technique that performs pre-detection light field manipulation. We present two novel optical structure designs: surface-wave-enabled darkfield aperture (SWEDA) and light field sensor. These structures can be directly incorporated onto optical sensors to accomplish pre-detection background suppression and wavefront sensing. We further demonstrate SWEDA’s ability to boost the detection sensitivity, with a contrast enhancement of 27 dB.</p
Engineering Nucleic Acid Mechanisms for Regulation and Readout of Gene Expression: Conditional Dicer Substrate Formation and Sensitive Multiplexed Northern Blots
Get PDFNucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.
RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.
Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.</p
Microglia in the Cerebral and Cerebellar Cortices in Individuals with Autism
Get PDFIn this thesis, we explore the density of the microglia in the cerebral and cerebellar cortices of individuals with autism to investigate the hypothesis that neuroinflammation is involved in autism. We describe in our findings an increase in microglial density in two disparate cortical regions, frontal insular cortex and visual cortex, in individuals with autism (Tetreault et al., 2012). Our results imply that there is a global increase in the microglial density and neuroinflammation in the cerebral cortex of individuals with autism.
We expanded our cerebellar study to additional neurodevelopmental disorders that exhibit similar behaviors to autism spectrum disorder and have known cerebellar pathology. We subsequently found a more than threefold increase in the microglial density specific to the molecular layer of the cerebellum, which is the region of the Purkinje and parallel fiber synapses, in individuals with autism and Rett syndrome. Moreover, we report that not only is there an increase in microglia density in the molecular layer, the microglial cell bodies are significantly larger in perimeter and area in individuals with autism spectrum disorder and Rett syndrome compared to controls that implies that the microglia are activated. Additionally, an individual with Angelman syndrome and the sibling of an individual with autism have microglial densities similar to the individuals with autism and Rett syndrome. By contrast, an individual with Joubert syndrome, which is a developmental hypoplasia of the cerebellar vermis, had a normal density of microglia, indicating the specific pathology in the cerebellum does not necessarily result in increased microglial densities. We found a significant decrease in Purkinje cells specific to the cerebellar vermis in individuals with autism.
These findings indicate the importance for investigation of the Purkinje synapses in autism and that the relationship between the microglia and the synapses is of great utility in understanding the pathology in autism. Together, these data provide further evidence for the neuroinflammation hypothesis in autism and a basis for future investigation of neuroinflammation in autism. In particular, investigating the function of microglia in modifying synaptic connectivity in the cerebellum may provide key insights into developing therapeutics in autism spectrum disorder.</p
Development of a Novel Ring Contraction Strategy and Application to the Total Synthesis of Presilphiperfolanol Natural Products
Get PDFBiologically active and structurally complex natural products provide a powerful driving force for the development of novel reaction methodology. Major advances can reshape the way chemists approach the construction of challenging chemical bonds.
In this work, we begin by describing the development of a catalytic asymmetric synthesis of five and seven-membered rings containing all-carbon quaternary stereocenters. Enantioselective Pd-catalyzed decarboxylative allylic alkylation reactions of β-ketoester substrates afforded a variety of chiral seven-membered α-quaternary vinylogous esters. Initial attempts to convert these compounds to γ-quaternary cycloheptenones led to the discovery of a two-carbon ring contraction reaction, which provided isomeric γ-quaternary acylcyclopentenes. Subsequent adjustment of reaction parameters provided divergent access to the originally targeted cycloheptenones. Numerous synthetic applications of the two versatile product types are demonstrated. The methodology expands on our previous investigations of six-membered ring scaffolds and provides additional chiral building blocks for asymmetric total synthesis.
The ring contraction approach to acylcyclopentenes was further developed in the total synthesis of the presilphiperfolanols, which are important intermediates in sesquiterpene biosynthesis. Key to our synthetic route to the tricyclic core was the application of intramolecular Diels–Alder and Ni-catalyzed 1,4-hydroboration reactions. From these efforts, the enantioselective total synthesis of presilphiperfolan-1β-ol was achieved. Future research efforts seek to extend the synthetic route to presilphiperfolan-9α-ol and study the synthetic compounds in biomimetic carbocation rearrangement processes.</p
Reactions of Small Molecules Facilitated by Metal-Acceptor Interactions
Get PDFThe role of metal-acceptor interactions arising from M–BR3 and M–PR3 bonding is discussed with respect to reactions between first-row transition metals and N2, H2, and CO. Thermally robust, S = 1/2 (TPB)Co(H2) and (TPB)Co(N2) complexes (TPB = tris(2- (diisopropylphosphino)phenyl)borane) are described and the energetics of N2 and H2 binding are measured. The H2 and N2 ligands are bound more weakly in the (TPB)Co complexes than in related (SiP3)M(L) complexes (SiP3 = tris(2- (diisopropylphosphino)phenyl)silyl). Comparisons within and between these two ligand platforms allow for the factors that affect N2 (and H2) binding and activation to be delineated. The characterization and reactivity of (DPB)Fe complexes (DPB = bis(2- (diisopropylphosphino)phenyl)phenylborane) in the context of N2 functionalization and E–H bond addition (E = H, C, N, Si) are described. This platform allows for the one-pot transformation of free N2 to an Fe hydrazido(-) complex via an Fe aminoimide intermediate. The principles learned from the N2 chemistry using (DPB)Fe are applied to CO reduction on the same system. The preparation of (DPB)Fe(CO)2 is described as well as its reductive functionalization to generate an unprecedented Fe dicarbyne. The bonding in this highly covalent complex is discussed in detail. Initial studies of the reactivity of the Fe dicarbyne reveal that a CO-derived olefin is released upon hydrogenation. Alternative approaches to uncovering unusual reactivity using metal- acceptor interactions are described in Chapters 5 and 6, including initial studies on a new π-accepting tridentate diphosphinosulfinyl ligand and strategies for designing ligands that undergo site-selective metallation to generate heterobimetallic complexes
Bandgap Engineering Silicon Nanopillars
No full textVertically oriented , bandgap engineered silicon nanopillars were fabricated and addressed. Devices were fabricated via a three dimensional etching process which created sub-5 nm constrictions in silicon radius upon oxidation. This effect was used to create a Coulomb blockade device. Devices were tested at room and liquid nitrogen temperatures. They showed a clear blockade effect distinctive of an asymmetric double tunnel junction at low temperatures which disappeared when tested at higher temperatures. Different device fabrication parameters were also tested to develop high-current devices, including chip anneal time. Furthermore, both device fabrication steps and current flow were modeled and simulated