Investigation of Lung Structure-Function Relationships Using Hyperpolarized Noble Gases

Magnetic Resonance Imaging (MRI) is an application of the nuclear magnetic resonance (NMR) phenomenon to non-invasively generate 3D tomographic images. MRI is an emerging modality for the lung, but it suffers from low sensitivity due to inherent low tissue density and short T2*. Hyperpolarization is a process by which the nuclear contribution to NMR signal is greatly enhanced to more than 100,000 times that of samples in thermal equilibrium. The noble gases 3He and 129Xe are most often hyperpolarized by transfer of light angular momentum through the electron of a vaporized alkali metal to the noble gas nucleus (called Spin Exchange Optical Pumping). The enhancement in NMR signal is so great that the gas itself can be imaged via MRI, and because noble gases are chemically inert, they can be safely inhaled by a subject, and the gas distribution within the interior of the lung can be imaged. The mechanics of respiration is an elegant physical process by which air is is brought into the distal airspaces of the lungs for oxygen/carbon dioxide gas exchange with blood. Therefore proper description of lung function is intricately related to its physical structure, and the basic mechanical operation of healthy lungs -- from pressure driven airflow, to alveolar airspace gas kinetics, to gas exchange by blood/gas concentration gradients, to elastic contraction of parenchymal tissue -- is a process decidedly governed by the laws of physics. This dissertation will describe experiments investigating the relationship of lung structure and function using hyperpolarized (HP) noble gas MRI. In particular HP gases will be applied to the study of several pulmonary diseases each of which demonstrates unique structure-function abnormalities: asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Successful implementation of an HP gas acquisition protocol for pulmonary studies is an involved and stratified undertaking which requires a solid theoretical foundation in NMR and hyperpolarization theory, construction of dedicated hardware, development of dedicated software, and appropriate image analysis techniques for all acquired data. The author has been actively involved in each of these and has dedicated specific chapters of this dissertation to their description. First, a brief description of lung structure-function investigations and pulmonary imaging will be given (chapter 1). Brief discussions of basic NMR, MRI, and hyperpolarization theory will be given (chapters 2 and 3) followed by their particular methods of implementation in this work (chapters 4 and 5). Analysis of acquired HP gas images will be discussed (chapter 6), and the investigational procedures and results for each lung disease examined will be detailed (chapter 7). Finally, a quick digression on the strengths and limitations of HP gas MRI will be provided (chapter 8)

Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles

Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development

Unzipping Dynamics of Long DNAs

The two strands of the DNA double helix can be `unzipped' by application of 15 pN force. We analyze the dynamics of unzipping and rezipping, for the case where the molecule ends are separated and re-approached at constant velocity. For unzipping of 50 kilobase DNAs at less than about 1000 bases per second, thermal equilibrium-based theory applies. However, for higher unzipping velocities, rotational viscous drag creates a buildup of elastic torque to levels above kBT in the dsDNA region, causing the unzipping force to be well above or well below the equilibrium unzipping force during respectively unzipping and rezipping, in accord with recent experimental results of Thomen et al. [Phys. Rev. Lett. 88, 248102 (2002)]. Our analysis includes the effect of sequence on unzipping and rezipping, and the transient delay in buildup of the unzipping force due to the approach to the steady state.Comment: 15 pages Revtex file including 9 figure

Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life

Performance of prototypes for the ALICE electromagnetic calorimeter

The performance of prototypes for the ALICE electromagnetic sampling calorimeter has been studied in test beam measurements at FNAL and CERN. A $4\times4$ array of final design modules showed an energy resolution of about 11% /$\sqrt{E(\mathrm{GeV})}$ $\oplus$ 1.7 % with a uniformity of the response to electrons of 1% and a good linearity in the energy range from 10 to 100 GeV. The electromagnetic shower position resolution was found to be described by 1.5 mm $\oplus$ 5.3 mm /$\sqrt{E \mathrm{(GeV)}}$. For an electron identification efficiency of 90% a hadron rejection factor of $>600$ was obtained.Comment: 10 pages, 10 figure

Statistical Analysis of Molecular Signal Recording

A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a “molecular ticker tape”, in which an engineered DNA polymerase (DNAP) writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1) the polymerase biochemical parameters, and (2) the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.United States. Defense Advanced Research Projects Agency. Living Foundries ProgramGoogle (Firm)New York Stem Cell Foundation. Robertson Neuroscience Investigator AwardNational Institutes of Health (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Transformative R01 1R01GM104948)National Institutes of Health (U.S.) (Single Cell Grant 1 R01 EY023173)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (CAREER Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS1042134)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award