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Mucosal Landscape of the Gut: Development and Application of 3D Imaging Tools for Interrogation of Host-Microbe Mucosal Interface in Mice and Humans
Get PDFMammalian gastrointestinal tract is inhabited by trillions of microbes that, in number, amount to the total number of cells in the human body. These microbes, collectively known as microbiota, are found on the skin and in body cavities, and come in close contact with the host on mucosal surfaces. Here, pivotal host-microbe interactions likely take place because close proximity to the host enhances the uptake of microbial metabolites by the host and enables direct contact. To aid the investigation of these interactions, we developed an imaging technology that preserves fragile mucosal structure, enables to explore large areas of mucosal surface, and image the structurally and biochemically complex host-microbe interface in 3D in a mouse. However, 3D imaging presents challenges, such as slow transport of large molecular weight reagents and low signal/background ratio at depth, and these challenges are further exacerbated in particularly thick samples, such as small intestinal samples with long finger-like villus protrusions and thick human gut samples. Therefore, we further advanced our technology to improve sensitivity and specificity at depth, and we have taken steps to translate our technology to precious resected human gut samples from inflammatory bowel disease patients. Finally, we applied these tools to interrogate Enterobacteriaceae – Bacteroidaceae interactions in the small intestine of a mouse weakened by malnutrition. Using complementary tools, we have first determined that Bacteroidaceae required malnutrition to increase in number in the jejunum digesta, whereas Enterobacteriaceae required both malnutrition and Bacteroidaceae. With imaging, we visualized that in malnourished mice not exposed to Enterobacteriaceae and Bacteroidaceae, bacteria were effectively cleared after digesta passage, whereas in exposed mice bacterial retention was detected, suggestive of bacterial adherence to and colonization of mucosa. Finally, we detected a rare event of abundant bacterial colonization of small intestinal mucosa and captured in 3D.</p
Spatial and Temporal Dynamics of Microorganisms Living Along Steep Energy Gradients and Implications for Ecology and Geologic Preservation in the Deep Biosphere
Get PDFThe deep biosphere represents a massive repository of life with unknown effects on global biogeochemical cycles. Even the fundamental life strategies of the endemic microorganisms that inhabit this biome remain enigmatic; some studies have indicated that subsurface organisms subsist in energetic regimes below the theoretical lower limit for life. A boom-bust life cycle, mediated by tectonic disturbances and subsurface fractures, may help explain these phenomena. This work addresses and expands on this question, first by exploring the response of continental deep biosphere microorganisms to an in situ organic matter amendment, then by analyzing the microbial community dynamics of the sediments and carbonate along a naturally-occurring energy gradient at a methane seep. Our experiments in the continental deep biosphere confirmed that mineralogical heterogeneity can drive differential colonization of the native microorganisms, implying that selection and adaptation to in situ conditions occurs, differentiating individual microbial niches. We also observed the formation of secondary framboidal iron sulfide minerals, a well-known phenomenon in marine sediments but not extensively observed in the deep subsurface, that were correlated to the presence of abundant sulfur-metabolizing microorganisms. Chapters 2 and 3 are instead focused on the microbial ecology of a methane seep on the Pacific margin of Costa Rica. Cold methane seeps themselves represent sharp boundaries between the generally low-energy background seafloor and abundant chemical energy in the form of methane. Chapter 2 describes that the microorganisms living at these seeps occupy a significantly narrower spatial scale than the endemic megafauna. In addition, by correlating community dissimilarity and geographic distance, the functional center of the seep was identified, allowing for insight into the ecological differentiation between clades of anaerobic methanotrophic archaea (ANME). Chapter 3 examines in greater detail the endolithic microbial community, principally composed of ANME-1. By conducting transplantation experiments of carbonates on the seafloor, we tested the response of the in situ endolithic communities and found that carbonates moved distinctly outside the active zone changed less than communities moved to regions of less activity.</p
Nanometer-sized Aerosol Particles in the Atmosphere: Measurement, Analysis, and Impact
Get PDFNew particle formation (NPF) from gaseous precursor vapors is frequently observed in the ambient environment and contributes to a major source of global cloud condensation nuclei (CCN). The survival and CCN activation of newly formed particles are highly dependent on particle growth below 10 nm. Characterizing and understanding nanoparticle early growth will therefore help to quantify the impact of NPF on cloud reflectivity and global energy budget. In this work, I first present a recently developed instrument, the Caltech nano-Scanning Electrical Mobility Spectrometer (nSEMS), which consists of a charge conditioner, a novel differential mobility analyzer (DMA), and a two-stage condensation particle counter (CPC). This new design, coupled with a data inversion method that combines empirical calibration and COMSOL simulation, can help to measure nanoparticle size distributions from 1.5 nm to 25 nm more accurately. This instrument was employed in the experiments conducted in the Cosmics Leaving OUtdoor Droplets (CLOUD) chamber at the European Organization for Nuclear Research (CERN) to better understand NPF, particle growth and survival. Multiple experimental parameters were varied to study the influence of different highly oxygenated molecules (HOMs) and inorganic trace gases, such as ammonia and nitrogen oxides on particle early growth. Experiment results have suggested a novel mechanism that may help to explain nanoparticle formation and growth in highly polluted urban environments or in the cold free troposphere. In as little as a few minutes, freshly nucleated particles as small as 2 nanometers in diameter can grow very rapidly due to simultaneous condensation of nitric acid and ammonia. This can help them to survive through the so-called "valley of death" where they would otherwise be lost to larger particles, and instead allow them to grow to sizes where they are less vulnerable to loss and can continue on to sizes where they influence local air quality or climate. Further, the laboratory results of nanoparticle growth were incorporated into the Global Model of Aerosol Processes (GLOMAP) model to study the impact of this extremely rapid growth on the global CCN budget. Having realized the importance of conducting well-controlled chamber experiments and of using chamber experimental data, we established an online data infrastructure, the Index of Chamber Atmospheric Research in the United States (ICARUS), for storing, sharing, and using chamber data. A combined effort of the described works contributes to better measuring the size distribution of nanoparticles and to understanding their impact on global climate.</p
Mechanisms and Consequences of Bacterial Resistance to Natural Antibiotics
Get PDFMany bacteria secrete natural antibiotics—toxic small molecules that can kill or inhibit the growth of other microorganisms. Several of these compounds have been commercialized as antimicrobial drugs, and the mechanisms and public health consequences of bacterial resistance to clinically-used antibiotics are well understood. By contrast, the role of bacterially-produced antibiotics in natural environments, where they have existed for millions of years, remains an open question. Besides potentially serving as tools of warfare between competing microbes, natural antibiotics have been proposed to serve less antagonistic functions ranging from the acquisition of nutrients to the transmission of signals between cells. Indeed, despite evidence that natural antibiotics can suppress sensitive microbes in environments such as the soil surrounding plant roots, the ecological significance of the toxicity of these molecules has sometimes been questioned. At the same time, for most natural antibiotics, the mechanisms and prevalence of resistance are either poorly characterized or entirely unknown.
This thesis addresses the molecular mechanisms and consequences of bacterial resistance to a particular class of redox-active natural antibiotics called phenazines. Phenazines are produced by a major opportunistic human pathogen, Pseudomonas aeruginosa, during infections, as well as by several bacterial species that associate with the roots of crops such as wheat, where they serve to protect their plant hosts against fungal pathogens. Resistance to this family of natural antibiotics is therefore potentially relevant to multiple sectors of human society. I begin by investigating the intrinsic phenazine resistance of a common soil bacterium, Agrobacterium tumefaciens, that does not itself produce phenazines. Using a functional genetics approach, I find that the composition of the respiratory electron transport chain plays a critical role in mitigating phenazine toxicity, one that cannot be compensated by increased expression of efflux pumps that transport phenazines out of the cell or oxidative stress responses that neutralize the toxic byproducts of phenazine redox-cycling. Subsequently, we turn to P. aeruginosa, the phenazine-producing opportunistic pathogen, and demonstrate that the defenses it activates against its own toxic phenazine, pyocyanin, collaterally accelerate the acquisition of resistance to certain clinical antibiotics. Other bacteria known to form multispecies infections with P. aeruginosa can also benefit from exposure to pyocyanin in the presence of these clinical antibiotics; we show that in at least one strain isolated from a patient, the effect of pyocyanin on the frequency of spontaneous antibiotic-resistant mutants rivals that of disruptions in DNA repair machinery. Importantly, a growing body of reports suggests that, besides pyocyanin, other metabolites produced by bacterial pathogens can also affect the efficacy of clinical antibiotics. We review the evidence for which types of bacterial metabolites alter susceptibility to antimicrobial drugs, as well as the mechanisms underlying this phenomenon. Finally, I examine the prevalence of bacterial resistance to an agriculturally-relevant phenazine in a wheat field where the use of native phenazine producers to control crop diseases has been studied for decades. I discover that while Gram-positive bacteria are generally more susceptible to this phenazine compared to Gram-negative bacteria, the sharpness of this distinction is pH-dependent; moreover, I uncover surprising heterogeneity in phenazine resistance within certain taxonomic groups. Taken together, these findings illuminate recurring themes in mechanisms of phenazine resistance and point to an underappreciated role for natural antibiotics in the resilience of opportunistic pathogens to clinical antibiotics. This thesis also lays the groundwork for developing a predictive model of phenazine resistance across diverse bacteria, with potential implications for optimizing the use of clinical antibiotics and improving agricultural sustainability.</p
Renewable Energy Generation and Storage: From Microwires to Macro-Energy Systems
Get PDFCurrent trajectories require urgent action to reach net-zero greenhouse gas emissions targets. This thesis focuses on efforts to support decarbonization through the development of solar fuels devices to produce green hydrogen and through macro-energy systems modeling.
Long-term stability of light-absorbing materials remains a substantial barrier to the viability of solar fuel devices. In this thesis, we identify corrosion pathways in TiO₂-protected silicon microwire arrays in a polymer membrane either attached to a substrate or free-standing. Both top-down and bottom-up corrosion processes were observed in both morphologies, with top-down corrosion arising from defects in the TiO₂ protection layer and bottom-up corrosion occurring through the substrate and membrane.
Moving to a systems perspective, we use a macro-scale energy model with historical demand in conjunction with hourly historical weather data to analyze the role of concentrated solar power (CSP) with thermal energy storage (TES) relative to photovoltaics (PV) and batteries in an idealized least-cost wind/solar/storage system that reliably meets hourly demand. We find that CSP+TES occupies a small niche providing valuable grid services by adding flexibility due to the favorable cost of storing energy in TES compared to batteries. Consequently, CSP does not compete directly with PV, but rather TES competes with short-duration storage from batteries, with the coupled CSP technology providing cost-effective grid services to achieve reliability. A cost-sensitivity analysis shows that penetration of CSP+TES in this idealized wind/solar/storage electricity system is primarily limited by the relatively high current CSP generation costs.</p
Neural Control of Male and Female Aggression in Drosophila
Get PDFAggression is essential for an individual’s survival, but it can also lead to unfavorable consequences when misregulated. It is thus important to study the neural basis of this behavior not only for learning how the nervous system is constructed to generate an innate behavior but also for finding the causality of misregulation. Although many circuit and molecular mechanisms underlying aggression have been revealed, our knowledge is mostly restricted to males. Given that sexual differences in aggression are seen in most if not all species, the mechanisms that we learned in one sex may not be directly applied to the other. Therefore, studying the neural basis of aggression in both sexes is necessary for gaining a full understanding of this behavior. Drosophila serves as a unique model for such studies because males and females differ not only in the level of aggressiveness but also in the motor patterns. Interestingly, the aggression-promoting neurons that have been identified so far are mostly sex-specific, raising the possibility that males and females adopt distinct circuits for controlling aggression. However, many sexually shared features of aggression also imply the existence of common circuit elements. My thesis work investigated whether any aggression circuit modules are shared by the two sexes and how the circuit is organized to generate sexually shared and dimorphic motor patterns. Through a behavioral screen and the genetic intersection approach, we identified a pair of sexually shared neurons, CAP, that regulates aggressive approach in both sexes, as well as a pair of male-specific neurons, MAP, whose activation promotes the transition from approach to male-specific attack. We subsequently identified the female homologue, fpC1 neurons, whose activation induces female aggression. Supported by the in vivo imaging and the behavioral epistasis results, we confirmed the functional connectivity between CAP and MAP/fpC1 in males and females, respectively. Lastly, we showed that the connectivity between CAP and MAP/fpC1 is strengthened in socially isolated flies, which exemplifies how circuits can be modified by social isolation to enhance aggression in both sexes. The connectivity between CAP and MAP/fpC1 provides a circuit logic for the control of sexually shared and dimorphic aggressive behaviors. It can be used as an entry point for circuit mapping as well as for further investigation of mechanisms underlying sexual differences in aggression.</p
Neurogenetic Analysis of C. elegans Developmental Decision-making
Get PDFHow does the neuronal genome dictate cellular physiology and function, which in turn impacts organismal development? This is the central question driving all the projects in this thesis. Here, we leveraged the well-defined nervous system and genetics of Caenorhabditis elegans to apply this genes to neurons to phenotype experimental approach. In Chapter 2, we delineate how a pair of first order amphid interneurons integrates conspecific cues and propagates this information via neuropeptidergic pathways to influence larval developmental fate. In Chapter 3, we identify and characterize a role for the evolutionarily-conserved forkhead transcription factor FKH-7/FOXP in regulating sensory neuron function during developmental decision-making. Our results show that perturbations of single genes encoding transcription factors, neuropeptides, and receptors can significantly alter neuronal function and ultimately have profound effects on an organism’s life history.</p
Integrated Spatial Genomics Reveals Organizational Principles of Single-Cell Nuclear Architecture
Get PDFThree-dimensional (3D) nuclear architecture plays key roles in many cellular processes such as gene regulation and genome replication. Recent sequencing-based and imaging-based single-cell studies have characterized a high variability of nuclear features in individual cells from a wide-range of measurement modalities, such as chromosome structures, subnuclear structures, chromatin states, and nascent transcription. However, the lack of technologies that allow us to interrelate those nuclear features simultaneously in the same single cells limits our understanding of nuclear architecture. To overcome this limitation, a technology that can examine 3D nuclear features across modalities from the same single cells is required. Here, we demonstrate integrated spatial genomics approaches, which enable genome-wide investigation of chromosome structures, subnuclear structures, chromatin states, and transcriptional states in individual cells. In Chapter 2, we introduce the "track first and identify later" approach, which enables multiplexed tracking of genomic loci in live cells by combining CRISPR/Cas9 live imaging and DNA sequential fluorescence in situ hybridization (DNA seqFISH) technologies. We demonstrate our approach by resolving the dynamics of 12 unique subtelomeric loci in mouse embryonic stem (ES) cells. In Chapter 3, we present the intron seqFISH technology, which enables transcriptome-scale gene expression profiling at their nascent transcription active sites in individual nuclei in mouse ES cells and fibroblasts, along with mRNA and lncRNA seqFISH and immunofluorescence. We show the transcription active sites position at the surfaces of chromosome territories with variable inter-chromosomal organization in individual nuclei. By building upon those technologies, in Chapter 4, we demonstrate integrated spatial genomics in mouse ES cells, which enables to image thousands of genomic loci by DNA seqFISH+, along with sequential immunofluorescence and RNA seqFISH in individual cells. We show "fixed loci" that are invariably associated with specific subnuclear structures across hundreds of single cells that can constrain nuclear architecture in individual nuclei. In addition, we find individual genomic loci appear to be pre-positioned to specific nuclear compartments with different frequencies, which are independent from nascent transcriptional states of single cells. Lastly, in Chapter 5, we demonstrate the integrated spatial genomics technology in the mouse brain cortex, enabling the investigation of single-cell nuclear architecture in a cell-type specific fashion as well as the exploration of common organizational principles of nuclear architecture across cell types. We reveal that inter-chromosomal organization and radial positioning of chromosomes are arranged with cell-type specific chromatin fixed loci and subnuclear structure organization in diverse cell types. We also uncover the variable organization of chromosome domain structures at the sub-megabase scale in individual cells, which can be obscured with bulk measurements. Together, these results demonstrate the ability of integrated spatial genomics to advance our overall understanding of single-cell nuclear architecture in various biological systems.</p
Navigating the Temporal Landscape of Trauma
Get PDFTrauma transforms time and narrative. Psychological trauma, the overwhelming mental response to distressing events, distorts the manner in which its victim perceives and experiences time. The representation of trauma is not uniform. Authors use different approaches to speak the unspeakable, to remember the unrememberable. This paper examines three novels and their depiction of time after trauma: Kurt Vonnegut’s Slaughterhouse-Five, Tom McCarthy’s Remainder, and Toni Morrison’s Beloved. Readings of these three novels engage with one another, enhancing the ways in which trauma can be understood. The synthesis of philosophical, scientific, and literary frameworks provides the multifaceted and necessary lens through which to examine the narrative of trauma. In each of these three novels, trauma leaves a mark—a remainder—that the passage of time cannot erase, and that even the altered temporal frameworks cannot fully represent
Thermal Ignition by Vertical Cylinders
Get PDFAccidental thermal ignition events present a significant hazard to the aviation industry. There is scarcity of experimental data on ignition by external natural convection flows for surface areas larger than 10 cm². In this work, thermal ignition of external natural convection flows by vertical cylinders is investigated. The effect of geometry is studied by resistively heating stainless steel cylinders of various sizes in a stoichiometric n-hexane and air mixture at 298 K and 1 bar. Cylinder lengths range from 12.7 to 25.4 cm, and cylinder surface areas vary from 25 to 200 cm². Logistic regression is used to provide statistical information about the ignition threshold (50% probability of ignition). The maximum ignition threshold found is 1117 K for a cylinder 12.7 cm long and 50 cm² in surface area. The minimum ignition threshold found is 1019 K for a cylinder 25.4 cm long and 200 cm² in surface area. The maximum uncertainty on these ignition thresholds is ±29 K, which comes from the maximum uncertainty on the pyrometer measurement used to record cylinder surface temperatures.
The dependence of ignition threshold on both surface area and length of a cylinder is found to be minor. High speed visualizations of ignition indicated that ignition occurs near the top edge of all cylinders.
The entire experimental setup is heated to allow for ignition tests with multi-component, heavy-hydrocarbon fuels including Jet A and two surrogate fuels, Aachen and JI. The cylinder used for all testing of heavier fuels is 25.4 cm long and 200 cm² in surface area. Hexane is also tested with the heated vessel to investigate the effect of ambient temperature on ignition. At an ambient temperature of 393 K, the ignition threshold of hexane is 933 K. Aachen has an ignition threshold of 947 K at an ambient temperature of 373 K. JI has an ignition temperature of 984 K at an ambient temperature of 393 K. Jet A has an ignition temperature of 971 K at an ambient temperature of 333 K. The maximum uncertainty on these thresholds is ±29 K. JI is found to be the most appropriate surrogate for Jet A.
From the experiments, two main conclusions are reached. Ignition threshold temperatures in external natural convection flows are very weakly correlated with surface area. The observed ignition thresholds do not show the drastic transition of ignition temperature with surface area that is observed in internal natural convection situations. Observed ignition thresholds for comparable surface areas (100 to 200 cm²) are 500 to 600 K higher for external natural convection than internal natural convection. Hexane was found to be a reasonable surrogate for Jet A (38 K difference in ignition threshold) in external natural convection ignition testing. The more complex multi-component JI surrogate, while having an ignition threshold more comparable to Jet A (13 K difference in ignition threshold), requires heating the experimental apparatus and associated difficulties of fuel handling as well as the soot generation by combustion.
Two simplified models of ignition are explored. The first is an investigation of ignition chemistry using a zero-dimensional reactor and a detailed kinetic mechanism for hexane. The temperature history of the reactor is prescribed by an artificial streamline whose rate of temperature increase is parametrically varied. The results from the zero-dimensional reactor computation reveal that a gradually heated streamline exhibits two-stage ignition behavior, while a rapidly heated streamline only experiences one ignition event. The second model of ignition is a one-dimensional simulation of ignition adjacent to a cylinder at a prescribed temperature. The formulation included diffusion of species and thermal energy as well as chemical reaction and employed Lagrangian coordinates. The chemistry is modeled with a reaction mechanism for hydrogen to reduce numerical demand. Heat flux and energy balance are analysed to gain insight into the ignition dynamics. Initially, heat transfer is from the wall into the gas, and a mostly nonreactive thermal boundary layer develops around the cylinder. As reaction in the gas near the surface begins to release energy, the heat transfer decreases, and, near the critical temperature for ignition, the direction of heat flux reverses and is from the gas into the wall. In a case where ignition takes place, there is rapid rise in temperature in the gas within the thermal layer, and a propagating flame is observed to emerge into surrounding cold gas. The heat transfer from the hot combustion products results in a continuous heat flux from the gas into the wall. In a case where ignition does not take place, no flame is observed and the heat flux at the wall is slightly positive. For the critical condition just below the ignition threshold, a balance between energy release and diffusion in the adjacent gas results in a small temperature rise in the thermal layer, but a propagating flame is not created. The Van't Hoff ignition criterion of vanishing heat flux at the ignition threshold is approximately but not exactly satisfied. Contrasting the two modeling ideas, we observe that modeling adiabatic flows along computed nonreactive streamlines is useful in examining the role of detailed chemistry but lacks important diffusion effects. Including mass and thermal transport provides more insight into important ignition dynamics but comes at the expense of increased computational complexity.</p