Browsing by Subject "Stoichiometry"

Now showing 1 - 6 of 6
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    Brightness analysis in finite geometries: probing protein interactions in cellular, cell-free and aqueous environments
    (2012-12) Macdonald, Patrick
    Fluorescence fluctuation spectroscopy (FFS) is a powerful technique for quantitatively analyzing protein interactions. Using brightness analysis methods, we are uniquely able to measure the stoichiometry of protein complexes. FFS is particularly valuable because it allows measurements within living cells. This thesis demonstrates that measuring in very small volumes, such as E. coli cells, introduces a bias into the measured brightness. We show that this bias is a result of accumulative sample loss, or photodepletion, and that we can account for this effect and recover correct brightness values. Similarly, very thin samples, such as cell cytoplasm, introduce a bias due to the sample being shorter along the vertical axis than the volume of the excitation light. We introduce z-scan FFS and theory to identify and model thin samples and to recover unbiased data. Although measuring in cells is a primary strength of the FFS technique, some studies require the greater degree of experimental control afforded by solution measurements. Thus, we characterize cell-free expression solution for FFS measurements, an environment that offers increased control but permits genetic fluorescent labeling. We take advantage of this system to perform chromophore maturation experiments as a function of temperature on three common fluorescent proteins: EGFP, EYFP and mCherry. Our results prove that EGFP has fast maturation and is a good reporter for fluorescence experiments. Finally, we apply FFS and brightness analysis to the enzyme, APOBEC3G. We reveal that APOBEC3G interactions with RNA and single-stranded DNA are sequence dependent, which has important implications for the mechanism by which APOBEC3G packages itself into HIV-1 viral particles and restricts the virus to prevent infection.
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    Consumer nutrient stoichiometry : patterns, homeostasis, and links with fitness.
    (2010-10) Hood II, James Michael
    The linkages between food webs and nutrient cycles are heterogeneous and often influenced by human activities. Ecological stoichiometry provides one framework for understanding and predicting these linkages. Yet, as it has been extended underlying assumptions are often not evaluated. This dissertation shows that examination of implicit and explicit assumptions reveals unknown mechanisms, interactions, and linkages. For instance, theory assumes that invertebrate stoichiometry does not vary with diet stoichiometry (i.e., strict homeostasis), even though many invertebrates are not strictly homeostatic. Chapters one and two examine the role of stoichiometric homeostasis in shaping the fitness of Daphnia species. Chapter one shows that the long-­‐term phosphorus (P) use efficiency of stoichiometrically flexible Daphnia species is higher in habitats with temporally variable diets, resulting in higher fitness relative to strictly homeostatic species. Chapter two shows that the P cost of a unit of growth increased with growth rate and structures tradeoffs among growth rate, sensitivity to P limitation, and stoichiometric flexibility. Stoichiometric theory can be extended to novel ecosystems, such as streams, to predict the role of consumers in food web and nutrient cycles. To do To do this, the balance between consumer and diet stoichiometries is a logical starting point. Chapter three examines intra-­‐specific variation in consumer-­‐resource stoichiometries at a suite of sites within a river network. In contrast to previous work, this chapter describes wide intra-­‐ specific variation in consumer stoichiometry, similar in magnitude to the variation among invertebrate taxa. Intra-­‐specific variation in nitrogen and phosphorus content was related to both ontogeny and diet. These results suggest that the role of a species in stream nutrient cycles could vary spatially with diet and temporally through ontogeny.Chapter four examines the influence of diet stoichiometry on nutrient release ratios of four stream detritivores. Predictions of nutrient release ratios from bulk diet stoichiometries were misleading for these detritivores, which selectively consumed a nutrient rich portion of the bulk diet. Selective feeding greatly reduced stoichiometric mismatches between these consumers and their diets. Taken together, this dissertation demonstrates that examination of stoichiometric assumptions improves our understanding of consumer-­‐resource dynamics, competition, and the role of consumers in nutrient cycles.
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    Decomposition and Production of Dissolved Organic Matter by Aquatic Bacteria
    (2019-06) Thompson, Seth
    Aquatic ecosystems transport large amounts of organic matter from the landscape to the oceans. Along this pathway, heterotrophic bacteria rapidly cycle these compounds by acting as both degraders and producers of organic compounds. Understanding the ultimate fate of the organic matter and predicting how increased organic matter exports from terres-trial ecosystems will impact its delivery to the ocean, requires a better understanding of the factors that influence organic matter degradation and production in freshwater systems. While many scientists have approached this problem by focusing on microbial modifica-tions of carbon (C), much less attention has been paid to other major elements found in or-ganic molecules (namely nitrogen (N) and phosphorus (P)). A more integrated approach that incorporates microbial processing of both C and major macronutrients such as N and P is needed to describe the biogeochemical transformations of organic matter in freshwaters. In this dissertation I examine the degradation and production of dissolved organic matter by heterotrophic bacteria, specifically focusing on dissolved organic phosphorus (DOP). In chapter 1, I quantify the degradation rates and overall bioavailability of DOP across 27 unique aquatic systems and explore important environmental and chemical regu-lators of these rates. Data from these systems show that DOP degradation rates are spatially variable, but are typically as high or higher than rates of degradation for C. Also, the chem-ical composition of organic matter was an important predictor of DOP bioavailability with DOP bioavailability being lowest when DOP was scarce relative to C. This relationship means that DOP is degraded by bacteria in systems that are more likely to experience P lim-itation, suggesting that DOP may be an important source of P to bacteria in these systems. Chapter 1 concludes by documenting the importance of incorporating estimates of organic matter bioavailability into estimates of resource imbalance experienced by aquatic bacteria. Accounting for the bioavailability of organic matter generally reduces the estimates of nutri-ent imbalance experienced by aquatic bacteria compared to estimates using bulk nutrient concentrations. This reduction in imbalance would result in more efficient C cycling by aquatic bacteria, which has important implications for understanding the composition of organic matter exported downstream and ultimately to the ocean. Chapter 2 goes on to explore the production of organic matter by heterotrophic bac-teria. It is well documented in marine systems that bacteria can produce an immensely di-verse set of organic molecules, even when they are only given a single carbon source to start from. However, the factors that control this production and the extent to which bacteria also produce DOP remains unclear. Previous work has shown that bacteria in freshwaters have different stoichiometric strategies for dealing with nutrient imbalance, with some strains of bacteria capable of changing the chemical composition of their cells to more closely match that of their resources. This flexibility in biomass nutrient composition has important implications for the recycling rates of multiple nutrients and therefore likely im-pacts the production of organic molecules by bacteria as well. Using previously isolated bacterial strains that have had their biomass flexibility quantified, I test the impact of these different stoichiometric strategies on the composition of the organic matter the strains pro-duce. In this chapter, I show that bacteria produce measurable amounts of dissolved organic phosphorus, even under strongly phosphorus limited conditions. Overall, bacteria convert-ed ~0.01%-10% of the phosphate in the original media to dissolved organic phosphorus, with the highest conversion efficiencies under carbon limited growth conditions. Interest-ingly, the conversion efficiency was higher under extreme phosphorus limitation than mod-erate phosphorus limitation. This pattern was driven primarily by relatively high conversion efficiencies by bacteria with flexible biomass stoichiometry in the most phosphorus limited conditions demonstrating the importance of physiological responses to nutrient imbalance. This chapter also explores the impact of bacterial biomass flexibility on the optical proper-ties of the organic matter produced by bacteria. I show that biomass flexibility is signifi-cantly and positively related to the specific ultraviolet absorbance at a wavelength of 254 nm, a measure of the aromaticity of the organic matter, when grown under extreme phos-phorus limitation. This suggested that bacteria with more flexible biomass stoichiometry produce more complex carbon molecules under strong phosphorus limitation than less flex-ible strains do. While more work is needed to fully understand how the physiological growth strategies of different microbial taxa impact the production of DOM, this chapter provides some important first insights into this question. In the final chapter, I transition away from research on aquatic ecology into what I consider to be another fundamental aspect of being a scientist: training the next generation of scientific thinkers. Over the last decade, there has been a clear call to shift the instruc-tional methods used for teaching undergraduate biology courses. We now know that active learning approaches to teaching science lead to better science outcomes for students. Fur-thermore, engaging undergraduate science students in undergraduate research experiences has been shown to have a number of important benefits for students such as increased stu-dent engagement, interest in science careers, and understanding of the scientific process. To offer the benefits of research experiences to a broader set of students, many institutions have started offering Course-based Undergraduate Research Experiences (CUREs) in la-boratory classes for students. It is common for these laboratory sections to be primarily fa-cilitated by undergraduate or graduate teaching assistants (TAs) rather than full-time faculty members. For these TAs to efficiently achieve the goals of these CUREs they must under-stand both (a) the philosophical underpinnings of discovery-based inquiry, and (b) strate-gies for facilitating inquiry, based on evidence-based practices, in the teaching laboratory. However, TAs are rarely trained in pedagogy, which likely limits their abilities to effective-ly facilitate inquiry in the laboratory. Chapter 3 is a case study documenting the results of a theoretically grounded professional development pilot program. This pilot program revealed that novice TAs are initially concerned primarily about the logistical aspects of teaching: classroom management, content preparation, grading assignments, etc. These concerns limit their readiness for engaging with the more complex pedagogical concepts of evidence-based instruction or inclusive teaching. This means that TA professional development needs to be designed to parallel the dynamic nature of TA concerns and that programing focused on advanced teaching techniques is only effective after TAs have established a sense of comfort and confidence in their own teaching.
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    Habitat selection and ecological stoichiometry: the role of seston C:P in Daphnia daytime spatial location.
    (2009-01) Forman, Mary Rebecca
    Diel vertical migration (DVM) of zooplankton has intrigued ecologists for over 100 years. Traditional DVM theory holds that Daphnia migrate into the hypolimnion during the daytime to escape predation pressure from visually-hunting predators, while nighttime ascent into the epilimnion facilitates feeding and growth. It has been demonstrated that Daphnia behavior may be altered by algal quantity, predator cues, and temperature or light levels. However, little was known about how algal quality (in terms of nutrient content) affects Daphnia vertical position. This dissertation examines the effects of algal quality on the daytime habitat selection of Daphnia through laboratory experiments and analysis of field data. Laboratory studies examined the movements of Daphnia in a thermally-stratified water column. When algal quality was high (C:P ~ 150) throughout the water column, mature Daphnia were found in the epilimnion during the daytime, despite the presence of a predator cue. However, when algal quality was low (C:P ~1500) throughout the water column, mature Daphnia tended to spend the daytime in the hypolimnion. These habitat preferences were not detected when the experiment was repeated using young juveniles (age 0-5 days), though at age 6 days, juveniles’ behavior appeared to begin to shift toward that of adults. As expected, body mass, fecundity, and net reproductive rate were found to be positively correlated with increased environmental temperature, and fecundity and net reproductive rate were positively correlated with increased food quality. A field study of 34 lakes revealed that habitat selection was not only food-quality dependent, but also species-specific. D. retrocurva and D. mendotae exhibited a preference for the upper lake layer when food quality was good throughout the water column, but not when food quality was poor in the upper layer. D. pulicaria did not exhibit any layer preference in either lake environment. The results from this research provide insight into Daphnia’s ability to balance the multiple factors associated with a heterogeneous vertical gradient in order to realize maximum productivity, and thus maximum fitness, in the presence of visually-hunting predators. The results further indicate that food quality is an important factor in determining habitat selection, productivity, and ultimately fitness.
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    A taxon for every treatment: Influence of nutrient and temperature shifts on Great Lakes phytoplankton community composition
    (2024-09) Loiselle, Reane
    The Laurentian Great Lakes hold over 20% of our global freshwater. Cyanobacterial harmful algal blooms (CHABs) have been some of the most significant environmental issues to threaten the health of the Great Lakes in recent decades, causing an estimated $65-71 million dollars per year in economic losses for Lake Erie alone (Bingham et al. 2015). The impact of increased temperature and nutrients on HABs has been extensively studied in recent decades. But still, little is known about how changes in temperature, nitrogen and phosphorus impact the relative abundance of specific bloom forming taxa in the Great Lakes or elsewhere. To better understand these shifts, I analyzed identical 28-day nutrient addition bioassays at ambient and plus 3 ℃ temperatures in Mawikwe Bay, Lake Superior and Sandusky Bay, Lake Erie during the summers of 2022 and 2023. This length of experiment was selected to allow for competition to take place over realistic ecological time scales to elucidate the impact of nutrients and temperature on phytoplankton competition. A FluoroProbe was used to track broad changes in phytoplankton groups every 3 days and microscopy was used to obtain detailed community information on days 0, 14, and 28. Further microscopy analyses were used to determine changes in relative abundance of dominant bloom forming species, shifts in major phytoplankton groups, and implications for associated traits.Lowering Lake Superior N:P ratios resulted in shifts from diatom dominance to increased green algae and cyanobacteria abundance, including the main bloom forming cyanobacterium Dolichospermum. This outcome indicated that the normally diatom-dominated phytoplankton community in Lake Superior is a result of intense competition for P, while P additions promote Dolichospermum abundance. In Lake Erie, Microcystis was significantly more abundant in treatments with higher N:P ratios while Dolichospermum was more abundant in treatments with lower N:P ratios. Low N:P favoring Dolichospermum in both lakes might be explained by the ability of Dolichospermum to fix nitrogen. It also suggests that P-only nutrient management in Lake Erie would likely shift the species composition of HABs from non-diazotrophic species to diazotrophic species, but not eliminate them. In contrast, P-only management in Lake Superior is likely to be effective. A several degree warming was not found to be a prominent factor in steering phytoplankton community composition in either lake, suggesting weak temperature effects on competitive outcomes over expected amounts of warming.
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    Transport and Magnetism in Bulk and Thin Film Strontium Titanate
    (2015-10) Ambwani, Palak
    SrTiO3 is a wide band-gap perovskite oxide semiconductor that is widely investigated in the bulk form, due to its remarkable electronic properties. These properties arise from its quantum paraelectric nature which enables unique features, such as, a high-mobility low-density metallic state, quantum transport in an unusual limit, and the most dilute superconducting state thus reported. Recent advances in deposition of oxide thin films and heterostructures have further led to some remarkable observations, such as, the strain-enhancement of mobility in doped thin films of SrTiO3, and the presence of 2D electron gases at interfaces and in delta-doped layers. The presence of magnetic moments and their possible ordering, and the simultaneous observation of quantum oscillations and superconductivity, have been reported in these 2D electron gases. While magnetism has been observed in heterostructures of SrTiO3, there have been limited reports on magnetism in bulk SrTiO3. The first part of this thesis (Chapter 3) discusses how circularly polarized light can induce an extremely long-lived magnetic moment in slightly oxygen-deficient but otherwise nominally pure SrTiO3-δ bulk crystals. These magnetic signals, which are induced at zero applied magnetic field and at low temperatures below ~ 18 K, can be controlled in both magnitude and sign by means of the circular polarization and wavelength of sub-bandgap illumination (400-500 nm), and point to the existence of optically polarizable "V" _"O" -related complexes in the forbidden gap of SrTiO3-δ, rather than collective or long-range magnetic order. The methods used to detect optically induced magnetization are also discussed (Appendix A). The phenomenal progress reported in thin films and heterostructures of SrTiO3 has been possible only by precise control of stoichiometry and defect density in SrTiO3 using techniques such as oxide/LASER MBE or high-temperature PLD. The next part of the thesis (Chapter 4) demonstrates that high pressure oxygen RF sputtering from a ceramic target is similarly capable of growth of high-quality, precisely stoichiometric thin films of SrTiO3. By employing homoepitaxy on SrTiO3(001) substrates, it is shown that optimization of deposition temperature (above 750 C), oxygen pressure (above 2.5 mBar) and deposition rate (below 1.5 Å/min) leads to films that are indistinguishable from the substrate via grazing incidence and wide-angle X-ray scattering. The importance of pre-annealing of substrates in oxygen above 900 C and polishing the target prior to deposition, to obtain bulk-like lattice parameters and eliminate interfacial scattering contrast, is reiterated. Detailed transport measurements were also performed on reduced films grown on LaAlO3(001) and LSAT(001) substrates. The films were found to be semiconducting with mobilities at least an order lower than bulk. Detection and quantification of trace impurities was carried out using PIXE, and the possible causes of low mobility and semiconducting transport characteristics are discussed. Despite the rapid recent progress in thin film deposition techniques, controlled dopant incorporation and attainment of high mobility in thin films of SrTiO3 remain problematic. The last part of the thesis (Chapter 5) discusses the use of analytical scanning transmission electron microscopy to study the local atomic and electronic structure of Nb-doped SrTiO3 both in ideally substitutionally-doped bulk single crystals, and epitaxial thin films. The films are deposited under conditions that would yield highly stoichiometric undoped SrTiO3, as discussed in the previous chapter, but are nevertheless insulating. The Nb incorporation in such films was found to be highly inhomogeneous on nanoscopic length-scales, with large quantities of what is deduced to be interstitial Nb. Electron energy loss spectroscopy reveals changes in the electronic density of states in Nb-doped SrTiO3 films compared to undoped SrTiO3, but without a clear shift in the Fermi edge, that is seen in bulk single crystal Nb-doped SrTiO3. Analysis of atomic-resolution annular dark-field images leads to the conclusion that the interstitial Nb is in the Nb0 state, confirming that it is electrically inactive. It is argued that this approach will enable future work establishing the vitally needed relationships between synthesis/processing conditions and electronic properties of Nb-doped SrTiO3 thin films.