Biomimetic modeling of the nitrogen-centered radical postulated to occur during the inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides

Ribonucleotide reductases (RNR) are essential enzymes that catalyze the reduction of ribonucleotides to 2'-deoxyribonucleotides, which is a critical step that produces precursors for DNA replication and repair. The inactivation of RNR, logically, would discontinue producing the precursors of the DNA of viral or cancer cells, which then would consequently end the cycle of DNA replication. Among different compounds that were found to be inhibitors of RNR, 2'-azido-2'-deoxynucleotide diphosphates (N3NDPs) have been investigated in depth as potent inhibitors of RNR. Decades of investigation has suggested that the inactivation of RNR by N3NDPs is a result of the formation of a nitrogen-centered radical (N·) that is covalently attached to the nucleotide at C3' and cysteine molecule C225 [3'-C(R-S-N·-C-OH)]. Biomimetic simulation reactions for the generation of the nitrogen-centered radicals similar to the one observed during the inactivation of the RNR by azionuclotides was investigated. The study included several modes: (i) theoretical calculation that showed the feasibility of the ring closure reaction between thiyl radicals and azido group; (ii) synthesis of the model azido nucleosides with a linker attached to C3' or C5' having a thiol or vicinal dithiol functionality; (iii) generation of the thiyl radical under both physiological and radiolysis conditions whose role is important in the initiation on RNR cascades; and (iv) analysis of the nitrogen-centered radical species formed during interaction between the thiyl radical and azido group by electron paramagnetic resonance spectroscopy (EPR). Characterization of the aminyl radical species formed during one electron attachment to the azido group of 2'-azido-2'-deoxyuridine and its stereospecifically labelled 1'-, 2'-, 3'-, 4'- or 5,6-[2H 2]-analogues was also examined. This dissertation gave insight toward understanding the mechanism of the formation of the nitrogen-centered radical during the inactivation of RNRs by azidonucleotides as well as the mechanism of action of RNRs that might provide key information necessary for the development of the next generation of antiviral and anticancer drugs.

The exchange of carbon, nitrogen, and phosphorus in dwarf and fringe mangroves of the oligotrophic southern Everglades

Water management has altered both the natural timing and volume of freshwater delivered to Everglades National Park. This is especially true for Taylor Slough and the C-111 basin, as hypersaline events in Florida Bay have been linked to reduced freshwater flow in this area. In light of recent efforts to restore historical flows to the eastern Everglades, an understanding of the impact of this hydrologic shift is needed in order to predict the trajectory of restoration. I conducted a study to assess the importance of season, water chemistry, and hydrologic conditions on the exchange of nutrients in dwarf and fringe mangrove wetlands along Taylor Slough. I also performed mangrove leaf decomposition studies to determine the contribution of biotic and abiotic processes to mass loss, the effect of salinity and season on degradation rates, and the importance of this litter component as a rapid source of nutrients. ^ Dwarf mangrove wetlands consistently imported total nutrients (C, N, and P) and released NO2− +NO3 −, with enhanced release during the dry season. Ammonium flux shifted from uptake to release over the study period. Dissolved phosphate activity was difficult to discern in either wetland, as concentrations were often below detection limits. Fluxes of dissolved inorganic nitrogen in the fringe wetland were positively related to DIN concentrations. The opposite was found for total nitrogen in the fringe wetland. A dynamic budget revealed a net annual export of TN to Florida Bay that was highest during the wet season. Simulated increases and decreases in freshwater flow yielded reduced exports of TN to Florida Bay as a result of changes in subsystem and water flux characteristics. Finally, abiotic processes yielded substantial nutrient and mass losses from senesced leaves with little influence of salinity. Dwarf mangrove leaf litter appeared to be a considerable source of nutrients to the water column of this highly oligotrophic wetland. To summarize, nutrient dynamics at the subsystem level were sensitive to short-term changes in hydrologic and seasonal conditions. These findings suggest that increased freshwater flow has the potential to lead to long-term, system-level changes that may reach as far as eastern Florida Bay. ^

Ecosystem responses to hydrologic change and mechanisms of nitrogen sequestration in seasonally flooded tree islands of the southern Everglades

In the Florida Everglades, tree islands are conspicuous heterogeneous elements in a complex wetland landscape. I investigated the effects of increased freshwater flow in southern Everglades seasonally flooded tree islands, and characterized biogeochemical interactions among tree islands and the marsh landscape matrix, specifically examining hydrologic flows of nitrogen (N), and landscape N sequestration capacity. I utilized ecological trajectories of key ecosystem variables to differentiate effects of increased sheetflow and hydroperiod. I utilized stable isotope analyses and nutrient content of tree island ecosystem components to test the hypothesis that key processes in tree island nitrogen cycling would favor ecosystem N sequestration. I combined estimates of tree island ecosystem N standing stocks and fluxes, soil and litter N transformation rates, and hydrologic inputs of N to quantify the net sequestration of N by a seasonally flooded tree island. ^ Results show that increased freshwater flow to seasonally flooded tree islands promoted ecosystem oligotrophy, whereas reduced flows allowed some plant species to cycle P less efficiently. As oligotrophy is a defining parameter of Everglades wetlands, and likely promotes belowground production and peat development, reintroducing freshwater flow from an upstream canal had a favorable effect on ecosystem dynamics of tree islands in the study area. Important factors influencing the stable isotopic composition of nitrogen and carbon were: (1) a contribution to soil N by soil invertebrates, animal excrement, and microbes, (2) a possible NO3 source from an upstream canal and an "open" ecosystem N cycle, and (3) greater availability of phosphorus in tree islands relative to the marsh landscape, suggesting that tree island N cycling favors N sequestration. Hydrologic sources of N were dominated by surface water loads of NO3- and NH 4+, and an important soil N transformation promoting the net loss of surface water DIN was nitrate immobilization associated with soils and surficial leaf litter. The net inorganic N sequestration capacity of a seasonally flooded tree island was 50 g yr-1 m -2. Thus, tree islands likely have an important function in landscape sequestration of inorganic N, and may reduce significant anthropogenic N loads to downstream coastal systems. ^

Quantifying nitrogen cycling rates in freshwater marshes of the southern Everglades using nitrogen-15 tracer techniques

This dissertation research project addressed the question of how hydrologic restoration of the Everglades is impacting the nutrient dynamics of marsh ecosystems in the southern Everglades. These effects were analyzed by quantifying nitrogen (N) cycle dynamics in the region. I utilized stable isotope tracer techniques to investigate nitrogen uptake and cycling between the major ecosystem components of the freshwater marsh system. I recorded the natural isotopic signatures (δ15N and δ 13C) for major ecosystem components from the three major watersheds of the Everglades: Shark River Slough, Taylor Slough, and C-111 basin. Analysis of δ15 N and δ13C natural abundance data were used to demonstrate the spatial extent to which nitrogen from anthropogenic or naturally enriched sources is entering the marshes of the Everglades. In addition, I measured the fluxes on N between various ecosystem components at both near-canal and estuarine ecotone locations. Lastly, I investigated the effect of three phosphorus load treatments (0.00 mg P m-2, 6.66 mg P m-2, and 66.6 mg P m-2) on the rate and magnitude of ecosystem N-uptake and N-cycling. The δ15N and δ13C natural abundance data supported the hypothesis that ecosystem components from near-canal sites have heavier, more enriched δ 15N isotopic signatures than downstream sites. The natural abundance data also showed that the marshes of the southern Everglades are acting as a sink for isotopically heavier, canal-borne dissolved inorganic nitrogen (DIN) and a source for "new" marsh derived dissolved organic nitrogen (DON). In addition, the 15N mesocosm data showed the rapid assimilation of the 15N tracer by the periphyton component and the delayed N uptake by soil and macrophyte components in the southern Everglades.

Nutrient content of the seagrass Thalassia testudinum reveals regional patterns of relative availability of nitrogen and phosphorus in the Florida Keys USA

Between 1992 and 2000, we sampled 504 randomly chosen locations in theFlorida Keys, Florida, USA, for the elemental content of green leaves of theseagrass Thalassia testudinum. Carbon content ranged from29.4–43.3% (dry weight), nitrogen content from 0.88–3.96%, andphosphorus content from 0.048–0.243%. N and P content of the samples werenot correlated, suggesting that the relative availability of N and P variedacross the sampling region. Spatial pattern in C:N indicated a decrease in Navailability from inshore waters to the reef tract 10 km offshore;in contrast, the pattern in C:P indicated an increase in P availability frominshore waters to the reef tract. The spatial pattern in N:P was used to definea P-limited region of seagrass beds in Florida Bay and near shore, and anN-limited region of seagrass beds offshore. The close juxtaposition ofN–and P-limited regions allows the possibility that N loading from thesuburban Florida Keys could influence the offshore, N-limited seagrass bedswithout impacting the more nearshore, P-limited seagrass beds. Carbonate - Nutrient lim

An assessment of the microbial contribution to aquatic dissolved organic nitrogen using amino acid enantiomeric ratios

There is increasing evidence that certain microbially-derived compounds may account for part of the aquatic dissolved organic nitrogen (DON) pool. Enantiomeric ratios of amino acids were used to assess the microbial input to the DON pool in the Florida Everglades, USA. Elevated levels of d-alanine, d-aspartic acid, d-glutamic acid and d-serine indicated the presence of peptidoglycan in the samples. The estimated peptidoglycan contribution to amino acid nitrogen ranged from 2.8 ± 0.1% to 6.4 ± 0.9%, increasing with salinity from freshwater to coastal waters. The distribution of individual d-amino acids in the samples suggests additional inputs to DON, possibly from archaea or from abiotic racemization of l-amino acids.

Lake metabolism and the diel oxygen technique: State of the science

Significant improvements have been made in estimating gross primary production (GPP), ecosystem respiration (R), and net ecosystem production (NEP) from diel, “free-water” changes in dissolved oxygen (DO). Here we evaluate some of the assumptions and uncertainties that are still embedded in the technique and provide guidelines on how to estimate reliable metabolic rates from high-frequency sonde data. True whole-system estimates are often not obtained because measurements reflect an unknown zone of influence which varies over space and time. A minimum logging frequency of 30 min was sufficient to capture metabolism at the daily time scale. Higher sampling frequencies capture additional pattern in the DO data, primarily related to physical mixing. Causes behind the often large daily variability are discussed and evaluated for an oligotrophic and a eutrophic lake. Despite a 3-fold higher day-to-day variability in absolute GPP rates in the eutrophic lake, both lakes required at least 3 sonde days per week for GPP estimates to be within 20% of the weekly average. A sensitivity analysis evaluated uncertainties associated with DO measurements, piston velocity (k), and the assumption that daytime R equals nighttime R. In low productivity lakes, uncertainty in DO measurements and piston velocity strongly impacts R but has no effect on GPP or NEP. Lack of accounting for higher R during the day underestimates R and GPP but has no effect on NEP. We finally provide suggestions for future research to improve the technique.

The Importance of Dissimilatory Nitrate Reduction to Ammonium (DNRA) in the Nitrogen Cycle of Coastal Ecosystems

Until recently, it was believed that biological assimilation and gaseous nitrogen (N) loss through denitrification were the two major fates of nitrate entering or produced within most coastal ecosystems. Denitrification is often viewed as an important ecosystem service that removes reactive N from the ecosystem. However, there is a competing nitrate reduction process, dissimilatory nitrate reduction to ammonium (DNRA), that conserves N within the ecosystem. The recent application of nitrogen stable isotopes as tracers has generated growing evidence that DNRA is a major nitrogen pathway that cannot be ignored. Measurements comparing the importance of denitrification vs. DNRA in 55 coastal sites found that DNRA accounted for more than 30% of the nitrate reduction at 26 sites. DNRA was the dominant pathway at more than one-third of the sites. Understanding what controls the relative importance of denitrification and DNRA, and how the balance changes with increased nitrogen loading, is of critical importance for predicting eutrophication trajectories. Recent improvements in methods for assessing rates of DNRA have helped refine our understanding of the rates and controls of this process, but accurate measurements in vegetated sediment still remain a challenge.