The influence of disparate levels of submersed aquatic vegetation on largemouth bass population characteristics in a Georgia reservoir

Population characteristics of largemouth bass (Micropterous salmoides L.) including growth, body condition (relative weight), size structure, survival, and fecundity were examined in relation to abundance of submersed aquatic vegetation (SAV) coverage (primarily hydrilla Hydrilla verticillata L.f. Royle) in three major embayments of Lake Seminole, Georgia. Relative weight, fecundity, and growth of large-mouth bass in the Spring Creek embayment (76% areal SAV coverage) was considerably less than measured in the Chattahoochee and Flint river arms that contained lower SAV coverages (26% and 32%). It took fish 1.8 years longer to reach 406 mm in Spring Creek compared to the Chattahoochee-Flint arms. Consequently, fish were smaller in Spring Creek than in the Chattahoochee-Flint arms. In addition, due to slower growth rates and lower fecundity-to-body weight relation, we predicted a 47% reduction in total potential ova production in Spring Creek compared to the other two reservoir embayments. The annual survival rate of 3 to 10 year old largemouth bass was higher in Spring Creek (84%) than in the Chattahoochee-Flint arms (72%) and suggested either lower harvest and/or lower accessibility of particularly larger fish to angling in dense vegetation. Contrary to our expectaions, the fit between number-at-age and age in a catch-curve regression was weaker for fish collected in Spring Creek and suggested greater recruitment variability has occurred over time in this highly vegetated embayment. In Lake Seminole, spatial differences in largemouth bass population characterstics were associated with disparate levels of SAV. Our data suggest that a reduction in hydrilla, but maintenance of an intermediate level of SAV in Spring Creek, should improve largermouth bass population in this arm of the reservoir.

A Method for siting and prioritizing the removal of derelict vessels in Florida Coastal Waters: test applications in the Florida Keys

Increased boating activities and new waterfront developments have contributed an estimated 3,000 dismantled, abandoned, junked, wrecked, derelict vessels to Florida coastal waters. This report outlines a method of siting and prioritizing derelict vessel removal using the Florida Keys as a test area. The data base was information on 240 vessels, obtained from Florida Marine Patrol files. Vessel location was plotted on 1:250,000 regional and 1:5,000 and 1:12,000 site maps. Type of vessel, length, hull material, engine, fuel tanks, overall condition, afloat and submerged characteristics, and accessibility, were used to derive parametric site indices of removal priority and removal difficulty. Results indicate 59 top priority cases which should be the focus of immediate clean up efforts in the Florida Keys. Half of these cases are rated low to moderate in removal difficulty; the remainder are difficult to remove. Removal difficulty is a surrogate for removal cost: low difficulty -low cost, high difficulty - high cost. The rating scheme offers coastal planners options of focusing removal operations either on (1) specific areas with clusters of high priority derelict vessels or on (2) selected targeted derelicts at various, specific locations. (PDF has 59 pages.)

The ecological basis of fishery yield of the Puerto Rico-Virgin Islands Insular Shelf: 1987 Assessment

A literature review was conducted to locate information on the flow of energy from primary producers to the fishery stocks of the Puerto Rican-Virgin Islands insular shelf. This report uses site-specific information to describe the major ecological subsystems, or habitats, of the region, to identify the more common species and the subsystems in which they occur, to quantify productivity and biomass, and to outline trophic relationships. Discussions on each topic and subsystem vary in substance and detail, being limited by the availability and accessibility of information. (PDF contains 189 pages) Seven distinct subsystems are described: mangrove estuary, seagrass bed, coral reef, algal plain, sand/mud bottom, shelf break, and overlying pelagic. Over 50 tables provide lists of species found in each habitat on various surveys dating back to 1956. Estimates of density, relative abundance, and productivity are provided when possible. We evaluated whether sufficient information exists to support an analysis of the energy basis of fishery production in the area, beginning with the design and development of an ecosystem model. Data needs in three categories - species lists, biomass, and trophic relations - were examined for each subsystem and for each of three species groups - primary producers, invertebrates, and fish. We concluded that adequate data, sufficient for modeling purposes, are available in 16 (25%) of 64 categories; limited data, those requiring greater extrapolation, are available in 35 (55%) categories; and no data are available in 13 (20%) categories. The best-studied subsystems are seagrass beds and coral reefs, with at least limited data in all categories. Invertebrates, the intermediate link in the food web between primary producers and fishes, are the least quantified group in the region. Primary production and fishes, however, are relatively well-studied, providing sufficient data to support an ecosystem-level analysis and to initiate a modeling effort.

Towards accessible e-book platforms

Outcomes of a piece of research into the accessibility of e-books. LTD

Towards accessible e-book platforms

A small scale project was funded to explore the accessibility of e-book platforms, offer specific confidential advice for participating partners and provide generic guidance for publishers and aggregators.

Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

227 págs.

Viability Conditions for a Compartmentalized Protometabolic System: A Semi-Empirical Approach

In this work we attempt to find out the extent to which realistic prebiotic compartments, such as fatty acid vesicles, would constrain the chemical network dynamics that could have sustained a minimal form of metabolism. We combine experimental and simulation results to establish the conditions under which a reaction network with a catalytically closed organization (more specifically, an (M, R)-system) would overcome the potential problem of self-suffocation that arises from the limited accessibility of nutrients to its internal reaction domain. The relationship between the permeability of the membrane, the lifetime of the key catalysts and their efficiency (reaction rate enhancement) turns out to be critical. In particular, we show how permeability values constrain the characteristic time scale of the bounded protometabolic processes. From this concrete and illustrative example we finally extend the discussion to a wider evolutionary context.

Delivery of small nucleic acids by conjugation to carbohydrates and lipids as novel research and therapeutic tools

399 p. : il., graf.

Characterization of rural livelihood of Lake Chad basin fishermen

The study examined the characterization of rural livelihood of the fishermen in the Nigeria portion of Lake Chad Basin are as part of European Commission (EC) founded project entitled "Sustainable development of continental fisheries; a regional study of policy options and policy formation mechanisms for the Lake Chad Basin" Wealth ranking exercise which was not based on real income but on production capacity of the fishermen was carried out in twenty (20) villages survey on the western part of the region using Rapid Rural Appraisal Technique with semi- structured interviews. The different activities carried out by the villagers for living were identified according to their socio-economic status. This was followed by an assessment of the socio-economic characterization within each wealth group. Series of comparative analysis of the ethnic composition, accessibility of fishing gear ownership by the population were done. The results show that the 3 wealth groups in the region include the rich (Group 1) the middle class (Group 2) and the poor (Group 3). It was identified that fishing is just one component of the socio-economic production system along side farming, livestock rearing and trading which are closely integrated. The diversified livelihood system being practiced in the Chad Basin region are not only less vulnerable but also more sustainable

The photochemistry of dioxorhenium(V)

The excited-state properties of trans-ReO₂(py)₄⁺ (ReO₂⁺) in acetonitrile solution have been investigated. The excited-state absorption spectrum of ReO₂⁺ is dominated by bleaching of the ground state MLCT and d-d systems. The reduction potential of ReO₂^2+/+* is estimated from emission and electrochemical data to be -0.7 V (SSCE). The ReO₂⁺ excited state efficiently reduces methylviologen and other pyridinium and olefin acceptors. The resulting Re(VI) species oxidizes secondary alcohols and silanes. Acetophenone is the product of sec-phenethyl alcohol oxidation.

The emission properties of ReO₂⁺ in aqueous solutions of anionic and nonionic surfactants have been investigated. The emission and absorption maxima of ReO₂⁺ are dependent on the water content of its environment. Emission lifetimes vary over four orders of magnitude upon shifting from aqueous to nonaqueous environments. The emission lifetime has a large (8.6) isotope effect (k(H₂O)/k(D₂O)) that reflects its sensitivity towards the environment. These properties have been used to develop a model for the interactions of ReO₂⁺ with sodium dodecyl sulfate (SDS). A hydrophobic ReO₂⁺ derivative, ReO₂(3-Ph-py)₄⁺, has been used to probe micelles of nonionic surfactants, and these results are consistent with those obtained with SDS.

The emission properties of ReO₂⁺ in Nafion perfluorosulfonated membranes have been investigated. Absorption and emission spectroscopy indicate that the interior of the membrane is quite polar, similar to ethylene glycol. Two well-resolved emission components show different lifetimes and different isotope effects, indicative of varying degrees of solvent accessibility. These components are taken as evidence for chemically distinct regions in the polymer film, assigned as the interfacial region and the ion cluster region.

The unsubstituted pyridine complex shows monophasic, τ = 1.7 µs, emission decay when bound to calf thymus DNA. Switching to the 3-Ph-py complex yields a biphasic emission decay (τ₁ = 2.4 µs, τ₂ = 10 µs) indicative of an additional, solvent-inaccessible binding mode. Photoinduced electron transfer to methylviologen leads to oxidative cleavage of the DNA as detected by gel electrophoresis. Electrochemical and spectrophotometric techniques used with organic substrates also can be used to monitor the oxidation of DNA. Abstraction of the ribose 4' hydrogen by ReO₂²⁺ is a possible mechanism.

Multiplexed DNA-mediated electrochemistry

The aromatic core of double helical DNA possesses the unique and remarkable ability to form a conduit for electrons to travel over exceptionally long molecular distances. This core of π-stacked nucleobases creates an efficient pathway for charge transfer to proceed that is exquisitely sensitive to even subtle perturbations. Ground state electrochemistry of DNA-modified electrodes has been one of the major techniques used both to investigate and to harness the property of DNA-mediated charge transfer. DNA-modified electrodes have been an essential tool for both gaining insights into the fundamental properties of DNA and, due to the exquisite specificity of DNA-mediated charge transfer for the integrity of the π-stack, for use in next generation diagnostic sensing. Here, multiplexed DNA-modified electrodes are used to (i) gain new insights on the electrochemical coupling of metalloproteins to the DNA π-stack with relevance to the fundaments of in vivo DNA-mediated charge transfer and (ii) enhance the overall sensitivity of DNA-mediated reduction for use in the detection of low abundance diagnostic targets.

First, Methylene Blue (MB′) was covalently attached to DNA through a flexible C12 alkyl linker to yield a new redox reporter for DNA electrochemistry measurements with enhanced sensitivity. Tethered, intercalated MB′ was reduced through DNA-mediated charge transport. The redox signal intensity for MB′-dT-C12-DNA was found to be at least 3 fold larger than that of previously used Nile Blue (NB)-dT-DNA, which is coupled to the base stack via direct conjugation. The signal attenuation, due to an intervening mismatch, and therefore the degree of DNA-mediated reduction, does, however, depend on the DNA film morphology and the backfilling agent used to passivate the surface. These results highlight two possible mechanisms for the reduction of MB′ on the DNA-modified electrode that are distinguishable by their kinetics: reduction mediated by the DNA base pair stack and direct surface reduction of MB′ at the electrode. The extent of direct reduction at the surface can be minimized by overall DNA assembly conditions.

Next, a series of intercalation-based DNA-mediated electrochemical reporters were developed, using a flexible alkane linkage to validate and explore their DNA-mediated reduction. The general mechanism for the reduction of distally bound redox active species, covalently tethered to DNA through flexible alkyl linkages, was established to be an intraduplex DNA-mediated pathway. MB, NB, and anthraquinone were covalently tethered to DNA with three different covalent linkages. The extent of electronic coupling of the reporter was shown to correlate with the DNA binding affinity of the redox active species, supporting an intercalative mechanism. These electrochemical signals were shown to be exceptionally sensitive to a single intervening π-stack perturbation, an AC mismatch, in a densely packed DNA monolayer, which further supports that the reduction is DNA-mediated. Finally, this DNA-mediated reduction of MB occurs primarily via intra- rather than inter duplex intercalation, as probed through varying the proximity and integrity of the neighboring duplex DNA. Further gains to electrochemical sensitivity of our DNA-modified devices were then achieved through the application of electrocatalytic signal amplification using these solvent accessible intercalative reporters, MB-dT-C8, and hemoglobin as a novel electron sink. Electrocatalysis offers an excellent means of electrochemical signal amplification, yet in DNA based sensors, its application has been limited due to strict assembly conditions. We describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low density DNA films. Electrocatalysis of MB that is covalently tethered to the DNA by a flexible alkyl linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of single CA mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: it is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.

Finally, we expanded the application of our multiplexed DNA-modified electrodes to the electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins that contain redox cofactors. Multiplexed analysis of EndonucleaseIII (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, elucidated subtle differences in the electrochemical behavior as a function of DNA morphology. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction. Closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. The electrochemical comparison of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster, was able to be quantitatively performed. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. Based on the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues, but also through changes in solvation near the cluster.

La cerámica de la Segunda Edad del Hierro en el País Vasco: Estudio tecnológico, funcional y social

670 p. Capítulos de introducción, metodología, discusión y conclusiones en castellano e inglés.

In vivo analysis of interactions between trans-acting factors and their target genes

The investigations presented in this thesis use various in vivo techniques to understand how trans-acting factors control gene expression. The first part addresses the transcriptional regulation of muscle creatine kinase (MCK). MCK expression is activated during the course of development and is found only in differentiated muscle. Several in vivo footprints are observed at the enhancer of this gene, but all of these interactions are limited to cell types that express MCK. This is interesting because two of the footprints appear to represent muscle specific use of general transcription factors, while the other two correspond to sites that can bind the myogenic regulator, MyoD1, in vitro. MyoD1 and these general factors are present in myoblasts, but can bind to the enhancer only in myocytes. This suggests that either the factors themselves are post-translationally modified (phosphorylation or protein:protein interactions), or the accessibility of the enhancer to the factors is limited (changes in chromatin structure). The in vivo footprinting study of MCK was performed with a new ligation mediated, single-sided PCR (polymerase chain reaction) technique that I have developed.

The second half of the thesis concerns the regulation of mouse metallothionein (MT). Metallothioneins are a family of highly conserved housekeeping genes whose expression can be induced by heavy metals, steroids, and other stresses. By adapting a primer extension method of genomic sequencing to in vivo footprinting, I've observed both metal inducible and noninducible interactions at the promoter of MT-I. From these results I've been able to limit the possible mechanisms by which metal responsive trans-acting factors induce transcription. These interpretations correlate with a second line of experiments involving the stable titration of positive acting factors necessary for induction of MT. I've amplified the promoter of MT to 10^2-10^3 copies per cell by fusing the 5' and 3' ends of the MT gene to the coding region of DHFR and selecting cells for methotrexate resistance. In these cells, there is a metal-specific titration effect, and although it acts at the level of transcription, it appears to be independent of direct DNA binding factors.

Environmental variables and fisheries diversity of the Naaf River Estuary, Bangladesh

The Naaf River estuary is one of the large estuaries in the Bangladesh coastal region not to have been affected by extensive human disturbance. This research provides information about the fisheries diversity status by Estuarine Set Bag Net (ESBN) sampling relation to physicochemical variables in both spatio-temporal scales. About 25 km of the lower estuary was divided into six zones for sample collection by considering the accessibility and availability of the ESBN operation, fish landing centers and location of the fishing villages. In total 48 samples have been analyzed which were taken throughout March to October 2006. To quantify the species diversity, all fisheries data were analyzed by using EstimateS and EcoSim software which accounts the different diversity indices viz., species richness, Shannon–Wiener diversity Index, Dominance and Evenness index. The research results demonstrate that the Naaf River estuary is a habitat of 161 (species richness, Sobs=161, Choa 1=162±2.34, ACE=161.73) different species which belong to 98 fin fishes, 23 shrimps and prawns,13 crabs, 11 molluscs, 3 echinoderms, 4 other crustaceans;while 9 remain unidentified. Results on the aquatic environment,mainly salinity and turbidity were found to have a major influence on their occurrence and distribution. All the findings indicated that the Naaf River estuary is a highly productive system and provides a favourable environment for large variety of estuarine species assemblages.

Investigations of DNA-mediated protein oxidation

DNA charge transport (CT) involves the efficient transfer of electrons or electron holes through the DNA π-stack over long molecular distances of at least 100 base-pairs. Despite this shallow distance dependence, DNA CT is sensitive to mismatches or lesions that disrupt π-stacking and is critically dependent on proper electronic coupling of the donor and acceptor moieties into the base stack. Favorable DNA CT is very rapid, occurring on the picosecond timescale. Because of this speed, electron holes equilibrate along the DNA π-stack, forming a characteristic pattern of DNA damage at low oxidation potential guanine multiplets. Furthermore, DNA CT may be used in a biological context. DNA processing enzymes with 4Fe4S clusters can perform DNA-mediated electron transfer (ET) self-exchange reactions with other 4Fe4S cluster proteins, even if the proteins are quite dissimilar, as long as the DNA-bound [4Fe4S]^3+/2+ redox potentials are conserved. This mechanism would allow low copy number DNA repair proteins to find their lesions efficiently within the cell. DNA CT may also be used biologically for the long-range, selective activation of redox-active transcription factors. Within this work, we pursue other proteins that may utilize DNA CT within the cell and further elucidate aspects of the DNA-mediated ET self-exchange reaction of 4Fe4S cluster proteins.

Dps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. One aspect of their protection involves ferroxidase activity, whereby ferrous iron is bound and oxidized selectively by hydrogen peroxide, thereby preventing formation of damaging hydroxyl radicals via Fenton chemistry. Understanding the specific mechanism by which Dps proteins protect the bacterial genome could inform the development of new antibiotics. We investigate whether DNA-binding E. coli Dps can utilize DNA CT to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate oxidative DNA damage via the flash-quench technique, which localizes to a low potential guanine triplet. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps which lack available reducing equivalents, significantly attenuates the yield of oxidative DNA damage at the guanine triplet. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA CT may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance.

Further work focused on spectroscopic characterization of the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation via the flash-quench technique. Upon irradiation with poly(dGdC)₂, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)₂ is substituted with poly(dAdT)₂, the yield of Dps oxidation is decreased significantly, indicating that guanine radicals facilitate Dps oxidation. The more favorable oxidation of Dps by guanine radicals supports the feasibility of a long-distance protection mechanism via DNA CT where Dps is oxidized to fill guanine radical holes in the bacterial genome produced by reactive oxygen species.

We have also explored possible electron transfer intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the ferroxidase site (W52 in E. coli Dps). In comparison to WT Dps, in EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, W52Y and W52A mutants were deficient in forming the characteristic EPR signal at g = 4.3, with a larger deficiency for W52A compared to W52Y. In addition to EPR, we also probed the role of W52 Dps in cells using a hydrogen peroxide survival assay. Bacteria containing W52Y Dps survived the hydrogen peroxide challenge more similarly to those containing WT Dps, whereas cells with W52A Dps died off as quickly as cells without Dps. Overall, these results suggest the possibility of W52 as a CT hopping intermediate.

DNA-modified electrodes have become an essential tool for the study of the redox chemistry of DNA processing enzymes with 4Fe4S clusters. In many cases, it is necessary to investigate different complex samples and substrates in parallel in order to elucidate this chemistry. Therefore, we optimized and characterized a multiplexed electrochemical platform with the 4Fe4S cluster base excision repair glycosylase Endonuclease III (EndoIII). Closely packed DNA films, where the protein has limited surface accessibility, produce EndoIII electrochemical signals sensitive to an intervening mismatch, indicating a DNA-mediated process. Multiplexed analysis allowed more robust characterization of the CT-deficient Y82A EndoIII mutant, as well as comparison of a new family of mutations altering the electrostatics surrounding the 4Fe4S cluster in an effort to shift the reduction potential of the cluster. While little change in the DNA-bound midpoint potential was found for this family of mutants, likely indicating the dominant effect of DNA-binding on establishing the protein redox potential, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we proposed that the electron transfer pathway in EndoIII can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.

While the 4Fe4S cluster of EndoIII is relatively insensitive to oxidation and reduction in solution, we have found that upon DNA binding, the reduction potential of the [4Fe4S]^3+/2+ couple shifts negatively by approximately 200 mV, bringing this couple into a physiologically relevant range. Demonstrated using electrochemistry experiments in the presence and absence of DNA, these studies do not provide direct molecular evidence for the species being observed. Sulfur K-edge X-ray absorbance spectroscopy (XAS) can be used to probe directly the covalency of iron-sulfur clusters, which is correlated to their reduction potential. We have shown that the Fe-S covalency of the 4Fe4S cluster of EndoIII increases upon DNA binding, stabilizing the oxidized [4Fe4S]³⁺ cluster, consistent with a negative shift in reduction potential. The 7% increase in Fe-S covalency corresponds to an approximately 150 mV shift, remarkably similar to DNA electrochemistry results. Therefore we have obtained direct molecular evidence for the shift in 4Fe4S reduction potential of EndoIII upon DNA binding, supporting the feasibility of our model whereby these proteins can utilize DNA CT to cooperate in order to efficiently find DNA lesions inside cells.

In conclusion, in this work we have explored the biological applications of DNA CT. We discovered that the DNA-binding bacterial ferritin Dps can protect the bacterial genome from a distance via DNA CT, perhaps contributing to pathogen survival and virulence. Furthermore, we optimized a multiplexed electrochemical platform for the study of the redox chemistry of DNA-bound 4Fe4S cluster proteins. Finally, we have used sulfur K-edge XAS to obtain direct molecular evidence for the negative shift in 4Fe4S cluster reduction potential of EndoIII upon DNA binding. These studies contribute to the understanding of DNA-mediated protein oxidation within cells.