Mi primera experiencia en la metodología del caso… y algo más

Capítulo 5 del libro: Guisasola, Jenaro ; Garmendia, Mikel (eds.) "Aprendizaje basado en problemas, proyectos y casos: diseño e implementación de experiencias en la universidad" (ISBN: 978-84-9860-959-2)

Effects of different types of fertilizers on plankton productivity in earthen ponds

Three fertilizer types (NPK, Super-phosphate and cow dung) were applied at two levels (Low, 0.3 kg/25m super(2)/2weeks and High, 0.7kg/25 m super(2)/2weeks) to 12 ponds with two ponds serving as control. Each pond had an area of 25 m super(2). Application of fertilizers and monitoring of plankton productivity and water quality parameters continued fortnightly for 52 days. Results obtained were subjected to Statistical Variance Analysis. The abundance of phytoplankton was in the order: Chlorophyceae > Bacillariophyceae > Cyanophyceae > Desmideaceae. While that of zooplankton followed the order: Crustacean > Rotifer > Protozoan. Primary productivity showed a variation between treatments with lowest value of 5592 mg/O sub(2)/m super(3)/day obtained in the control and cow dung low application rates (1.5 kg/25 m super(2)/2weeks). The highest value for primary productivity was obtained at M sub(2) (0.7 kg/25 m super(2)/2weeks, N.P.K) with primary productivity value of 7200 mg/O sub(2)/m super(3)/day, closely followed by M sub(4) (0.7 kg/25 m super(2)/2weeks, super phosphate) with 6792 mg/O sub(2)/m super(3)/day.

Anwendungsmöglichkeiten instrumenteller Texturanalyse zur Charakterisierung industriell hergestellter Farcen

Commercially processed and for industrial purpose destined minces of several fish species were characterised with regard to their texture using texture profile analysis (TPA) of boiled specimen and by measuring the penetration force on raw minces as well as with regard to their colour by instrumental measurements of CIELab values. Concerning the parameters investigated the minces were in most cases significantly different. It was evident that the fish species is responsible for the functional properties of these intermediate products. For the TPA the influence of the measurement conditions was demonstrated and an appropriate method derived. IEF of proteins was used to verify the declared fish species. Unfortunately, this was not possible in all cases due to the lack of comparable pattern. Both the DMA and FA content were found to be in the normal range except those for saithe.

The role of women in artisanal fisheries along the Lower Benue River

A questionnaire survey was carried out to assess the role of women in the artisanal fisheries of fish markets sector along the Lower Benue River. Four (4) fishing settlement communities of fish markets along the Lower Benue were selected. They are namely North Bank, Wadata, Abinse and Gbajimba . Structure and unstructured questionnaire were administered to cover areas of personal data, fish production activities, processing and marketing among others. The study revealed that women along the Lower Benue River participate activity in the artisanal fisheries sub-sector. However, the major role played by the women was in the area of processing and preservation, marketing and distribution. These accounted 52 and 42% respectively. The distribution of the women was not tribally based however, Jukun women accounted for 66.20% followed by Agatu women 13.00%. The age bracket of the women mostly involved in fisheries activities was between 31-40 years. About 86% of them was married. The initial capital base was low and mostly by personal saving accounting for 52%. Need for government assistance was the highlight in this study. About 86% sought for Federal Government intervention from the National Poverty Eradication Programme. This they felt will be a welcome development to boost women participation in the Artisanal fisheries sub-sector along the Lower Benue River

Report of the Inter-American Tropical Tuna Commission for the year 1971

ENGLISH: The Inter-American Tropical Tuna Commission operates under the authority and direction of a convention originally entered into by the Republic of Costa Rica and the United States of America. The convention, which came into force in 1950, is open to adherence by other governments whose nationals fish for tropical tunas in the eastern Pacific Ocean. Under this provision the Republic of Panama adhered in 1953, the Republic of Ecuador in 1961, the United Mexican States in 1964, Canada in 1968 and Japan in 1970. In 1967, Ecuador gave notice of her intent to withdraw from the Commission, and her withdrawal became effective on August 21,1968. SPANISH: La Comisión Interamericana del Atún Tropical está bajo la autoridad y dirección de una convención la cual fue originalmente formada por la República de Costa Rica y los Estados Unidos de América. La Convención, vigente desde 1950, está abierta a la afiliación de otros gobiernos cuyos nacionales pesquen atún en el Pacífico oriental tropical. Bajo esta medida la República de Panamá se afilió en 1953, la República del Ecuador en 1961, los Estados Unidos Mexicanos en 1964, Canadá en 1968 y el Japón en 1970. En 1967, el Ecuador anunció su intención de retirarse de la Comisión y la renuncia se hizo efectiva el 21 de agosto de 1968. (PDF contains 127 pages.)

Myxobolus aeglefini in Wittlingen (Merlangius merlangus)der Nordsee und der lrischen See

In June 1994 and 1995 stations in the North, Irish, Celtic Seas and the Channel were studied for the occurrence of Myxobolus aeglefini in whiting (Merlangius merlangus). The disease was visible externally as either white nodules of a few millimeters diameter in the upper mouth cavity, gill arches and the basis of pelvic fins and in severe cases also on the lower jaws or in the cornea and sclera of the eye. It was verified morphometrically in histological sections of infected eyes by size and shape of spores. Myxobolus aeglefini was present in low prevalences at two North Sea stations and high prevalences of up to 49 % in the Irish Sea (Solway Firth) during both cruises. Whiting between 23 and 55 cm were found to be infected. Neither length-age prevalences nor condition factors and gonado, spleen, liver somatic indices differed in diseased and healthy fishes.

New strategies for the total synthesis of aza-propellane natural products

The propellane alkaloids comprise a large class of natural products that possess varying degrees of structural complexity and biological activity. The earliest of these to be isolated was acutumine, a chlorinated alkaloid that has been shown to exhibit selective T-cell cytotoxicity and antiamnesic properties. Alternatively, the hasubanan family of natural products has garnered considerable attention from the synthetic community in part due to its structural similarities to morphine. While these alkaloids have been the subject of numerous synthetic studies over the last forty years, very few enantioselective total syntheses have been reported to date.

As part of a research program directed towards the synthesis of various alkaloid natural products, we have developed a unified strategy for the preparation of the hasubanan and acutumine alkaloids. Specifically, a highly diastereoselective 1,2-addition of organometallic reagents to benzoquinone-derived tert-butanesulfinimines was established, which provides access to enantioenriched 4-aminocyclohexadienone products. This methodology enabled the enantioselective construction of functionalized dihydroindolones, which were found to undergo intramolecular Friedel-Crafts conjugate additions to furnish the propellane cores of several hasubanan alkaloids. As a result of these studies, the first enantioselective total syntheses of 8-demethoxyrunanine and cepharatines A, C, and D were accomplished in 9-11 steps from commercially available starting materials.

More recent efforts have focused on applying the sulfinimine methodology to the synthesis of a more structurally complex propellane alkaloid, acutumine. Extensive studies have determined that a properly functionalized dihydroindolone undergoes a photochemical [2+2] cycloaddition followed by a lactone fragmentation/Dieckmann cyclization to establish the carbocyclic framework of the natural product. The preparation of more appropriately oxidized propellane intermediates is currently under investigation, and is anticipated to facilitate our synthetic endeavors toward acutumine.

Electron flow through cytochrome P450

The cytochromes P450 (P450s) are a remarkable class of heme enzymes that catalyze the metabolism of xenobiotics and the biosynthesis of signaling molecules. Controlled electron flow into the thiolate-ligated heme active site allows P450s to activate molecular oxygen and hydroxylate aliphatic C–H bonds via the formation of high-valent metal-oxo intermediates (compounds I and II). Due to the reactive nature and short lifetimes of these intermediates, many of the fundamental steps in catalysis have not been observed directly. The Gray group and others have developed photochemical methods, known as “flash-quench,” for triggering electron transfer (ET) and generating redox intermediates in proteins in the absence of native ET partners. Photo-triggering affords a high degree of temporal precision for the gating of an ET event; the initial ET and subsequent reactions can be monitored on the nanosecond-to-second timescale using transient absorption (TA) spectroscopies. Chapter 1 catalogues critical aspects of P450 structure and mechanism, including the native pathway for formation of compound I, and outlines the development of photochemical processes that can be used to artificially trigger ET in proteins. Chapters 2 and 3 describe the development of these photochemical methods to establish electronic communication between a photosensitizer and the buried P450 heme. Chapter 2 describes the design and characterization of a Ru-P450-BM3 conjugate containing a ruthenium photosensitizer covalently tethered to the P450 surface, and nanosecond-to-second kinetics of the photo-triggered ET event are presented. By analyzing data at multiple wavelengths, we have identified the formation of multiple ET intermediates, including the catalytically relevant compound II; this intermediate is generated by oxidation of a bound water molecule in the ferric resting state enzyme. The work in Chapter 3 probes the role of a tryptophan residue situated between the photosensitizer and heme in the aforementioned Ru-P450 BM3 conjugate. Replacement of this tryptophan with histidine does not perturb the P450 structure, yet it completely eliminates the ET reactivity described in Chapter 2. The presence of an analogous tryptophan in Ru-P450 CYP119 conjugates also is necessary for observing oxidative ET, but the yield of heme oxidation is lower. Chapter 4 offers a basic description of the theoretical underpinnings required to analyze ET. Single-step ET theory is first presented, followed by extensions to multistep ET: electron “hopping.” The generation of “hopping maps” and use of a hopping map program to analyze the rate advantage of hopping over single-step ET is described, beginning with an established rhenium-tryptophan-azurin hopping system. This ET analysis is then applied to the Ru-tryptophan-P450 systems described in Chapter 2; this strongly supports the presence of hopping in Ru-P450 conjugates. Chapter 5 explores the implementation of flash-quench and other phototriggered methods to examine the native reductive ET and gas binding events that activate molecular oxygen. In particular, TA kinetics that demonstrate heme reduction on the microsecond timescale for four Ru-P450 conjugates are presented. In addition, we implement laser flash-photolysis of P450 ferrous–CO to study the rates of CO rebinding in the thermophilic P450 CYP119 at variable temperature. Chapter 6 describes the development and implementation of air-sensitive potentiometric redox titrations to determine the solution reduction potentials of a series of P450 BM3 mutants, which were designed for non-native cyclopropanation of styrene in vivo. An important conclusion from this work is that substitution of the axial cysteine for serine shifts the wild type reduction potential positive by 130 mV, facilitating reduction by biological redox cofactors in the presence of poorly-bound substrates. While this mutation abolishes oxygenation activity, these mutants are capable of catalyzing the cyclopropanation of styrene, even within the confines of an E. coli cell. Four appendices are also provided, including photochemical heme oxidation in ruthenium-modified nitric oxide synthase (Appendix A), general protocols (Appendix B), Chapter-specific notes (Appendix C) and Matlab scripts used for data analysis (Appendix D).

Mechanisms of femtosecond laser-induced damage in magnesium fluoride

We studied the single-shot damage in magnesium fluoride irradiated by 800 nm femtosecond (fs) laser. The dependence of damage thresholds on the laser pulse durations from 60 to 750 fs was measured. The pump-probe measurements were carried out to investigate the time-resolved electronic excitation processes. A coupled dynamic model was applied to study the microprocesses in the interaction between fs laser and magnesium fluoride. The results indicate that both multiphoton ionization and avalanche ionization play important roles in the femtosecond laser-induced damage in MgF2. (C) 2006 Elsevier Ltd. All rights reserved.

Was wird aus dem Ostseedorsch

Die beiden Dorschbestände der Ostsee durchlaufen eine unterschiedliche Entwicklung. Sie haben allerdings gemeinsam, daß sie seit einigen Jahren immer kleiner werden. Die Arbeitsgruppe des Internationalen Rates für Meeresforschung, die für die Bodenfische in der Ostsee zuständig ist, hat sich in ihrer diesjährigen Sitzung wieder eingehend mit der augenblicklichen Situation befaßt.

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.