Comparative Study of Protein Unfolding in Aqueous Urea and Dimethyl Sulfoxide Solutions: Surface Polarity, Solvent Specificity, and Sequence of Secondary Structure Melting

Elucidation of possible pathways between folded (native) and unfolded states of a protein is a challenging task, as the intermediates are often hard to detect. Here, we alter the solvent environment in a controlled manner by choosing two different cosolvents of water, urea, and dimethyl sulfoxide (DMSO) and study unfolding of four different proteins to understand the respective sequence of melting by computer simulation methods. We indeed find interesting differences in the sequence of melting of alpha helices and beta sheets in these two solvents. For example, in 8 M urea solution, beta-sheet parts of a protein are found to unfold preferentially, followed by the unfolding of alpha helices. In contrast, 8 M DMSO solution unfolds alpha helices first, followed by the separation of beta sheets for the majority of proteins. Sequence of unfolding events in four different alpha/beta proteins and also in chicken villin head piece (HP-36) both in urea and DMSO solutions demonstrate that the unfolding pathways are determined jointly by relative exposure of polar and nonpolar residues of a protein and the mode of molecular action of a solvent on that protein.

Jacalin-carbohydrate interactions: distortion of the ligand molecule as a determinant of affinity

Jacalin is among the most thoroughly studied lectins. Its carbohydrate-binding site has also been well characterized. It has been postulated that the lower affinity of beta-galactosides for jacalin compared with beta-galactosides is caused by steric interactions of the substituents in the former with the protein. This issue has been explored energetically and structurally using different appropriate carbohydrate complexes of jacalin. It turns out that the earlier postulation is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in beta-galactosides. The difference in energy, and therefore in affinity, is caused by a distortion of the sugar ring in beta-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands for jacalin.

Environmental monitoring of bacterial contamination and antibiotic resistance patterns of the fecal coliforms isolated from Cauvery River, a major drinking water source in Karnataka, India

The present study focuses prudent elucidation of microbial pollution and antibiotic sensitivity profiling of the fecal coliforms isolated from River Cauvery, a major drinking water source in Karnataka, India. Water samples were collected from ten hotspots during the year 2011-2012. The physiochemical characteristics and microbial count of water samples collected from most of the hotspots exhibited greater biological oxygen demand and bacterial count especially coliforms in comparison with control samples (p <= 0.01). The antibiotic sensitivity testing was performed using 48 antibiotics against the bacterial isolates by disk-diffusion assay. The current study showed that out of 848 bacterial isolates, 93.51 % (n=793) of the isolates were found to be multidrug-resistant to most of the current generation antibiotics. Among the major isolates, 96.46 % (n=273) of the isolates were found to be multidrug-resistant to 30 antibiotics and they were identified to be Escherichia coli by 16S rDNA gene sequencing. Similarly, 93.85 % (n=107), 94.49 % (n=103), and 90.22 % (n=157) of the isolates exhibited multiple drug resistance to 32, 40, and 37 antibiotics, and they were identified to be Enterobacter cloacae, Pseudomonas trivialis, and Shigella sonnei, respectively. The molecular studies suggested the prevalence of blaTEM genes in all the four isolates and dhfr gene in Escherichia coli and Sh. sonnei. Analogously, most of the other Gram-negative bacteria were found to be multidrug-resistant and the Gram-positive bacteria, Staphylococcus spp. isolated from the water samples were found to be methicillin and vancomycin-resistant Staphylococcus aureus. This is probably the first study elucidating the bacterial pollution and antibiotic sensitivity profiling of fecal coliforms isolated from River Cauvery, Karnataka, India.

Raman and infra-red microspectroscopy: towards quantitative evaluation for clinical research by ratiometric analysis

Biomolecular structure elucidation is one of the major techniques for studying the basic processes of life. These processes get modulated, hindered or altered due to various causes like diseases, which is why biomolecular analysis and imaging play an important role in diagnosis, treatment prognosis and monitoring. Vibrational spectroscopy (IR and Raman), which is a molecular bond specific technique, can assist the researcher in chemical structure interpretation. Based on the combination with microscopy, vibrational microspectroscopy is currently emerging as an important tool for biomedical research, with a spatial resolution at the cellular and sub-cellular level. These techniques offer various advantages, enabling label-free, biomolecular fingerprinting in the native state. However, the complexity involved in deciphering the required information from a spectrum hampered their entry into the clinic. Today with the advent of automated algorithms, vibrational microspectroscopy excels in the field of spectropathology. However, researchers should be aware of how quantification based on absolute band intensities may be affected by instrumental parameters, sample thickness, water content, substrate backgrounds and other possible artefacts. In this review these practical issues and their effects on the quantification of biomolecules will be discussed in detail. In many cases ratiometric analysis can help to circumvent these problems and enable the quantitative study of biological samples, including ratiometric imaging in 1D, 2D and 3D. We provide an extensive overview from the recent scientific literature on IR and Raman band ratios used for studying biological systems and for disease diagnosis and treatment prognosis.

Growth variation and stock structure in North Pacific albacore

Working paper NPALB/87/21 submitted to the 10th North Pacific Albacore Workshop. Paper reports the results of ongoing research on validated age and growth models and the elucidation of stock structure for the North Pacific albacore. (Document pdf contains 22 pages)

Observations on the spawning of four species on tuna (Neothunnus macropterus, Katsuwonus pelamis, Auxis thazard and Euthynnus lineatus) in the Eastern Pacific Ocean, based on the distribution of their larvae and juveniles

ENGLISH: Knowledge of spawning habits is useful in the elucidation of the life history, ecology and population structure of tropical tunas, and is essential to the sound management of these resources. Until recently, little was known concerning the spawning of tunas, or about the distribution of their larval and juvenile stages, in the Eastern Pacific Ocean. Nichols and Murphy (1944) reported the capture off Colombia of young scombroids ultimately identified as frigate mackerel, Auxis thazard (Schaefer and Marr, 1948a). Fowler (1944) reported the capture off Manzanillo, Mexico of two young tunas, one of which is definitely and the other most likely Neothunnus macropterus (Klawe, 1959). In 1947, young of N. macropterus, K. pelamis, A. thazard and E. lineatus were caught offshore from Central America (Schaefer and Marr, 1948a, 1948b, and Schaefer, 1948). Further collections of young N. macropterus, A. thazard and E. lineatus were made in the same general area in the spring of 1949 (Mead, 1951). In January and February 1955, Clemens (1956) carried Out experiments in rearing young tunas, E. lineatus and A. thazard, in shipboard aquaria, using fish caught off Central America. Matsumoto (1958) reported captures of larval N. macropterus and K. pelamis in the area along the 120th meridian of west longitude. Klawe (1958 and 1961b) reported captures of larval N. macropterus and Auxis from the Revillagigedo Islands. Captures of young Auxis and E. lineatus in the Gulf of Panama in January 1922 during the Dana Expedition have recently been reported by Matsumoto (1959). Capture of juveniles of K. pelamis, E. lineatus and Auxis in the area off tropical Mexico and in the area of outlying islands during the SCOT Expedition has been reported by Klawe (1960a). SPANISH: El conocimiento sobre los hábitos del desove es útil para el esclarecimiento de la historia natural, ecología y estructura de las poblaciones de atunes tropicales, y es esencial para la acertada administración de estos recursos. Hasta hace poco tiempo no se sabía mucho sobre el desove de los atunes o acerca de la distribución de sus larvas y juveniles en el Océano Pacífico Oriental. Nichols y Murphy (1944) informaron sobre la captura frente a Colombia de escómbridos jóvenes últimamente identificados como melva, Auxis thazard (Schaefer y Marr, 1948a). Fowler (1944) también informó sobre la captura de dos atunes jóvenes frente a Manzanillo, México, uno de los cuales era definitivamente Neothunnus macropterus y el otro era lo más probable que también lo fuera (Klawe, 1959). En 1947 se capturaron especímenes juveniles de N. macropterus, K. pelamis, A. thazard y E. lineatus frente a la América Central (Schaefer y Marr, 1948a, 1948b, y Schaefer, 1948). Otras recolecciones de ejemplares jóvenes de N. macropterus, A. thazard y E. lineatus fueron hechas en la misma área general durante la primavera de 1949 (Mead, 1951). En enero y febrero de 1955, Clemens (1956) efectuó experimentos de crianza de atunes jóvenes, E. lineatus y A.. thazard, en acuarios a bordo para lo que empleó peces capturados frente a la América Central. Matsumoto (1958) informó sobre capturas de larvas de N. macropterus y K. pelamis en el área a lo largo del meridiano 120 de longitud oeste. Klawe (1958 y 1961b) ha dado cuenta también de capturas de larvas de N. macropterus y Auxis en las Islas Revillagigedo. Matsumoto (1959) ha informado recientemente acerca de capturas de ejemplares jóvenes de Auxis y E. lineatus en el Golfo de Panamá en enero de 1922 durante la Expedición Dana. Klawe (1960a) informó así mismo que durante la Expedición SCOT se capturaron juveniles de K. pelamis, E. lineatus y Auxis en el área frente a la zona tropical de México y en la región de las islas alejadas del continente.

Novel mechanisms for regulation of cell survival and migration by ceramide 1-phosphate

201 p. : gráf.

Morphometric comparison of skipjack from the Central and Eastern Tropical Pacific Ocean

ENGLISH: One of the important problems in the current investigations of the Inter-American Tropical Tuna Commission is the determination of the racial affinities of the stocks of yellowfin and skipjack inhabiting the Eastern Tropical Pacific fishing region. The nature of the problem is twofold. Primary concern is elucidation of the inter-regional relationship of stocks between the Eastern Tropical Pacific fishing region and those further to the westward. Hardly less important, however, is the intraregional relationship of stocks within the regions, particularly the Eastern Pacific region. This latter relationship must be at least partially understood in order properly to approach investigation of the former. SPANISH: Dentro de las las investigaciones que al presente efectúa la Comisión Interamericana del Atún Tropical, uno de los importantes problemas consiste en la determinación de las afinidades raciales de los stocks de atún aleta amarilla y barrilete que se encuentran en las regiones de pesca del Pacífico Oriental Tropical. La naturaleza del problema es doble. El interés primario es la elucidación de la relación inter-regional entre los stocks de la región pesquera del Pacífico Oriental Tropical y los de aquéllas más hacia el oeste de dicho océano. Apenas menos importante, sin embargo, es la relación intra-regional de los stocks dentro de las regiones, particularmente la del Pacífico del Este. Esta última relación debe ser por lo menos parcialmente conocida, a fin de abordar con propiedad la anteriormente citada. (PDF contains 63 pages.)

Geological evolution of two crustal scale shear zones. Part I. The Rand thrust complex, northwestern Mojave Desert, California. Part II. The Magdalena metamorphic core complex, north central Sonora, Mexico

The geology and structure of two crustal scale shear zones were studied to understand the partitioning of strain within intracontinental orogenic belts. Movement histories and regional tectonic implications are deduced from observational data. The two widely separated study areas bear the imprint of intense Late Mesozoic through Middle Cenozoic tectonic activity. A regional transition from Late Cretaceous-Early Tertiary plutonism, metamorphism, and shortening strain to Middle Tertiary extension and magmatism is preserved in each area, with contrasting environments and mechanisms. Compressional phases of this tectonic history are better displayed in the Rand Mountains, whereas younger extensional structures dominate rock fabrics in the Magdalena area.

In the northwestern Mojave desert, the Rand Thrust Complex reveals a stack of four distinctive tectonic plates offset along the Garlock Fault. The lowermost plate, Rand Schist, is composed of greenschist facies metagraywacke, metachert, and metabasalt. Rand Schist is structurally overlain by Johannesburg Gneiss (= garnet-amphibolite grade orthogneisses, marbles and quartzites), which in turn is overlain by a Late Cretaceous hornblende-biotite granodiorite. Biotite granite forms the fourth and highest plate. Initial assembly of the tectonic stack involved a Late Cretaceous? south or southwest vergent overthrusting event in which Johannesburg Gneiss was imbricated and attenuated between Rand Schist and hornblende-biotite granodiorite. Thrusting postdated metamorphism and deformation of the lower two plates in separate environments. A post-kinematic stock, the Late Cretaceous Randsburg Granodiorite, intrudes deep levels of the complex and contains xenoliths of both Rand Schist and mylonitized Johannesburg? gneiss. Minimum shortening implied by the map patterns is 20 kilometers.

Some low angle faults of the Rand Thrust Complex formed or were reactivated between Late Cretaceous and Early Miocene time. South-southwest directed mylonites derived from Johannesburg Gneiss are commonly overprinted by less penetrative north-northeast vergent structures. Available kinematic information at shallower structural levels indicates that late disturbance(s) culminated in northward transport of the uppermost plate. Persistence of brittle fabrics along certain structural horizons suggests a possible association of late movement(s) with regionally known detachment faults. The four plates were juxtaposed and significant intraplate movements had ceased prior to Early Miocene emplacement of rhyolite porphyry dikes.

In the Magdalena region of north central Sonora, components of a pre-Middle Cretaceous stratigraphy are used as strain markers in tracking the evolution of a long lived orogenic belt. Important elements of the tectonic history include: (1) Compression during the Late Cretaceous and Early Tertiary, accompanied by plutonism, metamorphism, and ductile strain at depth, and thrust driven? syntectonic sedimentation at the surface. (2) Middle Tertiary transition to crustal extension, initially recorded by intrusion of leucogranites, inflation of the previously shortened middle and upper crustal section, and surface volcanism. (3) Gravity induced development of a normal sense ductile shear zone at mid crustal levels, with eventual detachment and southwestward displacement of the upper crustal stratigraphy by Early Miocene time.

Elucidation of the metamorphic core complex evolution just described was facilitated by fortuitous preservation of a unique assemblage of rocks and structures. The "type" stratigraphy utilized for regional correlation and strain analysis includes a Jurassic volcanic arc assemblage overlain by an Upper Jurassic-Lower Cretaceous quartz pebble conglomerate, in turn overlain by marine strata with fossiliferous Aptian-Albian limestones. The Jurassic strata, comprised of (a) rhyolite porphyries interstratified with quartz arenites, (b) rhyolite cobble conglomerate, and (c) intrusive granite porphyries, are known to rest on Precambrian basement north and east of the study area. The quartz pebble conglomerate is correlated with the Glance Conglomerate of southeastern Arizona and northeastern Sonora. The marine sequence represents part of an isolated arm? of the Bisbee Basin.

Crosscutting structural relationships between the pre-Middle Cretaceous supracrustal section, younger plutons, and deformational fabrics allow the tectonic sequence to be determined. Earliest phases of a Late Cretaceous-Early Tertiary orogeny are marked by emplacement of the 78 ± 3 Ma Guacomea Granodiorite (U/Pb zircon, Anderson et al., 1980) as a sill into deep levels of the layered Jurassic series. Subsequent regional metamorphism and ductile strain is recorded by a penetrative schistosity and lineation, and east-west trending folds. These fabrics are intruded by post-kinematic Early Tertiary? two mica granites. At shallower crustal levels, the orogeny is represented by north directed thrust faulting, formation of a large intermontane basin, and development of a pronounced unconformity. A second important phase of ductile strain followed Middle Tertiary? emplacement of leucogranites as sills and northwest trending dikes into intermediate levels of the deformed section (surficial volcanism was also active during this transitional period to regional extension). Gravitational instabilities resulting from crustal swelling via intrusion and thermal expansion led to development of a ductile shear zone within the stratigraphic horizon occupied by a laterally extensive leucogranite sill. With continued extension, upper crustal brittle normal faults (detachment faults) enhanced the uplift and tectonic denudation of this mylonite zone, ultimately resulting in southwestward displacement of the upper crustal stratigraphy.

Strains associated with the two ductile deformation events have been successfully partitioned through a multifaceted analysis. R_f/Ø measurements on various markers from the "type" stratigraphy allow a gradient representing cumulative strain since Middle Cretaceous time to be determined. From this gradient, noncoaxial strains accrued since emplacement of the leucogranites may be removed. Irrotational components of the postleucogranite strain are measured from quartz grain shapes in deformed granites; rotational components (shear strains) are determined from S-C fabrics and from restoration of rotated dike and vein networks. Structural observations and strain data are compatable with a deformation path of: (1) coaxial strain (pure shear?), followed by (2) injection of leucogranites as dikes (perpendicular to the minimum principle stress) and sills (parallel to the minimum principle stress), then (3) southwest directed simple shear. Modeling the late strain gradient as a simple shear zone permits a minimum displacement of 10 kilometers on the Magdalena mylonite zone/detachment fault system. Removal of the Middle Tertiary noncoaxial strains yields a residual (or pre-existing) strain gradient representative of the Late Cretaceous-Early Tertiary deformation. Several partially destrained cross sections, restored to the time of leucogranite emplacement, illustrate the idea that the upper plate of the core complex bas been detached from a region of significant topographic relief. 50% to 100% bulk extension across a 50 kilometer wide corridor is demonstrated.

Late Cenozoic tectonics of the Magdalena region are dominated by Basin and Range style faulting. Northeast and north-northwest trending high angle normal faults have interacted to extend the crust in an east-west direction. Net extension for this period is minor (10% to 15%) in comparison to the Middle Tertiary detachment related extensional episode.

Fluorescence microscopy of nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson’s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent α5 subunit and subsequent experiments to elucidate the cellular mechanisms through which α5 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of α5 are also examined.

Additionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.

Structure-, stereochemistry-, and metal-regulated DNA binding/cleaving molecules

In the cell, the binding of proteins to specific sequences of double helical DNA is essential for controlling the processes of protein synthesis (at the level of DNA transcription) and cell proliferation (at the level of DNA replication). In the laboratory, the sequence-specific DNA binding/cleaving properties of restriction endonuclease enzymes (secreted by microorganisms to protect them from foreign DNA molecules) have helped to fuel a revolution in molecular biology. The strength and specificity of a protein:DNA interaction depend upon structural features inherent to the protein and DNA sequences, but it is now appreciated that these features (and therefore protein:DNA complexation) may be altered (regulated) by other protein:DNA complexes, or by environmental factors such as temperature or the presence of specific organic molecules or inorganic ions. It is also now appreciated that molecules much smaller than proteins (including antibiotics of molecular weight less than 2000 and oligonucleotides) can bind to double-helical DNA in sequence-specific fashion. Elucidation of structural motifs and microscopic interactions responsible for the specific molecular recognition of DNA leads to greater understanding of natural processes and provides a basis for the design of novel sequence-specific DNA binding molecules. This thesis describes the synthesis and DNA binding/cleaving characteristics of molecules designed to probe structural, stereochemical, and environmental factors that regulate sequence-specific DNA recognition.

Chapter One introduces the DNA minor groove binding antibiotics Netropsin and Distamycin A, which are di- and tri(N-methylpyrrolecarboxamide) peptides, respectively. The method of DNA affinity cleaving, which has been employed to determine DNA binding properties of designed synthetic molecules is described. The design and synthesis of a series of Netropsin dimers linked in tail-to-tail fashion (by oxalic, malonic, succinic, or fumaric acid), or in head-to-tail fashion (by glycine, β-alanine, and γ-aminobutanoic acid (Gaba)) are presented. These Bis(Netropsin)s were appended with the iron-chelating functionality EDTA in order to make use of the technique of DNA affinity cleaving. Bis(Netropsin)-EDTA compounds are analogs of penta(N-methylpyrrolecarboxamide)-EDTA (P5E), which may be considered a head-to-tail Netropsin dimer linked by Nmethylpyrrolecarboxamide. Low- and high-resolution analysis of pBR322 DNA affinity cleaving by the iron complexes of these molecules indicated that small changes in the length and nature of the linker had significant effects on DNA binding/cleaving efficiency (a measure of DNA binding affinity). DNA binding/cleaving efficiency was found to decrease with changes in the linker in the order β-alanine > succinamide > fumaramide > N-methylpyrrolecarboxamide > malonamide >glycine, γ-aminobutanamide > oxalamide. In general, the Bis(Netropsin)-EDTA:Fe compounds retained the specificity for seven contiguous A:T base pairs characteristic of P5E:Fe binding. However, Bis(Netropsin)Oxalamide- EDTA:Fe exhibited decreased specificity for A:T base pairs, and Bis(Netropsin)-Gaba-EDT A:Fe exhibited some DNA binding sites of less than seven base pairs. Bis(Netropsin)s linked with diacids have C₂-symmmetrical DNA binding subunits and exhibited little DNA binding orientation preference. Bis(Netropsin)s linked with amino acids lack C₂-symmetrical DNA binding subunits and exhibited higher orientation preferences. A model for the high DNA binding orientation preferences observed with head-to-tail DNA minor groove binding molecules is presented.

Chapter Two describes the design, synthesis, and DNA binding properties of a series of chiral molecules: Bis(Netropsin)-EDTA compounds with linkers derived from (R,R)-, (S,S)-, and (RS,SR)-tartaric acids, (R,R)-, (S,S)-, and (RS,SR)-tartaric acid acetonides, (R)- and (S)-malic acids, N ,N-dimethylaminoaspartic acid, and (R)- and (S)-alanine, as well as three constitutional isomers in which an N-methylpyrrolecarboxamide (P1) subunit and a tri(N-methylpyrrolecarboxamide)-EDTA (P3-EDTA) subunit were linked by succinic acid, (R ,R)-, and (S ,S)-tartaric acids. DNA binding/cleaving efficiencies among this series of molecules and the Bis(Netropsin)s described in Chapter One were found to decrease with changes in the linker in the order β-alanine > succinamide > P1-succinamide-P3 > fumaramide > (S)-malicamide > N-methylpyrrolecarboxamide > (R)-malicamide > malonamide > N ,N-dimethylaminoaspanamide > glycine = Gaba = (S,S)-tartaramide = P1-(S,S)-tanaramide-P3 > oxalamide > (RS,SR)-tartaramide = P1- (R,R)-tanaramide-P3 > (R,R)-tartaramide (no sequence-specific DNA binding was detected for Bis(Netropsin)s linked by (R)- or (S)-alanine or by tartaric acid acetonides). The chiral molecules retained DNA binding specificity for seven contiguous A:T base pairs. From the DNA affinity cleaving data it could be determined that: 1) Addition of one or two substituents to the linker of Bis(Netropsin)-Succinamide resulted in stepwise decreases in DNA binding affinity; 2) molecules with single hydroxyl substituents bound DNA more strongly than molecules with single dimethylamino substituents; 3) hydroxyl-substituted molecules of (S) configuration bound more strongly to DNA than molecules of (R) configuration. This stereochemical regulation of DNA binding is proposed to arise from the inherent right-handed twist of (S)-enantiomeric Bis(Netropsin)s versus the inherent lefthanded twist of (R)-enantiomeric Bis(Netropsin)s, which makes the (S)-enantiomers more complementary to the right-handed twist of B form DNA.

Chapter Three describes the design and synthesis of molecules for the study of metalloregulated DNA binding phenomena. Among a series of Bis(Netropsin)-EDTA compounds linked by homologous tethers bearing four, five, or six oxygen atoms, the Bis(Netropsin) linked by a pentaether tether exhibited strongly enhanced DNA binding/cleaving in the presence of strontium or barium cations. The observed metallospecificity was consistent with the known affinities of metal cations for the cyclic hexaether 18-crown-6 in water. High-resolution DNA affinity cleaving analysis indicated that DNA binding by this molecule in the presence of strontium or barium was not only stronger but of different sequence-specificity than the (weak) binding observed in the absence of metal cations. The metalloregulated binding sites were consistent with A:T binding by the Netropsin subunits and G:C binding by a strontium or barium:pentaether complex. A model for the observed positive metalloregulation and novel sequence-specificity is presented. The effects of 44 different cations on DNA affinity cleaving by P5E:Fe were examined. A series of Bis(Netropsin)-EDTA compounds linked by tethers bearing two, three, four, or five amino groups was also synthesized. These molecules exhibited strong and specific binding to A:T rich regions of DNA. It was found that the iron complexes of these molecules bound and cleaved DNA most efficiently at pH 6.0-6.5, while P5E:Fe bound and cleaved most efficiently at pH 7.5-8.0. Incubating the Bis(Netropsin) Polyamine-EDTA:Fe molecules with K₂PdCl₄ abolished their DNA binding/cleaving activity. It is proposed that the observed negative metalloregulation arises from kinetically inert Bis(Netropsin) Polyamine:Pd(II) complexes or aggregates, which are sterically unsuitable for DNA complexation. Finally, attempts to produce a synthetic metalloregulated DNA binding protein are described. For this study, five derivatives of a synthetic 52 amino acid residue DNA binding/cleaving protein were produced. The synthetic mutant proteins carried a novel pentaether ionophoric amino acid residue at different positions within the primary sequence. The proteins did not exhibit significant DNA binding/cleaving activity, but they served to illustrate the potential for introducing novel amino acid residues within DNA binding protein sequences, and for the development of the tricyclohexyl ester of EDTA as a superior reagent for the introduction of EDT A into synthetic proteins.

Chapter Four describes the discovery and characterization of a new DNA binding/cleaving agent, [SalenMn(III)]OAc. This metal complex produces single- and double-strand cleavage of DNA, with specificity for A:T rich regions, in the presence of oxygen atom donors such as iodosyl benzene, hydrogen peroxide, or peracids. Maximal cleavage by [SalenMn(III)]OAc was produced at pH 6-7. A comparison of DNA singleand double-strand cleavage by [SalenMn(III)]⁺ and other small molecules (Methidiumpropyl-EDTA:Fe, Distamycin-EDTA:Fe, Neocarzinostatin, Bleomycin:Fe) is presented. It was found that DNA cleavage by [SalenMn(III)]⁺ did not require the presence of dioxygen, and that base treatment of DNA subsequent to cleavage by [SalenMn(III)]⁺ afforded greater cleavage and alterations in the cleavage patterns. Analysis of DNA products formed upon DNA cleavage by [SalenMn(III)] indicated that cleavage was due to oxidation of the sugar-phosphate backbone of DNA. Several mechanisms consistent with the observed products and reaction requirements are discussed.

Chapter Five describes progress on some additional studies. In one study, the DNA binding/cleaving specificities of Distamycin-EDTA derivatives bearing pyrrole N-isopropyl substituents were found to be the same as those of derivatives bearing pyrrole N-methyl substituents. In a second study, the design of and synthetic progress towards a series of nucleopeptide activators of transcription are presented. Five synthetic plasmids designed to test for activation of in vitro run-off transcription by DNA triple helix-forming oligonucleotides or nucleopeptides are described.

Chapter Six contains the experimental documentation of the thesis work.

Investigations on saprolegniosis. II. Study on the process of penetrations showing evidence of a proteolytic enzyme and histopathological aspects [Translation from: Riv.ital.Piscicolt.Ittiopatol. 11(4), 109-117, 1976]

The study of enzymatic activity is of great importance in the immunology of fungi. Indeed, knowledge of biological activity of antigenic structures is important for the elucidation of host-parasite relations as well as in the search for a taxonomic factor permitting differential diagnoses. The authors used Saprolegnia cultures to analyse soluble antigenic fractions arising from the mycelium of cultures of 4 species of Saprolegnia, which are found most frequently in the parasitic state on fish: S. parasitica, S. ferax, S. delica, S. diclina. The authors conclude that in the study of saprolegniasis, the enzymatic approach affords new elements for the examination of the etiology of fungi as well as an element of gravity concerning the biochemical modifications necessary to the change of saprophytism to parasitism.

Indisciplinada: a arte em um contexto escolar carioca: uma pesquisa no/do cotidiano

Trabalho centrado em pesquisa de campo de tipo etnográfico realizada em três escolas públicas da rede estadual de ensino do Rio de Janeiro. A dissertação é resultado das inquietações geradas diante do distanciamento entre o legitimado mundo das artes e a periferizada situação da escola pública: uma instituição que tem tantas vezes corroborado com a lógica da distinção. É possível (ao professor) trabalhar dentro da disciplina Artes sem ratificar essa lógica excludente? Exaltamos uma docência atenta ao caráter estético expresso na dupla: Arte via Ensino e Ensino via Arte. Oferece-se um olhar sobre o panorama das condições cotidianas que configuram algumas das mais destacadas realidades do Ensino da Arte na Educação Básica dinamizadas por suas macropolíticas, suas práticas diárias e suas crenças. A metodologia sugerida associa os estudos do cotidiano à revisão bibliográfica centrada nos estudos culturais e em algumas das atuais correntes críticas da Arte e de seu Ensino que circulam a contrapelo das verdades hegemônicas desses dois universos. Um dos objetivos do trabalho que entende a vida cotidiana como manifestação estética do cidadão comum é o de contribuir com a elucidação das tensões que fazem da Arte e, especificamente de seu ensino, um importante campo de embate entre iniciativas emancipatórias e ações reguladoras

Lugares e personagens do universo buarqueano

A partir da década de 1970, a ciência geográfica passou a entender que seus conceitos, suas categorias analíticas, bem como seus objetos de pesquisa poderiam se utilizar dos discursos literários, em suas diversas possibilidades, incluindo o cinema, a pintura e, até mesmo, a música. Compreender os lugares a partir dos panoramas narrados pelos literatos {escritores ou compositores} e fomentar uma leitura de mundo repleta de sentidos existenciais, oferecida pelas maneiras que os autores apreendem, se configura como um relevante caminho de entendimento do mundo vivido a partir da interrelação entre a linguagem científica e artística. Neste sentido, a corrente humanística em geografia tem se dedicado a esta análise, uma vez que a linguagem literária tem a particularidade de comunicar aspectos da vida ou fatos e tempos da experiência humana, revelando, pois, o sentimento e o entendimento do literato frente à sociedade. No que tange à literatura musical, os versos permitem múltiplas interpretações. A música pode afetar, comover, causar estranheza, interesse ou reflexão, fazer o corpo se movimentar ou relaxar e, ainda, servir de base para análises filosófico-científicas a respeito dos espaços e lugares geográficos. Dito isto, esta dissertação segue a trilha da geografia humanística e nossa intenção é mostrar esta corrente configurando um caminho para a elucidação da complexidade exposta pela análise subjetiva de um elenco de letras capturadas do acervo de Chico Buarque, bem como o desvendar da plêiade de efemérides, sentimentos, mazelas e vibrações encerradas no cancioneiro buarqueano e que podem ser lidas através do conceito de lugar, este confundido com a trajetória da geografia humanística.

Intervenção direta do estado no domínio econômico: contribuição para uma delimitação das esferas pública e privada a partir da Constituição Federal Brasileira de 1988.

A Constituição Federal Brasileira de 1988, primeiro texto constitucional após o regime militar no país, é um texto plural, pleno de significados e ambivalências, resultado de um esforço na busca de consensos em uma assembleia constituinte heterogênea num país democraticamente jovem. A abrangência de significados da Magna Carta precisa, portanto, a fim de ser aplicada às realidades também plurais e heterogêneas de um país enorme como o Brasil, ser interpretada à luz dessas mesmas realidades, sempre situadas e datadas. Nesse diapasão, a dedução dogmática da esfera legítima da atuação estatal no domínio econômico só pode ser apreendida através da técnica ponderativa, levando em conta o fato da constituição econômica encravada no texto da Lei Maior estabelecer como regra o desenvolvimento da atividade econômica pela livre iniciativa da sociedade. Sob o signo da emancipação da sociedade e da liberdade de empreender por um lado, e da obrigação do Estado garantir as condições da liberdade e do posicionamento estratégico do Brasil no cenário geopolítico e econômico mundial de outro, serão relidas as modalidades interventivas estatais como a prestação de serviços públicos, as atuações monopolistas e em regime de concorrência com a iniciativa privada, produzindo um quadro com as condições que justifiquem a presença do Estado na economia, servindo de guia dinâmico para a elucidação das fronteiras sempre contingentes entre as esferas pública e privada no domínio econômico.