Habitat mapping effort at the Olympic Coast National Marine Sanctuary – Current status and future needs

With elevating interest to establish conservation efforts for groundfish stocks and continued scrutiny over the value of marine protected areas along the west coast, the importance of enhancing our knowledge of seabed characteristics through mapping activities is becoming increasingly more important, especially in a timely manner. Shortly after the inception of the Seabed Mapping Initiative instituted with the US Geological Survey (USGS), the National Marine Sanctuary Program (NMSP) assembled a panel of habitat mapping experts. They determined that the status of existing data sets and future data acquisition needs varied widely among the individual sanctuaries and that more detailed site assessments were needed to better prioritize mapping efforts and outline an overall joint strategy. To assist with that specific effort and provide pertinent information for the Olympic Coast National Marine Sanctuary’s (OCNMS) Management Plan Review, this report summarizes the mapping efforts that have taken place at the site to date; calculates a timeframe for completion of baseline mapping efforts when operating under current data acquisition limitations; describes an optimized survey strategy to dramatically reduce the required time to complete baseline surveying; and provides estimates for the needed vessel sea-days (DAS) to accomplish baseline survey completion within a 2, 5 and 10 year timeframe. (PDF contains 38 pages.)

Olympic Coast National Marine Sanctuary Habitat Mapping: Survey report and classification of side scan sonar data from surveys HMPR-114-2004-02 and HMPR-116-2005-01.

The Olympic Coast National Marine Sanctuary (OCNMS) continues to invest significant resources into seafloor mapping activities along Washington’s outer coast (Intelmann and Cochrane 2006; Intelmann et al. 2006; Intelmann 2006). Results from these annual mapping efforts offer a snapshot of current ground conditions, help to guide research and management activities, and provide a baseline for assessing the impacts of various threats to important habitat. During the months of August 2004 and May and July 2005, we used side scan sonar to image several regions of the sea floor in the northern OCNMS, and the data were mosaicked at 1-meter pixel resolution. Video from a towed camera sled, bathymetry data, sedimentary samples and side scan sonar mapping were integrated to describe geological and biological aspects of habitat. Polygon features were created and attributed with a hierarchical deep-water marine benthic classification scheme (Greene et al. 1999). For three small areas that were mapped with both side scan sonar and multibeam echosounder, we made a comparison of output from the classified images indicating little difference in results between the two methods. With these considerations, backscatter derived from multibeam bathymetry is currently a costefficient and safe method for seabed imaging in the shallow (<30 meters) rocky waters of OCNMS. The image quality is sufficient for classification purposes, the associated depths provide further descriptive value and risks to gear are minimized. In shallow waters (<30 meters) which do not have a high incidence of dangerous rock pinnacles, a towed multi-beam side scan sonar could provide a better option for obtaining seafloor imagery due to the high rate of acquisition speed and high image quality, however the high probability of losing or damaging such a costly system when deployed as a towed configuration in the extremely rugose nearshore zones within OCNMS is a financially risky proposition. The development of newer technologies such as intereferometric multibeam systems and bathymetric side scan systems could also provide great potential for mapping these nearshore rocky areas as they allow for high speed data acquisition, produce precisely geo-referenced side scan imagery to bathymetry, and do not experience the angular depth dependency associated with multibeam echosounders allowing larger range scales to be used in shallower water. As such, further investigation of these systems is needed to assess their efficiency and utility in these environments compared to traditional side scan sonar and multibeam bathymetry. (PDF contains 43 pages.)

Benthic habitat mapping in the Olympic Coast National Marine Sanctuary: Classification of side scan sonar data from survey HMPR-108-2002-01: Version I

In September 2002, side scan sonar was used to image a portion of the sea floor in the northern OCNMS and was mosaiced at 1-meter pixel resolution using 100 kHz data collected at 300-meter range scale. Video from a remotely-operated vehicle (ROV), bathymetry data, sedimentary samples, and sonar mapping have been integrated to describe geological and biological aspects of habitat and polygon features have been created and attributed with a hierarchical deep-water marine benthic classification scheme (Greene et al. 1999). The data can be used with geographic information system (GIS) software for display, query, and analysis. Textural analysis of the sonar images provided a relatively automated method for delineating substrate into three broad classes representing soft, mixed sediment, and hard bottom. Microhabitat and presence of certain biologic attributes were also populated into the polygon features, but strictly limited to areas where video groundtruthing occurred. Further groundtruthing work in specific areas would improve confidence in the classified habitat map. (PDF contains 22 pages.)

Indexação e taxonomia em sites de webjornalismo : um estudo para aprimorar a recuperação de informação no Portal Câmara Notícias

Monografia (especialização) -- Curso de Arquitetura e Organização da Informação, Câmara dos Deputados, Centro de Formação, Treinamento e Aperfeiçoamento (Cefor) e Universidade Federal de Minas Gerais, Escola de Ciência da Informação (ECI), 2013.

Acoustic signatures of the seafloor: Tools for predicting grouper habitat

Groupers are important components of commercial and recreational fisheries. Current methods of diver-based grouper census surveys could potentially benefit from development of remotely sensed methods of seabed classification. The goal of the present study was to determine if areas of high grouper abundance have characteristic acoustic signatures. A commercial acoustic seabed mapping system, QTC View Series V, was used to survey an area near Carysfort Reef, Florida Keys. Acoustic data were clustered using QTC IMPACT software, resulting in three main acoustic classes covering 94% of the area surveyed. Diver-based data indicate that one of the acoustic classes corresponded to hard substrate and the other two represented sediment. A new measurement of seabed heterogeneity, designated acoustic variability, was also computed from the acoustic survey data in order to more fully characterize the acoustic response (i.e., the signature) of the seafloor. When compared with diver-based grouper census data, both acoustic classification and acoustic variability were significantly different at sites with and without groupers. Sites with groupers were characterized by hard bottom substrate and high acoustic variability. Thus, the acoustic signature of a site, as measured by acoustic classification or acoustic variability, is a potentially useful tool for stratifying diver sampling effort for grouper census.

Survey of fish protective facilities at water withdrawal sites on the Snake and Columbia Rivers

Proliferation of water withdrawals and new pump intake and screen designs has occurred with the growth of irrigated agriculture along the Columbia and Snake Rivers. Concern for the protection of anadromous and resident fish populations resulted in formulation of a survey of the water withdrawal systems. The survey included distribution studies of juvenile fish near pump sites and field inspection of those sites to determine adequacy of screening for protection of fish. A total of 225 sites were inspected in 1979 and 1980, with a follow-up inspection of 95 sites in 1982. Results indicated a definite trend toward lack of concern for the condition of fish protective facilities. Only 4 out of 22 sites not meeting criteria in 1979 had been upgraded to acceptable conditions. Of more concern, 13 of the sites meeting criteria in 1979 were below criteria when reinspected in 1982. Some of the discrepancies included lack of protective screens, poorly maintained screens, and screens permitting excessive velocity that could result in impingement of larvae or small fish. A conclusion from these surveys is that if adequate protection for fish is to exist, screens for water withdrawals need to be properly installed, inspected, and maintained. (PDF file contains 40 pages.)

Towards a participatory integrated assessment approach for planning and managing Natura 2000 network sites

Managing protected areas implies dealing with complex social-ecological systems where multiple dimensions (social, institutional, economic and ecological) interact over time for the delivery of ecosystem services. Uni-dimensional and top-down management approaches have been unable to capture this complexity. Instead, new integrated approaches that acknowledge the diversity of social actors in the decision making process are required. In this paper we put forward a novel participatory assessment approach which integrates multiple methodologies to reflect different value articulating institutions in the case of a Natura 2000 network site in the Basque Country. It integrates within a social multi-criteria evaluation framework, both the economic values of ecosystem services through a choice experiment model and ecological values by means of a spatial bio-geographic assessment. By capturing confronting social and institutional conflicts in protected areas the participatory integrated assessment approach presented here can help decision makers for better planning and managing Natura 2000 sites.

Direct observation of adsorption sites of protein impurities and their effects on step advancement of protein crystals

We measured noninvasively step velocities of elementary two-dimensional (2D) islands on {110} faces of tetragonal lysozyme crystals, under various supersaturations, by laser confocal microscopy combined with differential interference contrast microscopy. We studied the correlation between the effects of protein impurities on the growth of elementary steps and their adsorption sites on a crystal surface, using three kinds of proteins: fluorescent-labeled lysozyme (F-lysozyme), covalently bonded dimers of lysozyme (dimer), and a 18 kDa polypeptide (18 kDa). These three protein impurities suppressed the advancement of the steps. However, they exhibited different supersaturation dependencies of the suppression of the step velocities. To clarify the cause of this difference, we observed in situ the adsorption sites of individual molecules of F-lysozyme and fluorescent-labeled dimer (F-dimer) on the crystal surface by single-molecule visualization. We found that F-lysozyme adsorbed preferentially on steps (i.e., kinks), whereas F-dimer adsorbed randomly on terraces. Taking into account the different adsorption sites of F-lysozyme and F-dimer, we could successfully explain the different effects of the impurities on the step velocities. These observations strongly suggest that 18 kDa also adsorbs randomly on terraces. Seikagaku lysozyme exhibited a complex effect that could not alone be explained by the two major impurities (dimer and 18 kDa) present in Seikagaku lysozyme, indicating that trace amounts of other impurities significantly affect the step advancement.

Brain type II calcium and calmodulin-dependent protein kinase: characterization of a brain-region specific isozyme and regulation by autophosphorylation

A variety of molecular approaches have been used to investigate the structural and enzymatic properties of rat brain type ll Ca^(2+) and calmodulin-dependent protein kinase (type ll CaM kinase). This thesis describes the isolation and biochemical characterization of a brain-region specific isozyme of the kinase and also the regulation the kinase activity by autophosphorylation.

The cerebellar isozyme of the type ll CaM kinase was purified and its biochemical properties were compared to the forebrain isozyme. The cerebellar isozyme is a large (500-kDa) multimeric enzyme composed of multiple copies of 50-kDa α subunits and 60/58-kDa β/β’ subunits. The holoenzyme contains approximately 2 α subunits and 8 β subunits. This contrasts to the forebrain isozyme, which is also composed of and β/β'subunits, but they are assembled into a holoenzyme of approximately 9 α subunits and 3 β/β ' subunits. The biochemical and enzymatic properties of the two isozymes are similar. The two isozymes differ in their association with subcellular structures. Approximately 85% of the cerebellar isozyme, but only 50% of the forebrain isozyme, remains associated with the particulate fraction after homogenization under standard conditions. Postsynaptic densities purified from forebrain contain the forebrain isozyme, and the kinase subunits make up about 16% of their total protein. Postsynaptic densities purified from cerebellum contain the cerebellar isozyme, but the kinase subunits make up only 1-2% of their total protein.

The enzymatic activity of both isozymes of the type II CaM kinase is regulated by autophosphorylation in a complex manner. The kinase is initially completely dependent on Ca^(2+)/calmodulin for phosphorylation of exogenous substrates as well as for autophosphorylation. Kinase activity becomes partially Ca^(2+) independent after autophosphorylation in the presence of Ca^(2+)/calmodulin. Phosphorylation of only a few subunits in the dodecameric holoenzyme is sufficient to cause this change, suggesting an allosteric interaction between subunits. At the same time, autophosphorylation itself becomes independent of Ca^(2+) These observations suggest that the kinase may be able to exist in at least two stable states, which differ in their requirements for Ca^(2+)/calmodulin.

The autophosphorylation sites that are involved in the regulation of kinase activity have been identified within the primary structure of the α and β subunits. We used the method of reverse phase-HPLC tryptic phosphopeptide mapping to isolate individual phosphorylation sites. The phosphopeptides were then sequenced by gas phase microsequencing. Phosphorylation of a single homologous threonine residue in the α and β subunits is correlated with the production of the Ca^(2+) -independent activity state of the kinase. In addition we have identified several sites that are phosphorylated only during autophosphorylation in the absence of Ca^(2+)/ calmodulin.

Synthetic molecular machines for active self-assembly : prototype algorithms, designs, and experimental study

Computer science and electrical engineering have been the great success story of the twentieth century. The neat modularity and mapping of a language onto circuits has led to robots on Mars, desktop computers and smartphones. But these devices are not yet able to do some of the things that life takes for granted: repair a scratch, reproduce, regenerate, or grow exponentially fast–all while remaining functional.

This thesis explores and develops algorithms, molecular implementations, and theoretical proofs in the context of “active self-assembly” of molecular systems. The long-term vision of active self-assembly is the theoretical and physical implementation of materials that are composed of reconfigurable units with the programmability and adaptability of biology’s numerous molecular machines. En route to this goal, we must first find a way to overcome the memory limitations of molecular systems, and to discover the limits of complexity that can be achieved with individual molecules.

One of the main thrusts in molecular programming is to use computer science as a tool for figuring out what can be achieved. While molecular systems that are Turing-complete have been demonstrated [Winfree, 1996], these systems still cannot achieve some of the feats biology has achieved.

One might think that because a system is Turing-complete, capable of computing “anything,” that it can do any arbitrary task. But while it can simulate any digital computational problem, there are many behaviors that are not “computations” in a classical sense, and cannot be directly implemented. Examples include exponential growth and molecular motion relative to a surface.

Passive self-assembly systems cannot implement these behaviors because (a) molecular motion relative to a surface requires a source of fuel that is external to the system, and (b) passive systems are too slow to assemble exponentially-fast-growing structures. We call these behaviors “energetically incomplete” programmable behaviors. This class of behaviors includes any behavior where a passive physical system simply does not have enough physical energy to perform the specified tasks in the requisite amount of time.

As we will demonstrate and prove, a sufficiently expressive implementation of an “active” molecular self-assembly approach can achieve these behaviors. Using an external source of fuel solves part of the the problem, so the system is not “energetically incomplete.” But the programmable system also needs to have sufficient expressive power to achieve the specified behaviors. Perhaps surprisingly, some of these systems do not even require Turing completeness to be sufficiently expressive.

Building on a large variety of work by other scientists in the fields of DNA nanotechnology, chemistry and reconfigurable robotics, this thesis introduces several research contributions in the context of active self-assembly.

We show that simple primitives such as insertion and deletion are able to generate complex and interesting results such as the growth of a linear polymer in logarithmic time and the ability of a linear polymer to treadmill. To this end we developed a formal model for active-self assembly that is directly implementable with DNA molecules. We show that this model is computationally equivalent to a machine capable of producing strings that are stronger than regular languages and, at most, as strong as context-free grammars. This is a great advance in the theory of active self- assembly as prior models were either entirely theoretical or only implementable in the context of macro-scale robotics.

We developed a chain reaction method for the autonomous exponential growth of a linear DNA polymer. Our method is based on the insertion of molecules into the assembly, which generates two new insertion sites for every initial one employed. The building of a line in logarithmic time is a first step toward building a shape in logarithmic time. We demonstrate the first construction of a synthetic linear polymer that grows exponentially fast via insertion. We show that monomer molecules are converted into the polymer in logarithmic time via spectrofluorimetry and gel electrophoresis experiments. We also demonstrate the division of these polymers via the addition of a single DNA complex that competes with the insertion mechanism. This shows the growth of a population of polymers in logarithmic time. We characterize the DNA insertion mechanism that we utilize in Chapter 4. We experimentally demonstrate that we can control the kinetics of this re- action over at least seven orders of magnitude, by programming the sequences of DNA that initiate the reaction.

In addition, we review co-authored work on programming molecular robots using prescriptive landscapes of DNA origami; this was the first microscopic demonstration of programming a molec- ular robot to walk on a 2-dimensional surface. We developed a snapshot method for imaging these random walking molecular robots and a CAPTCHA-like analysis method for difficult-to-interpret imaging data.

Sequence specific complexation of b DNA at sites containing g,c base pairs

A series of eight related analogs of distamycin A has been synthesized. Footprinting and affinity cleaving reveal that only two of the analogs, pyridine-2- car box amide-netropsin (2-Py N) and 1-methylimidazole-2-carboxamide-netrops in (2-ImN), bind to DNA with a specificity different from that of the parent compound. A new class of sites, represented by a TGACT sequence, is a strong site for 2-PyN binding, and the major recognition site for 2-ImN on DNA. Both compounds recognize the G•C bp specifically, although A's and T's in the site may be interchanged without penalty. Additional A•T bp outside the binding site increase the binding affinity. The compounds bind in the minor groove of the DNA sequence, but protect both grooves from dimethylsulfate. The binding evidence suggests that 2-PyN or 2-ImN binding induces a DNA conformational change.

In order to understand this sequence specific complexation better, the Ackers quantitative footprinting method for measuring individual site affinity constants has been extended to small molecules. MPE•Fe(II) cleavage reactions over a 10^5 range of free ligand concentrations are analyzed by gel electrophoresis. The decrease in cleavage is calculated by densitometry of a gel autoradiogram. The apparent fraction of DNA bound is then calculated from the amount of cleavage protection. The data is fitted to a theoretical curve using non-linear least squares techniques. Affinity constants at four individual sites are determined simultaneously. The distamycin A analog binds solely at A•T rich sites. Affinities range from 10^(6)- 10^(7)M^(-1) The data for parent compound D fit closely to a monomeric binding curve. 2-PyN binds both A•T sites and the TGTCA site with an apparent affinity constant of 10^(5) M^(-1). 2-ImN binds A•T sites with affinities less than 5 x 10^(4) M^(-1). The affinity of 2-ImN for the TGTCA site does not change significantly from the 2-PyN value. At the TGTCA site, the experimental data fit a dimeric binding curve better than a monomeric curve. Both 2-PyN and 2-ImN have substantially lower DNA affinities than closely related compounds.

In order to probe the requirements of this new binding site, fourteen other derivatives have been synthesized and tested. All compounds that recognize the TGTCA site have a heterocyclic aromatic nitrogen ortho to the N or C-terminal amide of the netropsin subunit. Specificity is strongly affected by the overall length of the small molecule. Only compounds that consist of at least three aromatic rings linked by amides exhibit TGTCA site binding. Specificity is only weakly altered by substitution on the pyridine ring, which correlates best with steric factors. A model is proposed for TGTCA site binding that has as its key feature hydrogen bonding to both G's by the small molecule. The specificity is determined by the sequence dependence of the distance between G's.

One derivative of 2-PyN exhibits pH dependent sequence specificity. At low pH, 4-dimethylaminopyridine-2-carboxamide-netropsin binds tightly to A•T sites. At high pH, 4-Me_(2)NPyN binds most tightly to the TGTCA site. In aqueous solution, this compound protonates at the pyridine nitrogen at pH 6. Thus presence of the protonated form correlates with A•T specificity.

The binding site of a class of eukaryotic transcriptional activators typified by yeast protein GCN4 and the mammalian oncogene Jun contains a strong 2-ImN binding site. Specificity requirements for the protein and small molecule are similar. GCN4 and 2-lmN bind simultaneously to the same binding site. GCN4 alters the cleavage pattern of 2-ImN-EDTA derivative at only one of its binding sites. The details of the interaction suggest that GCN4 alters the conformation of an AAAAAAA sequence adjacent to its binding site. The presence of a yeast counterpart to Jun partially blocks 2-lmN binding. The differences do not appear to be caused by direct interactions between 2-lmN and the proteins, but by induced conformational changes in the DNA protein complex. It is likely that the observed differences in complexation are involved in the varying sequence specificity of these proteins.

Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

Transition metal clusters supported by multinucleating ligand frameworks as models of biological active sites

This dissertation describes efforts to model biological active sites with small molecule clusters. The approach used took advantage of a multinucleating ligand to control the structure and nuclearity of the product complexes, allowing the study of many different homo- and heterometallic clusters. Chapter 2 describes the synthesis of the multinucleating hexapyridyl trialkoxy ligand used throughout this thesis and the synthesis of trinuclear first row transition metal complexes supported by this framework, with an emphasis on tricopper systems as models of biological multicopper oxidases. The magnetic susceptibility of these complexes were studied, and a linear relation was found between the Cu-O(alkoxide)-Cu angles and the antiferromagnetic coupling between copper centers. The triiron(II) and trizinc(II) complexes of the ligand were also isolated and structurally characterized.

Chapter 3 describes the synthesis of a series of heterometallic tetranuclear manganese dioxido complexes with various incorporated apical redox-inactive metal cations (M = Na⁺, Ca²⁺, Sr²⁺, Zn²⁺, Y³⁺). Chapter 4 presents the synthesis of heterometallic trimanganese(IV) tetraoxido complexes structurally related to the CaMn₃ subsite of the oxygen-evolving complex (OEC) of Photosystem II. The reduction potentials of these complexes were studied, and it was found that each isostructural series displays a linear correlation between the reduction potentials and the Lewis acidities of the incorporated redox-inactive metals. The slopes of the plotted lines for both the dioxido and tetraoxido clusters are the same, suggesting a more general relationship between the electrochemical potentials of heterometallic manganese oxido clusters and their “spectator” cations. Additionally, these studies suggest that Ca²⁺ plays a role in modulating the redox potential of the OEC for water oxidation.

Chapter 5 presents studies of the effects of the redox-inactive metals on the reactivities of the heterometallic manganese complexes discussed in Chapters 3 and 4. Oxygen atom transfer from the clusters to phosphines is studied; although the reactivity is kinetically controlled in the tetraoxido clusters, the dioxido clusters with more Lewis acidic metal ions (Y³⁺ vs. Ca²⁺) appear to be more reactive. Investigations of hydrogen atom transfer and electron transfer rates are also discussed.

Appendix A describes the synthesis, and metallation reactions of a new dinucleating bis(N-heterocyclic carbene)ligand framework. Dicopper(I) and dicobalt(II) complexes of this ligand were prepared and structurally characterized. A dinickel(I) dichloride complex was synthesized, reduced, and found to activate carbon dioxide. Appendix B describes preliminary efforts to desymmetrize the manganese oxido clusters via functionalization of the basal multinucleating ligand used in the preceding sections of this dissertation. Finally, Appendix C presents some partially characterized side products and unexpected structures that were isolated throughout the course of these studies.