Synopsis of biological data on the blue crab, Callinectes sapidus Rathbun

This synopsis reviews taxonomy, morphology, distribution, life history, commercial hard and soft shell crab fisheries, physiology, diseases, ecology, laboratory culture methodology, and influences of environmental pollutants on the blue crab, Callinecles sapidus. Over 300 selected, published reports up to and including 1982 are covered. (PDF file contains 45 pages.)

On the physical and biological oceanography near the entrance of the Gulf of California, October 1966-August 1967

ENGLISH: A study of the temporal and spatial distribution of larval tunas and the concomitant oceanic conditions was made in cooperation with the Direccion General de Pesca e Industrias Conexas of Mexico. Field work consisted of eight hydrographic cruises made from October 1966 through August 1967 near the entrance of the Gulf of California. From January through April, surface currents were southerly at velocities up to 20 cm/sec; currents in June were variable in direction and mostly less than 10 cm/sec; by August the surface current was northerly at 10-15 cm/sec. Surface winds were usually secondary to the distribution of mass as an influence on the surface circulation. Currents at 100 m were generally similar in direction to those at the surface, but the water moved more slowly. Between the surface and 100 m, southbound currents crossed the entrance of the Gulf at velocities of 5-10 cm/sec during January and April, forming frontal boundaries with the California Current water, which often occurred south of the entrance. From April to August, the median concentration of surface chlorophyll a increased from 0.65 to 0.97 mg/m3, while the median productivity increased from 5.6 mgC/m3/day in April to 17.8 mgC/m3/day in June before returning to 2.6 mgC/m3/day in August. Primary productivity was closely correlated with the concentration of surface chlorophyll a. Productivity was generally higher in the vicinity of the Gulf than that found for water in the open Pacific. Productivity was highest near Islas Las Tres Marias and second highest near Cabo San Lucas, both locations of local upwelling. The standing crop of phytoplankton was shown to be subjected to progressively heavier grazing pressure in the spring and summer by zooplankton. SPANISH: Un estudio de la distribución temporal y espacial de las larvas de atún y de las condiciones oceánicas concomitantes fue realizado en cooperación con la Dirección General de Pesca e Industrias Conexas de México. El trabajo experimental consistió en ocho cruceros hidrográficos realizados desde octubre 1966 hasta agosto 1967, cerca a la entrada del Golfo de California. De enero a abril, las corrientes superficiales fueron meridionales alcanzando velocidades hasta de 20 cm/seg; las corrientes en junio fueron variables en dirección y la mayoría con una velocidad de menos de 10 cm/seg; en agosto la corriente superficial fue septentrional a 10-15 cm/seg, Los vientos superficiales fueron por lo común secundarios a la dístríbucíón de la masa, como una influencia de la circulación superficial. Las corrientes a 100 m fueron generalmente similares en dirección a las de la superficie, pero el agua se movió más lentamente. Entre la superficie y los 100 m, las corrientes que se dirigen hacia el sur cruzaron la entrada del Golfo a velocidades de 5-10 cm/seg durante enero y abril formando límites frontales con el agua de la Corriente de California, que apareció a menudo al sur de la entrada. De abril a agosto, la concentración media de la clorofila a superficial aumentó de 0.65 a 0.97 mg/m3, mientras que la productividad mediana aumentó de 5.6 mgC/m3/día en abril hasta 17.8 mgC/m3/día en junio antes de regresar a 2.6 mgC/m3/día en agosto. La productividad primaria se correlacionó estrechamente con la concentración de clorofila a superficial. La productividad fue generalmente más alta en la vecindad del Golfo que aquella encontrada en el agua de alta mar del Pacífico. La productividad fue más alta cerca a las Islas Tres Marías, y el segundo máximo fue cerca al Cabo San Lucas, ambas localidades de afloramiento local. Se indicó que la reserva permanente de fitoplancton estaba sujeta por el zooplancton a una fuerta presión progresiva de apacentamiento en la primavera y el verano. (PDF contains 116 pages.)

Biological Platforms for Environmental Sensor technologies and their uses as indicators of the marine environment, Seward, Alaska, September 19-21, 2007: workshop proceedings

The Alliance for Coastal Technologies (ACT) Workshop entitled, "Biological Platforms as Sensor Technologies and their Use as Indicators for the Marine Environment" was held in Seward, Alaska, September 19 - 21,2007. The workshop was co-hosted by the University of Alaska Fairbanks (UAF) and the Alaska SeaLife Center (ASLC). The workshop was attended by 25 participants representing a wide range of research scientists, managers, and manufacturers who develop and deploy sensory equipment using aquatic vertebrates as the mode of transport. Eight recommendations were made by participants at the conclusion of the workshop and are presented here without prioritization: 1. Encourage research toward development of energy scavenging devices of suitable sizes for use in remote sensing packages attached to marine animals. 2. Encourage funding sources for development of new sensor technologies and animal-borne tags. 3. Develop animal-borne environmental sensor platforms that offer more combined systems and improved data recovery methodologies, and expand the geographic scope of complementary fixed sensor arrays. 4. Engage the oceanographic community by: a. Offering a mini workshop at an AGU ocean sciences conference for people interested in developing an ocean carbon program that utilizes animal-borne sensor technology. b. Outreach to chemical oceanographers. 5. Min v2d6.sheepserver.net e and merge technologies from other disciplines that may be applied to marine sensors (e.g. biomedical field). 6. Encourage the NOAA Permitting Office to: a. Make a more predictable, reliable, and consistent permitting system for using animal platforms. b. Establish an evaluation process. c. Adhere to established standards. 7. Promote the expanded use of calibrated hydrophones as part of existing animal platforms. 8. Encourage the Integrated Ocean Observing System (IOOS) to promote animal tracking as effective samplers of the marine environment, and use of animals as ocean sensor technology platforms. [PDF contains 20 pages]

Nutrients and biological production in lakes

This paper is a review of studies on effects of nutrients on biological productivity and efforts made so far at restoration of nutrients in lakes. It is to provide an understanding of the basis scientific process accruing in lakes, therefore of prime importance in maintaining water quality standards for propagation of effective lake management

Oceanographic observations in the Gulf of Guayaquil, 1962-1964. Part II. Biological, chemical and physical.

ENGLISH: During 1961 the government of Ecuador, with the financial assistance of the Special Fund of the United Nations and the technical assistance of FAO experts, initiated an extensive program of fisheries research centered in a fisheries institute established in Guayaquil. In cooperation with this program, and in connection with Ecuador's adherence in 1961 to the Convention for the Establishment of an Inter-American Tropical Tuna Commission, a two-and-a-half year investigation of the ecology of the Gulf of Guayaquil and adjacent waters was started by the Inter-American Tropical Tuna Commission. SPANISH: Durante 1961 el gobierno ecuatoriano con el apoyo financiero del Fondo Especial de las Naciones Unidas y la ayuda técnica de los expertos de la FAO, inició un programa extensivo de investigación pesquera, centralizado en el instituto pesquero establecido en Guayaquil. En cooperación con este programa y en conexión a la afiliaci6n del Ecuador a la Convención, en 1961, para el establecimiento de una Comisión Interamericana del Atún Tropical, Cue iniciada por la Comisión una investigación de dos aftos y medio sobre la ecología del Golfo de Guayaquil y de las aguas adyacentes. (PDF contains 501 pages.)

The biological sense of smell: olfactory search behavior and a metabolic view for olfactory perception

Part I of the thesis describes the olfactory searching and scanning behaviors of rats in a wind tunnel, and a detailed movement analysis of terrestrial arthropod olfactory scanning behavior. Olfactory scanning behaviors in rats may be a behavioral correlate to hippocampal place cell activity.

Part II focuses on the organization of olfactory perception, what it suggests about a natural order for chemicals in the environment, and what this in tum suggests about the organization of the olfactory system. A model of odor quality space (analogous to the "color wheel") is presented. This model defines relationships between odor qualities perceived by human subjects based on a quantitative similarity measure. Compounds containing Carbon, Nitrogen, or Sulfur elicit odors that are contiguous in this odor representation, which thus allows one to predict the broad class of odor qualities a compound is likely to elicit. Based on these findings, a natural organization for olfactory stimuli is hypothesized: the order provided by the metabolic process. This hypothesis is tested by comparing compounds that are structurally similar, perceptually similar, and metabolically similar in a psychophysical cross-adaptation paradigm. Metabolically similar compounds consistently evoked shifts in odor quality and intensity under cross-adaptation, while compounds that were structurally similar or perceptually similar did not. This suggests that the olfactory system may process metabolically similar compounds using the same neural pathways, and that metabolic similarity may be the fundamental metric about which olfactory processing is organized. In other words, the olfactory system may be organized around a biological basis.

The idea of a biological basis for olfactory perception represents a shift in how olfaction is understood. The biological view has predictive power while the current chemical view does not, and the biological view provides explanations for some of the most basic questions in olfaction, that are unanswered in the chemical view. Existing data do not disprove a biological view, and are consistent with basic hypotheses that arise from this viewpoint.

Improving the biological activity of pyrrole-imidazole polyamides

DNA is nature’s blueprint, holding within it the genetic code that defines the structure and function of an organism. A complex network of DNA-binding proteins called transcription factors can largely control the flow of information from DNA, so modulating the function of transcription factors is a promising approach for treating many diseases. Pyrrole-imidazole (Py-Im) polyamides are a class of DNA-binding oligomers, which can be synthetically programmed to bind a target sequence of DNA. Due to their unique shape complementarity and a series of favorable hydrogen bonding interactions that occur upon DNA-binding, Py-Im polyamides can bind to the minor groove of DNA with affinities comparable to transcription factors. Previous studies have demonstrated that these cell-permeable small molecules can enter cell nuclei and disrupt the transcription factor-DNA interface, thereby repressing transcription. As the use of Py-Im polyamides has significant potential as a type of modular therapeutic platform, the need for polyamides with extremely favorable biological properties and high potency will be essential. Described herein, a variety of studies have been performed aimed at improving the biological activity of Py-Im polyamides. To improve the biological potency and cellular uptake of these compounds, we have developed a next-generation class of polyamides bearing aryl-turn moieties, a simple structural modification that allows significant improvements in cellular uptake. This strategy was also applied to a panel of high-affinity cyclic Py-Im polyamides, again demonstrating the remarkable effect minor structural changes can have on biological activity. The solubility properties of Py-Im polyamides and use of formulating reagents with their treatment have also been examined. Finally, we describe the study of Py-Im polyamides as a potential artificial transcription factor.

Tissue engineering active biological machines : bio-inspired design, directed self-assembly, and characterization of muscular pumps simulating the embryonic heart

Biological machines are active devices that are comprised of cells and other biological components. These functional devices are best suited for physiological environments that support cellular function and survival. Biological machines have the potential to revolutionize the engineering of biomedical devices intended for implantation, where the human body can provide the required physiological environment. For engineering such cell-based machines, bio-inspired design can serve as a guiding platform as it provides functionally proven designs that are attainable by living cells. In the present work, a systematic approach was used to tissue engineer one such machine by exclusively using biological building blocks and by employing a bio-inspired design. Valveless impedance pumps were constructed based on the working principles of the embryonic vertebrate heart and by using cells and tissue derived from rats. The function of these tissue-engineered muscular pumps was characterized by exploring their spatiotemporal and flow behavior in order to better understand the capabilities and limitations of cells when used as the engines of biological machines.

Synthesis and biological activity of anticoagulant heparan sulfate glycopolymers

Heparin has been used as an anticoagulant drug for more than 70 years. The global distribution of contaminated heparin in 2007, which resulted in adverse clinical effects and over 100 deaths, emphasizes the necessity for safer alternatives to animal-sourced heparin. The structural complexity and heterogeneity of animal-sourced heparin not only impedes safe access to these biologically active molecules, but also hinders investigations on the significance of structural constituents at a molecular level. Efficient methods for preparing new synthetic heparins with targeted biological activity are necessary not only to ensure clinical safety, but to optimize derivative design to minimize potential side effects. Low molecular weight heparins have become a reliable alternative to heparin, due to their predictable dosages, long half-lives, and reduced side effects. However, heparin oligosaccharide synthesis is a challenging endeavor due to the necessity for complex protecting group manipulation and stereoselective glycosidic linkage chemistry, which often result in lengthy synthetic routes and low yields. Recently, chemoenzymatic syntheses have produced targeted ultralow molecular weight heparins with high-efficiency, but continue to be restricted by the substrate specificities of enzymes.

To address the need for access to homogeneous, complex glycosaminoglycan structures, we have synthesized novel heparan sulfate glycopolymers with well-defined carbohydrate structures and tunable chain length through ring-opening metathesis polymerization chemistry. These polymers recapitulate the key features of anticoagulant heparan sulfate by displaying the sulfation pattern responsible for heparin’s anticoagulant activity. The use of polymerization chemistry greatly simplifies the synthesis of complex glycosaminoglycan structures, providing a facile method to generate homogeneous macromolecules with tunable biological and chemical properties. Through the use of in vitro chromogenic substrate assays and ex vivo clotting assays, we found that the HS glycopolymers exhibited anticoagulant activity in a sulfation pattern and length-dependent manner. Compared to heparin standards, our short polymers did not display any activity. However, our longer polymers were able to incorporate in vitro and ex vivo characteristics of both low-molecular-weight heparin derivatives and heparin, displaying hybrid anticoagulant properties. These studies emphasize the significance of sulfation pattern specificity in specific carbohydrate-protein interactions, and demonstrate the effectiveness of multivalent molecules in recapitulating the activity of natural polysaccharides.

Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

New materials for biological applications prepared by olefin metathesis reactions

With the advent of well-defined ruthenium olefin metathesis catalysts that are highly active and stable to a variety of functional groups, the synthesis of complex organic molecules and polymers is now possible; this is reviewed in Chapter 1. The majority of the rest of this thesis describes the application of these catalysts towards the synthesis of novel polymers that may be useful in biological applications and investigations into their efficacy.

A method was developed to produce polyethers by metathesis, and this is described in Chapters 2 and 3. An unsaturated 12-crown-4 analog was made by template- directed ring-closing metathesis (RCM) and utilized as a monomer for the synthesis of unsaturated polyethers by ring-opening metathesis polymerization (ROMP). The yields were high and a range of molecular weights was accessible. In a similar manner, substituted polyethers with various backbones were synthesized: polymers with benzo groups along the backbone and various concentrations of amino acids were prepared. The results from in vitro toxicity tests of the unsubstituted polyethers are considered.

The conditions necessary to synthesize polynorbornenes with pendent bioactive peptides were explored as illustrated in Chapter 4. First, the polymerization of various norbornenyl monomers substituted with glycine, alanine or penta(ethylene glycol) is described. Then, the syntheses of polymers substituted with peptides GRGD and SRN, components of a cell binding domain of fibronectin, using newly developed ruthenium initiators are discussed.

In Chapter 5, the syntheses of homopolymers and a copolymer containing GRGDS and PHSRN, the more active forms of the peptides, are described. The ability of the polymers to inhibit human dermal fibroblast cell adhesion to fibronectin was assayed using an in vitro competitive inhibition assay, and the results are discussed. It was discovered that the copoymer substituted with both GRGDS and PHSR peptides was more active than both the GRGDS-containing homopolymer and the GRGDS free peptide.

Historically, one of the drawbacks to using metathesis is the removal of the residual ruthenium at the completion of the reaction. Chapter 6 describes a method where the water soluble tris(hydroxymethyl)phosphine is utilized to facilitate the removal of residual ruthenium from RCM reaction products.

Biological electron transfer in copper proteins

Nature has used a variety of protein systems to mediate electron transfer. In this thesis I examine aspects of the control of biological electron transfer by two copper proteins that act as natural electron carriers.

In the first study, I have made a mutation to one of the ligand residues in the azurin blue copper center, methionine 121 changed to a glutamic acid. Studies of intramolecular electron transfer rates from that mutated center to covalently attached ruthenium complexes indicate that the weak axial methionine ligand is important not only for tuning the reduction potential of the blue copper site but also for maintaining the low reorganization energy that is important for fast electron transfer at long distances.

In the second study, I begin to examine the reorganization energy of the purple copper center in the CuA domain of subunit II of cytochrome c oxidase. In this copper center, the unpaired electron is delocalized over the entire binuclear site. Because long-range electron transfer into and out of this center occurs over long distances with very small driving forces, the reorganization energy of the CuA center has been predicted to be extremely low. I describe a strategy for measuring this reorganization energy starting with the construction of a series of mutations introducing surface histidines. These histidines can then be labeled with a series of ruthenium compounds that differ primarily in their reduction potentials. The electron transfer rates to these ruthenium compounds can then be used to determine the reorganization energy of the CuA site.

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.