Vibrational population relaxation of carbon monoxide in the heme pocket of photolyzed carbonmonoxy myoglobin: Comparison of time-resolved mid-IR absorbance experiments and molecular dynamics simulations

The vibrational energy relaxation of carbon monoxide in the heme pocket of sperm whale myoglobin was studied by using molecular dynamics simulation and normal mode analysis methods. Molecular dynamics trajectories of solvated myoglobin were run at 300 K for both the δ- and ɛ-tautomers of the distal His-64. Vibrational population relaxation times of 335 ± 115 ps for the δ-tautomer and 640 ± 185 ps for the ɛ-tautomer were estimated by using the Landau–Teller model. Normal mode analysis was used to identify those protein residues that act as the primary “doorway” modes in the vibrational relaxation of the oscillator. Although the CO relaxation rates in both the ɛ- and δ-tautomers are similar in magnitude, the simulations predict that the vibrational relaxation of the CO is faster in the δ-tautomer with the distal His playing an important role in the energy relaxation mechanism. Time-resolved mid-IR absorbance measurements were performed on photolyzed carbonmonoxy hemoglobin (Hb13CO). From these measurements, a T1 time of 600 ± 150 ps was determined. The simulation and experimental estimates are compared and discussed.

The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity.

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.

Cetáceos en la Reserva Marina de las Islas Columbretes (Mediterráneo noroccidental): 20 años de avistamientos oportunistas

En el presente trabajo se recopilan veinte años de observaciones realizadas por los servicios de vigilancia de la Reserva Marina y Reserva Natural de las Islas Columbretes, así como aquellas notificadas por embarcaciones de recreo y pesca, desde la creación de la reserva en 1990. Las observaciones fueron realizadas durante todo el año en el interior de la Reserva Marina e inmediaciones. Por avistamiento se ha anotado la especie, el tamaño de grupo, hora y situación aproximada. Para cada especie se ha analizado la presencia a lo largo de los meses del año, la distancia a las islas y el tamaño de los grupos. Se han obtenido datos de un total de 366 observaciones y 4928 individuos. La especie más frecuente ha sido el delfín mular Tursiops truncatus con el 71 % de las observaciones totales, seguida por el rorcual común Balaenoptera physalus (20 %), delfín listado Stenella coeruleoalba (5 %), delfín común Delphinus delphis (1.4 %), y con porcentajes inferiores al 1%: calderón común Globicephala melas, calderón gris Grampus griseus, cachalote Physeter catodon y orca Orcinus orca. La presencia constante de delfín mular en la Reserva Marina durante estos 20 años es una evidencia de que la protección de estas aguas ha contribuido a la conservación de esta especie en la zona. Por otra parte, las observaciones de rorcual común al este de la Reserva Marina indican la existencia de una zona de paso de los individuos en su migración latitudinal.

Tailoring heme -thiolate proteins into efficient biocatalysts with high specificity and selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Spatial Relationships among Hydroacoustic, Hydrographic and Top Predator Patterns: Cetacean Distributions in the Mid-Atlantic Bight

Effective conservation and management of top predators requires a comprehensive understanding of their distributions and of the underlying biological and physical processes that affect these distributions. The Mid-Atlantic Bight shelf break system is a dynamic and productive region where at least 32 species of cetaceans have been recorded through various systematic and opportunistic marine mammal surveys from the 1970s through 2012. My dissertation characterizes the spatial distribution and habitat of cetaceans in the Mid-Atlantic Bight shelf break system by utilizing marine mammal line-transect survey data, synoptic multi-frequency active acoustic data, and fine-scale hydrographic data collected during the 2011 summer Atlantic Marine Assessment Program for Protected Species (AMAPPS) survey. Although studies describing cetacean habitat and distributions have been previously conducted in the Mid-Atlantic Bight, my research specifically focuses on the shelf break region to elucidate both the physical and biological processes that influence cetacean distribution patterns within this cetacean hotspot.

In Chapter One I review biologically important areas for cetaceans in the Atlantic waters of the United States. I describe the study area, the shelf break region of the Mid-Atlantic Bight, in terms of the general oceanography, productivity and biodiversity. According to recent habitat-based cetacean density models, the shelf break region is an area of high cetacean abundance and density, yet little research is directed at understanding the mechanisms that establish this region as a cetacean hotspot.

In Chapter Two I present the basic physical principles of sound in water and describe the methodology used to categorize opportunistically collected multi-frequency active acoustic data using frequency responses techniques. Frequency response classification methods are usually employed in conjunction with net-tow data, but the logistics of the 2011 AMAPPS survey did not allow for appropriate net-tow data to be collected. Biologically meaningful information can be extracted from acoustic scattering regions by comparing the frequency response curves of acoustic regions to theoretical curves of known scattering models. Using the five frequencies on the EK60 system (18, 38, 70, 120, and 200 kHz), three categories of scatterers were defined: fish-like (with swim bladder), nekton-like (e.g., euphausiids), and plankton-like (e.g., copepods). I also employed a multi-frequency acoustic categorization method using three frequencies (18, 38, and 120 kHz) that has been used in the Gulf of Maine and Georges Bank which is based the presence or absence of volume backscatter above a threshold. This method is more objective than the comparison of frequency response curves because it uses an established backscatter value for the threshold. By removing all data below the threshold, only strong scattering information is retained.

In Chapter Three I analyze the distribution of the categorized acoustic regions of interest during the daytime cross shelf transects. Over all transects, plankton-like acoustic regions of interest were detected most frequently, followed by fish-like acoustic regions and then nekton-like acoustic regions. Plankton-like detections were the only significantly different acoustic detections per kilometer, although nekton-like detections were only slightly not significant. Using the threshold categorization method by Jech and Michaels (2006) provides a more conservative and discrete detection of acoustic scatterers and allows me to retrieve backscatter values along transects in areas that have been categorized. This provides continuous data values that can be integrated at discrete spatial increments for wavelet analysis. Wavelet analysis indicates significant spatial scales of interest for fish-like and nekton-like acoustic backscatter range from one to four kilometers and vary among transects.

In Chapter Four I analyze the fine scale distribution of cetaceans in the shelf break system of the Mid-Atlantic Bight using corrected sightings per trackline region, classification trees, multidimensional scaling, and random forest analysis. I describe habitat for common dolphins, Risso’s dolphins and sperm whales. From the distribution of cetacean sightings, patterns of habitat start to emerge: within the shelf break region of the Mid-Atlantic Bight, common dolphins were sighted more prevalently over the shelf while sperm whales were more frequently found in the deep waters offshore and Risso’s dolphins were most prevalent at the shelf break. Multidimensional scaling presents clear environmental separation among common dolphins and Risso’s dolphins and sperm whales. The sperm whale random forest habitat model had the lowest misclassification error (0.30) and the Risso’s dolphin random forest habitat model had the greatest misclassification error (0.37). Shallow water depth (less than 148 meters) was the primary variable selected in the classification model for common dolphin habitat. Distance to surface density fronts and surface temperature fronts were the primary variables selected in the classification models to describe Risso’s dolphin habitat and sperm whale habitat respectively. When mapped back into geographic space, these three cetacean species occupy different fine-scale habitats within the dynamic Mid-Atlantic Bight shelf break system.

In Chapter Five I present a summary of the previous chapters and present potential analytical steps to address ecological questions pertaining the dynamic shelf break region. Taken together, the results of my dissertation demonstrate the use of opportunistically collected data in ecosystem studies; emphasize the need to incorporate middle trophic level data and oceanographic features into cetacean habitat models; and emphasize the importance of developing more mechanistic understanding of dynamic ecosystems.

The management of bone tools from the Bronze Age site of El Portalón of Cueva Mayor, Sierra de Atapuerca, Burgos

In this paper we analyze the set of Bronze Age bone tools recovered at the archaeological site of El Portalón of Cueva Mayor in the Sierra de Atapuerca (Burgos). The Bronze Age cultural period is the best represented in the cavity and its study has forced us to unify the different excavation and stratigraphical criteria undertaken from the earliest archaeological excavations developed by J.M. Apellániz during the 70s until the excavations of the current research team (EIA) since 2000. We propose here for the first time a relationship between the initial system of “beds” used by Apellániz and our recent sedimentary sequence that recognizes eleven stratigraphic levels radiometrically dated from the late Upper Pleistocene to the Middle Age. Within the bone industry assemblage we recognize a large variety of utensils and ornamental elements, with native and allochthonous features, that make evident a regional as well as long distance relationships of these populations of the interior of the Iberian Peninsula during the recent Prehistory.

A critical analysis of sperm donation practices : the personal and social effects of disrupting the unity of biological and social relatedness for the offspring

This thesis critically analyses sperm donation practices from a child-centred perspective. It examines the effects, both personal and social, of disrupting the unity of biological and social relatedness in families affected by donor conception. It examines how disruption is facilitated by a process of mediation which is detailed using a model provided by Sunderland (2002). This model identifies mediating movements - alienation, translation, re-contextualisation and absorption - which help to explain the powerful and dominating material, and social and political processes which occur in biotechnology, or in reproductive technology in this case. The understanding of such movements and mediation of meanings is inspired by the complementary work of Silverstone (1999) and Sunderland. This model allows for a more critical appreciation of the movement of meaning from previously inalienable aspects of life to alienable products through biotechnology (Sunderland, 2002). Once this mediation in donor conception is subjected to critical examination here, it is then approached from different angles of investigation. The thesis posits that two conflicting notions of the self are being applied to fertility-frustrated adults and the offspring of reproductive interventions. Adults using reproductive interventions receive support to maximise their genetic continuity, but in so doing they create and dismiss the corresponding genetic discontinuity produced for the offspring. The offspring’s kinship and identity are then framed through an experimental postmodernist notion, presenting them as social rather than innate constructs. The adults using the reproductive intervention, on the other hand, have their identity and kinship continuity framed and supported as normative, innate, and based on genetic connection. This use of shifting frameworks is presented as unjust and harmful, creating double standards and a corrosion of kinship values, connection and intelligibility between generations; indeed, it is put forward as adult-centric. The analysis of other forms of human kinship dislocation provided by this thesis explores an under-utilised resource which is used to counter the commonly held opinion that any disruption of social and genetic relatedness for donor offspring is insignificant. The experiences of adoption and the stolen generations are used to inform understanding of the personal and social effects of such kinship disruption and potential reunion for donor offspring. These examples, along with laws governing international human rights, further strengthen the appeal here for normative principles and protections based on collective knowledge and standards to be applied to children of reproductive technology. The thesis presents the argument that the framing and regulation of reproductive technology is excessively influenced by industry providers and users. The interests of these parties collide with and corrode any accurate assessments and protections afforded to the children of reproductive technology. The thesis seeks to counter such encroachments and concludes by presenting these protections, frameworks, and human experiences as resources which can help to address the problems created for the offspring of such reproductive interventions, thereby illustrating why these reproductive interventions should be discontinued.

Uniform Civil Procedure Rules r150 : orders relating to "property" – whether sperm extracted and stored may be characterised as “property”

In Bazley v Wesley Monash IVF Pty Ltd [2010] QSC 118 an order was made under r 250 of the Uniform Civil Procedure Rules 1999 (Qld) (“UCPR”) requiring the respondent to continue to hold and maintain straws of semen belonging to the applicant’s deceased husband. The decision includes a useful analysis of the development of the common law regarding property rights in human bodies and body parts.

Property rights in and entitlement to possession of sperm following death

In May 2011, the Supreme Court of New South Wales in Jocelyn Edwards; re the estate of the late Mark Edwards [2011] NSWSC 478 granted an application by a wife, in her capacity as administrator of her late husband's estate, to possession of sperm extracted from the body of her late husband.

Selection criteria in the search for a sperm donor: Behavioural traits versus physical appearance

Despite extensive literature on female mate choice, empirical evidence on women’s mating preferences in the search for a sperm donor is scarce, even though this search, by isolating a male’s genetic impact on offspring from other factors like paternal investment, offers a naturally ”controlled” research setting. In this paper, we work to fill this void by examining the rapidly growing online sperm donor market, which is raising new challenges by offering women novel ways to seek out donor sperm. We not only identify individual factors that influence women’s mating preferences but find strong support for the proposition that behavioural traits (inner values) are more important in these choices than physical appearance (exterior values). We also report evidence that physical factors matter more than resources or other external cues of material success, perhaps because the relevance of good character in donor selection is part of a female psychological adaptation throughout evolutionary history. The lack of evidence on a preference for material resources, on the other hand, may indicate the ability of peer socialization and better access to resources to rapidly shape the female decision process. Overall, the paper makes useful contributions to both the literature on human behaviour and that on decision-making in extreme and highly important situations.