I. The 3.7 Å crystal structure of horse heart ferricytochrome C. II. The application of the Karle-Hauptman tangent formula to protein phasing

I. The 3.7 Å Crystal Structure of Horse Heart Ferricytochrome C.

The crystal structure of horse heart ferricytochrome c has been determined to a resolution of 3.7 Å using the multiple isomorphous replacement technique. Two isomorphous derivatives were used in the analysis, leading to a map with a mean figure of merit of 0.458. The quality of the resulting map was extremely high, even though the derivative data did not appear to be of high quality.

Although it was impossible to fit the known amino acid sequence to the calculated structure in an unambiguous way, many important features of the molecule could still be determined from the 3.7 Å electron density map. Among these was the fact that cytochrome c contains little or no α-helix. The polypeptide chain appears to be wound about the heme group in such a way as to form a loosely packed hydrophobic core in the molecule.

The heme group is located in a cleft on the molecule with one edge exposed to the solvent. The fifth coordinating ligand is His 18 and the sixth coordinating ligand is probably neither His 26 nor His 33.

The high resolution analysis of cytochrome c is now in progress and should be completed within the next year.

II. The Application of the Karle-Hauptman Tangent Formula to Protein Phasing.

The Karle-Hauptman tangent formula has been shown to be applicable to the refinement of previously determined protein phases. Tests were made with both the cytochrome c data from Part I and a theoretical structure based on the myoglobin molecule. The refinement process was found to be highly dependent upon the manner in which the tangent formula was applied. Iterative procedures did not work well, at least at low resolution.

The tangent formula worked very well in selecting the true phase from the two possible phase choices resulting from a single isomorphous replacement phase analysis. The only restriction on this application is that the heavy atoms form a non-centric cluster in the unit cell.

Pages 156 through 284 in this Thesis consist of previously published papers relating to the above two sections. References to these papers can be found on page 155.

Ancient DNA from South-East Europe Reveals Different Events during Early and Middle Neolithic Influencing the European Genetic Heritage

The importance of the process of Neolithization for the genetic make-up of European populations has been hotly debated, with shifting hypotheses from a demic diffusion (DD) to a cultural diffusion (CD) model. In this regard, ancient DNA data from the Balkan Peninsula, which is an important source of information to assess the process of Neolithization in Europe, is however missing. In the present study we show genetic information on ancient populations of the South-East of Europe. We assessed mtDNA from ten sites from the current territory of Romania, spanning a time-period from the Early Neolithic to the Late Bronze Age. mtDNA data from Early Neolithic farmers of the Starcevo Cris culture in Romania (Carcea, Gura Baciului and Negrilesti sites), confirm their genetic relationship with those of the LBK culture (Linienbandkeramik Kultur) in Central Europe, and they show little genetic continuity with modern European populations. On the other hand, populations of the Middle-Late Neolithic (Boian, Zau and Gumelnita cultures), supposedly a second wave of Neolithic migration from Anatolia, had a much stronger effect on the genetic heritage of the European populations. In contrast, we find a smaller contribution of Late Bronze Age migrations to the genetic composition of Europeans. Based on these findings, we propose that permeation of mtDNA lineages from a second wave of Middle-Late Neolithic migration from North-West Anatolia into the Balkan Peninsula and Central Europe represent an important contribution to the genetic shift between Early and Late Neolithic populations in Europe, and consequently to the genetic make-up of modern European populations.

Temporal and spatial aspects of bottlenose dolphin occurrence in coastal and estuarine waters near Charleston, South Carolina

The spatial and temporal occurrence of Atlantic bottlenose dolphins (Tursiops truncatus) in the coastal and estuarine waters near Charleston, SC were evaluated. Sighting and photographic data from photo-identification (ID), remote biopsy, capture-release and radio-tracking studies, conducted from 1994 through 2003, were analyzed in order to further delineate residence patterns of Charleston area bottlenose dolphins. Data from 250 photo-ID, 106 remote biopsy, 15 capture-release and 83 radio-tracking surveys were collected in the Stono River Estuary (n = 247), Charleston Harbor (n = 86), North Edisto River (n = 54), Intracoastal Waterway (n = 26) and the coastal waters north and south of Charleston Harbor (n = 41). Coverage for all survey types was spatially and temporally variable, and in the case of biopsy, capture-release and radio-tracking surveys, data analyzed in this report were collected incidental to other research. Eight-hundred and thirty-nine individuals were photographically identified during the study period. One-hundred and fifteen (13.7%) of the 839 photographically identified individuals were sighted between 11-40 times, evidence of consistent occurrence in the Charleston area (i.e., site fidelity). Adjusted sighting proportions (ASP), which reflect an individual’s sighting frequency in a subarea relative to other subareas after adjusting for survey effort, were analyzed in order to evaluate dolphin spatial occurrence. Forty-three percent (n = 139) of dolphins that qualified for ASP analyses exhibited a strong subarea affiliation while the remaining 57% (n = 187) showed no strong subarea preference. Group size data were derived from field estimates of 2,342 dolphin groups encountered in the five Charleston subareas. Group size appeared positively correlated with degree of “openness” of the body of water where dolphins were encountered; and for sightings along the coast, group size was larger during summer months. This study provides valuable information on the complex nature of bottlenose dolphin spatial and temporal occurrence near Charleston, SC. In addition, it helps us to better understand the stock structure of dolphins along the Atlantic seaboard.

Spatial and temporal analysis of bottlenose dolphin strandings in South Carolina, 1992-2005

This CD contains summary data of bottlenose dolphins stranded in South Carolina using a Geographical Information System (GIS) and contains two published manuscripts in .pdf files. The intent of this CD is to provide data on bottlenose dolphin strandings in South Carolina to marine mammal researchers and managers. This CD is an accumulation of 14 years of stranding data collected through the collaborations of the National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research (CCEHBR), the South Carolina Department of Natural Resources, and numerous volunteers and veterinarians that comprised the South Carolina Marine Mammal Stranding Network. Spatial and temporal information can be visually represented on maps using GIS. For this CD, maps were created to show relationships of stranding densities with land use, human population density, human interaction with dolphins, high geographical regions of live strandings, and seasonal changes. Point maps were also created to show individual strandings within South Carolina. In summary, spatial analysis revealed higher densities of bottlenose dolphin strandings in Charleston and Beaufort Counties, which consist of urban land with agricultural input. This trend was positively correlated with higher human population levels in these coastal counties as compared with other coastal counties. However, spatial analysis revealed that certain areas within a county may have low human population levels but high stranding density, suggesting that the level of effort to respond to strandings is not necessarily positively correlated with the density of strandings in South Carolina. Temporal analysis revealed a significantly higher density of bottlenose dolphin strandings in the northern portion of the State in the fall, mostly due to an increase of neonate strandings. On a finer geographic scale, seasonal stranding densities may fluctuate depending on the region of interest. Charleston Harbor had the highest density of live bottlenose dolphin strandings compared to the rest of the State. This was due in large part to the number of live dolphin entanglements in the crab pot fishery, the largest source of fishery-related mortality for bottlenose dolphins in South Carolina (Burdett and McFee 2004). Spatial density calculations also revealed that Charleston and Beaufort accounted for the majority of dolphins that were involved with human activities. 1

A pilot study to determine the movements of buoy line used in the crab pot fishery to assess bottlenose dolphin entanglement

A pilot study on the characteristics of crab pot buoy line movements to assess bottlenose dolphin entanglement was conducted from 19 September to 30 September 2005 in the Charleston Harbor, Charleston, South Carolina. The objectives of this study were to determine: 1) the movements of the buoy line in the water at various tidal stages, current strengths, lengths of line, and water depth, 2) if lead-core rope was a better alternative to nylon rope, 3) and if the manner of deployment of the gear affected the suspension of the line in the water and on the bottom. Diamond braided nylon (#10) rope of varying length (20 ft. – 80 ft.) were used during 31 trials and stiffened (polypropylene lead-core) rope was used in four trials. Observations of the buoy line movements were captured with an Atlantis underwater camera attached to a Digital DPC-1000 video recorder. Results from this study showed that: 1) the method used for deployment was important in keeping the buoy line from arcing or coiling, 2) little to no arcing occurred in water current velocities of >0.20 m/s, 3) rope lengths of ≥50 ft. deployed in <10 ft. of water produced waving in the water column and arcing on the bottom, 4) slack tide was a period of increased risk of entanglement for bottlenose dolphins, and 5) poly lead-core rope was not a good alternative to nylon rope unless in deep water with strong water current velocities. This pilot study produced questions that can be used for future studies on the characteristics of buoy line movements in the crab pot fishery as it relates to bottlenose dolphin entanglements.

A GIS analysis of coastal development and trends in bottlenose dolphin strandings in Charleston, SC: implications for coastal marine spatial planning

Bottlenose dolphins (Tursiops truncatus) inhabit estuarine waters near Charleston, South Carolina (SC) feeding, nursing and socializing. While in these waters, dolphins are exposed to multiple direct and indirect threats such as anthropogenic impacts (egs. harassment with boat traffic and entanglements in fishing gear) and environmental degradation. Bottlenose dolphins are protected under the Marine Mammal Protection Act of 1972. Over the years, the percentage of strandings in the estuaries has increased in South Carolina and, specifically, recent stranding data shows an increase in strandings occurring in Charleston, SC near areas of residential development. During the same timeframe, Charleston experienced a shift in human population towards the coastline. These two trends, rise in estuarine dolphin strandings and shift in human population, have raised questions on whether the increase in strandings is a result of more detectable strandings being reported, or a true increase in stranding events. Using GIS, the trends in strandings were compared to residential growth, boat permits, fishing permits, and dock permits in Charleston County from 1994-2009. A simple linear regression analysis was performed to determine if there were any significant relationships between strandings, boat permits, commercial fishing permits, and crabpot permits. The results of this analysis show the stranding trend moves toward Charleston Harbor and adjacent rivers over time which suggests the increase in strandings is related to the strandings becoming more detectable. The statistical analysis shows that the factors that cause human interaction strandings such as boats, commercial fishing, and crabpot line entanglements are not significantly related to strandings further supporting the hypothesis that the increase in strandings are due to increased observations on the water as human coastal population increases and are not a natural phenomenon. This study has local and potentially regional marine spatial planning implications to protect coastal natural resources, such as the bottlenose dolphin, while balancing coastal development.

Effects of Cu-Wire Surface Fluctuations on Early Failures

Different chemical mechanical polishing （CMP） slurries are used to obtain single-damascene Cu-wires with different surface fluctuations as well as pre-existing surface-defects in wires with rougher surfaces. The presence of such pre-existing defects strongly increases the rate of early failures to almost 100%, reduces electromigration lifetime rapidly to the level of early failures, and changes the multimodal failure distribution into monomodal. The activation energy （0. 74±0.02eV） for the failure mechanism associated with these pre-existing defects confirms a dominant surface diffusion. It shows how a weakest link approximation analysis can he applied to a single wire by dividing the wire into relevant segments and assigning different failure mechanisms to the various segments. The analysis confirms that, although surface-defects are not the fastest early failure mechanism, the ten times higher surface-defectdensity in the rougher wires is responsible for the observed high early-failure rate and unreliable performance.

Early development of germlings of Sargassum thunbergii (Fucales, Phaeophyta) under laboratory conditions

Morphology and culture studies on germlings of Sargassum thunbergii (Mertens et Roth) Kuntze were carried out under controlled laboratory conditions. Growth characteristics of these germlings grown under different temperatures (from 10 to 25A degrees C), irradiances (from 9 to 88 mu mol photons m(-2) s(-1)), and under blue and white light conditions are described. The development of embryonic germlings follows the classic "8 nuclei 1 egg" type described for Sargassaceae. Fertilized eggs spent 5-6 h developing into multicellular germlings with abundant rhizoids after fertilization. Under conditions of 20A degrees C, 44 mu mol photons m(-2) s(-1) and photoperiod of 12 h, young germlings with one or two leaflets reached 2-3 mm in length after 8 weeks. Temperature variations (10, 15, 20, 25A degrees C) under 88 mu mol photons m(-2) s(-1) significantly influenced the growth rate within the first week, although this effect became less obvious after 8 weeks, especially at 15 and 20A degrees C. Variation in germling growth was highly significant under different irradiances (9, 18, 44, 88 mu mol photons m(-2) s(-1)) at 25A degrees C. Low temperature (10A degrees C) reduced germling growth. Growth of germlings cultured under blue light was lower than in white light. Optimal growth of these germlings occurred at 25A degrees C and 44 mu mol photons m(-2) s(-1).

Floral closure in response to temperature and pollination in Gentiana straminea Maxim. (Gentianaceae), an alpine perennial in the Qinghai-Tibetan Plateau

Floral closure may be induced by pollination and various other factors, but is rarely studied comprehensively. Different kinds of floral closure should have various effects on reproductive fitness of plants. Two contrasting types of floral closure were observed in the flowers of Gentiana straminea Maxim. in the eastern Qinghai-Tibetan Plateau. The first type occurred prior to pollination during both gender phases, in response mainly to decreasing air temperatures. Flowers closed when decreasing temperatures approached 20 degrees C and subsequently began to reopen the following day during mid-morning when air temperatures warmed to approximately 13-15 degrees C. This kind of floral closure can protect pollen grains on either stamens or stigmas, increasing fitness of both male and female. Following pollination, permanent floral closure occurred, although there was a delay between the dates of pollination and permanent closure, during which flowers continued to show temporary closure in response to low temperature episodes. The time required for permanent, pollination-induced closure varied according to the age of the gender phase, including a prolonged time before closure if pollination occurred early in the female phase. The retaining of permanent closed flowers increased both approaching (to inflorescences) and visiting (to unpollinated flowers) frequencies of individual plants when with fewer open flowers and the persisting corolla is further beneficial for seed sets of these pollinated flowers. Thus, two separate types of floral closure, one in response to environmental cues and the other in response to the age of each gender stage, appeared to have a strong influence on reproductive fitness in this species. These results revealed a different adaptive strategy of alpine plants in the sexual reproduction assurance in addition to the well-known elevated floral longevity, dominant role of more effective pollinators and increased reproduction allocation in the arid habitats.

Na-23, Si-29, and C-13 MAS NMR investigation of glass-forming reactions between Na2CO3 and SiO2

Winter, Rudolf; Jones, A.R.; Greaves, G.N.; Smith, I.H., (2005) 'Na-23, Si-29, and C-13 MAS NMR investigation of glass-forming reactions between Na2CO3 and SiO2', Journal of Physical Chemistry B 109(49) pp.23154-23161 RAE2008

IMPROMPTU: FROM PUBLISHER'S IMPULSE TO AN ESTABLISHED SMALL­ SCALE GENRE IN PIANO LITERATURE

The word 'impromptu' began to appear in music literature in the early 19th century, specifically as title for a relatively short composition written for solo piano. The first impromptus appear to have been named so by the publishers. However, the composers themselves soon embraced the title to indicate, for the most part, fairly short character pieces. Impromptus do not follow any specific structural pattern, although many are cast in ternary form. The formal design ranges from strict compound ternary in the early impromptus to through-composed and variation forms. The peak of impromptu's popularity undoubtedly came during the middle and late19th century. However, they are still being composed today, albeit much less frequently. Although there have been many variants of impromptus in relation to formal design and harmonic language over the years, the essence of impromptu remains the same: it is still a short character piece with a general feeling of spontaneity. Overall, impromptus may be categorized into several different groups: some appear as part of a larger cycle, such as Dvorak's G minor Impromptu from his Piano Pieces, B. 110; many others use an element of an additional genre that enhances the character ofthe impromptu, such as Liszt's Valse-Impromptu and Antonio Bibalo's Tango Impromptu; yet another group consists of works based on opera themes, such as Liszt's Impromptu Brillant sur des themes de Rossini et Spontini and Czerny's Impromptus et variations sur Oberon, Op. 134. My recording project includes well-known impromptus, such as Schubert's Op. 142 and the four by Chopin, as well as lesser known works that have not been performed or recorded often. There are four impromptus that have been recorded here for the first time, including those written by Leopold Godowsky, Antonio Bibalo, Altin Volaj, and Nikolay Mazhara. I personally requested the two last named composers to contribute impromptus to this project. My selection represents works by twenty composers and reflects the different types of impromptus that have been encountered through almost three hundred years of the genre's existence, from approximately 1817 (VoriSek) to 2008 (Volaj and Mazhara).

Using time-to-contact information to assess potential collision modulates both visual and temporal prediction networks

Accurate estimates of the time-to-contact (TTC) of approaching objects are crucial for survival. We used an ecologically valid driving simulation to compare and contrast the neural substrates of egocentric (head-on approach) and allocentric (lateral approach) TTC tasks in a fully factorial, event-related fMRI design. Compared to colour control tasks, both egocentric and allocentric TTC tasks activated left ventral premotor cortex/frontal operculum and inferior parietal cortex, the same areas that have previously been implicated in temporal attentional orienting. Despite differences in visual and cognitive demands, both TTC and temporal orienting paradigms encourage the use of temporally predictive information to guide behaviour, suggesting these areas may form a core network for temporal prediction. We also demonstrated that the temporal derivative of the perceptual index tau (tau-dot) held predictive value for making collision judgements and varied inversely with activity in primary visual cortex (V1). Specifically, V1 activity increased with the increasing likelihood of reporting a collision, suggesting top-down attentional modulation of early visual processing areas as a function of subjective collision. Finally, egocentric viewpoints provoked a response bias for reporting collisions, rather than no-collisions, reflecting increased caution for head-on approaches. Associated increases in SMA activity suggest motor preparation mechanisms were engaged, despite the perceptual nature of the task.

Early-type stars observed in the ESO UVES Paranal Observatory Project - III. Sub-parsec and AU-scale structure in the interstellar medium

UVES interstellar observations from the Paranal Observatory Project are presented for early-type stars located in the line of sight to the nearby open clusters IC 2391 (Omni Vel) and NGC 6475 (M7), with spectroscopic resolution R similar to 80 000 and signal-to-noise ratios in the Ti II (3383 angstrom), Ca II K, CH+ (4232 angstrom), Na I D and K I (7698 angstrom) lines of several hundred. The sightlines are a mixture of cluster and non-cluster objects. A total of 22 early-type stars (A and B type) are present in our sample towards IC 2391, with 21 towards NGC 6475/M7, and enable us to probe for differences in column density on scales from similar to 0.07 to 7.3 and similar to 0.05 to 4.9 pc in the respective clusters. Additionally, towards Praesepe the Na I D interstellar variation only is probed towards 13 sightlines and transverse scales of similar to 0.16-10.7 pc at R = 70 000. Towards IC 2391 variations are found in Ti II, Ca II K and Na I D column density in different sightlines of up to 0.7, 1.0 and 1.8 dex (excluding one star), respectively. This kind of variability correlates well with the Hipparcos parallax of the objects, and probes structure within the Local Bubble. For cluster-only objects the variations are 0.3, 0.3 and 0.5 dex, respectively. For the field of view towards NGC6475 the corresponding maximum variations are somewhat smaller, being 0.5, 0.3, 0.8 and 1.0 dex for Ti II, Ca II K, Na I and K I, respectively, for all objects and 0.4, 0.2, 0.6 and 0.7 dex for the cluster-only objects. These are uncorrelated with parallax, and again demonstrate that Ca II K tends to be more smoothly distributed than Na I D. A few likely cluster sightlines show evidence for CH+ and variations in this molecular species of a factor of 10 in equivalent width over sub-pc scales. Towards Praesepe variation in interstellar Na I D is small, being a maximum of only similar to 0.4 dex (including measurement errors), but with fewer sightlines studied. Overall, the scatter in the data is similar for the singly ionized species Ti II and Ca II, lending more support to the hypothesis that these two species sample similar parts of the interstellar medium (ISM). This also appears to be the case for the neutral species Na I D and K I in the one cluster studied. Finally, multiple-epoch observations from a variety of archive sources are used to search for astronomical unit (au) scale structure in the ISM towards 46 sightlines. There are tentative indications of structure on scales of tens to thousands of au for three sightlines. Future observations will confirm the veracity or otherwise of the time-variable components and others presented.

An evaluation of the effect of altering nutrition and nutritional strategies in early lactation on reproductive performance and estrous behavior of high-yielding Holstein-Friesian dairy cows

Reproductive performance in the high-yielding dairy cow has severely decreased in the last 40 yr. The aim of this study was to compare the effectiveness of 4 nutritional strategies in improving the reproductive performance of high-yielding dairy cows. It was hypothesized that offering cows a high-starch ration in early lactation would enhance the onset of luteal activity, and that decreasing the severity of negative energy balance in the early postcalving period would improve reproductive parameters. Nutritional regimens aimed at improving fertility were applied to 96 Holstein-Friesian dairy animals. Upon calving, animals were allocated in a balanced manner to one of 4 dietary treatments. Primiparous animals were balanced according to live weight, body condition score and calving date. Multiparous animals were balanced according to parity, previous lactation milk yield, liveweight, body condition score and calving date. Treatment 1 was based on an industry best practice diet (control) to contain 170 g of crude protein/kg of dry matter. Treatment 2 was an individual cow feeding strategy, whereby the energy balance (EB) of individual animals was managed so as to achieve a predetermined target daily EB profile (+/- 10 MJ/d). Treatment 3 was a high-starch/high-fat combination treatment, whereby an insulinogenic (high-starch) diet was offered in early lactation to encourage cyclicity and followed by a lipogenic (low-starch, high-fat) diet to promote embryo development. Treatment 4 was a low-protein diet, containing 140 g of crude protein/kg of dry matter, supplemented with protected methionine at an inclusion level of 40 g per animal per day. The nutritional strategies implemented in this study had no statistically significant effects on cow fertility measures, which included the onset of luteal activity, conception rate, in-calf rate, and the incidence of atypical cycles. The individual cow feeding strategy improved EB in early lactation but had no benefit on conception rate to first insemination. However, conception rate to second insemination, 100-d pregnancy rate (from the commencement of breeding), and overall pregnancy rate tended to be higher in this group. The high-starch/high-fat treatment tended to decrease the proportion of delayed ovulations and increase the proportion of animals cycling by d 50 postcalving. Animals that failed to conceive to first insemination had a significantly longer luteal phase in the first cycle postpartum and a longer inter-ovulatory interval in the second cycle postpartum. With regards to estrous behavior, results indicate that as the size of the sexually active group increased, the intensity of estrus and the expression of mounting or attempting to mount another cow also increased. Furthermore, cows that became pregnant displayed more intense estrous behavior than cows that failed to become pregnant.

Precursors to aggression are evident by 6 months of age

We tested the hypothesis that developmental precursors to aggression are apparent in infancy. Up to three informants rated 301 firstborn infants for early signs of anger, hitting and biting; 279 (93%) were assessed again as toddlers. Informants' ratings were validated by direct observation at both ages. The precursor behaviours were significantly associated with known risk factors for high levels of aggressiveness. Individual differences were stable from early infancy to the third year and predicted broader conduct problems. These findings suggest that some individuals set forth on the trajectory to high levels of aggression by 6 months of age. The findings have implications for developmental studies of aggression, clinical prevention and intervention strategies, and theoretical considerations regarding the detection of precursors in different domains of development.