In vivo regulation of the Vi antigen in Salmonella and induction of immune responses with an in vivo-inducible promoter.

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo-inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.

Analysis of volatility transmissions in integrated and interconnected markets: The case of the Iberian and French markets

This paper models the mean and volatility spillovers of prices within the integrated Iberian and the interconnected Spanish and French electricity markets. Using the constant (CCC) and dynamic conditional correlation (DCC) bivariate models with three different specifications of the univariate variance processes, we study the extent to which increasing interconnection and harmonization in regulation have favoured price convergence. The data consist of daily prices calculated as the arithmetic mean of the hourly prices over a span from July 1st 2007 until February 29th 2012. The DCC model in which the variances of the univariate processes are specified with a VARMA(1,1) fits the data best for the integrated MIBEL whereas a CCC model with a GARCH(1,1) specification for the univariate variance processes is selected to model the price series in Spain and France. Results show that there are significant mean and volatility spillovers in the MIBEL, indicating strong interdependence between the two markets, while there is a weaker evidence of integration between the Spanish and French markets. We provide new evidence that the EU target of achieving a single electricity market largely depends on increasing trade between countries and homogeneous rules of market functioning.

Bag and size limit analyses for red drum in Northern and Southern Regions of the U.S. Atlantic

Assessments of the Atlantic red drum for the northern (North Carolina and north) and southern (South Carolina through east coast of Florida) regions along the U. S. Atlantic coast were recently completed. The joint Red Drum Technical Committee (SAFMC/ASMFC) selected the most appropriate catch matrix (incorporating an assumption on size of recreationally-released fish), selectivity of age 3 relative to age 2, and virtual population analysis (FADAPT). Given gear- and age-specific estimates of fishing mortality (F) for the 1992-1998 period, analyses were made of potential gains in escapement through age 4 and static spawning potential ratio (SPR) from further reductions in fishing mortality due to changes in slot and bag limits. Savings from bag limits were calculated given a particular slot size for the recreational fishery, with no savings for the commercial fisheries in the northern region due to their being managed primarily through a quota. Relative changes in catch-at-age estimates were used to adjust age-specific F and hence calculated escapement through age 4 and static SPR. Adjustment was made with the recreational savings to account for release mortality (10%, as in the stock assessment). Alternate runs for the northern region commercial fishery considered 25% release mortality for lengths outside the slot (instead of 0% for the base run), and 0% vs. 10% gain or loss across legal sizes in F. These results are summarized for ranges of bag limits with increasing minimum size limit (for fixed maximum size), and with decreasing maximum size limit (for fixed minimum size limit). For the southern region, a bag limit of one-fish per angler trip would be required to attain the stated target of 40% static SPR if the current slot limit were not changed. However, for the northern region, a bag limit of one-fish per angler trip appears to be insufficient to attain the stated target of 40% static SPR while maintaining the current slot limit. (PDF contains 41 pages)

Conservation Biology of Elasmobranchs

Elasmobranchs are vital and valuable components of the marine biota. From an ecological perspective they occupy the role of top predators within marine food webs, providing a regulatory control that helps balance the ecosystem. From an evolutionary perspective, this group represents an early divergence along the vertebrate line that produced many unusual, but highly successful, adaptations in function and form. From man's perspective, elasmobranchs have been considered both an unavoidable nuisance, and an exploitable fishery resource. A few of the large shark species have earned a dubious notoriety because of sporadic attacks on humans that occur in coastal areas each year worldwide; the hysteria surrounding an encounter with a shark can be costly to the tourist industry. More importantly, elasmobranchs are often considered a detriment to commercial fishing operations; they cause significant economic damage to catches and fishing gear. On the other hand, consumer attitudes have changed concerning many previously unpopular food fishes, including elasmobranchs, and this group of fishes has been increasingly used by both recreational and commercial fishing interests. Many elasmobranchs have become a popular target of recreational fishermen for food and sport because of their abundance, size, and availability in coastal waters. Similarly, commercial fisheries for elasmobranchs have developed or expanded from an increased demand for elasmobranch food products. (PDF file contains 108 pages.)

Behavioral, neurochemical and morphological changes induced by the overexpression of munc18-1a in brain of mice: relevance to schizophrenia

Overexpression of the mammalian homolog of the unc-18 gene (munc18-1) has been described in the brain of subjects with schizophrenia. Munc18-1 protein is involved in membrane fusion processes, exocytosis and neurotransmitter release. A transgenic mouse strain that overexpresses the protein isoform munc18-1a in the brain was characterized. This animal displays several schizophrenia-related behaviors, supersensitivity to hallucinogenic drugs and deficits in prepulse inhibition that reverse after antipsychotic treatment. Relevant brain areas (that is, cortex and striatum) exhibit reduced expression of dopamine D-1 receptors and dopamine transporters together with enhanced amphetamine-induced in vivo dopamine release. Magnetic resonance imaging demonstrates decreased gray matter volume in the transgenic animal. In conclusion, the mouse overexpressing brain munc18-1a represents a new valid animal model that resembles functional and structural abnormalities in patients with schizophrenia.

Time and space-resolved measurement of a gas-puff laser-plasma x-ray source

The dynamic interaction processes between a nano-second laser pulse and a gas-puff target, such as those of plasma formation, laser heating, and x-ray emission, have been investigated quantitatively. Time and space-resolved x-ray and optical measurement techniques were used in order to investigate time-resolved laser absorption and subsequent x-ray generation. Efficient absorption of the incident laser energy into the gas-puff target of 17%, 12%, 38%, and 91% for neon, argon, krypton, and xenon, respectively, was shown experimentally. It was found that the laser absorption starts and, simultaneously, soft x-ray emission occurs. The soft x-ray lasts much longer than the laser pulse due to the recombination. Temporal evolution of the soft x-ray emission region was analyzed by comparing the experimental results to the results of the model calculation, in which the laser light propagation through a gas-puff plasma was taken into account. (C) 2003 American Institute of Physics.

Insights into the mechanism of human erythrocyte hexose transport : a transferred NOE study of glucose binding to GLUT1

This study examines binding of α- and β-D-glucose in their equilibrium mixture to the glucose transporter (GLUT1) in human erythrocyte membrane preparations by an ^1H NMR method, the transferred NOE (TRNOE). This method is shown theoretically and experimentally to be a sensitive probe of weak ligand-macromolecule interactions. The TRNOEs observed are shown to arise solely from glucose binding to GLUT1. Sites at both membrane faces contribute to the TRNOEs. Binding curves obtained are consistent with a homogeneous class of sugar sites, with an apparent K_D which varies (from ~30 mM to ~70 mM for both anomers) depending on the membrane preparation examined. Preparations with a higher proportion of the cytoplasmic membrane face exposed to bulk solution yield higher apparent KK_Ds. The glucose transport inhibitor cytochalasin B essentially eliminates the TRNOE. Nonlinearity was found in the dependence on sugar concentration of the apparent inhibition constant for cytochalasin B reversal of the TRNOE observed in the α anomer (and probably the β anomer); such nonlinearity implies the existence of ternary complexes of sugar, inhibitor and transporter. The inhibition results furthermore imply the presence of a class of relatively high-affinity (K_D < 2mM) sugar sites specific for the α anomer which do not contribute to NMR-observable binding. The presence of two classes of sugar-sensitive cytochalasin B sites is also indicated. These results are compared with predictions of the alternating conformer model of glucose transport. Variation of apparent K_D in the NMR-observable sites, the formation of ternary complexes and the presence of an anomer-specific site are shown to be inconsistent with this model. An alternate model is developed which reconciles these results with the known transport behavior of GLUT1. In this model, the transporter possesses (at minimum) three classes of sugar sites: (i) transport sites, which are alternately exposed to the cytoplasmic or the extracellular compartment, but never to both simultaneously, (ii) a class of sites (probably relatively low-affinity) which are confined to one compartment, and (iii) the high-affinity α anomer-specific sites, which are confined to the cytoplasmic compartment.

Revealing, illuminating, and modifying proteins in human diseases using noncanonical amino acids

To better understand human diseases, much recent work has focused on proteins to either identify disease targets through proteomics or produce therapeutics via protein engineering. Noncanonical amino acids (ncAAs) are tools for altering the chemical and physical properties of proteins, providing a facile strategy not only to label proteins but also to engineer proteins with novel properties. My thesis research has focused on the development and applications of noncanonical amino acids in identifying, imaging, and engineering proteins for studying human diseases. Chapter 1 introduces the concept of ncAAs and reveals insights to how I chose my thesis projects.

ncAAs have been incorporated to tag and enrich newly synthesized proteins for mass spectrometry through a method termed BONCAT, or bioorthogonal noncanonical amino acid tagging. Chapter 2 describes the investigation of the proteomic response of human breast cancer cells to induced expression of tumor suppressor microRNA miR-126 by combining BONCAT with another proteomic method, SILAC or stable isotope labeling by amino acids in cell culture. This proteomic analysis led to the discovery of a direct target of miR-126, shedding new light on its role in suppressing cancer metastasis.

In addition to mass spectrometry, ncAAs can also be utilized to fluorescently label proteins. Chapter 3 details the synthesis of a set of cell-permeant cyclooctyne probes and demonstration of selective labeling of newly synthesized proteins in live mammalian cells using azidohomoalanine. Similar to live cell imaging, the ability to selectively label a particular cell type within a mixed cell population is important to interrogating many biological systems, such as tumor microenvironments. By taking advantage of the metabolic differences between cancer and normal cells, Chapter 5 discusses efforts to develop selective labeling of cancer cells using a glutamine analogue.

Furthermore, Chapter 4 describes the first demonstration of global replacement at polar amino acid positions and its application in developing an alternative PEGylation strategy for therapeutic proteins. Polar amino acids typically occupy solvent-exposed positions on the protein surface, and incorporation of noncanonical amino acids at these positions should allow easier modification and cause less perturbation compared to replacements at the interior positions of proteins.

Breaking the 400 ppm barrier: Physical and Social implications of the recent CO2 rise

6 p.

Lynx1 Modulation of Nicotinic Acetylcholine Receptors

Nicotinic receptors are the target of nicotine in the brain. They are pentameric ion channels. The pentamer structure allows many combinations of receptors to be formed. These various subtypes exhibit specific properties determined by their subunit composition. Each brain region contains a fixed complement of nicotinic receptor subunits. The midbrain region is of particular interest because the dopaminergic neurons of the midbrain express several subtypes of nicotinic receptors, and these dopaminergic neurons are important for the rewarding effects of nicotine. The α6 nicotinic receptor subunit has garnered intense interest because it is present in dopaminergic neurons but very few other brain regions. With its specific and limited presence in the brain, targeting this subtype of nicotinic receptor may prove advantageous as a method for smoking cessation. However, we do not fully understand the trafficking and membrane localization of this receptor or its effects on dopamine release in the striatum. We hypothesized that lynx1, a known modulator of other nicotinic receptor subtypes, is important for the proper function of α6 nicotinic receptors. lynx1 has been found to act upon several classes of nicotinic receptors, such as α4β2 and α7, the two most common subtypes in the brain. To determine whether lynx1 affects α6 containing nicotinic receptors we used biochemistry, patch clamp electrophysiology, fast scan cyclic voltammetry, and mouse behavior. We found that lynx1 has effects on α6 containing nicotinic receptors, but the effects were subtle. This thesis will detail the observed effects of lynx1 on α6 nicotinic receptors.

I. A study of the dynamics of triplet excitons in molecular crystals. II. An investigation of delayed light emission from Chlorella pyrenoidosa

I. PREAMBLE AND SCOPE

Brief introductory remarks, together with a definition of the scope of the material discussed in the thesis, are given.

II. A STUDY OF THE DYNAMICS OF TRIPLET EXCITONS IN MOLECULAR CRYSTALS

Phosphorescence spectra of pure crystalline naphthalene at room temperature and at 77˚ K are presented. The lifetime of the lowest triplet ³B_1u state of the crystal is determined from measurements of the time-dependence of the phosphorescence decay after termination of the excitation light. The fact that this lifetime is considerably shorter in the pure crystal at room temperature than in isotopic mixed crystals at 4.2˚ K is discussed, with special importance being attached to the mobility of triplet excitons in the pure crystal.

Excitation spectra of the delayed fluorescence and phosphorescence from crystalline naphthalene and anthracene are also presented. The equation governing the time- and spatial-dependence of the triplet exciton concentration in the crystal is discussed, along with several approximate equations obtained from the general equation under certain simplifying assumptions. The influence of triplet exciton diffusion on the observed excitation spectra and the possibility of using the latter to investigate the former is also considered. Calculations of the delayed fluorescence and phosphorescence excitation spectra of crystalline naphthalene are described.

A search for absorption of additional light quanta by triplet excitons in naphthalene and anthracene crystals failed to produce any evidence for the phenomenon. This apparent absence of triplet-triplet absorption in pure crystals is attributed to a low steady-state triplet concentration, due to processes like triplet-triplet annihilation, resulting in an absorption too weak to be detected with the apparatus used in the experiments. A comparison of triplet-triplet absorption by naphthalene in a glass at 77˚ K with that by naphthalene-h₈ in naphthalene-d₈ at 4.2˚ K is given. A broad absorption in the isotopic mixed crystal triplet-triplet spectrum has been tentatively interpreted in terms of coupling between the guest ³B_1u state and the conduction band and charge-transfer states of the host crystal.

III. AN INVESTIGATION OF DELAYED LIGHT EMISSION FROM Chlorella Pyrenoidosa

An apparatus capable of measuring emission lifetimes in the range 5 X 10^-9 sec to 6 X 10^-3 sec is described in detail. A cw argon ion laser beam, interrupted periodically by means of an electro-optic shutter, serves as the excitation source. Rapid sampling techniques coupled with signal averaging and digital data acquisition comprise the sensitive detection and readout portion of the apparatus. The capabilities of the equipment are adequately demonstrated by the results of a determination of the fluorescence lifetime of 5, 6, 11, 12-tetraphenyl-naphthacene in benzene solution at room temperature. Details of numerical methods used in the final data reduction are also described.

The results of preliminary measurements of delayed light emission from Chlorella Pyrenoidosa in the range 10^-3 sec to 1 sec are presented. Effects on the emission of an inhibitor and of variations in the excitation light intensity have been investigated. Kinetic analysis of the emission decay curves obtained under these various experimental conditions indicate that in the millisecond-to-second time interval the decay is adequately described by the sum of two first-order decay processes. The values of the time constants of these processes appear to be sensitive both to added inhibitor and to excitation light intensity.

Use of biological reference points for the conservation of atlantic salmon (Salmo salar L.)in the River Lune, North West England.

This paper deals with the development and use of biological reference points for salmon conservation on the River Lune, England. The Lune supports recreational and net fisheries with annual catches in the region of 1,000 and 1356 salmon respectively. Using models transported from other river systems, biological reference points exclusive to the Lune were developed; specifically the number of eggs deposited and carrying capacity estimates for age 0+ and 1+ parr. The conservation limit was estimated at 11.9 million eggs and between 1989 and 1998 was exceeded in two years. Comparison of juvenile salmon densities in 1991 and 1997 with estimates of carrying capacity indicated that 0+ and 1+ parr densities were at around 60 % of carrying capacity and may relate to the number of eggs deposited in 1990 and 1996 being approximately 70% of the target value. The paper discusses the management actions taken in order to ensure that the management target of the conservation limit being met four years out of five is delivered. It also discusses the balance between conservation and exploitation and the socio-economic decisions made in order to ensure parity of impacts on the rod and net fisheries. The regulations have been enforced since 1999 and the paper concludes with an assessment of the actions taken to deliver the management targets, over the last five years.

Use of stereo camera systems for assessment of rockfish abundance in untrawlable areas and for recording pollock behavior during midwater trawls

We describe the application of two types of stereo camera systems in fisheries research, including the design, calibration, analysis techniques, and precision of the data obtained with these systems. The first is a stereo video system deployed by using a quick-responding winch with a live feed to provide species- and size- composition data adequate to produce acoustically based biomass estimates of rockfish. This system was tested on the eastern Bering Sea slope where rockfish were measured. Rockfish sizes were similar to those sampled with a bottom trawl and the relative error in multiple measurements of the same rockfish in multiple still-frame images was small. Measurement errors of up to 5.5% were found on a calibration target of known size. The second system consisted of a pair of still-image digital cameras mounted inside a midwater trawl. Processing of the stereo images allowed fish length, fish orientation in relation to the camera platform, and relative distance of the fish to the trawl netting to be determined. The video system was useful for surveying fish in Alaska, but it could also be used broadly in other situations where it is difficult to obtain species-composition or size-composition information. Likewise, the still-image system could be used for fisheries research to obtain data on size, position, and orientation of fish.

New information from fish diets on the importance of glassy flying squid (Hyaloteuthis pelagica) (Teuthoidea: Ommastrephidae) in the epipelagic cephalopod community of the tropical Atlantic Ocean

Squids of the family Ommastrephidae are a vital part of marine food webs and support major fisheries around the world. They are widely distributed in the open ocean, where they are among the most abundant in number and biomass of nektonic epipelagic organisms. In turn, seven of the 11 genera of this family (Dosidicus, Illex, Martialia, Nototodarus, Ommastrephes, Sthenoteuthis, and Todarodes) are heavily preyed upon by top marine predators, i.e., birds, mammals, and fish, and currently support fisheries in both neritic and oceanic waters (Roper and Sweeney, 1984; Rodhouse, 1997). Their commercial importance has made the large ommastrephids the target of many scientific investigations and their biology is consequently reasonably well-known (Nigmatullin et al., 2001; Zuyev et al., 2002; Bower and Ichii, 2005). In contrast, much less information is available on the biology and ecological role of the smaller, unexploited species of ommastrephids (e.g., Eucleoteuthis, Hyaloteuthis, Ornithoteuthis, and Todaropsis).

Adaptive evolution of SCML1 in primates, a gene involved in male reproduction

Background: Genes involved in male reproduction are often the targets of natural and/or sexual selection. SCML1 is a recently identified X-linked gene with preferential expression in testis. To test whether SCML1 is the target of selection in primates, we