In the catalytic cycle of the glycine cleavage system (GCS), the major physiological pathway for glycine degradation, the lipoic acid-containing hydrogen carrier protein (H-protein) plays a pivotal role as a mobile carrier of reaction intermediates. and as a regulator for glycine decarboxylase (P-protein). Defects in the GCS lead to the accumulation of glycine in all body tissues, resulting in the genetic disease glycine encephalopathy (GE). Defects in P-protein lead to more than 80% of reported cases of GE, therefore. routine biochemical analysis only tests P-protein for activity. Unlike other amino acid decarboxylases, P-protein is itself inactive, and requires H-protein for biochemical activity. Currently. researchers use H-protein purified from chicken liver extracts for the P-protein activity assay. However. extraction and purification of chicken H-protein is laborious. costly. and has poor yield, making the expression and purification of H-protein from an alternate source desirable. We have overexpressed recombinant human H-protein in the methylotropic yeast Pichia pastoris by utilizing the inducible alcohol oxidase (AOX1) promoter and the Saccharomyces cerevisiae a-mating factor secretion signal. Enhanced green fluorescent protein (EGFP) was used as a fusion partner to aid detection of H-protein during expression and purification. Secreted H-protein was detected from 24-96 hours post induction, and constituted the major protein species in the culture medium. H-protein was purified to apparent homogeneity in a single step using nickel-chelating affinity chromatography, and lipoylated using lipoate protein ligase from E. coli. Functional analysis of holo-H-protein using the NAD+ lipoamide dehydrogenase assay demonstrated biochemical activity with the artificial substrate, suggesting that human H-protein produced in P. pastoris may be an appropriate replacement for the chicken H-protein currently used in the biochemical diagnosis of GE.

A comprehensive worldwide review and revision of the genus Sternaspis (Polychaeta: Sternaspidae) is presented based on type material, or material collected from type localities. The phylogeny was reconstructed using morphological characters and PAUP* with maximum parsimony set as the criterion. The more important characters were: the form of pharyngeal spines or their apposition to each other within a row; the form or distribution of cuticular papillae; number of chaetal fascicles associated with the margins of the ventral shield; and the presence or position of the peg chaetae. Sternaspis scutata (Ranzani. 1817) is synonymised with three other species; S. costata Marenzeller, 1979 is synonymised with one other species. S. affinis Stimpson, 1864 and S. spinosa Sluiter, 1882 are emended. S. fossor var. africana Augener, 1918 is raised to species status as S. africana. Two new species, S. andamana and S. gudmundi are also described. Comments on all valid species are provided.

The focus of this dissertation is the use of bioinformatics in the identification of highly conserved sequences among a set of poxvirus genomes and the subsequent functional analysis of the conserved functions of these sequences. A novel algorithm, Java Pattern Finder, which identifies sequences of a user-specified length that are conserved with a user-specified number of allowed differences, was used to identify near-perfectly conserved sequences among a set of poxvirus genomes. A scoring method was established to quantify the degree of conservation of these sequences and used to show that the 11 most conserved sequences were significantly more conserved than control sequences. Functional analysis showed that explanations such as low codon degeneracy or the presence of conserved promoter elements partially – but not fully – accounted for the conservation observed in these sequences, suggesting that these highly conserved regions may have novel functions in the poxvirus genome that have yet to be uncovered.

Persistent organic pollutants (POPs) are ubiquitous environmental contaminants that are lipophilic, slow to degrade, bioaccumulate in aquatic food webs and threaten the health of both humans and wildlife. Predatory marine mammals, such as harbour seals (Phoca vitulina), are at particularly risk for the accumulation of high POP concentrations and resultant increased risk of toxic effects. EIevated levels of certain POPs have been implicated in the disruption of the endocrine system in marine mammals. The main purpose of this study was to assess whether current levels of POP are affecting thyroid hormone physiology of free-ranging harbour seals (Phoca vitulina) in British Columbia (BC), Canada and Washington State (WA), U.S.A. TH functions mainly by binding to nuclear thyroid hormone receptors (TRs) in target tissues and modulating specific gene expression programs. TR isoforms α (TRα) and β (TREβ) from harbour seals were isolated and quantified in internal and external organs. Harbour seals inhabiting industrialized regions exhibited a contaminant-related increase in blubber TRa and a decrease in circulating total thyroxine (TT4) concentrations. Our TRa expression results provide evidence of contaminant-related disruption of TH action at the Ievel of regulation of gene expression. Our findings of a metabolically active blubber layer. and a contaminant-related disruption of blubber TRa expression, suggest that, in addition to disruption of normal development, contaminant exposure could have important implications for lipid metabolism in seals. Consequently, the disruption of blubber TRa expression could influence such critical life processes as energy storage, thermoregulation, and buoyancy in marine mammals. In this study, the use of gene expression biomarkers in combination with a biopsy-based sampling approach was successfully applied to a small marine mammal (i.e. harbour seal) and demonstrates great promise for investigations in other sentinel species (i.e. cetaceans).

Relationships between the terms culture and education are often taken for granted in educational research. This study challenges some of the taken for granted assumptions around the term culture in educational contexts, particularly in secondary language arts education. It examines these assumptions through an analysis of three debates from the contemporary culture wars in education. The implications of these debates on uses of the term culture in secondary language arts education are examined through Othello as a secondary school text. I am arguing that these debates, namely, on the literary canon, multicultural education, and cultural literacy, represent intractable conflicts over definitions of the term culture. In light of these conflicts, the aim of this study is to provide language arts educators with analytical tools for developing greater theoretical rigour when defining the term culture in language arts education. Drawing on recent theoretical writings on culture, concepts of cultural capital, cultural rights, and cultural reproduction are proposed as analytical tools. I then apply these to develop a methodological approach by which to structure my analysis of Othello as a school text. The study makes a theoretical contribution by bringing into sharper focus ways in which the ideological opposition between expressions of cultural right versus cultural left perspectives is articulated in language arts education, as well as illustrating that claims about culture in the canon debate reflect competing normative assumptions; in the multicultural education debate they reflect competing essentialist constructions; and in the cultural literacy debate they reflect competing empowerment goals. Such cultural debates have a long history and thus the study also situates the contemporary culture wars in education within a wider historical context by tracing related conflicts in the history of literary criticism on and performances of Othello over the past four centuries.

Francisella tularensis is a highly infectious. Gram-negative coccobacillus that is the etiological agent of the acute. febrile. zoonotic disease tularemia. A ca. 35 kb Francisella pathogenicity island (FM) was previously discovered. Two genes. pdpA and pdpD were shown to be required for virulence. The FP1 gene pdpC encodes a protein that has no significant similarities to any motifs, domains, or homologues of known bacterial proteins. This gene of unknown function may encode a novel virulence factor involved in Francisella infection. The role of PdpC in F. novicida intracellular growth was investigated. Results from this study demonstrated that the erythromycin allelic replacement mutant of pdpC was more attenuated in intracellular growth in the murine macrophage-like J774A.1 cells than in bone marrow-derived macrophages from BALB/c mice and that complementation in trans partially complements this mutation. To further investigate the role of pdpC in virulence. partial deletion mutagenesis in the C-terminus of PdpC was performed which resulted in four mutants that showed slight attenuation in J774A.1 intramacrophage growth but behaved like wildtype F, novicida in bone marrow-derived macrophages. Chicken embryos were infected to evaluate the virulence of these pdpC mutants. The virulence of the Em allelic replacement mutant was significantly more attenuated than wildtype F. novicida and complementation partially restored virulence. Partial deletion mutants of pdpC exhibited greater virulence than the EmR mutant in chicken embryos and were able to cause 100% mortality at day 6. Furthermore, eukaryotic expression of triple FLAG-tagged PdpC in chicken embryo fibroblasts resulted in cells that exhibited different morphologies than uninfected fibroblasts which suggests that PdpC may play a role in cytoskeletal rearrangements by altering host cell signaling pathways.

Studies of individual cells via microscopy and microinjection are a key component in research on gene functions, cancer, stem cells, and reproductive technology. As biomedical experiments become more complex, there is an urgent need for robotic systems to: improve cell manipulation, increase throughput, reduce lost cells, and improve reaction detection. Automation of these tasks using visual servoing creates significant benefits for biomedical laboratories including repeatability of experiments, higher throughput, and a controlled environment capable of operating 24 hours a day. In this work the design and development of a new five degree-of-freedom biomanipulator designed for single-cell microinjection, is described. The biomanipulator employs three degrees of linear motion and two degrees of rotation. This provides the ability to manipulate/micro-inject cells at varying orientations, thereby increasing flexibility in dealing with complex operations such as injecting clustered cells. The capability of the biomanipulator is shown with preliminary experimental results using mouse myeloma cells.

The goal of this project is to create a new instrument: the E-Drumset. This new interface addresses the lack of expressivity in current electronic percussion devices. The project combines Electrical Engineering for implementing hardware and digital signal processing, Computer Science for implementing musical and mapping software, and Music to devise new playing techniques and ways to combine them into a pedagogy and language of transmission. Like an acoustic drumset, the E-Drumset consists of different components that can be arranged together as a whole. An acoustic drumset can be thought of as a collection of pedals, drums and cymbals. The E-Drumset consists of the E-Pedal, E-Drum and E-Cymbal. The technology utilized in the E-Drumset includes sensor technologies with newly developed technologies such as acoustically excited physical models and timbre-recognition based instruments. These new technologies are discussed and applied to situations beyond the E-Drumset. Just building a new controller is not enough. It needs to be thoroughly tested in musical situations and to take into account feedback from musicians (both the player and other members of the ensemble) during the evaluation of the instrument. Clear and attainable technical guidelines have not been devised for the E-Drumset. In the case of the radiodrum, a spatial controller, improvements can be summarized to be better resolution in space and time. In the case of the E-Drumset the goal is to offer a flexible interface to percussionists where electronic drums are often the bottleneck in bandwidth. There is no clear answer to questions such as how low the latency needs to be to satisfy a drummer; an issue that will be explored through the project. The goals of the project are to provide the percussionist with an interface that they may sit down and use existing skills. Utilizing the great variety of gesture available to the expert, the E-Drumset allows the percussionist to explore all manners of controllers between acoustic instruments and electronic. To provide a smoother transition to the E-Drumset, notation and exercises for E-Drumset specific gestures and techniques was devised. The E-Drumset is a new instrument. Most new interfaces are derived to help lesser players achieve virtuosic ends, while other projects make a controller that is massively configurable where a more static instrument is appropriate. This project provides insight into the theory and practice of new musical interfaces while delivering novel forms of synthesis and gesture recognition appropriate for the E-Drumset.

Evidence suggests that the basic neural elements controlling and coupling the arms and the legs in humans during coordinated rhythmic movements are similar to that observed in quadrupedal animals. Further, it is possible that these interlimb connections may be exploited to assist in locomotor rehabilitation after neurotrauma. Specifically, the effect of arm activity on leg neural circuitry has great implications for walking retraining. However, our understanding of the neuromechanics of rhythmic arm movement as well as the neuronal connections between arms and legs active during rhythmic movement is lacking. Greater knowledge on details of interlimb coupling and combined neural and mechanical measurement of rhythmic arm movement are necessary to optimize parameters of interlimb coupling for use in walking rehabilitation. The primary goals of this thesis were to further our understanding of neural interlimb connections during combined arm and leg rhythmic movement and conduct neuromechanical investigations of rhythmic arm movement. First, this thesis developed a method for multiple parameter analysis of the Hoffmanreflex recruitment curve. A sigmoid function was found to be a reliable analysis technique that mimics the physiologically based prediction of the input/output relation of the ascending limb of the recruitment curve. This technique provided a baseline for evaluation of neural interactions between the arms and the legs during rhythmic movement and was utilized during following experiments. Second, the effect of rhythmic leg cycling on reflexes within, and corticospinal projections to, stationary arm muscles was examined. Rhythmic leg cycling significantly suppressed H-reflexes in forearm muscles. Additionally, sub-threshold transcranial magnetic stimulation (TMS) facilitation of H-reflexes was similar during leg cycling as during static contraction suggesting a considerable sub-cortical component. These results supports the hypothesis of a loose, but significant, neural coupling between the arms and the legs during rhythmic movement. Third, we used a reduced walking model of combined arm and leg cycling to examine the separate and combined effects of rhythmic arm and leg movement on the modulation of lower limb H-reflexes with and without stimulating a nerve innervating the hand. The suppressive effect of arm movement was less than that for leg movement and combined arm and leg rhythmic movement, which were generally equivalent. For H-reflexes conditioned by cutaneous input to the hand, amplitudes during combined arm and leg movement instead were in between those for modulation produced by arm movement and leg movement alone. Further a significant contribution for arm movement was revealed only in trials when hand stimulation was used to condition H-reflex amplitudes. Therefore a measurable interaction between neural activity regulating arm and leg movement during locomotion is specifically enhanced when cutaneous input from the hand is present. Fourth, we explored interlimb interactions during a locomotor-like, 3 limb stepping paradigm involving movement of both arms and one leg while eliciting an H-reflex in the stationary test limb. The conditioning effect of contralateral leg movement, bilateral arm movement, and combined bilateral arm and contralateral leg movement on H-reflex amplitude was evaluated at different phases across all tasks. Significant interactions between arm and leg activity could be revealed using the 3-limb paradigm. Further, across phases we observed differential suppressive effects of separate and combined arm and leg movement suggesting phase dependent contributions of arm and leg activity to overall 3-limb suppression. These results support the role of the arms in modulating activity in the legs during human locomotor tasks. Fifth, the mechanical effects of stimulating a cutaneous nerve innervating the dorsum of the hand during arm cycling were quantified. The results show that mechanical responses to cutaneous stimulation of the hand during arm cycling are related to the task and phase and consistent with the anatomical location of the stimulus (local sign). Therefore, these responses are comparable to functionally relevant responses in the legs during lower limb rhythmic movement. However, unlike the responses in the lower limbs, the mechanical responses cannot be easily described in the neuromechanical context of arm cycling. Therefore we suggest that the superimposed task constraints and control variable of arm cycling limit the kinematic reflex expression and make it difficult to decipher the true functional role of the reflexes. Overall, these results provide evidence for mechanical correlates to neural responses during arm cycling and further support parallels between the neural regulation of arm and leg rhythmic movement. Sixth, a combined neural and mechanical measurement approach was used to compare three rhythmic arm movement tasks: arm cycling; arm swing while standing; and arm swing while treadmill walking. The results highlight important neural and mechanical features that distinguish differences between tasks. Overall, differences in neural control between tasks (i.e., pattern of muscle activity) reflected changes in the mechanical constraints unique to each task while the results are consistent with conserved common central motor control mechanisms operational for arm cycling, arm swing while walking, and arm swing alone yet appropriately sculpted to demands unique to each task. Taken together the data in this thesis suggest that, in addition to understanding details of neural interlimb coupling, mechanical considerations may play an important role in the coordination of locomotor movements. Additionally, the use of rhythmic arm movement as a locomotor adjunct in rehabilitation is revealed through combined neural and mechanical measurement.

This thesis examines the marking of Roman glass and ceramic vessels with stamps in the period from the first century B.C. through the second century A.D. The thesis establishes the context for the study of such makers' marks by first examining the early history of Roman glass. the changes brought on by the introduction of glassblowing, and the organization and working conditions of the industry. Next the thesis examines the roles played by stamps on glass in the ancient world. Then the organization and conditions of the ceramics industry are examined and the same questions are posed regarding the roles of stamps and what they can tell us. These stamps show us how the two industries were organized and reveal that Roman makers' marks served not only as proto-brand identifiers and artists signatures, but also as tools for industrial organization.