Theileria parva is an intracellular protozoan parasite and the causative agent of the lethal livestock disease East Coast fever (ECF). Research has shown that a protective cell-mediated immune response against parasite-infected lymphocytes is capable of clearing the host of T. parva (Pearson et al. 1979), leaving the host solidly immune to reinfection. The work presented in this thesis describes my attempts to develop a method for identification of major histocompatibility complex class I-associated T. parva peptides involved in eliciting this protective cell-mediated immune response. The soluble chaperone gp96 interacts with peptides destined for association with major histocompatibily complex class I molecules and is therefore a source of T. parva peptides that interact with extracellular immune effectors. Using sensitive mass spectrometry methods the gp96-chaperoned peptide proteome from model parasite infected T lymphocytes was compared to an uninfected T cell line. With our findings we have demonstrated proof of concept for a highly sensitive method for the elucidation of potentially immunogenic peptides capable of initiating a protective immune response against the intracellular parasite T parva.

Cationic Antimicrobial Peptides (CAPs) exhibit broad-spectrum activity against a variety of microbial pathogens at concentrations that are non-toxic to higher eukaryotes. These properties make them excellent candidates for both pharmaceutical and agricultural applications. Here BMAP18, a CAP with an especially high charge to mass ratio, is evaluated for efficient production of the peptide and enhancement of disease resistance in transgenic potato. In vitro analyses indicated that BMAP18 had potent activity against a variety of clinically and agriculturally relevant pathogens, including the protozoan parasite Trypanosoma brucei. In planta activity was assessed by transformation of potato (Solanum tuberosum L.) plants with a synthetic BMAP18 gene under control of the enhanced CaMV 35S promoter or the Douglas-fir lumina] Binding Protein (PmBiP) promoter. Stable transformants expressing the transgene were shown to accumulate the peptide and exhibited enhanced resistance to Fusarium wilt and bacterial soft rot caused by Erwinia caratovora.

Here we report vertical profiles of dissolved (0.2 um filtered) Aluminum (Al) for eight stations in the Beaufort Sea in the Canadian Arctic, six of which are along a transect extending from the coastal shelf northeast of the Mackenzie River delta out to the Beaufort Sea. Sampling was performed aboard the CCGS Sir Wilfrid Laurier in September 2007 and all analyses were performed in a Class 100 clean space at the University of Victoria. Vertical profiles of dissolved Al in the water column displayed surface maxima, subsurface minima and a general increase in concentration with depth as is characteristic of a “scavenged” trace element in seawater. Concentrations of dissolved Al for the upper 1000 m were generally low ranging from the < 1 nmol kg-1 observed in the sub-surface minimum corresponding to the Pacific inflow layer and increasing to ~6-10 nmol kg-1 with depth. The surface maxima at stations for Al was associated with relatively fresh surface water (26-30 PSS) that is believed to be the result of seasonal sea-ice melt. This correlation suggests that the melting of sea-ice with entrained sediments may be an important mechanism for the delivery of Al and associated trace metals to the water column of the Beaufort Sea. We also report measurements of “total Al” (unfiltered and acidified to pH 1.7 for two year prior to analysis) for the Arctic Ocean which indicate that a significant proportion of Al in the water column is present in the > 0.2 um fraction. These measurements and the hydrographic data along the transect indicate the transport of a cold, saline, metal enriched water mass off of the continental shelf into the Canada Basin. This water mass appears to reflect the return of cold, high salinity slope water originally emplaced on the shelf by upwelling favourable winds. Alternatively, this water mass may be the product of brine exclusion from sea-ice formation during the previous winter that was unable to vacate the shelf due to the persistent upwelling observed throughout 2007. This finding suggests that the convection of cold, dense shelf water may be a mechanism for supplying the deep waters of the Arctic with an injection of water containing a significant Al content. In addition, a low volume sequential injection analysis (SIA) method is proposed for determination of elevated concentrations of Al, like those typically observed in coastal and river waters. A thorough optimization of the chemistry and instrumental parameters was performed along with an extensive investigation into potential interferents. The method was found to be largely free of interferents at environmentally relevant concentrations and was determined to have a detection limit of 24 nM. The precision of the method was reported to be 2% at 75 nmol kg-1 and analysis of the SLRS-4 certified reference material validated the accuracy of the method. Analysis of several samples that were previously analyzed via flow injection analysis (FIA) and standardized with consensus values of an open ocean reference material indicated the method returned comparable values for the Al concentration in the samples. Development and optimization of the SIA has resulted in an accurate and precise low-cost method of analysis that is both sensitive and relatively free from interference for the detection of nano-molar levels of Al in coastal and natural waters.

The fundamental subunit of chromatin, known as a nucleosome, is comprised of DNA wrapped around two H2A-H2B dimers and one H3-H4 tetramer. This structure perpetuates itself and together with linker histones (histone H1) give rise to the chromatin fibre. This causes compaction of DNA within the nucleus of a eukaryotic cell, which have inhibitory effects in terms of both its accessibility and metabolism. By modifying chromatin structure, cells can regulate and fine-tune different cellular processes, such as DNA repair, replication, transcription and spermatogenesis. Chromatin structure can be modulated by three main mechanisms: covalent post-translational modification of histone tails, incorporation of histone variants and recruitment of chromatin remodelling complexes. In this thesis, the contribution of histone variants and their post-translational modifications to chromatin structure will be discussed. In Chapter 1, I review the role of H2A.X in DNA double stranded break (DSB) repair and other less studied cellular processes, such as transcription and cell cycle. In addition, this chapter also introduces a putative model for the role of H2A.X phosphorylation in DNA DSB repair. In Chapter 2, our results demonstrate that S139 and T136 of H2A.X are both phosphorylated during DNA DSB repair. These two post-translational modifications are functionally different in that S139E and T136A/S139E mutants partition to different chromatin fractions. Furthermore, we show that nucleosomes containing H2A.X are less stable compared to nucleosomes with canonical H2A. The destabilizing effect is more prominent in the nucleosomes containing H2A.X phosphorylated by DNA-dependent protein kinase suggesting that the post-translational modifications of histone variants and histone variant itself have a direct structural role in chromatin integrity. Recombinantly expressed H2A.Bbd has also been shown to modify chromatin structure by destabilizing nucleosomes in a way that resembles that of H2A.X. However, the native form of this H2A.Bbd has never been identified in vivo. Chapter 3 provides evidence for the presence of native H2A.Bbd in mammalian testis and human sperm. Histone variant hTSH2B, which was found in only a fraction of mature human sperm, has been characterized most recently. In Chapter 4, we report the structural characterization of this variant in the context of other core histones (histone octamer) and in a nucleosome. Although an hTSH2B-containing nucleosome did not show structural alterations compared to its canonical counterpart, hTSH2B octamers were shown to be less stable. Finally, we addressed the disagreement in the literature as to whether or not H1t, a linker histone variant specific to mammalian testis, is phosphorylated during spermatogenesis. Chapter 5 shows that native H1t is phosphorylated. The sites of phosphorylation of H1t were determined. The phosphorylation of histone H1 at the C-terminal domain has been shown to significantly weaken its affinity for the chromatin fibre thus inferences chromatin structure. It is not surprising that the newly identified phosphorylation sites of H1t within this region also serve similar function. The four histone variants analyzed in this thesis: H2A.X, H2A.Bbd, hTSH2B and H1t, are all expressed in mammalian germ cells and hence play an important role in spermiogenesis. Their structural contribution may help explain some of the complex chromatin transitions involved in this multifaceted cell differentiation process.

This Thesis aims to examine the effects of substituents on the adjacent proton accepting phenyl ring with respect to a new type of excited state intramolecular proton transfer (ESIPT) process discovered by Wan and co-workers. Therefore, a number of 2-phenylphenols 23-28 were synthesized with electron-donor and electron-acceptor substituents such as methyl, methoxy, and ketone moieties on the adjacent proton accepting phenyl ring. The results obtained from examination of photochemical deuterium exchange showed that all derivatives except for ketone 27 underwent deuterium exchange (Фex = 0.019 - 0.079), primarily at the 2’-position on photolysis in D2O-CH3CN. In general, compounds with methoxy moiety (ies) on the adjacent proton accepting ring showed higher deuterium exchange yields. Diol 28 has the potential to undergo photosolvolysis as well as ESIPT process since it has both a benzyl alcohol and a phenol chromophore on the same molecule. Irradiation of 28 in 1:1 H2O-CH3OH gave the corresponding methyl ether product in high yield. Photolysis of 28 in 1:1 D2O-CH3OH also showed that ESIPT competes very well with photosolvolysis. Thus, this work has established that ESIPT can compete efficiently with photosolvolysis. Semi-empirical AM1 (examination of HOMOs and LUMOs) calculations show a large degree of charge transfer in the electronic excited state (except 27), from the phenol ring to the attached phenyl ring of the studied compounds. The AM1 calculation for ketone 27 showed that the carbonyl oxygen is more basic than the carbon atoms of the benzene ring, which explains the lack of deuterium exchange observed for 27.

Functional and temporal-spatial studies of Netrin and its receptors have been reported in several species including, M. musculus, D. melanogaster and C. elegans. These studies indicate that Netrins are a family of evolutionarily conserved, secreted proteins that function to elicit the extension and turning responses of axons. Here, I describe the sequences for netrin and its receptors, unc5 and neogenin, in Strongylocentrotus purpuratus and show that the larval nervous system is patterned predictably with respect to cell body and axon location, early in its development. These findings led to a tentative hypothesis that Sp-Netrin functions to guide axonal growth in the larval nervous system. Quantitative PCR indicates that Sp-netrin and Sp-unc5 are expressed prior to neurogenesis, whereas Sp-neogenin is expressed close to the stage at which neurons differentiate. A polyclonal antibody to Sp-Netrin and in situ hybridizations reveal that Sp-Netrin is initially expressed in the vegetal plate, the archenteron and the protein is present on the basal surface of the oral ectoderm in early prism stage embryos. Suppression of Netrin expression, with a morpholino antisense oligonucleotide, results in loss of neurons, loss of ciliary band cells and loss of the oralectoderm markers, Chordin and Goosecoid. These findings suggest that Netrin is responsible for maintaining or differentiating oral and ciliary band ectoderm, which is necessary for neural specification or differentiation. Further study of this model is necessary to determine if Sp-Netrin retains a role in axon guidance.

The aqueous photochemistry of 4-hydroxy-3,5-dimethoxybenzoic acid (syringic acid) has been studied as a model humic substance in order to better understand the reactions that compounds of this type undergo in the natural environment. Syringic acid was chosen since it has been identified as a component of humic substances in the environment and bears many of chemical moieties found in structures of this type. In addition, there has been speculation that humic substances are responsible for some of the production of halomethanes that are released into the environment. Photolysis of these compounds in marine and estuarine waters may be responsible for the release of halomethanes which are known stratospheric ozone depleters. Photochemical product studies of syringic acid and related compounds along with UV-Vis spectrometry, laser flash photolysis and membrane introduction mass spectrometry were carried out in aqueous solutions to study its photochemical transformations. Syringic acid was found to form methanol at a 0.01 quantum yield upon its photolysis in basic solution. Other major photoproducts included 3-methoxygallic acid and 3,5-dimethoxybenzoic acid. Chloromethane was identified as a minor photoproduct in chloride enriched solution by following its production via membrane introduction mass spectrometry. The proposed mechanism for the formation of these photoproducts involves an initial photoprotonation of the benzene ring, resulting in a carbocation that can facilitate the nucleophilic attack by water or chloride, to produce methanol or chloromethane, respectively. The formation of 3,5-dimethoxybenzoic acid is via a novel pathway that involves the loss of the hydroxy group from the aromatic ring after the photoprotonation.

The goal of this thesis was to develop a method for efficiently synthesizing a large suite of asymmetric oligoester ion channel-forming compounds. A solid-phase organic synthesis (SPOS) approach on Wang resin was used to generate the ion channel candidates. A follow-on goal is to survey the compounds produced to uncover structure-related controls on ion transport activity. Two classes of building blocks were used to generate the oligoesters – head groups and cores. The core building blocks were three omega-hydroxy acid derivatives six, eight and twelve carbons in length and the alcohol protected as a tetrahydropyranyl ether. The head group building blocks were either a glutaric acid monoester derivative of varying lipophilicity (12 to 16 carbon long alkyl tail) or a beta-hydroxy acid derivative; these building blocks used a tert-butyldimethylsilyl ether for alcohol protection. Optimized conditions for building block coupling, deprotection, and product cleavage were first established by the generation of dimeric and trimeric products. The building blocks were coupled using diisopropylcarbodiimide/ dimethylaminopyridine conditions. The deprotection of the tetrahydropyranyl ether group from the alcohol used a dilute acid solution in methanol and dichloromethane. A fluoride solution (from tetrabutylammonium fluoride) in tetrahydrofuran was used to deprotect the tert-butyldimethylsilyl ether group. Cleavage of the product synthesized on Wang resin was achieved by treatment with a trifluoroacetic acid/dichloromethane or ethereal hydrogen chloride solution. The products were then isolated by gel filtration. Mass spectrometry was used to identify the minor impurities which were quantified by proton nuclear magnetic resonance integrations. With the nine building blocks, many tetrameric and pentameric structures can be made, but a directed-library approach was used to address structure-activity related questions. Three pentameric oligoester products were the largest products synthesized to determine the scope and limitations of the SPOS methodology. The oligoester ion channel candidates were tested for ion transport activity using a 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt fluorescence vesicle assay. For each compound a pseudo-first order rate constant was derived at a particular concentration. A more useful normalized rate constant was calculated for an interpolated transporter concentration which allowed for transport activity comparison between compounds. The results from the fluorescence assay showed that some compounds and some isomers were substantially more active than others. There appeared to be an optimal core length and lipophilicity for relatively high activity. The aggregation of the compounds in buffer solution was probed using a pyrene fluorescence experiment. The solid-phase methodology was extended to include coupling of amino acids. A tryptophan derivative was made from one of the most active SPOS oligoester ion channel-forming compounds. The integrity of the molecules synthesized by SPOS which contain the tryptophan group could then be determined by high performance liquid chromatography. The fluorescence of the indole is quenched by acrylamide. By first equilibrating the vesicles with the tryptophan-containing oligoesters and then adding a fluorescence quencher, the resulting indole fluorescence was monitored as a function of quencher concentration. A Stern-Volmer plot was derived based on the quenching data which reported the possible orientations of the tryptophan-containing oligoester within the vesicle.

The Electrochemical Quartz Crystal Microbalance (EQCM) was used as the main investigation tool coupled with other conventional electrochemical methods to study the electrocatalytic properties of polycrystalline Pt electrodes, including two separate projects. The first project studied the early stage of oxide film formation on the Pt surfaces and the inhibition of the catalytic properties by the oxide film. The inhibition of the fast electrode reaction of small molecules by the growth of oxide film allows those molecules to be used as probes for the nature of the oxide film. The hydrogen oxidation current, jox calculated by differencing the cyclic voltammetry currents with and without H₂ present showed a characteristic plateau-to-plateau profile, which implies a transition from the free Pt surface to the Pt surface completely covered by oxide film. This method allows determination of the onset potential for oxide formation and also the critical potential where a full monolayer of oxide is formed. This method applies to other fast surface reactions such as oxygen reduction reaction (ORR), and the results are enhanced by forced convection in the rotating disk electrode (RDE) experiments. The initial oxidation species was identified by charge and EQCM frequency analysis. Our results support the formation of a species with stoichiometry Pt₂O, for example, with an oxygen atom in the bridging position between two adjacent Pt atoms. In the second project, the stability of the Pt electrodes in acid media with Ag⁺ present was investigated. A substantial frequency drift (8.3 Hz cycle⁻¹, or 44 ng cm⁻² cycle⁻¹) was observed during Ag electrodeposition and stripping on the bare polycrystalline Pt surface. Cyclic voltammograms in pure HClO₄ solution showed nearly no frequency drift while the addition of 10⁻³ mol L⁻¹ Ag⁺ resulted in an immediate and characteristic frequency drift. The frequency drift appeared to be consistent with loss of material from the electrode surface and the ICP-MS detected a maximum Pt concentration of 2.3×10⁻⁶ mol L⁻¹ in solution due to Pt dissolution. The Pt concentration calculated from the EQCM frequency drift matched the ICP-MS results. This allowed the EQCM for direct investigation of Pt dissolution at different system temperatures, sweep rates, and potential ranges. The much higher rate of dissolution with Ag present than that in pure HClO₄ solution can be explained by the formation of Pt-Ag alloy during Ag underpotential deposition and the co-dissolution of Pt and Ag.

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.