African trypanosomes are protozoan parasites that cause African trypanosomiasis, diseases that affect humans and their livestock. Not only has trypanosomiasis had an overwhelming effect on the development of tropical Africa in the past, but it also constitutes one of the most significant present economic problems of the continent. Trypanosomes alternate between a mammalian host and a tsetse vector using a complex life cycle. In the mammalian host the trypanosomes live as bloodstream forms (BSFs) that are so proficient at antigenic variation, and thus host immune system evasion, that no suitable vaccine candidates have yet been identified. In contrast, the lifecycle stages that exist in the tsetse vector do not undergo antigenic variation. This potentially makes the vector-occupying trypanosomes much better targets for control if strategies can be devised to disrupt their lifecycle in the vector or to interfere with their transmission to mammalian hosts. The primary impediment to developing strategies for disruption of trypanosome life cycles in tsetse is a lack of understanding of the molecular basis of trypanosome-tsetse interactions. Although several major surface molecules have been identified on insect form trypanosomes, these have not been well studied due to a lack of appropriate antibody probes and to the difficulty in obtaining sufficient quantities of the different parasite life cycle stages required for such molecular studies. My thesis research was focused on developing and using monoclonal antibody probes for analysis of expression of major surface molecules of Trypanosoma congolense, a serious pathogen of cattle in Africa. I used this species of trypanosome since in addition to being a socioeconomically important parasite, all four of its major life cycle stages can be grown in vitro in amounts sufficient for immunochemical analysis. I successfully derived and characterized monoclonal antibodies that were useful for detecting the three major surface proteins of T. congolense insect forms: glutamic acid/alanine rich protein (GARP), the T. congolense heptapeptide repeat protein (TcHRP) and congolense epimastogote specific protein (CESP). Selected monoclonal antibody probes were then employed for expression analysis of these molecules throughout the parasite life cycle using in vitro grown trypanosomes and parasites taken directly from infected tsetse. In addition, I determined the peptide epitopes for two of my GARP-specific monoclonal antibodies and in collaboration with Dr. Martin Boulanger and Jeremy Mason was able to localize the epitopes on a high resolution three-dimensional structure obtained by X-ray crystallography. This allowed us to derive a model that describes the orientation of GARP in the trypanosome surface membrane and explains the possible structure-function relationships involved in replacement of the bloodstream form variant surface glycoprotein (VSG) by GARP as trypanosomes differentiate in the tsetse vector after a bloodmeal.

Dyestuffs are widely used industrial chemicals, yet surprisingly little is known about their fate in the environment. The potential modes of transformation and removal of reactive dyes in treatment and in the environment are principally through anaerobic and aerobic biodegradation and photodegradation. The research herein describes the use of LC-MS analysis with laboratory simulations to develop a better understanding of the occurrence and fate of reactive dyes and their degradation products in the aquatic environment. One reason for the lack of information on the environmental fate of reactive dyes has been the paucity of robust analytical methods suitable for the determination of dyes in aqueous samples. Robust analytical methods were optimised to provide LC-MS and MSMS identification of degradation products. Additionally, interpretation of the MSMS spectra of known reactive dyes provided novel characteristic fragment ions indicative of the triazine reactive group of reactive dyes . Fibre reactive dyes are designed to have a degree of photostability and therefore their photodegradation behaviour has not been widely investigated. Little is known of their stability to daylight over prolonged periods of irradiation in dilute aqueous solutions and in the presence of humic substances. The kinetics of photodegradation of an anthraquinone dye (Reactive Blue H4R) and azo dye (Reactive Yellow P5G) were evaluated. The former underwent rapid and extensive degradation 01/2 1.5 h). The major products formed were identified using LC-MSMS and a photodegradation pathway proposed. By comparison, the photodegradation of the azo dye was significantly slower, 01/2 30 h). The addition of humic substancesa ppearedt o have little effect on the rate of photodegradationu nder the conditions used. The reduction of azo dyes under anaerobic treatment has been extensively studied, but the subsequent fate of the initial reduction products when exposed to air are not understood. Three relatively simple azo dyes, Amaranth, Sunset Yellow and Naphthol Blue-Black, were reduced and their autoxidation products identified by LC-MS. These were subsequently used to predict the autoxidation products of a more complex azo reactive dye: Reactive Red 3.1. Additionally, a persistent degradation product from the anaerobicaerobic treatment of Reactive Red 3.1 was identified from LC-MS data. Azo reactive dyes are generally regarded as being resistant to aerobic degradation and there are few published data regarding degradation pathways for reactive anthraquinone dyes. Pure cultures of Pseudomonas docunhae, A 9046 and A texaco and mixed bacterial consortia (semi-continuous activated sludge, SCAS) aerobic degradation of azo and anthraquinone reactive dyes was studied. Two azo dyes were degraded by pure cultures of A docunhae and A 9046, suggesting that azo dyes can be aerobically degraded given favourable conditions. The antraquinone dye was extensively degraded by SCAS and pure culture biodegradation. Metabolites were identified by LC-MS and a degradation pathway proposed.

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).

DNA mismatch repair (MMR) is an essential part of genomic stability, guarding the integrity of the genome in virtually all cells. MMR corrects mismatched bases in DNA and is one of several DNA repair pathways conserved from bacteria to humans. In Escherichia coli, MutS and MutL are the key proteins in MMR. In prokaryotes, the MMR proteins function as homodimers whereas in eukaryotes the MMR proteins come together as heterodimers. In the absence of MMR there is a great increase in mutation frequency and a higher susceptibility to cancer in mammals. Previous studies in our laboratory have identified five MutS homologs and two MutL homologs in the single-celled ciliated protozoan, Tetrahymena thermophila. MMR repair has been extensively studied in E. coli but less is known in eukaryotes. T. thermophila 's biology and the recent sequencing of its genome make it an attractive eukaryotic research model. In this study, poly-histidine tagged MMR genes from T. thermophila and human thymine DNA glycosylase (TDG) were cloned into two different types of T thermophila expression plasmid. The integrated homologously recombinational T. thermophila vector approach and the espisomal rDNA vector approach which utilized Gateway® technology a cloning method based on the bacteriophage lambda site-specific recombination system. The homologous vector approach relies on mutant strains of T. thermophila harboring a negatively selectable allele of a P-tubulin gene producing sensitivity to paclitaxol. Upon knocking out the mutant J3-tubulin with the gene of interest the resistance of the Tetrahymena strain is then resorted and selected for. The various T. thermophila expression plasmids and DNA transformations techniques to follow such as biolistic bombardment and conjugated electroporation were carried out in order to optimize the technical aspect of working with T. thermophila. and finally lead to the purification of histidine tagged T. thermophila MMR proteins for antibody production and further studies. This study will lead to insight into the inner workings of DNA mismatch repair and technical aspect of working with the organism and will permit functional and structural studies of the MMR homolog proteins in T. thermophila.

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 discovery and mechanistic investigation of a new class of photochemical reactions of benzophenones and related compounds is documented in this Thesis. Their photobehaviour in aqueous solvent media varied dramatically from their well-known behaviour in organic solvents and suggests unique and unprecedented mechanistic pathways. The aqueous photoredox chemistry of various substituted benzophenones was initially explored. Particular attention was paid to 3-(hydroxymethyl)benzophenone (47), which upon photolysis in acidic aqueous media undergoes an intramolecular photoredox reaction to produce 3-formylbenzhydrol (61). Extensive investigation into the mechanistic behaviour of 3-(hydroxymethyl)benzophenone (47) produced evidence of a unique solvent-mediated, acid catalysed photoreaction. A mechanism has been proposed for the intramolecular photoredox reaction that proceeds via the protonated triplet state. This protonated triplet state subsequently promotes the deprotonation of the benzylic carbon before rearranging to form the redox product. The modification of the benzylic carbon with an alkyl group or with a phenyl group resulted in only slight changes in the photobehaviour. In both cases intramolecular photoredox reactions were observed although significantly more oligomeric side products were observed in some cases. To more fully elucidate the photobehaviour and to test the generality of the photoredox reaction, a variety of structurally related hydroxyalkyl aromatic carbonyls were synthesized and studied. Alternative chromophores were explored using xanthone and fluorenone derivatives. Both types of derivative compounds underwent an intramolecular photoredox reaction, supporting the assertion that the intramolecular photoredox reaction could be considered a general feature of aromatic carbonyls under aqueous conditions. However, significant differences in photoreactivity were also observed. It was found that 2-(hydroxymethyl)xanthone (53) exhibited sufficient photoactivity that the intramolecular photoredox reaction was observable even under neutral conditions whereas 2-(hydroxymethyl)fluorenone (54) was nearly photoinert. The last topic focuses on the extension of the electronic transmission from the carbonyl functional group to the benzylic alcohol by insertion of an additional phenyl group. The addition of the phenyl group also provided a bichromophoric molecule, rather than the monochromophoric substrates studied to this point. The substituent’s position played an important role in the photobehaviour, in that both of the meta- and ortho- substituted compounds underwent intramolecular photoredox reaction, while the para- substituted compound primarily exhibited photobehaviour indicative of hydrogen abstraction.

It has been frequently postulated that genome or domain-wide histone post-translational modifications induce structural changes to chromatin. Until recently, however, experimental evidence for this hypothesis was lacking. H4 K16 acetylation is the first, and only, of all of the possible post-translational modifications to be directly linked to changes in chromatin conformation. I was interested in clarifying the mechanism by which H4 K16Ac exerts its modulatory effect. To characterize the role of this modification I reconstituted mononucleosomes with H4 K16Ac isolated from chicken erythrocytes by weak cation-exchange HPLC. Analytical ultracentrifuge (AUC) and MgCI2 solubility data suggested that H4 K16Ac structurally relaxes the association of nucleosomal DNA with the histone octamer by weakening intra-nucleosomal DNA-histone electrostatic interaction. Similar to early studies on H4 K16 acetylation, evidence suggests that the phosphorylation of linker histones promotes chromatin decondensation and increases DNA accessibility. In an effort to initiate a study characterizing the structural effects of chromatin fibers containing phosphorylated linker histories, I have optimized a method of purification for nonphosphorylated and monophosphorylated linker histone H5 from erythrocytes of anemic chickens. Preliminary AUC data of oligonucleosomes containing the nonphosphorylated control linker histones (manipulated to the same degree as the experimental monophosphorylated linker histones) showed a salt-dependent sedimentation trend that was consistent with expected values at low ionic concentrations. The purification method described is valuable as it results in a high yield of pure post¬translationally modified H5 product suitable for oligonucleosome reconstitutions. In the third component of this thesis, I describe the creation of a DNA template, Pbsn-208(10), which contains a Rattus norvegicus probasin promoter nucleosome positioning sequence flanked on both sides by 5 tandem repeats of the Lytechinus variegatus nucleosome positioning sequence. Unlike other DNA templates used in the study of chromatin compaction in vitro, the Pbsn-208(10) allows not only the reconstitution of homogenous chromatin fibers but also the differentiation of the middle of the fiber from its flanking ends. Thus, the effects of PTMs on histone-DNA interactions and nucleosome conformation in the center of a chromatin fiber can now be easily investigated. In addition, the Pbsn-208(10) template can help to identify the precise location of H1/H5 within the nucleosome.

The WTI gene encodes for a transcription factor which is mutated in approximately 15% of sporadic Wilms' tumors (Orkin et al., 1984; Fearon et al., 1990). Mutations in WTI are also associated with a number of clinical disorders such as Denys-Drash and Frasier syndromes. which are distinguished by genitourinary malformation and kidney disease (McTaggart et al., 2001). The nucleic acid binding domain of WTI is comprised of four tandemly arranged C2H2 type zinc fingers (Haber et al., 1990; Rauscher et al.. 1990; Morris et al., 1991). The WT1 transcript is regulated by two different alternative splicing events. The first alternative splice introduces 17 amino acids between the proline-rich amino terminus and the zinc finger domain (Haber et al.. 1991). The second alternative splice inserts the amino acids KTS between zinc fingers 3 and 4 (Haber et al., 1991). The +KTS and -KTS isoforms of WT1 have different nucleic acid binding specificities. The .-KTS isoform readily binds to specific sequences in both DNA and RNA. while the +KTS isoform only binds to specific sequences in RNA (Zhai et al., 2001). The RNA binding capabilities of the +KTS variant combined with its presence in spliceosomes (Davies et al.. 1998) and nuclear poly(A)+ ribonucleoprotein (Ladomery et at., 1999), suggests that it is involved in RNA metabolism. WT1 has been shown to interact with RNA through the zinc finger domain (Caricasole et al, 1996), but as of yet. there is not a purified crystal structure of WT l interacting with RNA. There have been many experiments that have attempted to decipher the mechanism for WTI RNA binding (Caricasole et al, 1996; Bardeesy and Pelletier, 1998). One zinc finger knock-out experiment has proposed that it is zinc finger number 1 which plays the most important role in this RNA interaction (Caricasole et at, 1996) while another suggests that it is finger 4 (Bardeesy and Pelletier, 1998). In order to determine which zinc finger of WT 1 is the most crucial for RNA binding, WT 1 swap mutants and deletion mutants were created by PCR and a filter binding assay was employed to determine dissociation binding constants of the mutant proteins with the WT1-specific RNA aptamer Pe122. The mutant W12P8W4, which has finger 3 of WTI replaced with finger 8 of the zinc finger protein p43, demonstrated a relative affinity for Pe122 of less than 0.29. The deletion mutant WTlAF4 which had finger 4 of WTI deleted showed no affinity for Pe122 RNA, while the mutant Wp4 which had finger 4 of WTI replaced with finger 9 of p43 demonstrated a 10 times greater affinity for Pe122 than wild type WT1. These results suggest that finger 3 makes important residue-base contacts with the RNA and that perhaps finger 4 confers some sort of stability to the WT1-RNA complex.

Can one classify biochemical pathways based on their topology? What is the topology of a biochemical pathway? What are the fundamental principles underlying different biochemical pathways involved in similar functional areas? Will one be able to characterize pathway "motifs" similar to motifs in proteins - i.e. reoccurring patterns in pathways? This thesis describes an attempt to develop a quantitative framework for the general representation and comparison of biochemical pathways. This quantitative framework involves a mathematical model to represent biochemical pathways and a set of similarity criteria to compare these biochemical pathways. We anticipate that such a tool would allow biologists to answer important questions such as the ones mentioned above.

In order to elucidate the molecular and biochemical events occurring in embryogenesis in Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco), an essential gene expressed highly during early embryogenesis was identified, cloned and further characterized in this study. Douglas-fir LYR/LVR eDNA was obtained using RT-PCR with specific primers. followed by cloning and sequencing. Northern blot analysis showed higher amounts of LYR/LVR transcripts in early-cotyledonary embryonic stages and megagametophytes when compared with mid- and late-cotyledonary embryos. LYR/LVR transcript levels declined in seeds (mature embryos) and seedlings. Differential regulation of LYR/LVR gene expression with response to brassinosteroid treatment of Douglas-fir seeds was studied. LYR/LVR mRNA showed higher accumulation in seeds treated with different concentrations of brassinosteroids. Bioinformatic analysis showed that Douglas-fir LYR/LVR protein may be an essential inner mitochondrial protein, NADH oxidoreductase necessary for energy production. The phylogenetic tree analysis was used to investigate the evolutionary relationship of the newly identified Douglas-fir LYR/LVR protein with closely related proteins (LYR family) in different organisms. InterPro, UniProt and Pfam results showed the sequence similarity of Douglas-fir LYR/LVR protein with other related members in Arabidopsis thaliana and Oryza sativa, indicating that the LYR complex contains short stretches of closely related proteins that are essential for energy production. Amino acids 19-90 in the LYR/LVR protein were highly conserved and is likely the functional LYR motif necessary for oxidoreductase activity.