In order for solid tumors to metastasize, tumor cells must acquire the ability to invade the surrounding tissue and intravasate into blood- or lymph vessels, survive in the circulation and then extravasate at a distant site to form a new tumor. Overexpression of the glycosaminoglycan hyaluronan, and its adhesion receptor CD44, correlate with breast cancer progression. This thesis focuses on the role of hyaluronan in tumor invasion and metastasis. In paper I, we demonstrated that upregulation of the hyaluronan synthesizing enzyme hyaluronan synthase 2 (HAS2) was crucial for transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells. In paper II, we further demonstrated that silencing of HAS2 decreased the invasive behavior of bone-metastasizing breast cancer cells, via upregulation of tissue inhibitor for metalloproteinase 1 (TIMP1), and dephosphorylation of focal adhesion kinase (FAK). During tumorigenesis, stromal cells, such as fibroblasts, play important roles and several growth factors are synthesized, promoting crosstalk between different cell surface receptors. In paper III, we investigated the crosstalk between the hyaluronan receptor CD44 and the receptors for TGFβ and platelet-derived growth factor BB (PDGF-BB) in dermal fibroblasts. We found that the receptors for the three molecules form a ternary complex, and that PDGF-BB can activate the Smad pathway downstream of TGFβRI. Importantly, CD44 negatively modulated the signaling of both PDGF-BB and TGFβ. In paper IV, we studied the process by which breast cancer cells invade blood-vessels and the role of hyaluronan and CD44 in angiogenesis. Importantly, CD44, or the hyaluronan degrading enzyme hyaluronidase 2 (HYAL2), decreased the capacity of endothelial cells to form tubes in a 3D in vivo-like assay.  Collectively, our studies add to the understanding of the role of hyaluronan in tumor progression.

Starch is an important component of a wheat grain, comprising 50-70% of its dry weight. Its biosynthesis involves a complex pathway mediated by several enzymes, each of which is encoded by genes that have more than one family member. To better understand starch synthesis in wheat grains, this study characterized the sucrose-starch metabolic pathway using physiological, molecular, biochemical and metabolic approaches. These analyses led to the identification of genes that appear to have predominant expression during grain development in wheat including, TaSUT1, TaSuSy2, AGPL1, SSI, SSIIIa and SBEIIa, suggesting that these genes play a regulatory role in starch accumulation. This was further confirmed by comparative analyses of starch synthesis between cultivars with contrasting thousand kernel weights, which revealed a closer association of the expression of the same set of genes with starch accumulation in developing wheat grains. The effect on starch yield of one of the candidate genes identified, AGPase, was examined through a transgenic approach, which involved expression of a gene encoding modified version of maize AGPase large subunit, designated as Sh2r6hs, in wheat under the control of maize’s constitutive Ubiquitin1 promoter. This manipulation of the wheat AGPase activity produced wheat lines with increased AGPase activity, grain weight and grain starch level, suggesting that the wheat grain size can be enhanced through increasing the capacity of starch synthesis both in the source and sink tissues. The study also identified and characterized a partial fragment of wheat rbcS promoter, and indicated that the promoter fragment can potentially be used as a tool for targeting the expression of genes of interest in photosynthetic source tissues. / October 2014

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of L-lysine and meso-diaminopimelate biosynthesis, which is the condensation of (S)-aspartate-β-semialdehyde (ASA) and pyruvate into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS has been an attractive antibiotic target because L-lysine and meso-diaminopimelate are cross-linking components between peptidoglycan heteropolysaccharide chains in bacterial cell walls. Studies revealed that mutant auxotrophs for diaminopimelate undergo lysis in the absence of diaminopimelate in the medium; therefore the assumption is that strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and L-lysine biosynthesis in bacteria and would stop or decrease bacterial growth (eventually leading to bacterial death). In this work, the DHDPS inhibitor design is focused on the allosteric site of the enzyme. It was proposed that a compound mimicking binding of two L-lysine molecules at the allosteric site at the enzyme’s dimer-dimer interface would be a more potent inhibitor than the natural allosteric inhibitor of this enzyme, L-lysine. This inhibitor (R,R-bislysine) was synthesized as a racemic mixture, which was then separated with the aid of chiral HPLC. The mechanism of feedback inhibition of DHDPS from Campylobacter jejuni with its natural allosteric modulator, L-lysine, and its synthetic mimic, R,R-bislysine, is studied in detail. It is found that L-lysine is a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. R,R-bislysine is a mixed partial inhibitor with respect to pyruvate and a noncompetitive partial inhibitor with respect to ASA, with an inhibition constant of 200 nM. Kinetic evaluation of each DHDPS mutants (Y110F, H56A, H56N, H59A and H59N) has revealed amino acids responsible for the inhibitory effect of L-lysine, R,R-bislysine, and we have found that R,R-bislysine is a strong submicromolar inhibitor of Y110F, H56A, H56N and H59N.

The influence of body size on metabolic rate, muscle enzyme activities, and the underlying patterns of mRNA for these enzymes were explored in an effort to explain the genetic basis of allometric variation in metabolic enzymes. Two pairs of sister species of centrarchid fishes were studied: black bass (largemouth bass, Micropterus salmoides and smallmouth bass, Micropterus dolomieui), and sunfish (pumpkinseed, Lepomis gibbosus and bluegill, Lepomis macrochirus). The goal was to assess the regulatory basis of both intraspecific and interspecific variation in relation to body size, as well as gain insights into the evolutionary constraints within lineages. Whole animal routine metabolic rate showed scaling coefficients not significantly different from 1, ranging from +0.87 to +0.96. However, there were significant effects of body size on the specific activities of oxidative and glycolytic enzymes. Mass-specific activity of the oxidative enzyme citrate synthase (CS) scaled negatively with body size in each species, with scaling coefficients ranging from -0.15 to -0.19 whereas the glycolytic enzyme pyruvate kinase (PK) showed positive scaling, with scaling coefficients ranging from +0.08 to +0.23. The ratio of mass-specific enzyme activity in PK to CS increased with body size, whereas the ratio of mRNA transcripts of PK to CS was unaffected, suggesting the enzyme relationships were not due simply to transcriptional regulation of both genes. The mass-dependent differences in PK activities were best explained by transcriptional regulation of the muscle PK gene; PK mRNA was a good predictor of PK specific enzyme activity within species and between species. Conversely, CS mRNA did not correlate with CS specific enzyme activities, suggesting post-transcriptional mechanisms may explain the observed inter-specific and intraspecific differences in oxidative enzymes. / Thesis (Master, Biology) -- Queen's University, 2007-10-31 11:55:28.757

The purpose of this project was to determine how Dlc-2 and Rho signaling modulate the ceramide induction of PGP synthase. This induction was studied at the transcriptional, post-transcriptional, and post-translational levels using cell culture, Real-Time RT-PCR, protein purification, phage display, and western blotting techniques. We have demonstrated that the PGP synthase gene is not controlled at the transcriptional level by ceramide and Rho, nor is the mRNA stability of PGP synthase affected. However, ceramide and Rho do seem to exhibit translational or post-translational control over the PGP synthase protein. The relationships between Dlc-2 (and Rho), ceramide, and PGP synthase (and CL) are important to understand. All three are involved in cancer and apoptotic responses. The knowledge gained by the experiments discussed in this thesis will contribute to an understanding of how these proteins and lipids interact. This knowledge may then be used in the future to develop cancer treatments.

Isoflavonoids are a class of plant secondary metabolites which have multiple biological roles in plants as pest feeding deterrents, phytoalexins and signals to rhizobial microbes. Some isoflavonoids, or their breakdown products, are estrogenic when ingested by animals, and pastures with high levels of the isoflavonoid formononetin can cause sterility in ewes. White clover has low levels of isoflavonoids and is susceptible to pests like the clover root weevil. The overall aim of this project was to test whether isoflavonoids could be manipulated in white clover through metabolic engineering. The genes of the key isoflavonoid biosynthesis enzymes have been cloned from a range of legumes and three major genes, chalone reductase (CHR), isoflavone synthase (IFS) and isoflavonoid O-methyltransferase (IOMT), were cloned from white clover in this study. The white clover IFS2_12 gene was expressed in transgenic tobacco. Genistein, an isoflavonoid that is not naturally present in tobacco, was detected in the IFS-expressing tobacco, thus confirming the functionality of the IFS2_12 gene. Tobacco plants were transformed with ANT1, a transcription factor that induces the production of anthocyanins that share precursors with the isoflavonoid biosynthesis pathway. When IFS was expressed in red tobacco leaves, where anthocyanin biosynthesis was occurring, the levels of genistein were greater than in anthocyanin-free green leaves. White clover was transformed to overexpress the cloned IFS2_12 gene and some transformants had greater levels of IFS gene expression, up to 12.9 times the average wild type level. However, these transformants did not produce formononetin levels greater than the wild-type. A gene fusion of alfalfa chalcone isomerase (CHI), which produces the precursors naringenin and liquiritigenin, and soybean IFS, which converts the precursors to genistein and daidzein, respectively, was received from the Noble Foundation. Transgenic white clover plants expressing IFS/CHI were produced using a novel method that also regenerated wild-type clones of the transgenic plants. When compared with their wild-type clones, two IFS/CHI transformants produced higher levels of formononetin, thus supporting the suggestion that isoflavonoid levels can be increased in white clover through overexpression of isoflavonoid biosynthesis genes.

Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.

Acetolactate synthase (ALS) inhibiting herbicides are commonly used to eliminate weeds from mature turfgrasses. Field trials were conducted from 2004-2006, testing ALS herbicides for preemergence and early postemergence activity on newly seeded turfgrasses, using four species: Riviera bermuda, Zenith and Companion zoysia, L- 93 creeping bentgrass, and Poa annua L. Data collected were phytotoxicity and percent turf cover. Bermuda and zoysia herbicides were metsulfuron-methyl (42 g ha-1), trifloxysulfuron (29 g ha-1), flazasulfuron (53 g ha-1), foramsulfuron (30 g ha-1), bispyribac-sodium (112 g ha-1), and rimsulfuron (35 g ha-1). Treatments occurred the day of seeding and two-three weeks after seeding. Flazasulfuron, trifloxysulfuron and bispyribac-sodium caused significant damage in all treatments. Data suggests that bermuda and zoysia are tolerant of seedling treatments of foramsulfuron, rimsulfuron, and metsulfuron-methyl at these rates. Bentgrass and P. annua herbicides were foramsulfuron (15 and 30 g ha-1), siduron (2803 g ha-1), bispyribac-sodium (49 g ha-1), and paclobutrazol (281 g ha-1). Treatments occurred the day of seeding, two and four weeks after seeding. Foramsulfuron at 15 and 30 g ha-1 caused significant damage regardless of when it was applied. Data suggests that bentgrass and P. annua are tolerant of seedling treatments of siduron, paclobutrazol, and bispyribac-sodium at these rates.

Extreme thermophiles and hyperthermophiles are microorganisms capable of growing optimally at 65-79??C and 80??C plus, respectively. Many of the enzymes isolated from them are thermostable, which makes them a potential resource for research and industrial applications. An increasing number of hyper/thermophiles is shown to be able to produce ethanol as an end-metabolite. Despite characterization of many alcohol dehydrogenases (ADHs) with a potential role in the production of ethanol, to date there has been no significant progress in identifying the enzymes responsible for the production of acetaldehyde, which is an intermediate in production of ethanol from pyruvate.<br> Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles is a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus.<br> The bifunctional and TPP-containing POR/PDC enzyme was isolated and characterized from the ethanol-producing hyperthermophilic archaeon Thermococcus guaymasensis (Topt=88??C), as well as the bacteria Thermotoga hypogea (Topt=70??C) and Thermotoga maritima (Topt=80??C). The T. guaymasensis enzyme was purified anaerobically to homogeneity as judged by SDS-PAGE analysis. POR and PDC activities were co-eluted from each of the chromatographic columns, and the ratio of POR to PDC activities remained constant throughout the purification steps. All of the enzyme activities were CoA- and TPP-dependent and highly sensitive toward exposure to air. The apparent kinetic parameters were determined for the main substrates, including pyruvate and CoA for each activity. Since the genome sequence of T. guaymasensis and T. hypogea were not available, sequences of the genes encoding POR were determined via primer walking and inverse PCR.<br> A novel enzyme capable of catalyzing the production of acetaldehyde from pyruvate in hyperthermophiles was also characterized. The enzyme contained TPP and flavin and was expressed as recombinant histidine-tagged protein in the mesophilic host Escherichia coli. The new enzyme was a bifunctional enzyme catalyzing another reaction as the major reaction besides catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde.<br> Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g. Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles used in this study.<br> In conclusion, no commonly-known PDCs was found in hyperthermophiles, but two types of acetaldehyde-producing enzymes were present in various bacterial and archaeal hyperthermophiles. Although the deduced amino acid sequences from different hyperthermophiles are quite similar, the levels of POR and PDC activities appeared to vary significantly between the archaeal and bacterial enzymes, which most likely reflects the different physiological implications of each activity.

The purpose of this project was to determine how Dlc-2 and Rho signaling modulate the ceramide induction of PGP synthase. This induction was studied at the transcriptional, post-transcriptional, and post-translational levels using cell culture, Real-Time RT-PCR, protein purification, phage display, and western blotting techniques. We have demonstrated that the PGP synthase gene is not controlled at the transcriptional level by ceramide and Rho, nor is the mRNA stability of PGP synthase affected. However, ceramide and Rho do seem to exhibit translational or post-translational control over the PGP synthase protein. The relationships between Dlc-2 (and Rho), ceramide, and PGP synthase (and CL) are important to understand. All three are involved in cancer and apoptotic responses. The knowledge gained by the experiments discussed in this thesis will contribute to an understanding of how these proteins and lipids interact. This knowledge may then be used in the future to develop cancer treatments.