Six isoflavones derived from Millettia griffoniana namely, 4’-methoxy-7-O-[(E)-3-methyl-7hydroxymethyl-2,6 octadienyl]isoflavone (7-O-DHF), Griffonianone C (Griff C), 7-O-geranylformononetin (7-O-GF), 3’,4’-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO), Griffonianone E (Griff E), 4’-O-geranylisoliquiritigenin (4-O-GIQ) were tested for potential oestrogenic activities in three different oestrogen receptor alpha (ERα) dependent assays, namely a recombinant yeast assay, a reporter gene assay based on stably transfected MCF-7 cells (MVLN cells) and the induction of alkaline phosphatase in Ishikawa cells. The oestrogenic activities of isoflavones from Millettia griffoniana could be completely suppressed by the pure oestrogen antagonist, fulvestrant. The expression of Ki-67, proliferating cell nuclear antigen (PCNA) and cyclin D1 (CD1) mRNA used as indicator of cell proliferation in MCF-7 cells was assayed. Based on these in vitro results, Griff C was further tested in vivo. The main objective of this part of the work was to study the mechanistic basis of the oestrogenicity Three different doses of Griff C (2, 10, or 20 mg/kg BW) of Griff C in ovariectomised Wistar rats. 17β-oestradiol (E2: 10 µg/kg BW) was used as positive control. They were treated daily for three consecutive days and sacrificed 24 hours after receiving the last dose. The whole uterus was removed and weight. Liver and vena cava fragments were also collected and stored together with uteri in liquid nitrogen for subsequent real-time PCR to evaluate the effects of Griff C on the regulation of some relevant oestrogen–responsive genes in the uterus, the liver and the vena cava. The role of Griff C in apoptosis or in cell survival, through mediation of the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) signaling pathway, was also investigated. Western blot analysis revealed that Griff C slightly increased the phosphorylation of Akt at its serine 473 residue. In this work, oestrogenic properties of the isoflavones derived from Millettia griffoniana are described using reporter gene assays and the oestrogen-inducible alkaline phosphatase Ishikawa model for the first time. These in vitro data were verified in vivo showing the regulation of the expression of various relevant oestrogen-responsive genes by Griff C. The spectrum of its activity was clearly similar to that of 17β-oestradiol on uterine hepatic and vena cava tissues of ovariectomised rats except for the proliferative response. However Griff C remained 100 to 1000 times less effective than oestradiol. These findings confirmed that some of the biological effects attributed to Millettia griffoniana are closely related to oestrogen-mediated action.

Fascioloides magna (the giant liver fluke) originated from North America, is known in the Czech Republic since 1930s. This pathogenic fluke invades mostly cervids, but livestock too. Excretory-secretory products (ES products) contain number of esential biomolecules which are produced by excretory and secretory system of the fluke. These molecules play key role in many biological process during the life cycle not only of fascioloid flukes (e.g. migration in the host tissues, immune evasion and digestion). Due to their antigenic properties they could be also used in immunodiagnostics. Excretory-secretory proteins from adult Fascioloides magna and comparative related species Fasciola hepatica were purified and separated by the basic biochemical methods (1D, 2D electrophoresis, ion-exchange chromatography) and their activity was confirmed by specific (fluorogenic peptide) and nonspecific (gelatine) substrates. By using the mass spectrometry methods (MALDI TOF/TOF), the most abundant peptidolytically active proteins from ES products of F. magna were identified as cathepsin L (FmCL). Recombinant analog of FmCL was expressed in Pichia pastoris expression system. The peptidolytic activity was again confirmed using the synthetic fluorogenic substrates; the specifity of recombinant FmCL active site was...

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Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria. / Bookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry. / 1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC