Hemocytes are important as storage and producers of proteins of the innate immune defence, as well as actors of the cellular immune response. Therefore the hematopoietic process is critical for survival of most invertebrates. In order to search for molecules of importance for hemocyte development in crayfish we investigated proteins in crayfish plasma, which were increased after microbial challenge. As a result we were able to identify, purify and characterize a new invertebrate cytokine named astakine, and could clearly show that this protein is important for hematopoietic development in vivo as well as in an in vitro cell culture system. Astakine contains a prokineticin (PK) domain shown for the first time in an invertebrate, however, unlike the vertebrate PKs, astakine binds to a cell surface F1 ATP synthase β subunit located on the hematopoietic tissue (hpt) cell membranes. Extracellular ATP synthases as receptors have earlier been reported in different vertebrate cells and here we show that extracellular ATP synthase β subunit acts as a receptor for an invertebrate cytokine and is involved in hematopoiesis. We also found two other groups of proteins, which were increased in plasma after microbial challenge and they were further characterized. A great number of different Kazal type proteinase inhibitors were produced by the hemocytes and this type of proteinase inhibitors have variable reactive sites determining the specificity of their inhibition. In crayfish Kazal inhibitors with similar reactive sites were found as a response to specific microorganisms suggesting that the crayfish Kazal proteinase inhibitors may provide enough variability to participate in diverse innate immune reactions against different pathogens. Antimicrobial peptides were synthesized by the hemocytes and were likewise released in high amount upon microbial infection and we have characterized the main group of cystein-rich crustin-like antimicrobial peptides and investigated their tissue distribution and expression pattern.

Some yeast strains secrete extracellular polypeptide toxins known to have potential growth inhibitory activity on sensitive yeast cells. These yeast strains are known as killer yeasts and their toxins are named as killer toxins or killer proteins. Yeast killer proteins are found inhibitory to Gram-positive bacteria in several studies which were based on microbial interactions of the producer strains tested with sensitive strains. K5 type yeast killer protein produced by Pichia anomala NCYC 434 was previously purified and characterized in our laboratory. The protein is glycosilated and has a pI value of 3,7 and molecular mass of 49 kDa, with exo &amp / #946 / -1,3-glucanase activity. Antibacterial activity of the pure K5 type yeast killer protein was tested against 19 clinical isolates of gram-positive bacteria causing skin infections and 2 quality control strains and found to have inhibitory activity on the isolates of Methicillin-sensitive Staphylococcus aureus (MSSA) and Enterococcus faecium. Toxin MIC and MBC ranges were 32 - 256 &micro / g/ml and 64 - &gt / 512 &micro / g/ml respectively. Cell killing analysis revealed that toxin has a bacteriostatic activity and the inhibitory effect starts between 8. and 12. hours. Regrowth of the bacteria is retarded with the increased dose of the toxin. K5 type yeast killer protein might be used as a topical antibacterial agent with its bacteriostatic activity for skin and wound infections caused by MSSA and Enterococcus faecium with appropriate formulation studies upon the antibacterial spectrum determination of the toxin in this study.

Zeolites are nanoporous crystalline aluminosilicates that are tasteless, odorless and nontoxic to humans. They can be tailored into antibacterial agents that are more cost effective than other conventional alternatives. Considering the increasing demand for enduring antibacterial agents, the potential uses of antibacterial zeolites are numerous in medical applications and for everyday household products. To produce antibacterial zeolites, the extra framework cations in the zeolite structures can be exchanged with silver ion (Ag+), the most commonly used antibacterial heavy metal ion due to its high stability, strong activity and broad spectrum. Utilization of antibacterial zeolite powders can be diversified when they are used as fillers in a polymer matrix. Polyurethanes (PU) are a class of polymers which can be prepared in wide range of physical structures with excellent mechanical properties. Ag+ loaded zeolites used as fillers in the PU matrix would contribute to the diversity and efficiency of the PU utilization in many applications including biomedical uses and consumer products. In this study, three types of zeolites, namely / zeolite Beta, X and A with different pores sizes and SiO2/Al2O3 ratios were synthesized hydrothermally and treated with Ag+ containing solution for the exchange of cations. Composites were prepared by incorporation of sieved Ag+ exchanged zeolite particles into biomedical grade PU prepolymers which were prepared either in film or as sponge forms. Films were prepared by molding and foams were prepared in the presence of water as the blowing agent. Liquid media antibacterial tests showed that all of the Ag+-zeolite powders were effective against E. coli at a concentration of 500 ppm zeolite in deionized water. To assess the antibacterial effect of composites against E. coli, disc diffusion tests were carried out. Bacterial growth inhibition zones formed around the composite samples were the evidence of the antibacterial activity in the vicinity of the surface. All three kinds of zeolites successfully introduced the desired antibacterial property to the biomedical grade PU both in elastomeric film and in the foam form. Mechanical characterization of the composites yield higher ultimate tensile strength, modulus of elasticity and elongation at break values compared to control PU. No significant change in thermal properties of the composites was observed. Hence mechanical and thermal characterization of the composites showed that zeolites serve for the reinforcement of the mechanical properties of the polymer and did not cause any deterioration in thermal properties.

Flora and fauna from terrestrial and marine environments provide libraries of natural compounds for drug discovery. The last four decades have seen major advances in ocean exploration that have allowed chemists and biologists to explore previously inaccessible and rare marine organisms. The study of under-explored marine organisms can result in the discovery of structurally novel and unusual natural products with drug potential. Prior to 2005, no natural products had been reported from the Fijian red macroalgae Callophycus serratus or Neurymenia fraxinifolia. As a result of the work described in this thesis and others in the same research group, 33 unique brominated meroditerpenes have been isolated and elucidated alpha-pyrone natural products were discovered from N. fraxinifolia, enriching the natural product library for drug development. Several natural products isolated from C. serratus exhibited sub-micromolar inhibition against the human malaria parasite Plasmodium falciparum, including a drug-resistant strain. Derivatization of the natural product bromophycolide A into fluorescent probes allowed the determination of a non-enzymatic mechanism of action against the human malaria parasite P. falciparum. Through a combination of detailed SAR mapping, molecular fluorescent imaging of live cells, UV-vis spectroscopic analyses, and protein affinity techniques, the mechanism of action of bromophycolide A against P. falciparum was determined to involve inhibition of heme crystallization. These studies identify a new class of natural products that target heme detoxification in both drug-sensitive and drug-resistant P. falciparum and suggest an avenue to circumvent drug resistance.

This thesis aimed to investigate the features and properties of the ground states, transient species and photoproducts involved in the photophysical and photochemical processes for four kinds of drug compounds: lomefloxacin (LF), norfloxacin (NF), tiaprofenic acid (TPA), and flurbiprofen (Fp). The investigation used femtosecond transient absorption (fs-TA), nanosecond transient absorption (ns-TA), UV/Vis absorption spectra (UV/Vis), nanosecond transient resonance Raman (ns-TR2) and nanosecond time-resolved resonance Raman spectroscopy (ns-TR3), as well as density functional theory (DFT) calculations. Although many previous investigations have indicated that photo-defluorination or photo-decarboxylation reactions may account for the phototoxicity for these compounds, detailed information on the mechanisms remains unclear. In this thesis, the photo-defluorination reaction of LF was explored in neutral water at pH 7.2. The fs-TA results revealed that the lowest lying excited singlet state species (S1) partially decayed into the ground state through fluorescence emission and partially underwent cleavage of the carbon-fluorine bond at position 8 to generate into a singlet aryl cation. Subsequently, intersystem crossing (ISC) allowing the transformation from singlet cation to triplet carbene was observed. Finally, a cyclization reaction with the N-ethyl chain took place for the triplet carbene to generate the final product. The mechanism underlying NF phototoxicity involves a photo-defluorination reaction in neutral water (pH=7.2). The fs-TA spectra indicated that the S1 underwent efficient ISC to swiftly transform into lowest excited triplet (T1) The ns-TA gained under nitrogen-saturated condition observed a new transient species produced from T1 that was proposed to be a transient species derived from the photo-defluorination reaction involving a SN2Ar* mechanism. The photo-defluorinated product ultimately experienced an ISC process to produce the final product. The photo-decarboxylation mechanism of TPA was studied in a neutral phosphate buffered solution (PBS). The fs-TA data revealed that S1 went through an efficient ISC to rapidly transform into T1 that then undergoes a photo-decarboxylation reaction to produce a triplet biradical species (denoted as TB3). The ns-TA and ns-TR3 results supplied evidence of the protonation process of TB3 that produces the neutral species (denoted as TBP3) that then decayed through ISC to give rise to the singlet TBP species, which underwent further reaction to make the final product (DTPA). The photo-decarboxylation reaction of Fp was explored in pure acetonitrile (MeCN). The second excited singlet (S2) went through internal conversion (IC) to decay to S1. Intriguingly, three different pathways for S1 decay co-exist. One pathway is fluorescence emission and the second is an ISC process. The third pathway is the homolysis of the carbon α bond reaction that proceeds to generate two radical species, one being a carboxyl species and the other being the residual, denoted as FpR that was liable to be oxidized under an oxygen-saturated condition to yield a new radical species with the addition of one oxygen molecule which is denoted as FOR that then experienced intramolecular hydrogen transfer (IHT) and dehydroxylation (DHO) to produce the final product. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Microbe resistence is a serious issue, especially as they have become resistant to most well known drugs. Therefore this is considered as a global problem and is now dealt with at a poitical level. Since no new classes of antimicrobial agents have been discovered in the past three deacdes, the development of new drugs is extremely urgent. Therefore the aim of this project was to synthesise derivatives of benzimidazole, and then assesses their antimicrobial activities in vitro by using disc (well) diffusion and MICs tests. A total of 69 benzimidazole derivatives, with substituents at positions 1, 2, and 5, were synthesised, characterised and tested against selected bacteria and fungi. In addition, six bezimidazole silver complexes were prepared and evaluated for their antimicrobial behavior. The SAR showed that the antimicrobial activity of the compounds depended on the substituents attached to the bicyclic heterocycle. Some promising results were obtained. In particular, 5 compounds displayed antibacterial activity against two MRSA strains with MIC values corresponding to ciprofloxacin, which can be considered significant. The compounds have some common features; four possess 5-chloro or 5-bromo substituents; two are derivatives of (S)-2- ethanaminebenzimidazole and the others are derivative of one 2-(chloromethyl)-1Hbenzo[d]imidazole, (1H-benzo[d]imidazol-2-yl)methanethiol and 2-(methoxymethyl)-1-methyl-1H-benzo[d]imidazole. The results from the antifungal screening were very interesting as there were 26 compounds, including two silver complexes, which were potent fungicides against the selected fungal species. They showed equivalent or greater potentency in their MIC values than amphotericin B. In particular, the 5-fluoro, 5-chloro and 5-bromo benzimidazole showed broad spectrum activity.

A novel approach of using near infrared region (NIR) dyes is applied to elucidate the binding interaction between human serum albumin (HSA) and mangiferin (MGF). HSA is a blood carrier protein used for drug delivery, while mangiferin is a natural polyphenol found in mangoes that possesses numerous beneficial health properties. The NIR dyes are used as a probe to investigate MGF binding interaction with HSA via monitoring fluorescence of Trp-214 residue. Molecular modeling is used for docking and semi-empirical analysis. The investigation of the binding interaction between Trp-214 and MGF is significant, for it may offer broader pharmacological insight and applications for the polyphenol. Mangiferin in proposed to bind with a 2:1 stoichiometric ratio with HSA to the Trp-214 residue in subdomain IIA and another possible binding site to be determined in future studies. Spectral changes suggest a stabilized protein conformation upon mangiferin binding with the addition of NIR dye E-06 and MHI-06.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

This thesis describes the synthesis and biological evaluation of highly substituted, ring-fused 2-pyridones. The utility of the bicyclic 2-pyridones to gain fundamental insights into the disease processes of bacterial infections and Alzheimer’s disease has been investigated. The 2-pyridones have mainly been studied as a new class of anti-infective agents termed pilicides. The function of the pilicides has been explored using uropathogenic E. coli (UPEC) as a prototype pathogen and urinary tract infection as a model disease. The pilicides target the infectious ability of UPEC by inhibiting key proteins (chaperones) in the so-called chaperone-usher pathway, thus preventing the assembly of bacterial surface organelles (pili/fimbriae). Synthetic pathways to aminomethylate the 2-pyridones have been developed in order to increase their aqueous solubility while retaining biological activity. Also, the importance of a carboxylic acid has been demonstrated in studies with various carboxylate derivatives and by bioisosteric replacement. Moreover, synthetic procedures to extend the backbone of the rigid, dipeptide-mimicking 2-pyridones have been established. This rendered peptidomimetic building blocks and structures that alongside their potential use as pilicides are of more general interest in peptidomimetic-related research. The potential pilicides have been screened for chaperone affinity using relaxation-edited 1H-NMR spectroscopy. In addition, their ability to inhibit pilus biogenesis in E. coli has been demonstrated by assays of hemagglutination, biofilm formation and attachment to bladder cells, as well as with electron and atomic force microscopy. Moreover, it has been confirmed that pilicides regulate the expression of pili without affecting the biofunctional properties of the pilus rod. This was verified by measurements of individual P pili, on living bacteria, using force measuring optical tweezers. The pilicide binding site was investigated using NMR spectroscopy and X-ray crystallography of a pilicide-chaperone complex. Based on the results obtained, a mechanism whereby the pilicides may inhibit pilus assembly was proposed, which was subsequently experimentally supported by surface plasmon resonance assays and genetic analysis. Finally, based on the generic 2-pyridone scaffold, a new collection of substituted compounds has been synthesized and validated as inhibitors of Amyloid β (Aβ)-peptide aggregation, which has been suggested to be involved in Alzheimer’s disease.

The O-acetylation of peptidoglycan (PG) is currently known to occur in greater than 50 eubacterial species, including numerous pathogens. This modification, which occurs at the C-6 hydroxyl of the N-acetylmuramoyl residues within the heteropolymer’s glycan backbone, serves as a cell wall autolytic regulatory mechanism, and contributes to pathogenesis and persistence within a host. Despite these significant physiological and pathobiological roles however, the identity of the pathway(s) responsible for the modification was only recently elucidated, for which two unrelated systems were identified, viz., the O-acetylpeptidoglycan (OAP) cluster-encoded multi-component system typical of Gram-negative species and the singular OatA of Gram-positives. As part of the OAP PG O-acetylation system, our group previously identified O-acetylpeptidoglycan esterase (Ape) as an enzyme responsible for the removal of the modification, permitting the continued metabolism of the PG sacculus. Herein, studies were performed to assess the postulated viability of this class of enzyme as a novel antibacterial target. Specifically, recombinant Ape1 from Neisseria gonorrhoeae was purified to homogeneity and the inhibitory effect of purpurin, a natural product identified as such, evaluated in detail. Kinetic analysis demonstrated that the compound elicited a competitive mode of inhibition (Kic ~3.7 μM), while the in vivo treatment of an array of environmental and pathogenic species was found to result in growth arrest for those cells containing both O-acetylPG and Ape. Evaluation of modification levels, cell wall morphology, and viability indicated a bacteriostatic effect. Taken together these data provide proof of principle that this class of enzyme presents a worthy therapeutic target. In addition to the presence of an Ape, the OAP system further differs from that of OatA through the use of two PG O-acetyltransferases. While purported to be mutually exclusive and evolutionarily divergent, in silico genomic analyses indicated their potential copresence in Bacillus anthracis and other closely related organisms. Indeed, purpurin-mediated differential growth inhibition between several such isolates and other bacilli indicated Ape activity therein. To investigate this possibility, the hypothetical Ape3 protein from Bacillus cereus ATCC 10987 was overproduced, purified, and its function assessed. Data from activity assays involving natural and synthetic substrates indicated that the protein possesses basal esterase activity in vitro. Phenotypic analysis of B. anthracis mutants deficient in each of the organism’s putative integral membrane PG O-acetyltranslocases subsequently indicated that Ape3 preferentially functions as a PG O-acetyltransferase (Pat) in vivo and that the OAP-mediated system is required for the separation of daughter cells following division. In addition, the presence of an Oat homologue was also confirmed. Thus, this is the first report of a bacterium known to possess both types of PG O-acetylation systems. / Natural Sciences and Engineering Research Council of Canada (NSERC)