The gulf toadfish (Opsanus beta) is a member of a group of teleosts that have retained their ornithine urea cycle (OUC) allowing them to excrete nitrogenous waste in the form of urea (ureotely). Urea-­N for the entire day is excreted in 1-­2 quick pulsing events (1-­3 h). This study evaluated the hypothesis that urea-­N pulsing events in gulf toadfish can be triggered by social signals from conspecifics via a specific waterborne messenger. Using a crowding protocol, we found that pre-­conditioned seawater induced a secondary urea pulsing event in naïve conspecifics. Furthermore, it was revealed that other factors such as signal concentration and donor body mass relay information to recipients as well. Fractionation of pre-­conditioned seawater was carried out to narrow possible signal candidates and the aqueous portion was found to contain the active molecule. Ammonia was found to be an important factor controlling the response of toadfish to pre-conditioned seawater.

Type 2 diabetes is now recognized as a worldwide epidemic. Pancreatic beta-cell decompensation in the presence of insulin resistance is a major mechanism for the development of type 2 diabetes and may be triggered by mitochondrial dysfunction. Alcoholism is a known risk factor for type 2 diabetes. Excessive or chronic alcohol consumption leads to increased oxidative stress and mitochondrial dysfunction in beta-cells. Prohibitin is a multifunctional protein that also regulates mitochondrial biogenesis and function. Although it has anti-oxidant effects in some cell types, its role in pancreatic beta-cells is not known. This study has investigated the effects of prohibitin in ethanol treated pancreatic beta-cells using RINm5F and INS-1E cell lines. Prohibitin was found to be expressed in pancreatic beta-cells with localization to the nucleus and the perinuclear area. Ethanol increased the expression of prohibitin and induced its translocation from the nucleus to the mitochondria. Ethanol, through its metabolism by alcohol dehydrogenase (ADH), increased oxidative stress and altered mitochondrial membrane potential, decreased the activity of mitochondrial respiratory complexes I and IV, and uncoupled energy production with resulting reduction in ATP production. This was associated with activation of the proinflammatory enzyme c-Jun N-terminal kinase and proapoptotic proteins Bax and caspase-3, leading to beta-cell apoptosis. Ethanol also reduced glucose induced insulin secretion without alteration of the beta-cell transcription factors PDX-1 and MafA. Treatment with exogenous prohibitin or cellular overexpression of endogenous prohibitin attenuated ADH activity, prevented the deleterious effects of ethanol on mitochondrial function and reduced apoptosis, whereas prohibitin knockdown enhanced ethanol-induced apoptosis. In addition, prohibitin per se increased PDX-1 and MafA levels. Through the above mechanisms, prohibitin restored glucose induced insulin secretion in ethanol exposed beta-cells. In brief, ethanol causes mitochondrial dysfunction and induces apoptosis in beta-cells, which result in a reduction of insulin secretion; whereas prohibitin prevents mitochondrial dysfunction, apoptosis, and -cell failure by stabilizing mitochondrial complexes I and IV and inhibiting ADH activity during ethanol metabolism. In addition, prohibitin in itself increases the levels of beta-cell transcription factors. As a consequence, prohibitin maintains normal pancreatic beta-cell function and could be useful in diabetes prevention and treatment.

The spread of resistance to the cephalosporins in the Enterobacteriaceae and more specifically within E. coli is a continuing cause of public health concern, with such resistance increasingly seen in community- and nosocomial-acquired infections. Extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase (AmpC) enzymes cause most cephalosporin resistance in E. coli by hydrolysis of the antimicrobial and continue to jeopardize patient outcome. The purpose of this thesis was to determine the prevalence of ESBL-producing Enterobacteriaceae and to molecularly characterize ESBL and AmpC producers found to be associated with the increasing cephalosporin resistance among E. coli within Canadian hospitals from 2005 to 2009. Isolates were collected as part of the Canadian Intensive Care Unit and Canadian Ward surveillance studies. ESBL and AmpC producers were molecularly characterized for resistance genes, virulence factors and phylogenetic groups. All strains were typed using PFGE and ESBL-producing E. coli were further typed by MLST. Plasmids bearing the ESBL and AmpC genes were characterized by BglII RFLP analysis and a multiplex PCR for replicon typing. ESBL-producing E. coli and K. pneumoniae and AmpC-producing E. coli were found to be firmly established in Canadian hospitals; whereas, ESBL-producing K. oxytoca and P. mirabilis have yet to emerge. Increasing resistance to several unrelated antimicrobials leading to multi-drug resistance among these pathogens is concerning. The successful dissemination of ESBL-producing E. coli in Canada occurs through a diversity of different mechanisms and does not correspond to a single ESBL determinant, or a single clone, or a single plasmid but rather through the combination of clonal spread of virulent strains and the acquisition of diverse ESBL-bearing plasmids. However, the predominance of CTX-M-15-producing E. coli in this study was mainly due to the virulent ST131 clone and the diverse IncFII plasmids bearing the blaCTX-M-15 gene. Whereas, horizontal transfer of genetically similar IncI1, IncA/C and IncK/B plasmids bearing blaCMY-2 and the clonal spread of virulent strains, including ST131 with ampC promoter/attenuator mutations, appears to be playing a role in the spread of AmpC-producing E. coli isolates in Canadian hospitals. The increasing prevalence of these multi-drug resistant pathogens in Canadian hospitals demonstrates the need for increased surveillance and understanding of these emerging pathogens. The continued surveillance will help guide proper infection control procedures and identify optimal treatment of these clinically important pathogens in Canadian hospitals.

This thesis is concerned with the influence of acute and long-term beta-adrenergic blockade on myocardial infarction in man. An original statistical evaluation of all published and some available unpublished clinical trials is presented in Chapter I. Chapter III and TV concern the measurement and evolution of infarct size in man. In Chapter III, praecordial ECG mapping and the standard 12 lead ECG have been correlated with cumulative release of the MB isomer of creatine kinase. Using these techniques, I have found that approximately 50% of eventual infarction is complete in 6 hours; implying that interventions designed to salvage ischaemic myocardium may be feasable (Chapter IV). In Chapter V, I have demonstrated delayed beta-adrenergic blockade after oral administration of atenolol and that an initial intravenous dose is essential to achieve early and effective beta-blockade. In a randomised control trial of 215 patients, atenolol administered intravenously within 12 hours of pain, prevented infarction in treated patients with initial threatened infarcts and reduced infarct size and morbidity in those with initial definite infarcts (Chapter VI). Patients with anterior myocardial infarction, randomised to receive atenolol for a year, showed significantly greater R wave recovery and Q wave disappearance on serial praecordial maps compared to placebo patients. In a further study, this was demonstrated to be due to lowering the heart rate. This phenomenon of improved EGG recovery with atenolol was reproduced in experimental infarction and was shown to be due to improved scar shrinkage (Chapter VII). The implications of these studies are discussed. It is likely that both early and long-term beta-blockade will be beneficial to patients with myocardial infarction.

Chapter 1 is the introduction to the thesis. The general principles of radical cyclisation reactions are highlighted with a focus upon the reactivity of iminyl radicals. A more detailed discussion follows on radical cyclisations onto nitriles including examples of tandem cyclisations. The final section is concerned with cyano migration reactions, and provides evidence for the reversibility of these translocations. The investigation into tandem radical cyclisations of nitriles is discussed in Chapter 2. The aim of the project was to form bicyclic nitrogen heterocycles from acyclic precursors by utilising the nitrile function as a radical acceptor which could then undergo further cyclisation onto a suitably placed alkene. We found a surprising chemoselectivity for 1,5-exo-cyclisation of alkyl, aryl and vinyl radicals onto the cyano group over 1,6-exo-cyclisation onto suitably placed alkenes. The presence of an electron-withdrawing group on the carbon a- to the nitrile group resulted in an overall 1,4-cyano migration reaction. The intermediate cyclic iminyl radical does not undergo further cyclisation, nor does it abstract hydrogen from tributyltin hydride. Instead, fragmentation by β-scission yields a stabilised radical, e.g. by an ester or nitrile group. In order to investigate the effect of substituents on the cyclisation of aryl radicals onto nitriles, and the β-scission reactions of the iminyl radical intermediates, a series of aryl radical precursors were prepared. a-Nitrile, amide, sulfone and phenyl groups favoured β-scission and a-alkyl groups favoured cyclisation or reduction of the aryl radicals. The study indicated the existence of a Thorpe-Ingold effect on the cyclisation of aryl radicals onto nitriles. In Chapter 3 the largely unsuccessful studies of the reversibility of radical cyclisations onto nitriles is reported. Finally, in Chapter 4, the results obtained from our research into vinyl radicals as precursors for tandem radical cyclisations of nitriles are presented. A vinyl iodide underwent complete conversion when standard radical cyclisation conditions were applied; the cyclic ketone resulting from a single 5-exo cyclisation was isolated in the absence of any other products. The experimental relevant to the discussion is detailed in Chapter 5.

Alzheimer’s disease (AD), a progressive age-related neurodegenerative disorder is characterized by impairments in memory and cognitive functions. The two main pathogenic hallmarks associated with the progression of this multifactorial disease include accumulation of amyloid-beta (Aβ) plaques and the deterioration of the cholinergic system in the brain. Using cost-effective synthetic procedures, mono-, di-, and tri- substituted sym-triazine derivatives incorporating acetylcholine substrate analogues and aromatic phenyl moieties were synthesized for the targeted modulation of Aβ aggregation and acetylcholinesterase (AChE) activity. A subset of these sym-triazines demonstrated dual inhibition of Aβ fibrillization and AChE hydrolytic activity in vitro studies. These highly effective compounds were also shown to be well tolerated by differentiated human neuronal cells in cell viability tests. These novel compounds have the potential to undergo future in vivo pharmaceutical analysis and have a positive impact on the quality of life of the people living with this devastating disease and their caretakers.

Alzheimer’s disease (AD), a progressive age-related neurodegenerative disorder is characterized by impairments in memory and cognitive functions. The two main pathogenic hallmarks associated with the progression of this multifactorial disease include accumulation of amyloid-beta (Aβ) plaques and the deterioration of the cholinergic system in the brain. Using cost-effective synthetic procedures, mono-, di-, and tri- substituted sym-triazine derivatives incorporating acetylcholine substrate analogues and aromatic phenyl moieties were synthesized for the targeted modulation of Aβ aggregation and acetylcholinesterase (AChE) activity. A subset of these sym-triazines demonstrated dual inhibition of Aβ fibrillization and AChE hydrolytic activity in vitro studies. These highly effective compounds were also shown to be well tolerated by differentiated human neuronal cells in cell viability tests. These novel compounds have the potential to undergo future in vivo pharmaceutical analysis and have a positive impact on the quality of life of the people living with this devastating disease and their caretakers.

A beta-galactosidase gene from Bifidobacterium breve B24 which showed the higher hydrolytic and synthetic activity was cloned in E. coli. The complete beta-galactosidase gene contained 2076 bp nucleotides and encoded 691 amino acids which had a high homology to the other Bifidobacterium species. This beta-galactosidase was homologous to that of the LacA family. The galA gene was successfully over-expressed in E. coli ER2566. To observe any change in the recombinant enzyme, beta-galactosidases from Bifidobacterium breve B24 and recombinant E. coli ER2566 were purified to homogeneity by ion exchange chromatography (Mono-Q) and gel-filtration chromatography (Superose-12 and Superdex 200) columns. The molecular mass of both beta-galactosidases was estimated to be 75 kDa on SDS-PAGE. Activity staining on non-denaturing Native-PAGE and Superose-12 gel-filtration chromatography showed that the enzymes are composed of a dimer with a molecular mass of 150 kDa. / The optimum pHs of the native and recombinant enzymes for hydrolyzing O-nitrophenyl-beta-D-galactopyranose (ONPG) were pH 6.0 and 7.0, respectively, and they were stable over the pH range of 5-8 and 6-9, respectively. The optimum temperature of both enzymes for hydrolyzing ONPG was similar at 45 °C and they were stable over the temperature range of 20-45 °C. Both enzymes were stable up to 45 °C during 5 h of incubation at pH 6.5. The recombinant enzyme was slightly activated by bivalent metal ions, Mg2+, Mn2+, and Zn2+ at 1 mM but strongly inhibited by Hg2+ and p-chloromercuribenzoic acid (PCMB). The K m values of both native and recombinant beta-galactosidases for ONPG were 2.77 and 1.82 mM, respectively, and the Vmax values were 1.02 and 1.39 mM/min, respectively. / The two beta-galactosidase activities were also tested with lactose as substrate. The optimum pH of the native and recombinant enzymes for hydrolyzing lactose was similar at pH 6.0. Both enzymes had more than 80 % of their activity in the range of pH 6-8, indicating that the enzymes were stable at neutral pH. However, the native beta-galactosidase had around 40 % of its activity at pH 5.0, whereas the recombinant enzyme had no activity at this pH. On the other hand, the recombinant enzyme had over 50 % of its activity at pH 9.0, while the native beta-galactosidase showed lower than 5 % of its activity. The optimum temperature of both enzymes was at 45 °C. The profiles of both enzyme activities were very similar except at the temperature of 10 °C. The recombinant beta-galactosidase still had around 20 % of its enzyme activity at 10 °C, while no enzyme activity from the native enzyme was detected at this temperature.

Current cancer therapies have low specificity for tumor cells and have serious toxic side effects. Targeting drugs to the cancer cells can help improve the outcome of existing cancer therapies. In recent years, a number of peptides have been identified by peptide phage display for targeting different tumor types. Peptides identified from the phage display for targeting cancer cells can be further improved for specific binding and metabolic stability by chemical manipulation of their structures. The aims of this work were: (i) to develop a peptide array-whole cell binding assay for screening peptides with specific binding affinity for cancer cells (ii) design of novel peptidomimetics to improve their properties as drug candidates. First, peptide arrays based on the lead peptide sequences NGR and p160 were designed and synthesized. A direct peptide-cell binding assay using CyQUANT dye allowed identification of several new peptides with higher affinity for MDA-MB-435 and MCF-7 cancer cells compared to the wild type p160. These peptides did not recognize the normal endothelial HUVEC cells. Three p160 peptide analogues, namely, 11 (RGDPAYQGRFL), 18 (WXEAAYQRFL), and 40 (WXEPAYQRKL), that displayed highest affinity for the cancer cells were manually synthesized and labelled with FITC. The binding ability of these peptides was confirmed using fluorescence imaging and flow cytometry. The results confirmed the high and specific affinity of peptides 11 and 18 for the cancer cells. The peptide array-cell binding assay established in this study is not only useful for the identification of cancer targeting peptides. It can also be used for the generation of diagnostic tools for cancer. Secondly, two new classes of -peptides, 3- and 2-peptides derived from L-Asp and L-Dap monomers, respectively, were synthesized. The methodology allowed independent buildup of the -peptide backbone and the introduction of sequential side chain substitutions. It is shown that / mixed peptide increases target recognition and retains the proteolytic stability. Moreover, -peptides impart no cytotoxicity, which will expand their potential application in the design of biologically active peptides. As a result, these compounds represent good candidates for new drugs and as tools to gain further insight into protein folding and molecular recognition processes. / Pharmaceutical Sciences

Type 1 diabetes is an autoimmune disease in which patients' insulin-secreting beta cells in pancreatic islets are destroyed by their own immune system, leading to unregulated blood glucose levels and severe complications. Its only treatment is intensive insulin therapy, which carries the risk of hypoglycemic episodes and can result in seizures, coma, and even death. Islet transplantation has recently become an alternative, albeit experimental, treatment for type 1 diabetes patients. More than one donor graft is usually required to render recipients insulin independent, making the shortage of donor tissue an extremely important challenge in islet transplantation. Identifying the cell type that has the ability to differentiate into islet-like tissue is an important area of study. In this study, I hypothesized that insulin secreting human islet-like clusters could be generated from pancreatic ductal cells, a potential pancreatic progenitor cell type. Islet-like clusters were generated using crude exocrine tissue from human cadaveric donors. This crude exocrine tissue contained a large number of ductal cells, as well as other pancreatic cell types. To evaluate insulin secretion by human islet-like clusters, a static incubation system was set up and tested using Min6 cells, a known insulin-secreting cell line. Using static incubation, significant increases in insulin secretion by islet-like clusters were observed when the clusters were exposed to higher glucose levels and GLP-1, a known insulin secretagogue. Presence of corresponding C-peptide secretion demonstrated that de novo insulin secretion occurred. Furthermore, basal insulin secretion increased as culture stages progressed. An attempt was made to generate islet-like clusters using ductal cells purified by fluorescent activated cell sorting or magnetic activated cell sorting. Nevertheless, it was difficult to ensure survival and proliferation of purified ductal cells. Further studies will be necessary to confirm the role of ductal cells in the generation of islet-like clusters using the crude exocrine tissue, as well as to identify factors that can promote ductal cells proliferation after cell sorting.