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No description available.

No description available.

No description available.

$ beta$-Galactosidase (E.C. 3.2.1.23) or lactase was produced by the growth of a selected Bacillus subtilis strain (KL88) which was adapted to grow at 10$ sp circ$C. The growth and enzyme production were maximal at 2% (w/v) lactose supplemented with 0.2% (w/v) yeast extract. A Fast Protein Liquid Chromatography system (FPLC) was used for $ beta$-galactosidase purification. The enzyme was purified to 44-fold over the crude extract with a recovery of $ sim$54%. Native-PAGE and SDS-PAGE using "PhastSystem" showed the presence of two isoenzymes having molecular weights of 88 and 170 kD. The purified enzyme showed high activity at low temperatures (10$ sp circ$C) and recorded an optimum pH of 7.0. The K$ sb{ rm m}$ values were found to be 2.21 mM and 28.08 mM for o-nitrophenyl-$ beta$-D-galactopyranoside (ONPG) and lactose, respectively. / $ beta$-Galactosidase from psychrotrophic Bacillus subtilis was specific to the $ beta$-D-glycosidic linkage normally present in lactose. / To investigate the possibility of producing proteinase-free $ beta$-galactosidase from this psychrotrophic microorganism, FPLC was used for the rapid separation of $ beta$-galactosidase.

This thesis assessed the prevalence, patterns of antibiotic resistance, and molecular characteristics of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli (EC) and Klebsiella pneumoniae (KPN) isolated from Canadian hospitals. Bacterial isolates were collected as part of the CANWARD national surveillance study. The prevalence of ESBL-EC [2007: 3.4%, 2012: 7.6%], AmpC-EC [2007: 0.7%, 2012: 2.2%], and ESBL-KPN [2007: 1.5%, 2012: 3.6%] increased between 2007 and 2012. Antimicrobials demonstrating the greatest activity against isolates in this study were colistin, amikacin, ertapenem, and meropenem, while 78.8%, 34.9%, and 66.7% of ESBL-EC, AmpC-EC, and ESBL-KPN, respectively, were multidrug resistant. Isolates were generally unrelated by PFGE; however, ST-131 was identified among 56.9% and 31.7% of ESBL-EC and AmpC-EC, respectively. CTX-M-15 was the dominant genotype in ESBL-EC (66.5%) and ESBL-KPN (48.1%), while the dominant genotype in AmpC-EC was CMY-2 (53.2%). Carbapenemase production was identified in 0.03% of EC and 0.05% of KPN, all of which produced KPC-3.

Barley has been receiving increased attention as a human food due to the health benefits associated with β-glucan fiber and its potential as a low glycemic index (GI) functional food. Research has shown a relationship between the physico-chemical properties of β-glucan and the physiological effects, which may be altered by processing. However, it is not known if the physiological effects of consuming barley are affected by variations in chemical composition among cultivars or by common processing methods such as pearling or milling. The primary objective of this thesis was to characterize the effects of differences in cultivar starch and fibre content, level of pearling and milling on the GI, satiety and the physico-chemical properties of β-glucan. Nine barley cultivars varying in starch-type and β-glucan content were studied in three experiments in separate groups of ten healthy participants. Blood glucose and satiety ratings were measured and the GI was calculated. Total starch, total fibre, β-glucan, molecular weight (MW), solubility and β-glucan viscosity were determined in vitro. Results showed that GI varied by cultivar (CDC-Fibar, 26 ± 3 vs. AC-Parkhill, 35 ± 4, P < 0.05) and pearling (WG, 26 ± 4 vs. WP 35 ± 3, P < 0.05). When two cultivars were milled and processed to wet pasta the GI increased by 184% (P < 0.05). The pearled wet pasta had a significantly lower GI compared to the whole grain (P < 0.05). Boiled barley kernels tended to elicit greater satiety than white bread, but the difference was not significant. In both the boiled barley kernels and the wet pasta, pearling did not affect the MW, viscosity and solubility. MW did not significantly differ between cultivars but solubility and viscosity did (P < 0.05). The wet pasta had significantly lower MW, solubility, viscosity but not β-glucan content than the boiled barley kernels (P < 0.05). In conclusion, pearling did not have an effect but milling and extruding resulted in significant reduction in MW, solubility and viscosity. The GI of barley is influenced significantly by cultivar, pearling and milling. Further studies are required to determine the effect on satiety.

Barley has been receiving increased attention as a human food due to the health benefits associated with β-glucan fiber and its potential as a low glycemic index (GI) functional food. Research has shown a relationship between the physico-chemical properties of β-glucan and the physiological effects, which may be altered by processing. However, it is not known if the physiological effects of consuming barley are affected by variations in chemical composition among cultivars or by common processing methods such as pearling or milling. The primary objective of this thesis was to characterize the effects of differences in cultivar starch and fibre content, level of pearling and milling on the GI, satiety and the physico-chemical properties of β-glucan. Nine barley cultivars varying in starch-type and β-glucan content were studied in three experiments in separate groups of ten healthy participants. Blood glucose and satiety ratings were measured and the GI was calculated. Total starch, total fibre, β-glucan, molecular weight (MW), solubility and β-glucan viscosity were determined in vitro. Results showed that GI varied by cultivar (CDC-Fibar, 26 ± 3 vs. AC-Parkhill, 35 ± 4, P < 0.05) and pearling (WG, 26 ± 4 vs. WP 35 ± 3, P < 0.05). When two cultivars were milled and processed to wet pasta the GI increased by 184% (P < 0.05). The pearled wet pasta had a significantly lower GI compared to the whole grain (P < 0.05). Boiled barley kernels tended to elicit greater satiety than white bread, but the difference was not significant. In both the boiled barley kernels and the wet pasta, pearling did not affect the MW, viscosity and solubility. MW did not significantly differ between cultivars but solubility and viscosity did (P < 0.05). The wet pasta had significantly lower MW, solubility, viscosity but not β-glucan content than the boiled barley kernels (P < 0.05). In conclusion, pearling did not have an effect but milling and extruding resulted in significant reduction in MW, solubility and viscosity. The GI of barley is influenced significantly by cultivar, pearling and milling. Further studies are required to determine the effect on satiety.

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.