Adhesion and production of degrading enzymes by bacteria isolated from biofilms in raw milk cooling tanks

Biofilms in milk cooling tanks compromise product quality even on farms. Due to the lack of studies of this topic, this study evaluated the microbiological conditions of raw milk cooling tanks on farms and characterized the microorganisms isolated from these tanks. Samples were wiped off with sterile swabs from seven milk cooling tanks in three different points in each tank. Mesophiles and psychrotrophic counts were performed in all samples. The isolation of Pseudomonas spp., Bacillus cereus and atypical colonies formed on selective media were also performed, totalizing 297 isolates. All isolates were tested for protease and lipase production and biofilm formation. Of the total isolates, 62.9% produced protease, 55.9% produced lipase, and 50.2% produced biofilm. The most widespread genus inside the milk cooling tank was Pseudomonas since it was not possible to associate this contamination with a single sampling point in the equipment. High counts of microorganisms were found in some cooling tanks, indicating poor cleaning of the equipment and providing strong evidences of microbial biofilm presence. Moreover, it is worth mentioning the milk potential contamination with both microbial cells and their degrading enzymes, which compromises milk quality.

Screening and kinetics of glutaminase and glutamate decarboxylase producing lactic acid bacteria from fermented Thai foods

L-glutaminase and glutamic acid decarboxylase (GAD) catalyzes the hydrolysis of L-glutamine and glutamate, respectively. L-glutaminase widely used in cancer therapy along with a combination of other enzymes and most importantly these enzymes were used in food industries, as a major catalyst of bioconversion. The current investigation was aimed to screen and select L-glutaminase, and GAD producing lactic acid bacteria (LAB). A total of 338 LAB were isolated from fermented meat, fermented fish, fermented soya bean, fermented vegetables and fruits. Among 338 isolates, 22 and 237 LAB has been found to be positive for L-glutaminase and GAD, respectively. We found that 30 days of incubation at 35 ºC and pH 6.0 was the optimum condition for glutaminase activity by G507/1. G254/2 was found to be the best for GAD activity with the optimum condition of pH 6.5, temperature 40 ºC and ten days of incubation. These LAB strains, G507/1 and G254/2, were identified as close relative of Lactobacillus brevis ATCC 14869 and Lactobacillus fermentum NBRC 3956, respectively by 16S rRNA sequencing. Further, improvements in up-stream of the fermentation process with these LAB strains are currently under development.

Synthesis and characterization of nanoperforated metal oxide thin films

For advanced devices in the application fields of data storage, solar cell and biosensing, one of the major challenges to achieve high efficiency is the fabrication of nanopatterned metal oxide surfaces. Such surfaces often require both precise structure at the nanometer scale and controllable patterned structure at the macro scale. Nowadays, the dominating candidates to fabricate nanopatterned surfaces are the lithographic technique and block-copolymer masks, most of which are unfortunately costly and inefficient. An alternative bottom-up approach, which involves organic/inorganic self-assembly and dip-coating deposition, has been studied intensively in recent years and has proven to be an effective technique for the fabrication of nanoperforated metal oxide thin films. The overall objective of this work was to optimize the synthesis conditions of nanoperforated TiO2 (NP-TiO2) thin films, especially to be compatible with mixed metal oxide systems. Another goal was to develop fabrication and processing of NP-TiO2 thin films towards largescale production and seek new applications for solar cells and biosensing. Besides the traditional dip-coating and drop-casting methods, inkjet printing was used to prepare thin films of metal oxides, with the advantage of depositing the ink onto target areas, further enabling cost-effective fabrication of micro-patterned nanoperforated metal oxide thin films. The films were characterized by water contact angle determination, Atomic Force Microscopy, Scanning Electron Microscopy, X-ray Photoelectron Spectroscopy and Grazing Incidence XRay Diffraction. In this study, well-ordered zinc titanate nanoperforated thin films with different Zn/Ti ratios were produced successfully with zinc precursor content up to 50 mol%, and the dominating phase was Zn2Ti3O8. NP-TiO2 structures were also obtained by a cost-efficient means, namely inkjet printing, at both ambient temperature and 60 °C. To further explore new biosensing applications of nanoperforated oxide thin films, inkjet printing was used for the fabrication of both continuous and patterned polymeric films onto NP-TiO2 and perfluorinated phosphate functionalized NP-TiO2 substrates, respectively. The NP-TiO2 films can be also functionalized with a fluoroalkylsilane, resulting in hydrophobic surfaces on both titania and silica. The surface energy contrast in the nanoperforations can be tuned by irradiating the films with UV light, which provides ideal model systems for wettability studies.

Novel cancer-related regulatory targets for the signalling sphingolipids sphingosine-1-phosphate and sphingosylphosphorylcholine

The signalling sphingolipid sphingosine-1-phosphate (S1P) is necessary for development of the immune system and vasculature and on a cellular level regulates migration, proliferation and survival. Due to these traits S1P has an important role in cancer biology. It is considered a primarily cancer-promoting factor and the enzyme which produces it, sphingosine kinase (SphK), is often over-expressed in tumours. S1P is naturally present in the blood, lymph, tissue fluids and cell cytoplasm and functions through its cell surface receptors (S1P1-5) and as an intracellular second messenger. Sphingosylphosphorylcholine (SPC) is closely related to S1P and has similar regulatory functions but has not been extensively studied. Both S1P and SPC are able to evoke either stimulatory or inhibitory effects on cancer cells depending on the context. The aim of this thesis work was to study novel regulatory targets of S1P and SPC, which mediate the effects of S1P/SPC signalling on cancer cell behaviour. The investigated targets are the transcription factor hypoxia-inducible factor 1 (HIF-1), the intermediate filament protein vimentin and components of the Hippo signalling pathway. HIF-1 has a central role in cancer biology, as it regulates a multitude of cancer-related genes and is potently activated by intratumoural hypoxia through stabilization of the regulatory subunit HIF-1α. Tumours typically harbour high HIF-1α levels and HIF-1, in turn, facilitates tumour angiogenesis and metastasis and regulates cancer cell metabolism. We found S1P to induce follicular thyroid cancer cell migration in normal oxygen conditions by increasing HIF-1α synthesis and stability and subsequently HIF-1 activity. Vimentin is a central regulator of cell motility and is also commonly over-expressed in cancers. Vimentin filaments form a cytoskeletal network in mesenchymal cells as well as epithelial cancer cells which have gone through epithelial-mesenchymal transition (EMT). Vimentin is heavily involved in cancer cell invasion and gives tumours metastatic potential. We saw both S1P and SPC induce phosphorylation of vimentin monomers and reorganization of the vimentin filament network in breast and anaplastic thyroid cancer cells. We also found vimentin to mediate the anti-migratory effect of S1P/SPC on these cells. The Hippo pathway is a novel signalling cascade which controls cancer-related processes such as cellular proliferation and survival in response to various extracellular signals. The core of the pathway consists of the transcriptional regulators YAP and TAZ, which activate predominantly cancer-promoting genes, and the tumour suppressive kinases Lats1 and Lats2 which inhibit YAP/TAZ. Increased YAP expression and activity has been reported for a wide variety of cancers. We found SPC to regulate Hippo signalling in breast cancer cells in a two-fold manner through effects on phosphorylation status, activity and/or expression of YAP and Lats2. In conclusion, this thesis reveals new details of the signalling function of S1P and SPC and regulation of the central oncogenic factors HIF-1 and vimentin as well as the novel cancer-related pathway Hippo.

Timing of di-ammonium phosphate addition to fermenting chardonnay must : effect on nitrogen utilization, ethyl-carbamate formation, and CAR1 expression by yeast /

The maximum amount of ethyl carbamate (EC), a known animal carcinogen produced by the reaction of urea and ethanol, allowed in alcoholic beverages is regulated by legislation in many countries. Wine yeast produce urea by the metabolism of arginine, the predominant assimilable amino acid in must. This action is due to arginase (encoded by CARl). Regulation of CARl, and other genes in this pathway, is often attributed to a well-documented phenomenon known as nitrogen catabolite repression. The effect of the timing of di-ammonium phosphate (DAP) additions on the nitrogen utilization, regulation of CARl, and EC production was investigated. A correlation was found between the timing of DAP addition and the utilization of nitrogen. When DAP was added earlier in the fermentations, less amino nitrogen and more ammonia nitrogen was sequestered from the media by the cells. It was also seen that early DAP addition led to more total nitrogen being used, with a maximal difference of ~25% between fermentations where no DAP was added versus addition at the start of the fermentation. The effect of the timing ofDAP addition on the expression of CARJ during fermentation was analyzed via northern transfer and the relative levels of CARl expression were determined. The trends in expression can be correlated to the nitrogen data and be used to partially explain differences in EC formation between the treatments. EC was quantified at the end of fermentation by GC/MS. In Montrachet yeast, a significant positive correlation was found between the timing of DAP addition, from early to late, and the final EC concentration m the wine (r = 0.9226). In one of the fermentations, EC levels of 30.5 ppb was foimd when DAP was added at the onset of fermentation. A twofold increase (69.5 ppb) was observed when DAP was added after 75% of the sugars were metabolized. When no DAP was added, the ethyl carbamate levels are comparable at a value of 38 ppb. In contrast, the timing of DAP additions do not affect the level EC produced by the yeast ECU 18 in this manner. The study of additional yeast strains shows that the effect of DAP addition to fermentations is strain dependent. Our results reveal the potential importance of the timing of DAP addition to grape must with respect to EC production, and the regulatory effect of DAP additions on the expression of genes in the pathway for arginine metabolism in certain wine yeast strains.

Syntrophic relationships between algae and bacteria in a fresh-water stream and in culture /

Interactions between freshwater algae and bacteria were examined in a natural stream habitat and a laboratory model. Field observations provided circumstantial evidence, in statistical correlation for syntrophy between the microbial populations. This relation is probably subject to control by the temperature and pH of the aquatic environment. Several species of a pond community were isolated in axenic culture and tests were performed to determine the nature of mixed species interactions. Isolation procedures and field studies indicated that selected strains of Chlorella and Azotobacter were closely associated in their natural habitat. With the suspected controlling parameters, pH and temperature, held constant, mixed cultures of algae and bacteria were compared to axenic cultures of the same organisms, and a mutual stimulation of growth was observed. A mixed pure culture apparatus was designed in this laboratory to study the algal-bacterial interaction and to test the hypothesis that such an interaction may take place through a diffusable substance or through certain medium-borne conditions, Azotobacter was found to take up a Chlorella-produced exudate, to stimulate protein synthesis, to enhance chlorophyll production and to cause a numerical increase in the interacting Chlorella population. It is not clear whether control is at the environmental, cellular or genetic level in these mixed population interactions. Experimental observations in the model system, taken with field correlations allow one to state that there may be a direct relationship governing the population fluctuations of these two organisms in their natural stream surroundings.

Ecological interactions between zooplankton and photosynthetic bacteria in Crawford Lake, Ontario

The present study was carried out to test the hypothesis that photosynthetic bacteria contribute a large portion of the food of filter feeding zooplankton populations in Crawford Lake, Ontario. The temporal and spatial variations of both groups of organisms are strongly dependent on one another. 14 By using C-Iabelled photosynthetic bacteria. the ingestion and clearance rates of Daphnia pulex, ~. rosea, and Keratella spp were estimated during summer and fall of 1982. These quantitative estimations of zooplankton ingestion and clearence rates on photosynthetic bacteria comprised an original addition to the literature. Photosynthetic bacteria comprised a substantial portion of the diet of all four dominant zooplankton species. The evidence for this is based on the ingestion and clearance rates of the dominant zooplankton species. Ingestion rates of D. pulex and D. rosea ranged 5 5 -1 -1 - -- 5 - -- 5 from 8.3X10 -1 to 14.6XlO -1 cells.ind. hr and 8.1X10 to 13.9X10 cells.ind. hr • Their clearance rates ranged from 0.400 to 1.000 -1 -1 -1 -1 ml.ind. hr. and 0.380 to 0.930 ml.ind. hr • The ingestion and clearance -1 -1 -1 -1 rates of Keratella spp were 600 cell.ind. hr and 0.40 ul.ind. hr respectively. Clearance rates were inversely proportional to the concentration of food cells and directly proportional to the body size of the animals. It is believed that despite the very short reg~neration times of photosynthetic bacteria (3-8 hours) their population densities were controlled in part by the feeding rates of the dominant zooplankton in Crawford Lake. By considering the regeneration times of photosynthetic bacteria and the population clearance rates of zooplankton, it was estimated that between 16 to 52% and 11 to 35% of the PHotosynthetic bacteria were' consumed· by Daphnia· pulex. and Q.. rosea per day. The temporal and spatial distribution of Daphnia pulex, !.. rosea, Keratella quadrata, K. coChlearis and photosynthetic bacteria in Crawford Lake were also investigated during the period of October, 1981 to December, 1982. The photosynthetic bacteria in the lake, constituted a major food source for only those zooplankton Which tolerate anaerobic conditions. Changes in temperature and food appeared to correlate with the seasonal changes in zooplankton density. All four dominant species of zooplankton were abundant at the lake's surface (O-4m) during winter and spring and moved downwards with the thermocline as summer stratification proceeded. Photosynthetic bacteria formed a 2 m thick layer at the chemocline. The position of this photosynthetic bacterial J-ayer changed seasonally. In the summer, the bacterial plate moved upwards and following fall mixing it moved downwards. A vertical shift of O.8m (14.5 to 15.3m) was recorded during the period of June to December. The upper limit of the photosynthetic bacteria in the water column was controlled by dissolved oxygen, and sulfide concentrations While their lower limit was controlled by light intensity. A maximum bacterio- 1 chlorophyll concentration of 81 mg Bchl.l was recorded on August 9, 1981. The seasonal distribution of photosynthetic bacteria was controlledinpart' by ·theg.-"z1ai'_.Q;~.zoopl. ank:tCm;-.Qther -ciactors associated with zooplankton grazing were oxygen and sulfide concentrations.

Factors affecting DNA uptake by mammalian cells

The ability to introduce DNA and express custom DNA sequences in bacteria opened the door for improvements in a large number of fields including agriculture, pharmacology, medicine, nutrition, etc. The ability to introduce foreign DNA sequences into mammalian cells in an efficient manner would have a large impact on therapeutic applications especially gene therapy. The methods in use today suffer from low efficiencies and sometimes toxicity. In this work a number of factors were evaluated for their effect onONA uptake efficiency. The factors studied included exposure to sublethal concentration of hydrogen peroxide which have been show to lead to destabilisation ofthe lysosomes. These exposures have proven to be very toxic to cells when combined with either the calcium phosphate or the lipofectAMINE® transfection methods. Another factor evaluated was exposure to Electro-Magnetic Fields (EMF). This was fuelled by the fact that EMF have been shown to mediate a number of effects on cell structure and/or physiology. EMF exposure by itself was not sufficient to induce the cells to pick up the DNA, therefore its effect on calcium phosphate and lipofectAMINE® was tested. Although some positive results were obtained, the variability of these results exceeded by far any observed enhancements which discouraged any further work on EMF. Also tested was the possible effect the presence of the cytomegalovirus (CMV) sequence might have on DNA uptake (based on previous results in this lab). It was found that the presence ofCMV in the DNA sequence does not enhance uptake or slow down degradation of the internalised DNA. The final factor tested was the effect of basic amino acids on transfection efficiency. It was found that arginine can enhance DNA uptake by about 170% v/ith calcium phosphate and about 200% with LipofectAMINE®. A model was proposed to explain the effect of arginine as well as the lack of effect from other amino acids.

Factors influencing the seasonal changes in primary productivity of the photosynthetic bacteria of Crawford Lake, Ontario

A naturally occurring population of photosynthetic bacteria, located in the meromictic Crawford Lake, was examined during two field seasons (1979-1981). Primary production, biomass, light intensity, lake transparency, pH and bicarbonate concentration were all monitored during this period at selected time intervals. Analysis of the data indicated that (l4C) bacterial photosynthesis was potentially limited by the ambient bicarbonate concentration. Once a threshold value (of 270 mg/l) was reached a dramatic (2 to 10 fold) increase in the primary productivity of the bacteria was observed. Light intensity appeared to have very little effect on the primary productivity of the bacteria, even at times when analyses by Parkin and Brock (1980a) suggested that light intensity could be limiting (i.e., 3.0-5.0 ft. candles). Shifts in the absorption maxima at 430 nrn of the .bacteriochlorophyll spectrum suggested that changes in the species or strain composition of the photosynthetic bacteria had occurred during the summer months. It was speculated that these changes might reflect seasonal variation in the wavelength of light reaching the bacteria. Chemocline erosion did not have the same effect on the population size (biomass) of the photosynthetic bacteria in Crawford Lake (this thesis) as it did in Pink Lake (Dickman, 1979). In Crawford Lake the depth of the chemocline was lowered with no apparent loss in biomass (according to bacteriochlorophyll data). A reverse current was. proposed to explain the observation. The photosynthetic bacteria contributed a significant proportion (10-60%) of the lake1s primary productivitya Direct evidence was obtained with (14C) labelling of the photosynthetic bacteria, indica.ting that the zooplankton were grazing the photosynthetic bacteria. This indicated that some of the photosynthetic bacterial productivity was assimilated into the food chain of the lake. Therefore, it was concluded that the photosynthetic bacteria made a significant contribution to the total productivity of Crawford Lake.

Diversity among bacteria causing blotch disease on the commercial mushroom, agaricus bisporus /

A total of 251 bacterial isolates were isolated from blotched mushroom samples obtained from various mushroom farms in Canada. Out of 251 stored isolates, 170 isolates were tested for pathogenicity on Agaricus bisporus through mushroom rapid pitting test with three distinct pathotypes observed: dark brown, brovm and yellow/yellow-brown blotch. Phenotypic analysis of 83 isolates showed two distinct proteinase K resistant peptide profiles. Profile group A isolates exhibited peptides with masses of 45, 18, 16 and 14 kDa and fiirther biochemical tests identified them as Pseudomonasfluorescens III and V. Profile group B isolates lacked the 16-kDa peptide and the blotch causing bacterial isolates of this group was identified as Serratia liquefaciens and Cedecea davisae. Comparative genetic analysis using Amplified Fragment Length Polymorphism (AFLP) on 50 Pseudomonas sp. isolates (Group A) showed that various blotch symptoms were caused by isolates distributed throughout the Pseudomonas sp. clusters with the exception of the Pseudomonas tolaasii group and one non-pathogenic Pseudomonas fluorescens cluster. These results show that seven distinct Pseudomonas sp. genotypes (genetic clusters) have the ability to cause various symptoms of blotch and that AFLP can discriminate blotch causing from non-blotch causing Pseudomonasfluorescens. Therefore, a complex of diverse bacterial organisms causes bacterial blotch disease

Modifications to the Suzuki reaction and mechanistic insights on the NBS mediated cleavage of benzylidene acetals /

One of the most challenging tasks for a synthetic organic chemist today, is the development of chemo, regio, and stereoselective methodologies toward the total synthesis of macromolecules. r . The objective of my thesis was to develop methodologies towards this end. The first part of my project was to develop highly functionalized chirons from D-glucose, a cheap, chiral starting material, to be utilized in this capacity. The second part of the project dealt with modifying the carbon-carbon bond forming Suzuki reaction, which is utilized quite often as a means of combining molecular sub units in total synthesis applications. As previously stated the first area of the project was to develop high value chirons from D-glucose, but the mechanism of their formation was also investigated. The free radical initiated oxidative fragmentation of benzylidene acetals was investigated through the use of several test-case substrates in order to unravel the possible mechanistic pathways. This was performed by reacting the different acetals with N-bromosuccinimide and benzoyl peroxide in chlorobenzene at 70^C in all cases. Of the three mechanistic pathways discussed in the literature, it was determined, from the various reaction products obtained, that the fragmentation of the initial benzylic radical does not occur spontaneously but rather, oxidation proceeds to give the benzyl bromide, which then fragments via a polar pathway. It was also discovered that the regioselectivity of the fragmentation step could be altered through incorporation of an allylic system into the benzylidene acetal. This allows for the acquisition of a new set of densely functionalized. chiral, valuable synthetic intermediates in only a few steps and in high yields from a-Dglucose. The second part of the project was the utilization of the phosphonium salt room temperature ionic liquid tetradecyltrihexylphosphonium chloride (THPC) as an efficient reusable medium for the palladium catalyzed Suzuki cross-coupling reaction of aryl halides, including aryl chlorides, under mild conditions. The cross-coupling reactions were found to proceed in THPC containing small amounts of water and toluene using potassium phosphate and 1% Pd2(dba)3. Variously substituted iodobenzenes, including electron rich derivatives, reacted efficiently in THPC with a variety of arylboronic acids and afforded complete conversion within 1 hour at 50 ^C. The corresponding aryl bromides also reacted under these conditions with the addition of a catalytic amount of triphenylphosphine that allowed for complete conversion and high isolated yields. The reactions involving aryl chlorides were considerably slower, although the addition of triphenylphosphine and heating at 70 ^C allowed high conversion of electron deficient derivatives. Addition of water and hexane to the reaction products results in a triphasic system in which the top hexane phase contained the biaryl products, the palladium catalyst remained fully dissolved in the central THPC layer, while the inorganic salts were extracted into the lower aqueous phase. The catalyst was then recycled by removing the top and bottom layers and adding the reagents to the ionic liquid which was heated again at 50 ^C; resulting in complete turnover of iodobenzene. Repetition of this procedure gave the biphenyl product in 82-97% yield (repeated five times) for both the initial and recycled reaction sequences.

Some ecological factors affecting the input and population level of total and faecal coliforms and salmonella in Twelve Mile Creek, Lake Ontario and sewage waters near St. Catharines, Ontario

Some Ecological Factors Affecting the Input and Population Levels of Total and Faecal Coliforms and Salmonella in Twelve Mile Creek, Lake Ontario and Sewage Waters Near St. Catharines, Ontario. Supervisor: Dr. M. Helder. The present study was undertaken to investigate the role of some ecological factors on sewage-Dorne bacteria in waters near St. Catharines, Ontario. Total and faecal coliform levels and the presence of Salmonella were monitored for a period of a year along with determination of temperature, pH, dissolved oxygen, total dissolved solids, nitrate N, total phosphate P and ammonium N. Bacteriological tests for coliform analysis were done according to APHA Standard Methods by the membrane filtration technique. The grab sampling technique was employed for all sampling. Four sample sites were chosen in the Port Dalhousie beach area to determine what bacteriological or physical relationship the sites had to each other. The sample sites chosen were the sewage inflow to and the effluent from the St. Catharines (Port Dalhousie) Pollution Control Plant, Twelve Mile Creek below the sewage outfall and Lake Ontario at the Lakeside Park beach. The sewage outfall was located in Twelve Mile Creek, approximately 80 meters from the creek junction with the beach and piers on Lake Ontario. Twelve Mile Creek normally carried a large volume of water from the WeIland Canal which was diverted through the DeCew Generating Station located on the Niagara Escarpment. An additional sample site, which was thought to be free of industrial wastes, was chosen at Twenty Mile Creek, also in the Niagara Region of Ontarioo 3 There were marked variations in bacterial numbers at each site and between each site, but trends to lower_numbers were noted from the sewage inflow to Lake Ontario. Better correlations were noted between total and faecal coliform population levels and total phosphate P and ammonium N in Twenty Mile Creek. Other correlations were observed for other sample stations, however, these results also appeared to be random in nature. Salmonella isolations occurred more frequently during the winter and spring months when water temperatures were minimal at all sample stations except the sewage inflow. The frequency of Salmonella isolations appeared to be related to increased levels of total and faecal coli forms in the sewage effluent. However, no clear relationships were established in the other sample stations. Due to the presence of Salmonella and high levels of total and faecal coliform indicator organisms, the sanitary quality of Lake Ontario and Twelve Mile Creek at the sample sites seemed to be impaired over the major portion of the study period.