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Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a human intestinal pathogen, which can cause severe disease. EHEC O157:H7 is responsible for outbreaks of diarrhea and hemorrhagic colitis. EHEC produces a potent cytotoxin known as Vero (Shiga-like) cytotoxin, which causes diarrhea-associated hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Current treatment remains predominantly supportive in nature because antibiotics and non-steroidal anti-inflammatory drugs exacerbate the condition. Therefore, alternative therapeutic approaches that will prevent the EHEC colonization without the release of toxins need to be delineated. Understanding the pathobiology of disease is likely to yield novel approaches to interrupt the infectious process. My hypothesis was that pathogen-derived effectors associate with lipid rafts and, thereby, promote the recruitment of host signal transduction proteins to lipid rafts in response to EHEC O157:H7 infection. In this thesis, specific host signalling pathways hijacked by EHEC O157:H7, through lipid raft signalling platforms, to elicit pathogenic effects are studied using complementary approaches, including epithelial model cell lines and an animal model of infection (Citrobacter rodentium challenge of mice). A lack of osteopontin resulted in decreased attaching effacing lesions and reduced colonic epithelial cell hyperplasia in response to C. rodentium infection. These findings suggest that C. rodentium, mimicking EHEC O157:H7 infection, is capable of utilizing host cell components to elicit its pathogenic effects. In vitro data showed that EHEC O157:H7 effector proteins manipulate cell signalling through lipid rafts employed as platforms to recruit and activate host second messengers. PKC and PI3K activation led to attaching and effacing lesions, disruption of tight junctions, and the initiation of both innate and adaptive host immune responses. The results pointed towards a role for atypical PKC in EHEC-induced attaching and effacing lesion formation. The role of lipid rafts in EHEC O157:H7 pathogenesis was also studied using Citrobacter rodentium-infected Niemann-pick type C (NPC) mice. Infection of NPC mice, which lack lipid rafts, with C. rodentium resulted in delayed colonization and delayed onset of attaching-effacing lesion formation, compared with infected wild type mice. C. rodentium-infected NPC mice also demonstrated reduced colonic epithelial hyperplasia and decreased secretion of the pro-inflammatory cytokine, interferon-γ. Taken together, the findings presented in this thesis highlight the importance of host cell signal transduction cascades in EHEC O157:H7 disease pathogenesis, and demonstrate a role for lipid rafts and OPN in mediating host cell signaling responses to non-invasive enteric microbial pathogens.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a human intestinal pathogen, which can cause severe disease. EHEC O157:H7 is responsible for outbreaks of diarrhea and hemorrhagic colitis. EHEC produces a potent cytotoxin known as Vero (Shiga-like) cytotoxin, which causes diarrhea-associated hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Current treatment remains predominantly supportive in nature because antibiotics and non-steroidal anti-inflammatory drugs exacerbate the condition. Therefore, alternative therapeutic approaches that will prevent the EHEC colonization without the release of toxins need to be delineated. Understanding the pathobiology of disease is likely to yield novel approaches to interrupt the infectious process. My hypothesis was that pathogen-derived effectors associate with lipid rafts and, thereby, promote the recruitment of host signal transduction proteins to lipid rafts in response to EHEC O157:H7 infection. In this thesis, specific host signalling pathways hijacked by EHEC O157:H7, through lipid raft signalling platforms, to elicit pathogenic effects are studied using complementary approaches, including epithelial model cell lines and an animal model of infection (Citrobacter rodentium challenge of mice). A lack of osteopontin resulted in decreased attaching effacing lesions and reduced colonic epithelial cell hyperplasia in response to C. rodentium infection. These findings suggest that C. rodentium, mimicking EHEC O157:H7 infection, is capable of utilizing host cell components to elicit its pathogenic effects. In vitro data showed that EHEC O157:H7 effector proteins manipulate cell signalling through lipid rafts employed as platforms to recruit and activate host second messengers. PKC and PI3K activation led to attaching and effacing lesions, disruption of tight junctions, and the initiation of both innate and adaptive host immune responses. The results pointed towards a role for atypical PKC in EHEC-induced attaching and effacing lesion formation. The role of lipid rafts in EHEC O157:H7 pathogenesis was also studied using Citrobacter rodentium-infected Niemann-pick type C (NPC) mice. Infection of NPC mice, which lack lipid rafts, with C. rodentium resulted in delayed colonization and delayed onset of attaching-effacing lesion formation, compared with infected wild type mice. C. rodentium-infected NPC mice also demonstrated reduced colonic epithelial hyperplasia and decreased secretion of the pro-inflammatory cytokine, interferon-γ. Taken together, the findings presented in this thesis highlight the importance of host cell signal transduction cascades in EHEC O157:H7 disease pathogenesis, and demonstrate a role for lipid rafts and OPN in mediating host cell signaling responses to non-invasive enteric microbial pathogens.

Individuals with abdominal obesity are at higher risk for developing type 2 diabetes, predisposing cardiovascular events and insulin resistance. Low glycemic index (GI) diets may be beneficial in the management of insulin resistance. Insulin resistance is associated with increased intramyocellular lipid (IMCL) content as measured by proton nuclear magnetic resonance spectroscopy (1H-MRS). The primary objective of this thesis was to determine whether a low GI diet can improve insulin sensitivity by reducing IMCL of skeletal muscle. One hundred and twenty-one male and female participants aged 30 to 70 years (mean+SD, 53+10)) with abdominal obesity, entered a 4 to 6 week weight-maintaining, low-fat dietary advice run-in phase. Of the 121 eligible participants, 95 completed the run-in phase and were randomly assigned to either a low-GI (LGID, n=48) or high-GI diet (HGID, n=47) for 24 weeks. Participants underwent a 75g oral glucose tolerance test (OGTT) and had soleus-muscle IMCL measured by 1H-MRS at the beginning and end of the intervention period. Insulin sensitivity was assessed by the homeostatic model assessment index (HOMA) and the insulinogenic index (ISI) was calculated for insulin secretion. At the end of the run-in phase, there were significant reductions in serum total-, LDL-, and HDL-cholesterol (all, p<0.0001) and an increase in fasting plasma glucose (p<0.05). In 57 participants who wore a continuous glucose monitoring system for 24 hours during the run-in period, a total of 30% (p<0.001) of the variation in the incremental area under the blood glucose curve after self-selected breakfast meals was explained by GI. After 24 weeks, diet GI was significantly lower in the LGID than HGID group (55.5+3.1 vs 63.9+3.1, p<0.0001). Plasma glucose 60 minutes after the OGTT was significantly lower on the LGID than at baseline (p<0.05) and there was a non-significant trend towards an increase in ISI (p=0.07). On the HGID, ISI increased significantly from baseline (p<0.01). It is concluded that the LGID reduced 60 minute plasma glucose but did not significantly affect IMCL or insulin sensitivity in individuals with abdominal obesity.

The Arctic is considered as a focal region in the ongoing climate change debate. The currently observed and predicted climate warming is particularly pronounced in the high northern latitudes. Rising temperatures in the Arctic cause progressive deepening and duration of permafrost thawing during the arctic summer, creating an ‘active layer’ with high bioavailability of nutrients and labile carbon for microbial consumption. The microbial mineralization of permafrost carbon creates large amounts of greenhouse gases, including carbon dioxide and methane, which can be released to the atmosphere, creating a positive feedback to global warming. However, to date, the microbial communities that drive the overall carbon cycle and specifically methane production in the Arctic are poorly constrained. To assess how these microbial communities will respond to the predicted climate changes, such as an increase in atmospheric and soil temperatures causing increased bioavailability of organic carbon, it is necessary to investigate the current status of this environment, but also how these microbial communities reacted to climate changes in the past. This PhD thesis investigated three records from two different study sites in the Russian Arctic, including permafrost, lake shore and lake deposits from Siberia and Chukotka. A combined stratigraphic approach of microbial and molecular organic geochemical techniques were used to identify and quantify characteristic microbial gene and lipid biomarkers. Based on this data it was possible to characterize and identify the climate response of microbial communities involved in past carbon cycling during the Middle Pleistocene and the Late Pleistocene to Holocene. It is shown that previous warmer periods were associated with an expansion of bacterial and archaeal communities throughout the Russian Arctic, similar to present day conditions. Different from this situation, past glacial and stadial periods experienced a substantial decrease in the abundance of Bacteria and Archaea. This trend can also be confirmed for the community of methanogenic archaea that were highly abundant and diverse during warm and particularly wet conditions. For the terrestrial permafrost, a direct effect of the temperature on the microbial communities is likely. In contrast, it is suggested that the temperature rise in scope of the glacial-interglacial climate variations led to an increase of the primary production in the Arctic lake setting, as can be seen in the corresponding biogenic silica distribution. The availability of this algae-derived carbon is suggested to be a driver for the observed pattern in the microbial abundance. This work demonstrates the effect of climate changes on the community composition of methanogenic archae. Methanosarcina-related species were abundant throughout the Russian Arctic and were able to adapt to changing environmental conditions. In contrast, members of Methanocellales and Methanomicrobiales were not able to adapt to past climate changes. This PhD thesis provides first evidence that past climatic warming led to an increased abundance of microbial communities in the Arctic, closely linked to the cycling of carbon and methane production. With the predicted climate warming, it may, therefore, be anticipated that extensive amounts of microbial communities will develop. Increasing temperatures in the Arctic will affect the temperature sensitive parts of the current microbiological communities, possibly leading to a suppression of cold adapted species and the prevalence of methanogenic archaea that tolerate or adapt to increasing temperatures. These changes in the composition of methanogenic archaea will likely increase the methane production potential of high latitude terrestrial regions, changing the Arctic from a carbon sink to a source. / Die Arktis ist in den gegenwärtigen Diskussionen zum Klimawandel von besonderem Interesse. Die derzeitig beobachtete globale Erwärmung ist in den hohen nördlichen Breiten besonders ausgeprägt. Dies führt dazu, dass ehemals gefrorene Böden zunehmend tiefer auftauen und daher im Boden enthaltene Kohlenstoffquellen für die mikrobielle Umsetzung und Mineralisierung zur Verfügung stehen. Aufgrund dieser Prozesse entstehen klimarelevant Gase, darunter Kohlendioxid und Methan, die aus den Böden und Sedimenten freigesetzt werden können. Wenn man bedenkt, dass in den nördlichen Permafrostgebieten die Hälfte des weltweit unter der Bodenoberfläche gelagerten Kohlenstoffs gelagert ist, wird die Bedeutung dieser Region für das Verständnis des globalen Kohlenstoffkreislaufes und der möglichen Treibhaus-gasemissionen sichtbar. Trotz dieser Relevanz, sind die am Kohlenstoffumsatz beteiligten Mikroorganismen in der Arktis wenig untersucht und ihre Anpassungsfähigkeit an die gegenwärtigen Klimaveränderungen unbekannt. Die vorliegende Arbeit untersucht daher, wie sich Klimaveränderungen in der Vergangenheit auf die Anzahl und Zusammensetzung der mikrobiellen Gemeinschaften ausgewirkt haben. Dabei liegt ein besonderer Fokus auf die methanbildenden Archaeen, um das Verständnis der mikrobiellen Methandynamik zu vertiefen. Im Rahmen dieser Arbeit wurden drei Bohrkerne aus zwei verschiedenen Standorten in der russischen Arktis untersucht, darunter terrestrischer Permafrost und Seesedimente aus Sibirien und Chukotka, Russland. Mittels der Identifikation und Quantifizierung von mikrobiellen Genen und charakteristischen Bestandteilen der mikrobiellen Zellmembran war es möglich, fossile mikrobielle Gemeinschaften in Seesedimenten mit einem Alter von bis zu 480 000 Jahren und in Permafrostablagerungen mit einem Alter von bis zu 42 000 Jahren zu rekonstruieren. Es wurde gezeigt, dass es während vergangener warmen Perioden zu einem Wachstum von Bakterien und Archaeen in allen untersuchten Standorten gekommen ist. Dieser Trend konnte auch für die Gemeinschaft der methanogenen Archaeen gezeigt werden, die während warmen und insbesondere feuchten Klimabedingungen in großer Anzahl und Diversität vorhanden waren, was wiederrum Rückschlüsse auf mögliche Methanemissionen erlaubt. In den terrestrischen Permafroststandorten wird der Temperaturanstieg als direkte Ursache für die gefundene Reaktion der mikrobiellen Gemeinschaft vermutet. Im Gegenzug dazu, führte der Temperaturanstieg im untersuchten arktischen See wahrscheinlich zu einer erhöhten Primärproduktion von organischem Kohlenstoff, die wiederum das Wachstum der Mikroorganismen antrieb. Weiterhin konnte im Rahmen dieser Arbeit gezeigt werden, dass Methanosarcina-verwandte Spezies in der Russischen Arktis weit verbreitet sind und sich an veränderte Umweltbedingungen gut anpassen können. Im Gegensatz dazu stehen Vertreter von Methanocellales und Methano-microbiales, die nicht in der Lage sich an veränderte Lebensbedingungen anzupassen. Im Rahmen dieser Arbeit konnte erstmalig gezeigt werden, dass es in früheren Warmphasen zu einem vermehrten Wachstum der an der Umsetzung des organischen Kohlenstoffs beteiligten Mikroorganismen in der Russischen Arktis gekommen ist. Im Zusammenhang mit der zukünftigen Erwärmung der Arktis kann also von einer Veränderung der am Kohlenstoffkreislauf beteiligten Mikroorganismen ausgegangen werden kann. Mit den steigenden Temperaturen werden sich einige Vertreter der methanproduzierenden Mikroorganismen an die veränderten Bedingungen anpassen können, während Temperatur-empfindliche Vertreter aus dem Habitat verdrängt werden. Diese Veränderungen in der mikrobiellen Gemeinschaft können die Methanproduktion der hohen noerdlichen Breiten erhoehen und dazu beitragen, dass aus der Arktis als eine Kohlenstoffsenke eine Kohlenstoffquelle wird.

Chemical, physical and sensory properties of omega-3 fatty acid enriched chicken frankfurters developed with flax oil and microencapsulated fish oil at 1.2%, 2.4% and 3.6% were evaluated. Four replicate batches of frankfurters were produced for texture profile analysis and TBARS for assessment of lipid oxidation over four weeks of refrigerated storage. Gas chromatograph analysis indicated that omega-3 fatty acid levels increased (p<0.05) with flax and fish oils treatments resulting in a shift in omega-6/omega-3 with no increase in lipid oxidation over the storage period. The two highest levels of fish oil resulted in increased redness, hardness, gumminess and chewiness (p<0.05) with the highest fish oil having the lowest rating for acceptability. 1.2 and 2.4% flax oil and 1.2% fish oil samples were softer and juicier than commercial frankfurters. Addition of oils high in omega-3 fatty acids to chicken-based frankfurters can result in product resistant to oxidation and acceptable to consumers.

Although quality of commercial fish oil is of the upmost importance to both suppliers and consumers, it can be difficult to maintain due to rapid lipid oxidation attributable to the high levels of EPA and DHA. Fish oil quality can be assessed in a number of ways; this paper focuses on ethyl ester (EE) content and oxidation products. Fish oil supplements are sold as both triacylglycerols (TAG) and EE. TAG products are more resistant to oxidation, have better bioavailability and are generally considered to be of higher quality. Here, a method is described to quantify EE in fish oil using solid phase microextraction (SPME) headspace analysis and gas chromatography-mass spectrometry (GCMS). A related aspect of quality is state of oxidation and although the causes of oxidation in fish oil are well known, there is little research on the kinetics of the oxidation process. The work presented here monitors hydroperoxides to model the kinetics of oxidation in two commercially available fish oil supplements by fitting the data to the Arrhenius model. It was determined that the same mechanisms of oxidation hold at temperatures ? 40 °C and thus, this temperature was used in the final stages of this work where accelerated stability testing of fish oil was conducted. Currently, taste panels are the only reliable method to assess the sensory properties of fish oil, but these are costly and subjective. Described here is an alternative method using SPME-GCMS to monitor volatile oxidation products. Two different statistical methods were used to identify oxidative volatiles that correlate with sensory characteristics of fish oil. First, stepwise discriminant function analysis (DFA) was used to identify volatiles that could be used to classify oil as acceptable or unacceptable based on sensory analysis. Principal component analysis (PCA) and linear regression were then applied with greater success. Both techniques identified similar oxidative volatiles as being important to sensory properties. It is anticipated that these methods could be adopted by fish oil manufacturers as measures of quality.

During nutrient-replete growth of microalgae, new photosynthate is allocated toward three different biochemical pools: light harvesting compounds, the biosynthetic apparatus and energy storage. The mechanisms governing allocation of photosynthate between the energy storage compounds carbohydrate and lipid are not well understood. For biofuel production, it is desirable to identify conditions and algal strains that allocate maximum amounts of photosynthate to lipid. This thesis assessed the allocation of photosynthate toward the energy storage pool, and to lipid vs. carbohydrate, at two light levels and during ongoing nitrogen-starvation, for two diatoms and two chlorophytes. Nitrogen-starvation resulted in an increase in the photosynthate allocated toward energy storage, however the magnitude of change was determined by a combination of species and light level. Of the four species studied, the diatom Chaetoceros muelleri, grown in high light, accumulated lipid during N-starvation at a relatively high rate, making it a good candidate for biofuel production.

A common strategy employed to destroy harmful bacteria is to disrupt the bacterial membrane through the action of pore-forming anti-microbial peptides. The manner in which the peptides arrange themselves spatially to form a pore in the membrane, which is important for understanding both the mechanism of pore formation and pore function, is a topic of current debate. We contrast the response of a model membrane bilayer to the presence of solid, cylindrical nanoparticle insertions, when the bilayer is composed of persistent worm-like chains and when it is composed of flexible Gaussian chains. We use self-consistent field theory, with the appropriate single-chain propagator, to describe the amphiphilic star-like triblock copolymers composing the membrane and the solvent. The nanoparticle surfaces are designed to have patches that prefer either the solvent or the tail groups of the copolymers, and the nanoparticles are fixed in space. Using this model with polymers in the lamellar phase, we investigate the question of pore-formation, nanoparticle insertion and hydrophobic mismatch in lipid bilayers and the effect that chain rigidity has on these particular interactions. We find that the main effect of increased chain rigidity is that it increases the free energy scaling and the significance of the energy barriers associated with these pore-forming processes. These results demonstrate the importance of using a more realistic persistent chain when modelling pore formation. / NSERC, CFI, SHARCNET