Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

Changes in milk fat composition influence its nutritional quality as well as the technological properties of butter. The impact of feed on fat composition is well known; however, limited information is available on the genetic variability of fatty acids in bovine milk. The overall aim of this PhD thesis was to study the genetic variability of fatty acid profile in bovine milk and fat. This type of research needs a large amount of data. Expensive reference analysis is used to measure the fatty acid contents in fat. The first objective of this thesis was to develop an alternative method that could be faster and cheaper than traditional methods. Calibration equations predicting the contents of fatty acid from mid-infrared spectrum were established. The contents of saturated and monounsaturated fatty acids, omega-9, fatty acids with short, medium and long chain were the best predicted. Thanks to the implementation of this method in the Walloon routine milk recording, more than 20,000 milk samples were analyzed. This database permitted to model the variation of fatty acid contents in milk and fat. From these models, the genetic variability of fatty acid profile was shown. The complexity of models increased throughout this project due to the increase of new available data. Differences across 7 dairy breeds were estimated using single and multi-trait mixed models. Milk fat and delta-9 desaturase activity of Jersey and dual purpose Belgian Blue differed significantly from Holsteins. Therefore, the choice of a given breed could modify the fat composition. Heritability values obtained for studied fatty acids with multi-trait mixed model ranged from 0.05 to 0.42. Higher values were observed for saturated compared to unsaturated fatty acids. Moderate heritability estimates were observed for the activity of delta-9 desaturase (0.20) and the hardness of butter (0.27). These two traits were estimated by specific fatty acid ratios. The heritability observed using a multi-trait random regressions test day model for the content of saturated fatty acids (0.42) was similar to the one observed for the percentage of fat (0.37). Considering the impact of selection on fat content, the selection could have a great impact on fat composition. High genetic correlations were observed between some fatty acids having similarities in their synthesis. Heritability and correlations varied through the duration of the lactation. Due to the large number of fatty acids, the estimation of an index, which includes the proper fatty acid profile, could be interesting for a future selection program. This PhD thesis provides the background required by future studies to estimate the impact of animal selection on milk fat composition.

Significant seasonal variation in size at settlement has been observed in newly settled larvae of Dreissena polymorpha in Lake Constance. Diet quality, which varies temporally and spatially in freshwater habitats, has been suggested as a significant factor influencing life history and development of freshwater invertebrates. Accordingly, experiments were conducted with field-collected larvae to test the hypothesis that diet quality can determine planktonic larval growth rates, size at settlement and subsequent post-metamorphic growth rates. Larvae were fed one of two diets or starved. One diet was composed of cyanobacterial cells which are deficient in polyunsaturated fatty acids (PUFAs), and the other was a mixed diet rich in PUFAs. Freshly metamorphosed animals from the starvation treatment had a carbon content per individual 70% lower than that of larvae fed the mixed diet. This apparent exhaustion of larval internal reserves resulted in a 50% reduction of the postmetamorphic growth rates. Growth was also reduced in animals previously fed the cyanobacterial diet. Hence, low food quantity or low food quality during the larval stage of D. polymorpha lead to irreversible effects for postmetamorphic animals, and is related to inferior competitive abilities.

The primary function of adipose tissue is to store energy in the form of triacylglycerol, which is hydrolyzed to fatty acids to supply other tissues with energy. While insulin promotes the storage of triacylglycerol, catecholamines stimulate its hydrolysis. The development of type II diabetes is strongly associated with obesity, indicating a role of triacylglycerol metabolism in the pathogenesis of diabetes. Caveolae are plasma membrane invaginations found in most cells but are highly abundant in adipocytes. Insulin receptors are localized in caveolae and their function depends on intact caveolae structures. In the present thesis work, mass spectrometry-based methodology allowed identification of a number of new proteins and their posttranslational modifications in caveolae of human adipocytes. Variable N-terminal acetylation and phosphorylation of caveolin-1α and caveolin-1β were identified, which might regulate the function of caveolae. The transcription regulator protein PTRF was identified as the major caveolae associated protein. Specific proteolytic modifications of PTRF at the cytosolic surface of caveolae and phosphorylation on nine serine and one threonine residues were identified. Moreover, insulin induced translocation of PTRF from the plasma membrane to the nucleus. PTRF was previously shown to regulate the activity of both RNA polymerase I and polymerase II, thus a role of PTRF in mediating the anabolic action of insulin on protein synthesis and gene transcription is proposed. PTRF was also involved in an extranuclear function in the hormonal regulation of triacylglycerol metabolism in caveolae. PTRF was colocalized with the triacylglycerol regulator proteins perilipin and hormone-sensitive lipase (HSL) in the triacylglycerol-synthesizing caveolae subclass. We showed that, while perilipin was translocated to the plasma membrane, both PTRF and HSL were translocated from the plasma membrane to the cytosol as a complex in response to insulin. The perilipin recruited to the plasma membrane was highly threonine phosphorylated. By mass spectrometry, three phosphorylated threonine residues were identified and were located in an acidic domain in the lipid droplet targeting domain of perilipin. The insulin-induced recruitment of perilipin to the plasma membrane might, therefore be phosphorylation-dependent. Isoproterenol, which stimulates hydrolysis of triacylglycerol, induced a complete depletion of perilipin B from the plasma membrane, suggesting a function of perilipin B to protect newly synthesized triacylglycerol in caveolae from being hydrolyzed by HSL. The location of PTRF and HSL was not affected by isoproterenol, indicating that insulin is acting against a default presence of PTRF and HSL in caveolae. Taken together, this thesis expands our knowledge about caveolae and provided valuable information about their involvement in novel roles, particularly in the hormonal regulation of triacylglycerol metabolism.

Anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert most of their actions by binding to cannabinoid receptors. The effects of the endocannabinoids are short-lived due to rapid cellular accumulation and metabolism, for AEA, primarily by the enzymes fatty acid amide hydrolase (FAAH). This has led to the hypothesis that by inhibition of the cellular processing of AEA, beneficial effects in conditions such as pain and inflammation can be enhanced. The overall aim of the present thesis has been to examine the mechanisms involved in the cellular processing of AEA and how they can be influenced pharmacologically by both synthetic natural compounds. Liposomes, artificial membranes, were used in paper I to study the membrane retention of AEA. The AEA retention mimicked the early properties of AEA accumulation, such as temperature-dependency and saturability. In paper II, FAAH was blocked by a selective inhibitor, URB597, and reduced the accumulation of AEA into RBL2H3 basophilic leukaemia cells by approximately half. Treating intact cells with the tyrosine kinase inhibitor genistein, an isoflavone found in soy plants and known to disrupt caveolae-related endocytosis, reduced the AEA accumulation by half, but in combination with URB597 no further decrease was seen. Further on, the effects of genistein upon uptake were secondary to inhibition of FAAH. The ability to inhibit the accumulation and metabolism of AEA was shared by several flavonoids (shown in paper III). In paper IV, the isoflavone biochanin A and URB597 had effects in vivo, in a model of persistent pain, effects decreased by the cannabinoid receptor 1 antagonist AM251. In paper VI, the cellular processing of the endocannabinoid metabolites following degradation was examined, a mechanism poorly understood. It was found that nitric oxide (NO) donors significantly increased the retention of tritium in cell membranes following incubation with either tritiated AEA or 2-AG. Further experiments revealed that the effect of NO donors mainly involves the arachidonate part of the molecules. Inhibition of FAAH completely reduced the effect of NO donors in cells with a large FAAH component, indicating that the effects were downstream of the enzyme. These results suggest that the cellular processing of endocannabinoids can be affected in a manner of different ways by pharmacological manipulation in vitro and that naturally occurring flavonoid compounds can interact with the endocannabinoid system.

Feeding diets rich in rapidly fermentable non-structural carbohydrates can lead to the development of ruminal acidosis. This study was conducted to determine if the duration of time that cattle are fed a high-grain diet affects their absorption of short-chain fatty acids (SCFA) and susceptibility to, and recovery from, ruminal acidosis. Sixteen Angus heifers (BW ± SEM, 261 ± 6.1 kg) were assigned to 1 of 4 blocks, and fed a backgrounding diet consisting of 60% barley silage, 30% barley grain, and 10% supplement (DM basis). Within block, cattle were randomly assigned to 1 of 2 treatments differing in the number of days they were fed the high-grain diet prior to an acidosis challenge: 34 d for long-adapted (LA) and 8 d for short-adapted (SA). All cattle were exposed to the same 20-d dietary transition using 5 dietary steps until achieving the final diet that contained 9% barley silage, 81% barley grain, and 10% supplement (DM basis). Data were collected during an 8-d baseline period (BASE), on the d of the acidosis challenge (CHAL), and during two consecutive 8 d recovery periods (REC1 and REC2). Ruminal acidosis was induced by restricting feed to 50% of DMI:BW for 24 h followed by an intraruminal infusion of ground barley at 10% DMI:BW. Cows were then given their regular diet allocation 1 h after the intraruminal infusion. The duration of time fed the high-grain diet did not affect ruminal pH, lactate, or SCFA concentrations (P > 0.050). However, during BASE and on the day of CHAL the SA heifers experienced greater linear (P = 0.031), quadratic (P = 0.016), and cubic (P = 0.008) between day change in the duration of time that pH was < 5.5 than LA heifers. Relative to BASE, inducing acidosis increased daily duration (531 to 1020 min/d; P < 0.001) and area (176 to 595 (min × pH)/d; P < 0.001) that pH was < 5.5. Inducing ruminal acidosis also increased the daily mean (0.3 to 11.4 mM; P = 0.013) and maximum (1.3 to 29.3 mM; P = 0.008) rumen fluid lactate concentrations relative to BASE, suggesting that an acute bout of ruminal acidosis was induced. In addition, a treatment × day interaction for the duration that pH was < 5.5 during REC1 suggests that LA cattle tended to recover from the CHAL more rapidly than SA cattle (P = 0.085). Indeed, analysis of covariance confirmed that the LA heifers experienced a quicker linear (P = 0.019) recovery over time from CHAL. The greater rate of recovery possibly resulted from the LA heifers having greater rates of both fractional butyrate (45 vs. 36 %/h; P = 0.019) and propionate absorption (42 vs. 34 %/h; P = 0.045), and tending to have greater rates, on an absolute basis, of butyrate absorption (94 vs. 79 mmol/h; P = 0.087) iii and, on a fractional basis, of total SCFA absorption (37 vs. 32 %/h; P = 0.100). Treatment × period interactions revealed that LA heifers had greater serum D-lactate concentrations (P = 0.003), and fractional rates of lactate absorption (P = 0.024) than SA heifers, during CHAL and REC1, respectively. When treatments were pooled, the absorption (%/h and mmol/h) of acetate, propionate, butyrate, and total SCFA increased between REC1 and REC2, with intermediate values for BASE (P ≤ 0.05). Corresponding to a reduction in absorption during REC1 (2 d post CHAL), saliva production (kg/h; P = 0.018) increased between BASE and REC1, with intermediate values for REC2. These results indicate that the duration of time cattle are fed a high-grain diet may stabilize rumen pH, both prior to and after an induced bout of acute ruminal acidosis, likely through increased ruminal absorptive capacity for SCFA and lactate. In addition, this study found evidence to suggest that beef cattle possess the ability to increase saliva secretion in order to compensate for decreased absorptive capacity.

While all women hope to deliver a healthy baby, approximately 3-5% babies are affected by birth defects. Birth defects can occur naturally or be induced by teratogens. Alcohol is a known teratogen that causes fetal alcohol spectrum disorder (FASD), the most commonly known cause of neurobehavioural and neurodevelopmental deficits. Individuals affected with FASD are likely to be involved with or require additional assistance from healthcare, education, social services, and justice sectors. Due to this immense burden, effective prevention of FASD can have a major public impact. Prevention of FASD can occur at different levels: primary prevention (preventing alcohol-induced birth defects from occurring in the first place); secondary prevention (preventing alcohol-induced birth defects from developing or progressing); tertiary prevention (improving the outcome of individuals affected with FASD); and quaternary prevention (preventing another child from being affected with FASD). The objective of this thesis was to explore a multilevel birth defect and FASD prevention strategy. Primary prevention by was investigated by maternal multivitamin supplementation to optimize fetal growing conditions, as alcoholics are commonly deficient in nutrients. A meta-analysis of maternal multivitamin supplementation demonstrated a decreased risk for certain congenital anomalies and pediatric cancers. Secondary prevention was investigated by a randomized double-blinded placebo-controlled evaluating the ability of high doses of antioxidants (vitamin C and vitamin E) to mitigate the effects of prenatal alcohol exposure. The study was ceased due to safety concerns regarding high doses of vitamin C and vitamin E in preeclamptic studies. Tertiary prevention was investigated by anonymous meconium screening of babies of Grey-Bruce, Ontario residents delivering at or transferred to St. Joseph’s Health Care in London, Ontario. A 30% prevalence of fatty acid ethyl esters (FAEE) positive meconium was observed at this high-risk unit. Meconium screening is also a means of quaternary prevention since positive screens also identify mothers who were unable to stop consuming alcohol after 13 weeks of pregnancy, and therefore are at risk of delivering another child who is prenatally exposed to alcohol. The identification and engagement of these mothers into treatment programs constitutes primary prevention of FASD in subsequent pregnancies.

Mit dem Beginn der Industrialisierung stieg in den westlichen Kulturen rasant die Prävalenz von Krankheitsbildern wie Adipositas, arterieller Hypertonie, Typ-2-Diabetes Mellitus und Hyperlipidämie, die als Cluster eines multifaktoriellen Krankheitsbildes namens „Metabolisches Syndrom“ (MTS) verstanden werden. Tierstudien, in denen durch die Inhibition der Fettsäuresynthase (FASN) ein rapider Abfall des Körpergewichts in Mäusen erzeilt wurden, bestätigen zunehmend genetische Prädispositionen als ursächlich für die Ausbildung des MTS. Um herauszufinden ob und in welchem Ausmaß das FASN-Gen mit humanen Merkmalen des MTS assoziiert ist, wurde das Gen in 48 nicht verwandten ostdeutschen Probanden sequenziert. Acht repräsentative tagging-SNPs wurden dabei identifiziert, anschließend in 1311 deutschen Probanden (Erwachsene) genotypisiert und in Fall-Kontroll-Studien zwischen 389 schlanken Probanden (BMI ≤ 25kg/m²) vs. 446 adipösen Teilnehmern (BMI ≥ 30kg/m²) sowie zwischen 502 glukosetoleranten Probanden (NGT) vs. 640 Probanden mit Typ-2-Diabetes (T2D) miteinader verglichen. Für den Polymorphismus rs2229422 (P = 1.3x10-5 adjustiert auf Alter, Geschlecht und Diabetes-status) konnten die stärksten Assoziationen mit BMI und weiteren Merkmalen der Fettleibigkeit identifiziert werden (adjustiert P < 0.05). Des Weiteren wurde der zuvor in der Literatur beschriebene protektive Einfluss der Val1483Ile Substitution (rs2228305) gegenüber Adipositas, sowie der geschlechts-spezifische Effekt auf den BMI bestätigt (adjustiert, P = 0.03).

Adoption of no-till (NT) and reduced tillage management is widespread on the Canadian prairies and together form the basic platform of soil management upon which most crop production is based. Elimination of tillage in cropping systems changes the physical and chemical characteristics of the soil profile and can affect crop growth and ultimately yield. As such, understanding how soil biota, as drivers of nutrient turnover, adapt to NT is important for maximizing crop productivity and mitigating environmental damage in agroecosystems. This work aims to achieve a greater understanding of microbial community structure and function in long-term NT versus conventionally tilled (CT) soils. Community phospholipid and DNA fingerprinting did not reveal any consistent tillage-induced shifts in microbial community structure, but demonstrated a clear influence of depth within the soil profile. While tillage did not result in broad changes in the community structure, total, bacterial and fungal biomass was consistently greater near the surface of NT soils. Further examination at one site near Swift Current, SK revealed differences in microbial biomass and community structure in NT and CT in field-formed aggregate size fractions. Measurement of mineralization and nitrification at the same site indicated that differences in the early-season turnover of N may be related to physical rather than microbial differences in NT and CT soils. Potential nitrification was higher prior to seeding than mid-season, was not affected by tillage and was correlated with ammonia oxidizer population size of archaea, but not bacteria. This work indicates that edaphic soil properties and spatial distribution of resources in the soil profile, rather than tillage management, are the primary factors driving microbial community structure in these soils.

The nutritional value of flaxseed meal (FSM), a by-product of the flax crushing industry, has not been evaluated properly for use within swine rations. A series of experiments were conducted to determine the nutritional profile of this novel feed ingredient for pigs.<p>The analysis of FSM revealed that it contains, on a dry matter (DM) basis, 133 g/kg ether extract (EE), 345 g/kg crude protein (CP), 60 g/kg ash, 164 g/kg ADF, 250 g/kg NDF, 102 g/kg crude fibre, 14 g/kg starch and 9 g/kg phosphorus. The gross energy (GE) content of the meal was 5.2 Mcal/kg DM. The ether extract fraction was characterized by, as a percent of total fat, 46.6% á-linolenic acid, an omega-3 fatty acid. Palmitic, stearic, oleic and linoleic acids accounted for 9.5, 4.8, 20.7 and 18.4% of the total fat content respectively. The crude protein content was well balanced for all amino acids with the exception of lysine (4.1% of CP), the level of which falls below that of the requirements for growing pigs (5.3% of CP for pigs 20-50 kg). The apparent digestibility of DM, nitrogen, ash, EE and GE as well as determination of the DE and NE content of FSM was determined for both growing pigs (32 pigs, initial weight 70 ± 3 kg) and gestating sows (26 pigs, parities 2 4). Animals were fed wheat/barley based diets containing 0, 10, 20 or 30% FSM. Faecal grab samples were collected for 3 days after a dietary adaptation period. The apparent digestibility of nutrients in FSM was determined both by regression and by difference calculations. As calculated by difference, the apparent digestibility coefficients for DM, nitrogen, ash, and GE were 63.0, 60.8, 22.3 and 60.5% respectively for growing pigs. The values obtained for sows were 64.1, 58.8, 20.8, 94.9 and 65.4% for DM, nitrogen, ash, EE and GE respectively. The DE content was 3.37 Mcal/kg for growing pigs and 3.52 Mcal/kg for sows. Net energy was then estimated by use of a prediction equation to be 2.34 and 2.44 Mcal/kg for growing pigs and sows. <p>An experiment was conducted to evaluate the growth performances and carcass fatty acid profiles of pigs fed with graded levels of FSM. A total of 200 pigs (100 barrows, 100 gilts; initial weight 32 ± 4 kg) were blocked by gender and housed in groups of 5 pigs per pen. The experiment was divided into three phases for pigs 32-60 kg, 60-85 kg and 85-115 kg. Each group was assigned to one of four dietary treatments containing 0, 5, 10 or 15% FSM at the expense of wheat and soybean meal. At the time of market, 6 pigs per treatment group were randomly selected for carcass fatty acid analysis, and backfat and rib-end loin samples were collected. The average daily gains, average daily feed intakes and gain to feed ratios were not affected by dietary treatment (P > 0.05). Inclusion of 15% dietary FSM increased the ALA content from 11 to 47 (± 0.8) mg/g of backfat (P < 0.001) and from 5 to 10 (± 0.4) mg/g of loin tissue (P < 0.001). Increasing dietary FSM decreased the saturated fatty acid content of backfat (P < 0.01). <p> The final experiment was designed to determine the availability of phosphorus in semi-synthetic diets containing FSM, and to determine the effects of microbial phytase inclusion of this availability. Five treatment groups, 8 barrows (45 ± 4 kg initial weight) each, were fed a diet containing 30% FSM with increasing levels of phytase (0, 575, 1185, 2400 and 2570 FTU/kg). Apparent P digestibility increased from 20.6 to 61.3% with the inclusion of up to 2570 FTU/kg microbial phytase (P < 0.001), and followed a quadratic response pattern with an R2 value of 0.96. A broken-line analysis estimated the optimal phytase inclusion level to be 1415 FTU/kg of diet. Inclusion of just 575 FTU/kg accounted for half of the response, improving the apparent P digestibility by 20% and reducing P excretion by 850 mg/kg dry matter intake.