Preeclampsia (PE) is a potentially fatal complication of pregnancy characterized by an increase in blood pressure (>140/90 mmHg) and proteinuria (>300 mg/24 hrs), often accompanied by edema. Symptoms of PE start after 20 weeks of gestation. If PE remains untreated, it can lead to eclampsia, grand-mal seizures responsible for most fatalities. PE is believed to affect 2-10% of pregnancies worldwide, and claims the lives of over 75,000 mothers and 500,000 newborns yearly. No therapeutic agents have been developed to prevent or cure PE. Part of the reason for this is the absence of a complete understanding of the pathogenesis of this disease. PE has long been regarded as a “disease of theories”, and the pathophysiology of PE continues to be the subject of debate. Nonetheless, several abnormalities have been observed to precede established, clinical PE and have in turn been proposed to be involved in the causation of this disease, all with involvement of the mother's placenta as a central feature. Removal of placenta is the only cure for PE and results in a rapid resolution of the symptoms. Thus, the placenta remains an organ of substantial interest and many research groups have attempted to identify abnormal placental features occurring in PE. None of these studies have focused on less abundant, low molecular weight (LMW) biomolecules, which play important roles in the pathophysiology of many diseases. There are a number of alterations that are believed to affect the placenta and contribute to the pathogenesis of PE. The most widely accepted ones include hypoxia, oxidative stress, and an increase of pro-inflammatory mediators in the mother's placenta. The goal of my initial study was to identify which of these hypothesized causative pathways has a significance in the etiology of this syndrome as well as to investigate which less abundant, low molecular weight biomolecules change in response to these abnormalities. For this purpose, we first adapted and optimized a previously developed methodology that studied LMW biomolecules in tissue specimens to study placental biomolecules. This approach involved a tissue homogenization step followed by protein depletion using acetonitrile. We compared two regions of human placenta: the chorionic plate and the basal plate to find differences in the LMW fraction. We discovered 16 species with statistically significant differences between the two sides, and identified 12 of them using tandem mass spectrometry. In the second study we collected normal human term placentas from elective C-section deliveries and exposed explants to each of the above-mentioned provocative agents or stress conditions for 48 hrs. Other explants without any stressors were cultured in parallel for the same amount of time. The processing of explants was divided into five steps: 1) explant culture; 2) tissue homogenization; 3) acetonitrile precipitation to remove high abundance, high molecular weight proteins; 4) injection of the protein-depleted specimen into a capillary liquid chromatography–mass spectrometer; 5) analysis of MS data to identify quantitative differences between cases (stressed explants) and controls (normal explants). In total, we observed 146 molecules changed in abundance between the treated explants and the controls with 75 of these molecules changed in response to hypoxic treatment, 23 changed due to hypoxia-reoxygenation, a process generating reactive oxygen species, and 48 changed due to tumor necrosis factor–alpha (TNFα), a pro-inflammatory cytokine. We were successful in identifying 45% of all these molecules by tandem MS. Statistical modeling that applied LASSO analysis allowed for the development of a model that used 16 of the 146 differentially expressed biomolecules to accurately classify and differentiate each of the 4 stressed conditions. In my third study, I then submitted actual preeclamptic and non-diseased placental tissue to our established homogenization and acetonitrile precipitation protocol to see if any of the differences in LMW biomolecules produced under stress conditions in normal placenta were recapitulated in actual diseased placenta. In a preliminary statistical analysis, 8 of the original 146 differentially expressed species, displayed significant or near significant changes in the actual disease placenta. After applying two stringent statistical tests that eliminated any potential influence of gestational age, four out of the 146 biomarkers previously studied, continued to be differentially expressed in both stringent analyses. Of the four, 1 biomarker (m/z 649.49 (+1)) showed an increased abundance in hypoxic placental explants as well as in PE placenta; 2 (461.06 (+1), 476.24 (+1)) were increased in response to TNFα-exposed placental explants and in these PE placentas and 1 (426.35 (+1)) increased in response to hypoxia-reoxygenation-treated placental explants was also increased in PE placenta. We have chemically characterized 2 of the 4 biomarkers. One was a phospholipid (m/z 476.24) while the other was an acyl-carnitine (m/z 426.35). This suggests that features of PE appear to arise from the predicted early abnormalities that affect the placenta. In conclusion, I was successful in developing an ‘omics’ approach to study less abundant, low molecular weight biomolecules in human placenta as well as investigate which biomarkers show differential expression in human placenta when exposed to proposed abnormalities of PE and have data to suggest that these same responses are present in PE placenta.

Des travaux récents ont montré que des revêtements polymères étaient réalisables par procédé connu sous le nom de Cold-Spray. Ces travaux sont particulièrement importants pour le polyéthylène de très haute masse molaire (UHMWPE) qui ne peuvent pas être mis en forme par les méthodes conventionnelles. Mais les mécanismes d'obtention des dépôts n'avaient pas été analysés. Cette thèse présente une analyse expérimentale mécanique détaillée du comportement à l'impact de particules d'UHMWPE et des mécanismes de formation du revêtement polymère sur un substrat en aluminium (Al) sous l'effet de la pulvérisation à froid de ces particules. La formation du revêtement se décompose en deux étapes : (1) se rapporte à la formation de la première couche de particules (interaction polymère-substrat), (2) à la croissance (par ajout de couches successives) du revêtement polymère (interaction polymère-polymère). La première étape de la formation du revêtement a été étudiée grâce à une technique expérimentale développée dans le cadre de cette thèse et appelée "Méthode de dépôt de particules isolées" (IPD). Il s'agit de déposer des particules isolées de UHMWPE sur un substrat en Al, en utilisant la même machine Cold-Spray, tout en contrôlant de manière précise la température du gaz et la teneur en FNA. Grâce à l'utilisation d'une caméra rapide, il a été possible de déterminer la vitesse des particules d'UHMWPE et par le calcul d'évaluer leur température avant et pendant leur impact. L'efficacité augmente avec d'une part la température et d'autre part la fraction de FNA. Cette efficacité dépend de la compétition entre l'énergie cinétique stockée sous forme d'énergie élastique Eel et l'énergie d'adhésion Ead du polymère à la surface du substrat. Pour que le film polymère se forme, il faut que Ead > Eel. Par ailleurs, Ead dépend de l'interface entre polyéthylène et aluminium, et de la présence de FNA. Les liaisons H disponibles sur la surface des FNA contribuent à l'adhésion, ce qui augmente le domaine de température favorable à la réalisation du dépôt. Concernant la croissance du revêtement, il s'agit cette fois-ci de l'adhésion polyéthylène-polyéthylène, avec toujours l'effet des nanoparticules FNA. Les études microstructurales et mécanique ont montré qu'en frittage conventionnel du UHMWPE sous forte pression, l'ajout de FNA renforce la cohésion des matériaux obtenus, mais que l'effet inverse est observé pour un frittage incomplet (sous faible pression). Enfin, afin d'explorer l'effet de vitesse de sollicitation de l'UHMWPE comparable à celles observées pour le Cold-Spray, des expériences utilisant des barres dites de Split-Hopkinson (SHPB) ont été menées. Les courbes obtenues permettent de disposer d'une cartographie complète du comportement thermo-viscoélasto-plastique de ce polymère, pur ou additionné de FNA. / Recent developments showed polymer coatings to be feasible by cold spray (CS) technique on different surfaces. This is especially important for Ultra-High Molecular Weight Polyethylene (UHMWPE) which cannot be classically processed. But the mechanisms behind coating formation was not largely understood. The thesis presents a mechanistic understanding of high strain rate impact behavior of Ultra-High Molecular Weight Polyethylene and the mechanism of coating formation during CS. The coating formation is first broken down into two major categories: 1. Interaction of UHMWPE with Al substrate (impacting particle-substrate interaction) during a high-speed impact and interaction of UHMWPE with already deposited UHMWPE particles (impacting particle-deposited particles) leading to a buildup in the coating. First stage of coating formation was understood from a technique developed for this work called Isolated Particle Deposition (IPD). In the experimental IPD process, effects of gas temperature and FNA content were calibrated empirically by depositing UHMWPE particles in an isolated manner on an Al substrate. The Deposition efficiency increased with gas temperature and FNA content. The use of an ultrafast video-camera helped to determine the particle velocity, and theoretical calculations helped to evaluate the temperature of UHMWPE particles before and during the impact process. Mechanical response of UHMWPE at different temperatures were understood by calculating elastic strain energy of UHMWPE which decreased with increasing material temperature and increased with the strain rate. Rebound of UHMWPE particles on Al surface depended upon whether UHMWPE particles after impact furnished a contact area with an interfacial bond stronger than elastic strain energy of the particle. External contributions like H-bonds on the FNA surface provide sufficiently strong extra bonds at the contact surface to increase the window of deposition at higher temperatures, which was otherwise very low. Second stage of coating formation was understood from the mechanism of welding of UHMWPE grains at different interfacial loading conditions and at varying FNA contents. The morphological and mechanical characterization showed that when UHMWPE was processed under high loading conditions (using classical sintering technique), FNA particles reinforced the UHMWPE interface. On the contrary, when UHMWPE was processed under low loading conditions, FNA particles weakened the interface. Last to be discussed in the thesis is the strain rate effect of UHMWPE using Split-Hopkinson Pressure Bar (SHPB) experiments, in order to approach comparable conditions to what happens during particle impacts. This part of the study discussed in detail the effects a high strain-rate compression has on UHMWPE by analyzing its stress-strain curves, with and without FNA. Thus, the mechanical response data with the inclusion 0%, 4% and 10% FNA to UHMWPE is also presented and discussed.

<p>This study evaluated the use of multi-lead ST-monitoring in the early assessment of patients with suspected or confirmed unstable coronary artery disease (UCAD).</p><p>At continuous 12-lead ECG (c12ECG), the definition of an ischemic episode as a transient ST-deviation ¡Ý0 for at least 1 minute resulted in a good observer agreement (kappa=0.72) and an acceptable incidence of postural ST-changes.</p><p>When c12ECG was performed from admission and for 12 hours in 630 patients with suspected UCAD, 16% had ischemic episodes. At 30 days, patients with episodes had a higher risk of cardiac death or myocardial infarction (MI) (10% vs. 1.5%). In a multivariate analysis, troponin T¡Ý0.10¦Ìg/l and presence of ischemic episodes were independent predictors of cardiac death or MI. When ST-monitoring and troponin T status were combined, patients could be divided into a low-, intermediate-, and high-risk group with 1%, 4% and 12% risk for cardiac death or MI at 30 days of follow up.</p><p>As a part of a multicenter trial, including patients with UCAD, 1016 patients underwent ST-monitoring with c12ECG or continuous vectorcardiography (cVCG). Ischemia was detected in 32% and 35%, respectively. When the groups with ischemia were compared, the groups were similar with respect to several clinical variables. Thus, these methods identify the same high-risk population.</p><p>Of the 629 patients treated non-invasively with extended treatment of low-molecular- weight heparin (LMWH) or placebo, 34% had ischemic episodes. In this group at 3 months, patients administered LMWH had a significantly lower risk of death, MI, or revascularization than patients treated with placebo (35.2% vs. 53.4%). In patients without transient ischemic episodes, the outcome in the LMWH and placebo group was similar.</p><p>Thus, multi-lead monitoring provides important prognostic information early after admission in this population, and seems to identify patients who benefit most from extended antithrombotic treatment.</p>

<p>Three chiral selectors (diketogulonic acid, benzoxycarbonylglycylproline and ketopinic acid) have been introduced for enantioseparation of pharmacologically active amines in non-aqueous capillary electrophoresis. The use of organic solvents, instead of aqueous buffers in the background electrolyte facilitated ion-pair formation between the analytes and the chiral selectors. The enantioresolution was strongly affected by the choice of selector and organic solvent but also depended on the other electrolytes. The most important parameter for the enantioresolution, apart from the choice of chiral selector, was the direction and magnitude of the electro-osmosis. Thus, covalently coated capillaries were used to suppress and to reverse this flow. Furthermore, the alkali metal hydroxide added to the background electrolyte had a great influence on the electro-osmosis. Exchanging LiOH for NaOH, was found to decrease the electro-osmotic flow. Interestingly, the flow was altered from cathodic to anodic, with KOH, RbOH or CsOH added to the ethanolic BGE. The occurrence of a reversed electro-osmosis had a great positive effect on the enantioresolution. An appropriate choice of solvent and electrolytes promoted also fast chiral separations, e.g., the enantiomers of isoprenaline were resolved within one minute. </p><p>The capillary electrophoresis systems developed within this work were applied for enantiomeric purity determinations of different pharmaceutical forms of drug products. A detection limit of 0.033 % was achieved for <i>1S,2R</i>-ephedrine, the enantiomeric impurity in Efedrin®, when diketogulonic acid was used as the selector. </p><p>By using the pre-concentration technique, transient isotachophoresis, the peak efficiency was enhanced for the enantiomers of timolol. This facilitated the introduction of a higher concentration of the sample into the capillary electrophoretic system containing ketopinic acid as the selector, and lowered the detection limit from 2.5 % to 0.2 % for the enantiomeric impurity <i>R</i>-timolol compared with injection without transient isotachophoresis.</p><p>The volatility of the non-aqueous media in capillary electrophoresis facilitated the hyphenation to mass spectrometry. The partial filling technique ensured that the selector did not contaminate the mass spectrometer, and the separated enantiomers of e.g., pronethalol were detected in the selector-free zone. </p>

<p>Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity.</p><p>In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation<i> in vivo</i>. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF <i>in vitro</i> efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated <i>in vivo</i> in patients transplanted with isolated islets.</p><p>There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets <i>in vitro</i> and<i> in vivo</i>, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials. </p>

Three chiral selectors (diketogulonic acid, benzoxycarbonylglycylproline and ketopinic acid) have been introduced for enantioseparation of pharmacologically active amines in non-aqueous capillary electrophoresis. The use of organic solvents, instead of aqueous buffers in the background electrolyte facilitated ion-pair formation between the analytes and the chiral selectors. The enantioresolution was strongly affected by the choice of selector and organic solvent but also depended on the other electrolytes. The most important parameter for the enantioresolution, apart from the choice of chiral selector, was the direction and magnitude of the electro-osmosis. Thus, covalently coated capillaries were used to suppress and to reverse this flow. Furthermore, the alkali metal hydroxide added to the background electrolyte had a great influence on the electro-osmosis. Exchanging LiOH for NaOH, was found to decrease the electro-osmotic flow. Interestingly, the flow was altered from cathodic to anodic, with KOH, RbOH or CsOH added to the ethanolic BGE. The occurrence of a reversed electro-osmosis had a great positive effect on the enantioresolution. An appropriate choice of solvent and electrolytes promoted also fast chiral separations, e.g., the enantiomers of isoprenaline were resolved within one minute. The capillary electrophoresis systems developed within this work were applied for enantiomeric purity determinations of different pharmaceutical forms of drug products. A detection limit of 0.033 % was achieved for 1S,2R-ephedrine, the enantiomeric impurity in Efedrin®, when diketogulonic acid was used as the selector. By using the pre-concentration technique, transient isotachophoresis, the peak efficiency was enhanced for the enantiomers of timolol. This facilitated the introduction of a higher concentration of the sample into the capillary electrophoretic system containing ketopinic acid as the selector, and lowered the detection limit from 2.5 % to 0.2 % for the enantiomeric impurity R-timolol compared with injection without transient isotachophoresis. The volatility of the non-aqueous media in capillary electrophoresis facilitated the hyphenation to mass spectrometry. The partial filling technique ensured that the selector did not contaminate the mass spectrometer, and the separated enantiomers of e.g., pronethalol were detected in the selector-free zone.

This study evaluated the use of multi-lead ST-monitoring in the early assessment of patients with suspected or confirmed unstable coronary artery disease (UCAD). At continuous 12-lead ECG (c12ECG), the definition of an ischemic episode as a transient ST-deviation ¡Ý0 for at least 1 minute resulted in a good observer agreement (kappa=0.72) and an acceptable incidence of postural ST-changes. When c12ECG was performed from admission and for 12 hours in 630 patients with suspected UCAD, 16% had ischemic episodes. At 30 days, patients with episodes had a higher risk of cardiac death or myocardial infarction (MI) (10% vs. 1.5%). In a multivariate analysis, troponin T¡Ý0.10¦Ìg/l and presence of ischemic episodes were independent predictors of cardiac death or MI. When ST-monitoring and troponin T status were combined, patients could be divided into a low-, intermediate-, and high-risk group with 1%, 4% and 12% risk for cardiac death or MI at 30 days of follow up. As a part of a multicenter trial, including patients with UCAD, 1016 patients underwent ST-monitoring with c12ECG or continuous vectorcardiography (cVCG). Ischemia was detected in 32% and 35%, respectively. When the groups with ischemia were compared, the groups were similar with respect to several clinical variables. Thus, these methods identify the same high-risk population. Of the 629 patients treated non-invasively with extended treatment of low-molecular- weight heparin (LMWH) or placebo, 34% had ischemic episodes. In this group at 3 months, patients administered LMWH had a significantly lower risk of death, MI, or revascularization than patients treated with placebo (35.2% vs. 53.4%). In patients without transient ischemic episodes, the outcome in the LMWH and placebo group was similar. Thus, multi-lead monitoring provides important prognostic information early after admission in this population, and seems to identify patients who benefit most from extended antithrombotic treatment.

Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity. In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation in vivo. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF in vitro efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated in vivo in patients transplanted with isolated islets. There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets in vitro and in vivo, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials.

Aquatic systems worldwide receive large amounts of organic carbon from terrestrial sources. This ‘allochthonous’ organic carbon (AlloOC) affects critical physical and chemical properties of freshwater ecosystems, with consequences for food web structures and exchange of greenhouse gases with the atmosphere. In the boreal region, loadings of AlloOC are particularly high due to leaching from huge organic deposits in boreal forest, mire and tundra soils. A main process of AlloOC turnover in aquatic systems is its use by heterotrophic bacteria. Applying a bioassay approach, I measured the respiration and growth (production) of bacteria in northern Sweden, in streams and lakes almost totally dominated by AlloOC. The objective was to elucidate how variations in AlloOC source, age, composition and concentration impact on its use by aquatic bacteria, and how AlloOC properties, in turn, are regulated by landscape composition and by hydrology. The bacterial respiration (30-309 µg C L-1 d-1) was roughly proportional to the concentration of AlloOC (7-47 mg C L-1), but not significantly related to AlloOC source or character. Bacterial production (4-94 µg C L-1 d-1), on the other hand, was coupled to the AlloOC character, rather than concentration. A strong coupling to AlloOC character was also found for bacterial growth efficiency (0.06-0.51), i.e. production per unit of assimilated carbon. Bacterial production and growth efficiency increased with rising concentrations of low molecular weight AlloOC (carboxylic acids, free amino acids and simple carbohydrates). While the total AlloOC concentrations generally were the highest in mire-dominated catchments, low molecular weight AlloOC concentrations were much higher in forested catchments, compared to mire-dominated. These patterns were reflected in a strong landscape control of aquatic bacterial metabolism. Moreover, high flow episodes increased the export of organic carbon from forests, in relation to the export from mires, stimulating the bacterial production and growth efficiency in streams with mixed (forest and mire) catchments. The potential of AlloOC to support efficient bacterial growth decreased on time-scales of weeks to months, as the AlloOC was aged in laboratory or lake in situ conditions. To conclude, landscape, hydrology and conditions which determine AlloOC age have large influence on bacterial metabolism in boreal aquatic systems. Considering the role of bacteria in heterotrophic food chains, these factors can have spin-off effects on the structure and function of boreal aquatic ecosystems.

The microstructure of polyolefins determines their macroscopic properties. Consequently, it is essential to predict how polymerization conditions will affect polyolefin microstructure. The most important microstructural distributions of ethylene/alfa-olefin copolymers made with coordination catalysts are their molecular weight (MWD), chemical composition (CCD), and comonomer sequence length (CSLD). Several mathematical models have been developed to predict these microstructural distributions; reliable techniques to estimate parameters for these models, however, are still poorly developed, especially for catalysts that have multiple site types, such as heterogeneous Ziegler-Natta complexes. Most commercial polyolefins are made with heterogeneous Ziegler-Natta catalysts, which make polyolefins with broad MWD, CCD, and CSLD. This behavior is attributed to the presence of several active site types, leading to a final product that can be seen as a blend of polymers made on the different catalyst site types. The main objective of this project is to develop a methodology to estimate the most important parameters needed to describe the microstructure of ethylene/alfa-olefin copolymers made with these multiple site-type catalysts. To accomplish this objective, we developed the Integrated Deconvolution Estimation Model (IDEM). IDEM estimates ethylene/alf-olefin reactivity ratios for each site type in two-steps. In the first step, the copolymer MWD, measured by high-temperature gel permeation chromatography, is deconvoluted into several Flory’s most probable distributions to determine the number of site types and the weight fractions of copolymer made on each of them. In the second estimation step, the model uses the MWD deconvolution information to fit the copolymer triad distributions measured by 13C NMR and estimate the reactivity ratios per site type. This is the first time that MWD and triad distribution information is integrated to estimate the reactivity ratio per site type of multiple site-type catalysts used to make ethylene/alfa-olefin copolymers. IDEM was applied to two sets of ethylene-co-1-butene copolymers made with a commercial Ziegler-Natta catalyst, covering a wide range of 1-butene fractions. In the first set of samples (EBH), hydrogen was used as a chain transfer agent, whereas it was absent in the second set (EB). Comparison of the reactivity ratio estimates for the sets of samples permitted the quantification of the hydrogen effect on the reactivity ratios of the different site types present in the Ziegler-Natta catalyst used in this thesis. Since 13C NMR it is an essential analytical step in IDEM, triad distributions for the EB and EBH copolymers were measured in two different laboratories (Department of Chemistry at the University of Waterloo, and Dow Chemical Research Center at Freeport, Texas). IDEM was applied to both set of triad measurements to find out the effect of interlaboratory 13C NMR analysis on reactivity ratio estimation.