<p>The acceptance of blood contacting implants creating favorable conditions <i>in vivo</i> is decisively determined by their interaction with proteins that mediate inter cellular interactions with synthetic substrates. Adsorbed proteins can activate blood cascade systems like coagulation and complement that may result in serious blood clots, and/or immunological reactions. Poly (ethylene glycol) (PEG), heparin, and phosphoryl choline (PC) functional poly (methacrylates) are previously used polymers with known non-adhesive properties in blood contacting events.</p><p>This thesis contributes to this extensive research by introducing a novel type of biomaterial that equips biodegradable polymers with biomimetic functionalities. The phospholipid mimetic material is synthesized by combining biodegradable polymers with various functional polar end-groups consisting of zwitterionic phosphoryl choline (PC), anionic succinates, and cationic quaternary ammonium. The polymer backbone provides mechanical stability and biodegradability whilst the various head groups provide a variety of functions. The careful evaluation of the synthesis has allowed reaction conditions to be optimized leading to complete conversion at each step and subsequently high yields. Initially, poly (e-caprolactone) (PCL) was used since it provided a suitable synthetic starting point. However, the synthesis has also included poly (trimethylene carbonate) (PTMC) to provide a material that allows spontaneous surface enrichment of the polar PC group. This was achieved with an added hydrophilic environment. </p><p>Through the synthesis of multi PC functional PTMC, additional bulk organisation by the formation of zwitterionomers (PC ionomer) was achieved. Low modulus elasticity and water uptake were some of the properties of the formed material. As a result it was shown that the PC ionomer could be used for protein/drug loading and subsequent release. Furthermore, the material possessed non-adhesive properties in different biological environments.</p><p>Importantly, the result suggests that a versatile synthetic platform has been established that may provide a smorgasbord of different functional polymers, or combinations of such. This is indeed important since it was shown that the polymer in many ways dictates how the material may take advantage of an added functionality. </p><p>Such materials should be interesting for a variety of biomedical applications including the production of soft hemocompatible tissue.</p>

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In this study the formation of supported membranes onto planar solid supports has been investigated. The stability and activity of reconstituted membrane receptors has been studied. The potential use of such preparations in biosensor applications is discussed. The lipid films were made by the Langmuir Blodgett and by the liposome fusion techniques. These supported films were characterised by ellipsometry, atomic force microscopy, surface plasmon resonance (SPR) and resonant mirror techniques. The thickness of the films was in agreement with that of a cell membrane. The kinetics of formation of the lipid films was studied and discussed. The proteins, bacteriorhodopsin, cytochrome oxidase, acetylcholinesterase and the nicotinic acetylcholine receptor were reconstituted into the supported membrane. The subsequent analysis showed that the proteins were individually distributed and that the activity was retained, in some cases for several weeks after immobilisation. The glycolipids, GM1, GM2, GD1b, asialo-GM1, globotriaosylceramide, lactosylceramide and galactosylceramide, were also reconstituted into the supported membranes. Their specific interaction with the toxin ricin or with its B-chain was examined using SPR. The affinity of intact toxin and of its B-chain differed markedly and was pH dependent. The carbohydrate chain length and charge density of the glycolipids also influenced the affinity.

The acceptance of blood contacting implants creating favorable conditions in vivo is decisively determined by their interaction with proteins that mediate inter cellular interactions with synthetic substrates. Adsorbed proteins can activate blood cascade systems like coagulation and complement that may result in serious blood clots, and/or immunological reactions. Poly (ethylene glycol) (PEG), heparin, and phosphoryl choline (PC) functional poly (methacrylates) are previously used polymers with known non-adhesive properties in blood contacting events. This thesis contributes to this extensive research by introducing a novel type of biomaterial that equips biodegradable polymers with biomimetic functionalities. The phospholipid mimetic material is synthesized by combining biodegradable polymers with various functional polar end-groups consisting of zwitterionic phosphoryl choline (PC), anionic succinates, and cationic quaternary ammonium. The polymer backbone provides mechanical stability and biodegradability whilst the various head groups provide a variety of functions. The careful evaluation of the synthesis has allowed reaction conditions to be optimized leading to complete conversion at each step and subsequently high yields. Initially, poly (e-caprolactone) (PCL) was used since it provided a suitable synthetic starting point. However, the synthesis has also included poly (trimethylene carbonate) (PTMC) to provide a material that allows spontaneous surface enrichment of the polar PC group. This was achieved with an added hydrophilic environment. Through the synthesis of multi PC functional PTMC, additional bulk organisation by the formation of zwitterionomers (PC ionomer) was achieved. Low modulus elasticity and water uptake were some of the properties of the formed material. As a result it was shown that the PC ionomer could be used for protein/drug loading and subsequent release. Furthermore, the material possessed non-adhesive properties in different biological environments. Importantly, the result suggests that a versatile synthetic platform has been established that may provide a smorgasbord of different functional polymers, or combinations of such. This is indeed important since it was shown that the polymer in many ways dictates how the material may take advantage of an added functionality. Such materials should be interesting for a variety of biomedical applications including the production of soft hemocompatible tissue.

Pulses are an important component in crop rotations in the semiarid Brown soil zone of southern Saskatchewan, Canada. Besides their capability to fix nitrogen, pulse crops establish a strong symbiotic relationship with arbuscular mycorrhizal fungi (AMF), which have been shown to increase nutrient and water uptake through hyphal extensions in the soil. Incorporating strongly mycorrhizal crops in a rotation may increase inoculum levels in the soil and benefit the growth of a subsequent crop. The objective of this study was to determine if AMF potential and colonization of a durum crop is significantly affected by cropping history and to assess the impact of pulses in crop rotations on the abundance and diversity of AMF communities in the soil. In 2004 and 2005, soil, plant, and root samples were taken on Triticum turgidum L. (durum) with preceding crops of Pisum sativum L. (pea), Lens culinaris Medik (lentil), Cicer arietinum L. (chickpea), Brassica napus L. (canola) or Triticum turgidum L. (durum). Although there were few differences in soil N and P levels, previous crop had a significant effect (p<0.05) on durum yields in both years. A previous crop of pea was associated with the highest yields, while the durum monocultures were lowest. Arbuscular mycorrhizal potential and colonization were significantly affected (p<0.05) by cropping history, but not consistently as a result of inclusion of a pulse crop. Phospholipid and neutralipid fatty acids (PLFA/NLFA) were completed to analyse the relative abundance of AMF (C16:1ù5), saprophytic fungi (C18:2ù6), and bacteria in the soil. The effect of treatment on the abundance of AMF, saprotrophic fungi and bacteria were not significant (p<0.05), but the changes over time were. These results demonstrate that although previous crop may play a role in microbial community structure, it is not the only influencing factor.

Pulses are an important component in crop rotations in the semiarid Brown soil zone of southern Saskatchewan, Canada. Besides their capability to fix nitrogen, pulse crops establish a strong symbiotic relationship with arbuscular mycorrhizal fungi (AMF), which have been shown to increase nutrient and water uptake through hyphal extensions in the soil. Incorporating strongly mycorrhizal crops in a rotation may increase inoculum levels in the soil and benefit the growth of a subsequent crop. The objective of this study was to determine if AMF potential and colonization of a durum crop is significantly affected by cropping history and to assess the impact of pulses in crop rotations on the abundance and diversity of AMF communities in the soil. In 2004 and 2005, soil, plant, and root samples were taken on Triticum turgidum L. (durum) with preceding crops of Pisum sativum L. (pea), Lens culinaris Medik (lentil), Cicer arietinum L. (chickpea), Brassica napus L. (canola) or Triticum turgidum L. (durum). Although there were few differences in soil N and P levels, previous crop had a significant effect (p<0.05) on durum yields in both years. A previous crop of pea was associated with the highest yields, while the durum monocultures were lowest. Arbuscular mycorrhizal potential and colonization were significantly affected (p<0.05) by cropping history, but not consistently as a result of inclusion of a pulse crop. Phospholipid and neutralipid fatty acids (PLFA/NLFA) were completed to analyse the relative abundance of AMF (C16:1ù5), saprophytic fungi (C18:2ù6), and bacteria in the soil. The effect of treatment on the abundance of AMF, saprotrophic fungi and bacteria were not significant (p<0.05), but the changes over time were. These results demonstrate that although previous crop may play a role in microbial community structure, it is not the only influencing factor.

This thesis entitled ‘ Unusual Acylation Properties of Type II Fatty Acid Biosynthesis Acyl Carrier Proteins’ describes the discovery of self-acylation and malonyl transferase activity in acyl carrier proteins involved in type II fatty acid biosynthesis and assigns a physiological role to these processes inside the cellular milieu. Acyl carrier protein (ACP) is one of the most abundant proteins present inside the cell and almost 4% enzymes require it as a cofactor. Acyl carrier proteins can exist either as discrete proteins or as domains of large functional proteins. They function in a variety of synthases as central molecules to which growing acyl intermediates and nascent product molecules are covalently tethered during the elongation and modification steps required to produce the final product. A prototypical bacterial ACP is composed of 70-80 amino acids and is generally expressed in the apo form. It is post-translationally modified to active holo form by the addition of 4'-phosphopantetheine moiety to an absolutely conserved serine residue in a reaction catalyzed by holo-ACP synthase or 4'-phosphopantetheine transferase. Chapter 1 surveys literature related to carrier proteins inside the cell and describes the thesis objective. It also presents an overview of the acyl carrier proteins and their involvement in various metabolic pathways inside the cell. The chapter details the structural organization of acyl carrier proteins from various sources revealing the conservation in their structure and also details the molecular basis of interaction of ACP with other enzymes inside the cell. The discovery of unusual self-acylation property in acyl carrier proteins involved in polyketide biosynthesis and its absence in acyl carrier proteins involved in fatty acid biosynthesis prompted me to investigate the reasons for this selective behavior. Discovery of self-acylation property in acyl carrier proteins Plasmodium falciparum and chloroplast targeted Brassica napus acyl carrier proteins involved in type II fatty acid biosynthesis and the mechanism of this reaction forms the basis of Chapter 2. In this chapter it has been shown that self-acylation property is intrinsic to a given acyl carrier protein and is not dependent on the pathway in which it is involved. Based on primary sequence analysis and site directed mutagenesis studies presence of an aspartate/glutamate has been identified to be critical for the self-acylation event. Furthermore, it has also been shown that the self-acylation event in type II fatty acid biosynthesis acyl carrier proteins is highly specific in nature employing only dicarboxylic acid –CoAs as substrates unlike the polyketide biosynthesis acyl carrier proteins which utilize both dicarboxylic acid and β-keto acid thiol ester -CoAs as substrates. The detailed kinetics of these reactions has also been worked out. Combining all the results a plausible mechanism for the self-acylation reaction has been proposed. Chapter 3 describes the discovery of a novel malonyl transferase behavior in acyl carrier proteins involved in type II fatty acid biosynthesis. Malonyl transferase property in ACPs of type II FAS from a bacterium (Escherichia coli), a plant (Brassica napus) and a parasitic protozoon (Plasmodium falciparum) were investigated to present a unifying paradigm for the mechanism of malonyl transferase behavior in ACPs. Identification of malonyl transferase property in Plasmodium falciparum ACP and Escherichia coli ACP (EcACP) and the absence of this property in Brassica napus ACP has been described in this chapter. Detailed investigations demonstrated that presence of an arginine or a lysine in loop II and an arginine or glutamine at the start of helix III as the residues that are critical for the transferase activity. In order to assign a physiologic function to these unusual acylation properties, fabD(Ts) mutant strain of Escherichia coli was utilized for heterologous complementation by the various wild type and mutant ACPs that are able to catalyze either or both of the activities. Growth of the mutant strain at non-permissive temperature, when complemented with ACPs catalyzing both the reactions confirmed that these properties have a physiologic relevance. Extensive mutagenesis experiments in conjunction with complementation studies allowed me to propose a plausible mechanism on how the self-malonylation and malonyl transferase properties operate in tandem. Chapter 4 describes the thermodynamic characterization of self-acylation process using Isothermal Titration Calorimetry. Isothermal Titration Calorimetric studies on the binding of malonyl, succinyl, butyryl and methylmalonyl –CoA to Plasmodium falciparum and Brassica napus acyl carrier proteins were performed to investigate the role of thermodynamic parameters in the specificity of self-acylation reaction. Calculation of the parameters showed that the thermodynamics does not control the self-acylation reaction. The evolution of self-acylation property in various acyl carrier proteins and its possible significance in the evolution of various metabolic events is described in Chapter 5. Extensive bioinformatics search was performed and phylogenetic analysis on acyl carrier proteins from 60 different taxa was done using the MEGA4 program. Analysis showed that this property was first found in cyanobacterium. Later, during the course of evolution this property was lost in most acyl carrier proteins, and was retained either in acyl carrier proteins that are targeted to organelles of cyanobaterial orgin viz. apicoplast in apicomplexans and chlorplasts in plants or in acyl carrier proteins involved in secondary metabolic events such as polyketide biosynthesis. Chapter 6 summarizes the findings of the thesis. Acyl carrier protein from Plasmodium falciparum, Brassica napus and Escherichia coli were characterized for their self-acylation and malonyl transferase properties and a combined mechanism for these two properties is proposed. The work done also provides an in vivo rationale to these in vitro processes. Furthermore, the evolutionary significance of the self-acylation behavior is also discussed in the thesis. The thesis also probes into the thermodynamics of the self-acylation reaction in Plasmodium falciparum and Brassica napus acyl carrier proteins. Thus, the thesis adds a new dimension to the much unexplored ACP biology and paves the way to study in vivo roles of these processes in detail. Appendix I describes the Isothermal Titration calorimetric characterization of binding of various acyl-PO4 molecules to Escherichia coli PlsX (Acyl-phosphate acyltransferase). PlsX, the first enzyme of phosphatidic acid biosynthesis pathway catalyzes the conversion of acyl-ACP into acyl-PO4, which is further used by other enzymes leading to the formation of phosphatidic acid. ITC results presented in this section show that longer chain length acyl-PO4 molecules show better binding to PlsX, as compared to the smaller ones demonstrating that long chain acyl molecules serve as better substrates for phosphatidic acid synthesis.

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Title from first page of PDF document. Includes bibliographical references (p. 22-27).

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