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Take-all is the name given to the disease caused by a soilborne fungus Gaeumannomyces graminis (Sacc.) von Arx and Olivier var. tritici Walker (Ggt), an ascomycete of the family Magnaportheaceae (Cook, 2003). This fungus is an aggressive soil-borne pathogen causing root rot of wheat (primary host), barley and rye crops (secondary host). The flowering, seedling, and vegetative growth stages can be affected by the infection of the whole plant, leaves, roots, and stems. Infections of roots result in losses in crop yield and quality primarily due to a lowering in nutrient uptake. Take-all is most common in regions where wheat is cultivated without adequate crop rotation. Crop rotation allows time between the planting dates of susceptible crops, which causes a decrease in the inoculum potential of soilborne plant pathogens to levels below an economic threshold by resident antagonistic soil microbial communities. Soilborne disease suppressiveness is an inherent characteristic of the physical, chemical, and/or biological structure of a particular soil which might be induced by agricultural practices and activities such as the cultivation of crops, or the addition of organisms or nutritional amendments, causing a change in the microfloral environment. Disturbances of soil ecosystems that impact on the normal functioning of microbial communities are potentially detrimental to soil formation, energy transfers, nutrient cycling, and long-term stability. In this regard, an overview of soil properties and processes indicated that the use of microbiological and biochemical soil properties, such as microbial biomass, the analysis of microbial functional diversity and microbial structural diversity by the quantification of community level physiological profiles and signature lipid biomarkers are useful as indicators of soil ecological stress or restoration properties because they are more responsive to small changes than physical and chemical characteristics. In this study, the relationship between physico-chemical characteristics, and different biological indicators of soil quality of agricultural soils conducive, suppressive, and neutral with respect to take-all disease of wheat as caused by the soilborne fungus Gaeumannomyces graminis var. tritici (Ggt), were investigated using various techniques. The effect of crop rotation on the functional and structural diversity of soils conducive to take-all disease was also investigated. Through the integration of quantitative and qualitative biological data as well as the physico-chemical characteristics of the various soils, the functional and structural diversity of microbial IV communities in the soils during different stadia of take-all disease of wheat were characterised. All results were evaluated statistically and the predominant physical and chemical characteristics that influenced the microbiological and biochemical properties of the agricultural soils during different stadia of take-all disease of wheat were identified using multivariate analyses. Although no significant difference @ > 0.05) could be observed between the various soils using conventional microbiological enumeration techniques, the incidence of Gliocladium spp. in suppressive soils was increased. Significant differences @ < 0.05) were observed between agricultural soils during different stadia of take-all disease of wheat. Although no clear distinction could be made between soils suppressive and neutral to take-all disease of wheat, soils suppressive and conducive to take-all disease of wheat differed substantially in their community level physiological profiles (CLPPs). Soils suppressive / neutral to take-all disease were characterised by enhanced utilisation of carboxylic acids, amino acids, and carbohydrates, while conducive soils were characterised by enhanced utilisation of carbohydrates. Shifts in the functional diversity of the associated microbial communities were possibly caused by the presence of Ggt and associated antagonistic fungal and bacterial populations in the various soils. It was evident that the relationships amongst the functionality of the microbial communities within the various soils had undergone changes through the different stages of development of take-all disease of wheat, thus implying different substrate utilisation capabilities of present soil microbial communities. Diversity indices were calculated as Shannon's diversity index (H') and substrate equitability (J) and were overall within the higher diversity range of 3.6 and 0.8, respectively, indicating the achievement of very high substrate diversity values in the various soils. A substantial percentage of the carbon sources were utilised, which contributed to the very high Shannon-Weaver substrate utilisation indices. Obtained substrate evenness (equitability) (J) indices indicated an existing high functional diversity. The functional diversity as observed during crop rotation, differed significantly (p < 0.05) from each other, implying different substrate utilisation capabilities of present soil microbial communities, which could possibly be ascribed to the excretion of root exudates by sunflowers and soybeans. Using the Sorenson's index, a clear distinction could be made between the degrees of substrate utilisation between microbial populations in soils conducive, suppressive, and neutral to take-all disease of wheat, as well as during crop rotation. Furthermore, the various soils could also be differentiated on the basis of the microbial community structure as determined by phospholipid fatty acid (PLFA) analysis. Soil suppressive to take-all disease of wheat differed significantly (p < 0.05) from soils conducive, and neutral to take-all disease of wheat, implying a shift in relationships amongst the structural diversity of microbial communities within the various soils. A positive association was observed between the microbial phospholipid fatty acid profiles, and dominant environmental variables of soils conducive, suppressive, and neutral to take-all disease of wheat. Soils conducive and neutral to take-all disease of wheat were characterised by high concentrations of manganese, as well as elevated concentrations of monounsaturated fatty acids, terminally branched saturated fatty acids, and polyunsaturated fatty acids which were indicative of Gram-negative bacteria, Gram-positive bacteria and micro eukaryotes (primarily fungi), respectively. These soils were also characterised by low concentrations of phosphorous, potassium, percentage organic carbon, and percentage organic nitrogen, as well as low soil pH. Soil suppressive to take-all disease of wheat was characterised by the elevated levels of estimated of biomass and elevated concentrations of normal saturated fatty acids, which is ubiquitous to micro-organisms. The concentration of normal saturated fatty acids in suppressive soils is indicative of a low structural diversity. This soil was also characterised by high concentrations of phosphorous, potassium, percentage organic carbon, and percentage organic nitrogen, as well as elevated soil pH. The relationship between PLFAs and agricultural soils was investigated using principal component analysis (PCA), redundancy analysis (RDA) and discriminant analysis (DA). Soil suppressive to take-all disease of wheat differed significantly (p < 0.05) from soils conducive, and neutral to take-all disease of wheat, implying a shift in relationships amongst the structural diversity of microbial communities within the various soils. A positive association was observed between the microbial phospholipid fatty acid profiles, and dominant environmental variables of soils conducive, suppressive, and neutral to take-all disease of wheat. Hierarchical cluster analysis of the major phospholipid fatty acid groups indicated that the structural diversity differed significantly between soils conducive, suppressive, and neutral to take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. The results indicate that the microbial community functionality as well as the microbial community structure was significantly influenced by the presence of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici, and that the characterisation of microbial functional and structural diversity by analysis of community level physiological profiles and phospholipid fatty acid analysis, respectively, could be successfully used as an assessment criteria for the evaluation of agricultural soils conducive, suppressive, and neutral to take-all disease of wheat, as well as in crop rotation systems. This methodology might be of significant value in assisting in the management and evaluation of agricultural soils subject to the prevalence of other soilborne diseases. / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2004.

Nonequilibrium work methods for determining potentials of mean force (PMF) w(z) have recently gained popularity as an alternative to standard equilibrium based methods. Introduced by Kosztin et al., the forward-reverse (FR) method is a bidirectional work method in that it requires the work to be sampled in both forward and reverse directions along the reaction coordinate z. This bidirectional sampling leads to much faster convergence than other nonequilibrium methods such as the Jarzynski equality, and the calculation itself is extremely simple, making the FR method an attractive way of determining the PMF. Presented here is an extension to the FR method that deals with sampling problems along essentially irreversible reaction coordinates. By oscillating a particle as it is steered along a reaction coordinate, both forward and reverse work samples are obtained as the particle progresses. Dubbed the oscillating forward-reverse (OFR) method, this new method overcomes the issue of irreversibility that is present in numerous soft-matter and biological systems, particularly in the stretching or unfolding of proteins. The data analysis of the OFR method is non-trivial however, and to this end a software package named the ‘OFR Analysis Tool’ has been created. This software performs all of the complicated analysis necessary, as well as a complete error analysis that considers correlations in the data, thus streamlining the use of the OFR method for potential end users. Another attractive feature of the FR method is that the dissipative work is collected at the same time as the free energy changes, making it possible to also calculate local diffusion coefficients, D(z), from the same simulation as the PMF through the Stokes-Nernst-Einstein relation Fdrag = −γv, with γ = kB T /D. While working with the OFR method, however, the D(z) results never matched known values or those obtained through other methods, including the mean square displacement (or Einstein) method. After a reformulation of the procedure to obtain D(z), i.e. by including the correct path length and particle speeds, results were obtained that were much closer to the correct values. The results however showed very little variation over the length of the reaction coordinate, even when D(z) was known to vary drastically. It seemed that the highly variable and noncontinuous velocity function of the particle being steered through the “stiff-spring” method was incompatible with the macroscopic definition of the drag coefficient, γ. The drag coefficient requires at most a slowly varying velocity so that the assumption of a linearly related dissipative work remains valid at all times. To address this, a new dynamic constraint steering protocol (DCP) was developed to replace the previously used “stiff-spring” method, now referred to as a dynamic restraint protocol (DRP). We present here the results for diffusion in bulk water, and both the PMF and diffusion results from the permeation of a water molecule through a DPPC membrane. We also consider the issue of ergodicity and sampling, and propose that to obtain an accurate w(z) (and D(z)) from even a moderately complex system, the final result should be a weighted average obtained from numerous pulls. An additional utility of the FR and OFR methods is that the permeability across lipid bilayers can be calculated from w(z) and D(z) using the inhomogeneous solubility-diffusion (ISD) model. As tests, the permeability was first calculated for H2O and O2 through DPPC. From the simulations, the permeability coefficients for H2O were found to be 0.129 ± 0.075 cm/s and 0.141 ± 0.043 cm/s, at 323 K and 350 K respectively, while the permeability coefficients for O2 were 114 ± 40 cm/s and 101 ± 27 cm/s, again at 323 K and 350 K respectively. As a final, more challenging system, the permeability of tyramine – a positively charged trace amine at physiological pH – was calculated. The final value of P = 0.89 ± 0.24 Ang/ns is over two orders of magnitude lower than that obtained from experiment (22 ± 4 Ang/ns), although it is clear that the permeability as calculated through the ISD is extremely sensitive to the PMF, as scaling the PMF by ∼ 20% allowed the simulation and experimental values to agree within uncertainty. With accurate predictions for free energies and permeabilities, the OFR method could potentially be used for many valuable endeavors such as rational drug design.

A l'heure de l'acquisition de donn'ees 'a haut d'ebit concernant le m'etabolisme cellulaire et son 'evolution, il est absolument n'ecessaire de disposer de mod'eles permettant d'int'egrer ces donn'ees en un ensemble coh'erent, d'en interpr'eter les variations m'etaboliques r'ev'elatrice, les 'etapes clefs o'u peuvent s'exercer des r'egulations, voire mˆeme d'en r'ev'eler des contradictions apparentes met- tant en cause les bases sur lesquelles le mod'ele lui-mˆeme est construit. C'est ce type de travail que j'ai entrepris 'a propos de donn'ees exp'erimentales obtenues dans le laboratoire biologique sur le m'etabolisme de cellules tu- morales en r'eponse 'a un traitement anti-canc'ereux. Je me suis attach'ee 'a la mod'elisation d'un point particulier de ce m'etabolisme. Il concerne le m'etabolisme des glyc'erophospholipides qui sont de bons marqueurs de la prolif'eration cellulaire. Les phospholipides constituent l'essentiel des mem- branes d'une cellule et l''etude de leur synth'ese (en particulier chez les cellules de mammif'eres) est de ce fait un sujet important. Ici, nous avons pris le parti de mettre en place un mod'ele math'ematique par 'equations diff'erentielles ordinaires, qui est essentiellement bas'e sur des 'equations hyperboliques (Michaelis-Menten), mais aussi sur des cin'etiques type loi d'action de masse et diffusion. Le mod'ele, compos'e de 8 'equations diff'erentielles, donc de 8 substrats d'int'erˆet, comporte naturellementdes param'etres inconnus in vivo, et certains d'ependents des conditions cellulaires (diff'erentiations de cellules, pathologies, . . .). Le mod'ele s'epare la structure du r'eseau m'etabolique, l''ecriture de la matrice de stoechiom'etrie, celles des 'equations de vitesse et enfin des 'equations diff'erentielles. Le mod'ele choisi est le mod'ele murin (souris/rat), parce qu'il est lui-mˆeme un mod'ele de l'homme. Plusieurs con- ditions sont successivement consid'er'ees pour l'identification des param'etres, afin d''etudier les liens entre la synth'ese de phospholipides et le cancer : - le foie sain du rat, - le m'elanome B16 et le carcinome de la lign'ee 3LL chez la souris, respectivement sans traitement, en cours de traitement 'a la Chloro'ethyl-nitrosour'ee et apr'es traitement, - enfin le m'elanome B16 chez la souris sous stress de privation de m'ethionine. En r'esum'e, ce tra- vail fourni une interpr'etation nouvelle des donn'ees exprimentales en mon- trant le rˆole essentiel de la PEMT et la nature superstable de l''etat sta- tionnaire de fonctionnement du r'eseau m'etabolique des phospholipides lors de la canc'erog'en'ese et du traitement des cancers. Il montre bien l'avantage de l'utilisation d'un mod'ele math'ematique dans l'interpr'etation de donn'ees m'etaboliques complexes.

The human genome project has allowed for the rapid identification of a large number of protein families based on similarities in their genetic sequences. The acyl-glycerol phosphate acyltransferase (AGPAT) family of enzymes have been largely identified through sequence homology, with eleven isoforms identified in both mice and humans. Interestingly, very little work has been done on the characterization of AGPAT isoform 4. In the present study, I report the functional characterization of AGPAT4 as an acyl-CoA: lysocardiolipin acyltransferase (ALCAT), which we have renamed ALCAT2. Although ALCAT2 is present in most tissues, it is abundant in multiple brain regions including olfactory bulbs, hippocampus, cerebellum, cortex, and brain stem, and is detectable in both primary neurons and glial cells. In assays performed in vitro, ALCAT2 significantly increased the incorporation of [14C]oleoyl-CoA into phosphatidylinositol and CL using either lysophosphatidylinositol, or monolysocardiolipin or dilysocardiolipin as acyl acceptors, respectively. ALCAT2 did not display significant acyltransferase activity with lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, or lysophosphatidylglycerol acyl acceptors. Overexpressing ALCAT2 in HEK-293 cells increased the total CL content, but did not significantly affect levels of other glycerophospholipids including phosphatidylinositol. Analysis of the fatty acyl profile of CL from ALCAT2-overexpressing cells indicated increased total saturated fatty acids, particularly stearate, palmitate, and myristate, and increased levels of n-3 polyunsaturated fatty acids α-linolenic acid (18:3n-3), eicosatrienoic acid (20:3n-3), and eicosapentanoic acid (20:5n-3). In accordance with its observed role in cardiolipin remodeling, ALCAT2 localized predominately to the mitochondria. ALCAT2 was also regulated during embryogenesis, and in varying metabolic states. In summary, ALCAT2 is a new enzyme in CL remodeling with a potential role in mitochondrial function.

Take-all is the name given to the disease caused by a soilborne fungus Gaeumannomyces graminis (Sacc.) von Arx and Olivier var. tritici Walker (Ggt), an ascomycete of the family Magnaportheaceae (Cook, 2003). This fungus is an aggressive soil-borne pathogen causing root rot of wheat (primary host), barley and rye crops (secondary host). The flowering, seedling, and vegetative growth stages can be affected by the infection of the whole plant, leaves, roots, and stems. Infections of roots result in losses in crop yield and quality primarily due to a lowering in nutrient uptake. Take-all is most common in regions where wheat is cultivated without adequate crop rotation. Crop rotation allows time between the planting dates of susceptible crops, which causes a decrease in the inoculum potential of soilborne plant pathogens to levels below an economic threshold by resident antagonistic soil microbial communities. Soilborne disease suppressiveness is an inherent characteristic of the physical, chemical, and/or biological structure of a particular soil which might be induced by agricultural practices and activities such as the cultivation of crops, or the addition of organisms or nutritional amendments, causing a change in the microfloral environment. Disturbances of soil ecosystems that impact on the normal functioning of microbial communities are potentially detrimental to soil formation, energy transfers, nutrient cycling, and long-term stability. In this regard, an overview of soil properties and processes indicated that the use of microbiological and biochemical soil properties, such as microbial biomass, the analysis of microbial functional diversity and microbial structural diversity by the quantification of community level physiological profiles and signature lipid biomarkers are useful as indicators of soil ecological stress or restoration properties because they are more responsive to small changes than physical and chemical characteristics. In this study, the relationship between physico-chemical characteristics, and different biological indicators of soil quality of agricultural soils conducive, suppressive, and neutral with respect to take-all disease of wheat as caused by the soilborne fungus Gaeumannomyces graminis var. tritici (Ggt), were investigated using various techniques. The effect of crop rotation on the functional and structural diversity of soils conducive to take-all disease was also investigated. Through the integration of quantitative and qualitative biological data as well as the physico-chemical characteristics of the various soils, the functional and structural diversity of microbial IV communities in the soils during different stadia of take-all disease of wheat were characterised. All results were evaluated statistically and the predominant physical and chemical characteristics that influenced the microbiological and biochemical properties of the agricultural soils during different stadia of take-all disease of wheat were identified using multivariate analyses. Although no significant difference @ > 0.05) could be observed between the various soils using conventional microbiological enumeration techniques, the incidence of Gliocladium spp. in suppressive soils was increased. Significant differences @ < 0.05) were observed between agricultural soils during different stadia of take-all disease of wheat. Although no clear distinction could be made between soils suppressive and neutral to take-all disease of wheat, soils suppressive and conducive to take-all disease of wheat differed substantially in their community level physiological profiles (CLPPs). Soils suppressive / neutral to take-all disease were characterised by enhanced utilisation of carboxylic acids, amino acids, and carbohydrates, while conducive soils were characterised by enhanced utilisation of carbohydrates. Shifts in the functional diversity of the associated microbial communities were possibly caused by the presence of Ggt and associated antagonistic fungal and bacterial populations in the various soils. It was evident that the relationships amongst the functionality of the microbial communities within the various soils had undergone changes through the different stages of development of take-all disease of wheat, thus implying different substrate utilisation capabilities of present soil microbial communities. Diversity indices were calculated as Shannon's diversity index (H') and substrate equitability (J) and were overall within the higher diversity range of 3.6 and 0.8, respectively, indicating the achievement of very high substrate diversity values in the various soils. A substantial percentage of the carbon sources were utilised, which contributed to the very high Shannon-Weaver substrate utilisation indices. Obtained substrate evenness (equitability) (J) indices indicated an existing high functional diversity. The functional diversity as observed during crop rotation, differed significantly (p < 0.05) from each other, implying different substrate utilisation capabilities of present soil microbial communities, which could possibly be ascribed to the excretion of root exudates by sunflowers and soybeans. Using the Sorenson's index, a clear distinction could be made between the degrees of substrate utilisation between microbial populations in soils conducive, suppressive, and neutral to take-all disease of wheat, as well as during crop rotation. Furthermore, the various soils could also be differentiated on the basis of the microbial community structure as determined by phospholipid fatty acid (PLFA) analysis. Soil suppressive to take-all disease of wheat differed significantly (p < 0.05) from soils conducive, and neutral to take-all disease of wheat, implying a shift in relationships amongst the structural diversity of microbial communities within the various soils. A positive association was observed between the microbial phospholipid fatty acid profiles, and dominant environmental variables of soils conducive, suppressive, and neutral to take-all disease of wheat. Soils conducive and neutral to take-all disease of wheat were characterised by high concentrations of manganese, as well as elevated concentrations of monounsaturated fatty acids, terminally branched saturated fatty acids, and polyunsaturated fatty acids which were indicative of Gram-negative bacteria, Gram-positive bacteria and micro eukaryotes (primarily fungi), respectively. These soils were also characterised by low concentrations of phosphorous, potassium, percentage organic carbon, and percentage organic nitrogen, as well as low soil pH. Soil suppressive to take-all disease of wheat was characterised by the elevated levels of estimated of biomass and elevated concentrations of normal saturated fatty acids, which is ubiquitous to micro-organisms. The concentration of normal saturated fatty acids in suppressive soils is indicative of a low structural diversity. This soil was also characterised by high concentrations of phosphorous, potassium, percentage organic carbon, and percentage organic nitrogen, as well as elevated soil pH. The relationship between PLFAs and agricultural soils was investigated using principal component analysis (PCA), redundancy analysis (RDA) and discriminant analysis (DA). Soil suppressive to take-all disease of wheat differed significantly (p < 0.05) from soils conducive, and neutral to take-all disease of wheat, implying a shift in relationships amongst the structural diversity of microbial communities within the various soils. A positive association was observed between the microbial phospholipid fatty acid profiles, and dominant environmental variables of soils conducive, suppressive, and neutral to take-all disease of wheat. Hierarchical cluster analysis of the major phospholipid fatty acid groups indicated that the structural diversity differed significantly between soils conducive, suppressive, and neutral to take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. The results indicate that the microbial community functionality as well as the microbial community structure was significantly influenced by the presence of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici, and that the characterisation of microbial functional and structural diversity by analysis of community level physiological profiles and phospholipid fatty acid analysis, respectively, could be successfully used as an assessment criteria for the evaluation of agricultural soils conducive, suppressive, and neutral to take-all disease of wheat, as well as in crop rotation systems. This methodology might be of significant value in assisting in the management and evaluation of agricultural soils subject to the prevalence of other soilborne diseases. / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2004.

Este estudo reúne os principais resultados de fluorescência estática e resolvida no tempo sobre a interação da porfirina meso-tetrakis (4-metilpiridil), na forma de base livre (TMPyP) e complexada com Zn2+ (ZnTMPyP), com vesículas de fosfolipídio. Adicionalmente foram utilizadas as técnicas de potencial zeta e espalhamento de luz dinâmico (DLS, do inglês \"dynamic light scattering\"). As vesículas de fosfolipídio foram formadas por dois conjuntos de fosfolipídios: saturados e insaturados. O primeiro grupo é formado pela mistura dos fosfolipídios zwiteriônico 1,2-dipalmitoil-sn-glicero-3-fosfocolina (DPPC) e aniônico 1,2-dipalmitoil-sn-3-glicero-[fosfo-rac-(1- glicerol)] (DPPG), a diferentes razões molares. Os estudos utilizando tais sistemas foram realizados abaixo (25oC) e acima (50oC) da temperatura de transição de fase gel-líquido cristalino destes fosfolipídios (~ 41oC). O segundo grupo é formado pela mistura dos fosfolipídios zwiteriônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfocolina (POPC) e aniônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfo(1-rac-glicerol) (POPG). Como a transição de fase destes dois fosfolipídios ocorre a temperaturas negativas, todos os experimentos foram realizados a 25oC (vesículas no estado líquido cristalino). Todos os sistemas foram preparados através do método de extrusão para a obtenção de vesículas grandes unilamelares (LUV, do inglês \"large unilamellar vesicles\"). As análises dos dados de fluorescência indicaram que a atração eletrostática entre os substituíntes (positivamente carregados) das porfirinas TMPyP e ZnTMPyP e o grupo das cabeças polares (camada de Stern) das vesículas de fosfolipídio desempenha um papel fundamental na associação da porfirina. A distribuição da TMPyP entre o meio aquoso (tampão) e as vesículas de fosfolipídio foi evidenciada pela coexistência de um tempo de vida de fluorescência mais curto (~ 5 ns) e outro mais longo (~ 9-11 ns), respectivamente. Baseado nos valores das constantes pré-exponenciais, estudos adicionais mostram que a distribuição acima é afetada pela concentração de sal na solução. Os resultados de supressão de fluorescência com o supressor iodeto de potássio (KI) indicaram que ambas porfirinas estão localizadas, preferencialmente, na região da camada de Stern. Este resultado foi confirmado pelos estudos de potencial zeta e de DLS, os quais mostraram uma neutralização parcial das cargas negativas na superfície das vesículas devido à associação da porfirina. / This study presents time-resolved and steady-state fluorescence results on the interaction of the meso-tetrakis (4-methylpyridil) porphyrin, in free base form (TMPyP), and complexed with Zn2+ (ZnTMPyP), with phospholipid vesicles. Zeta potential and dynamic light scattering (DLS) techniques were also used. Phospholipid vesicles were formed by two phospholipid systems: saturated and unsaturated. The first group is a mixture of zwiterionic dipalmitoyl-sn-glycero-3-phosphocoline (DPPC) and anionic 1,2-dipalmitoyl-sn-3-glycero-[phospho-rac-(1-glycerol)] (DPPG) phospholipids, at different molar ratios. Measurements were performed bellow (25oC) and above (50oC) the main gel-liquid crystalline phase transition temperature (~ 41oC). The second group is constituted by a mixture of zwiterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1-rac-glycerol) (POPG) phospholipids, at different molar ratios. Since the gel-liquid crystalline phase transition of these phospholipids occurs at a very low temperature value, all experiments were performed at 25oC (liquid crystalline state vesicles). All phospholipid systems were prepared through the extrusion method in order to obtain large unilamellar vesicles (LUV). The fluorescence data analyses indicated that the electrostatic attraction between the porphyrin substituents (positively charged) and the polar head groups of the phospholipid vesicles (Stern layer) plays an important role on the porphyrin binding affinity. The distribution of TMPyP between the aqueous medium (buffer) and the phospholipid vesicles was characterized by the coexistence of a shorter (~ 5 ns) and a longer (~ 9-11 ns) fluorescence lifetimes, respectively. Based on the pre- exponential values, additional time-resolved experiments showed a redistribution of the porphyrin at increasing salt concentration. The quenching studies, using potassium iodide (KI) as quencher, indicated that both TMPyP and ZnTMPyP are preferentially located at the Stern layer region. This result is in agreement with the zeta potential and DLS findings, which demonstrated a partial neutralization of the negative charges at the vesicle surface due to the porphyrin association.

Cette thèse est une étude théorique et expérimentale sur la formation de liposomes par des procédés utilisant le dioxyde de carbone (CO2) supercritique. Les liposomes sont des vésicules sphériques nano- ou micrométriques dont la paroi est composée d’une ou plusieurs bicouches concentriques de phospholipides séparant un milieu aqueux d’un autre. L’efficacité des procédés supercritiques, comme alternative aux méthodes conventionnelles pour former des liposomes, a déjà été démontrée. Une synthèse critique des résultats de la littérature a été réalisée au cours de ce travail. Parallèlement, une étude fondamentale sur le comportement des systèmes ternaires CO2/eau/surfactant sous pression a été menée et il a été démontré qu’en fonction du type d’émulsions formées au cours du procédé sous pression, les caractéristiques des liposomes produits lors de la dépressurisation sont différentes. Le procédé Supercritical Anti-Solvent a été utilisé pour microniser des phospholipides. Des liposomes sphériques, multilamellaires et de tailles comprises entre 0,1 et 1µm ont ensuite été formés de manière reproductible par hydratation des phospholipides micronisés. Dans les mêmes conditions (même solvant), la méthode conventionnelle de Bangham n’a pas donné des résultats reproductibles et les liposomes formés n’étaient pas tous sphériques. Le résultat majeur de ce travail est la mise au point d’un procédé supercritique innovant. Innovant car continu et en une seule étape, le procédé Continuous Anti-Solvent permet de former de manière reproductible des liposomes sphériques, multilamellaires et de tailles comprises entre 10 et 100µm. / The present thesis is dedicated to liposome formation using supercritical carbon dioxide (CO2). Liposomes are spherical vesicles composed of one or more concentric phospholipid bilayers surrounding an aqueous core. Dense gas processes offer reliable alternatives to conventional methods in liposome formation. We present a review of the literature and we summarized all of the results in a discussion section, with particular attention to emulsion formation under pressure. As it has been shown, the phase behaviour of the ternary CO2/water/surfactant system under pressure greatly influences liposome formation during depressurization. The Supercritical Anti-Solvent process has been used to micronized phospholipids. Then, the micronized particles were hydrated to form spherical and multilamellar liposomes with diameters between 0.1 and 1µm in a reproducible way. Using the same conditions (the same solvent), the conventional Bangham method did not provide reproducible assay results and formed liposomes were not all spherical. The main result of this work is the design of a new supercritical process dedicated to liposome formation. Unlike the current dense gas technologies, the Continuous Anti-Solvent method breaks new ground because it is a single step and continuous process. Liposomes prepared with the Continuous Anti-Solvent method are spherical and multilamellar with diameters between 10 and 100µm.

<p>Controlling the surface chemistry of 2D materials is critical for the development of next generation applications including nanoelectronics and organic photovoltaics (OPVs). Further, next generation nanoelectronics devices require very specific 2D patterns of conductors and insulators with prescribed connectivity and repeating patterns less than 10 nm. However, both top-down and bottom-up approaches currently used lack the ability to pattern materials with sub 10-nm precision over large scales. Nevertheless, a class of monolayer chemistry offers a way to solve this problem through controlled long-range ordering with superior sub-10 nm patterning resolution. Graphene is most often functionalized noncovalently, which preserves most of its intrinsic properties (<i>i.e.,</i> electronic conductivity) and allows spatial modulation of the surface. Phospholipids such as 1,2-bis(10,12-tricsadiynoyl)-<i>sn­</i>-glycero-3-phosphoethanolamine (diyne PE) form lying down lamellar phases on graphene where both the hydrophilic head and hydrophobic tail are exposed to the interface and resemble a repeating cross section of the cell membrane. Phospholipid is made up of a complex headgroup structure and strong headgroup dipole which allows for a diverse range of chemistry and docking of objects to occur at the nonpolar membrane, these principals are equally as important at the nonpolar interface of 2D materials. A key component in the development of nanoelectronics is the integration of inorganic nanocrystals such as nanowires into materials at the wafer scale. Nanocrystals can be integrated into materials through templated growth on to surface of interest as well as through assembly processes (i.e. interfacial adsorption). </p> <p>In this work, I have demonstrated that gold nanowires (AuNWs) can be templated on striped phospholipid monolayers, which have an orientable headgroup dipoles that can order and straighten flexible 2-nm diameter AuNWs with wire lengths of ~1 µm. While AuNWs in solution experience bundling effects due to depletion attraction interactions, wires adsorb to the surface in a well separated fashion with wire-wire distances (e.g. 14 or 21 nm) matching multiples of the PE template pitch. This suggests repulsive interactions between wires upon interaction with dipole arrays on the surface. Although the reaction and templating of AuNWs is completed in a nonpolar environment (cyclohexane), the ordering of wires varies based on the hydration of the PE template in the presence of excess oleylamine, which forms hemicylindrical micelles around the hydrated headgroups protecting the polar environment. Results suggest that PE template experience membrane-mimetic dipole orientation behaviors, which in turn influences the orientation and ordering of objects in a nonpolar environment.</p> <p>Another promising material for bottom-up device applications is MoS₂substrates due to their useful electronic properties. However, being able to control the surface chemistry of different materials, like MoS₂, is relatively understudied, resulting in very limited examples of MoS₂substrates used in bottom-up approaches for nanoelectronics devices. Diyne PE templates adsorb on to MoS₂­in an edge-on conformation in which the alkyl tails stack on top of each other increasing the overall stability of the monolayer. A decrease in lateral spacing results in high local concentrations of orientable headgroups dipoles along with stacked tails which could affect the interactions and adsorption of inorganic materials (i.e. AuNW) at the interface. </p> <p>Here, I show that both diyne PE/HOPG and diyne PE/MoS₂ substrates can template AuNW of various lengths with long range ordering over areas up to 100 µm². Wires on both substrates experience repulsive interactions upon contact with the headgroup dipole arrays resulting in wire-wire distances greater than the template pitch (7 nm). As the wire length is shortened the measured distance between wires become smaller eventually resulting in tight packed ribbon phases. Wires within these ribbon phases have wire-wire distances equal to the template. Ribbon phases occur on diyne PE/HOPG substrates when the wire length is ~50 nm, whereas wire below ~600 nm produce ribbon phases on diyne PE/MoS_2­substrates. </p> <p>Another important aspect to future scientific development is the catalysis of organic reactions, specifically oxygenation of organic sulfides. Sulfide oxygenation is important for applications such as medicinal chemistry, petroleum desulfurization, and nerve agent detoxification. Both reaction rates and the use of inexpensive oxidants and catalysts are important for practical applications. Hydrogen peroxide and <i>tert</i>-butyl hydroperoxide are ideal oxidants due to being cost efficient and environmentally friendly. Hydrogen peroxide can be activated through transition metal base homogeneous catalysts. Some of the most common catalysts are homo- and hetero-polyoxometalates (POMs) due their chemical robustness. Heptamolybdate [Mo₇O₂₄]^6-is a member of the isopolymolybdate family and its ammonium salt is commercially available and low in cost.²² Heteropolyoxometalates have been widely studied as a catalyst for oxygenation reactions whereas heptamolybdate has been rarely studied in oxygenation reactions. </p> <p> Here I report sulfide oxygenation activity of both heptamolybdate and its peroxo derivate [Mo₇O₂₂(O₂)₂]^6-. Sulfide oxygenation of methyl phenyl sulfide (MPS) by H₂O₂to sulfoxide and sulfone occurs rapidly with 100 % utility of H₂O₂ in the presence of [Mo₇O₂₂(O₂)₂]^6-, suggesting the peroxo adduct is an efficient catalyst. However, heptamolybdate is a faster catalyst compared to [Mo₇O₂₂(O₂)₂]^6- for MPS oxygenation and all other sulfides tested under identical conditions. Pseudo-first order <i>k</i>_cat constants from initial rate kinetics show that [Mo₇O₂₄]^6-catalyzes sulfide oxygenation faster. The significant difference in the <i>k</i>_cat suggests differences in the active catalytic species, which was characterized by both UV-Vis and electrospray ionization mass spectrometry. ESI-MS suggest that the active intermediate of [Mo₇O₂₄]^6-under catalytic reaction conditions for sulfide oxygenation by H₂O₂ is [Mo₂O₁₁]^2-. These results show that heptamolybdate is a highly efficient catalyst for H₂O₂oxygenation of organic sulfides.</p>

Phospholipid vesicles are common model systems for cell membranes. Important aspects of the membrane function relate to its mechanical properties. Here we have investigated the deformation behaviour of phospholipid vesicles in a dual-beam laser trap, also called an optical stretcher. This study explicitly makes use of the inherent heating present in such traps to investigate the dependence of vesicle deformation on temperature. By using lasers with different wavelengths, optically induced mechanical stresses and temperature increase can be tuned fairly independently with a single setup. The phase transition temperature of vesicles can be clearly identified by an increase in deformation. In the case of no heating effects, a minimal model for drop deformation in an optical stretcher and a more specific model for vesicle deformation that takes explicitly into account the angular dependence of the optical stress are presented to account for the experimental results. Elastic constants are extracted from the fitting procedures, which agree with literature data. This study demonstrates the utility of optical stretching, which is easily combined with microfluidic delivery, for the future serial, high-throughput study of the mechanical and thermodynamic properties of phospholipid vesicles.