Due to increasing problems with bacterial resistance development, there is a growing need for identifying new types of antibiotics. Antimicrobial peptides constitute an interesting group of substances for this purpose, since they are believed to act mainly by disrupting the bacterial membrane, which is a fast and non-specific mechanism. In order to understand the details on this action simplified phospholipid model membranes based on liposomes, monolayers and bilayers, were employed in this thesis. By in situ ellipsometry studies on supported lipid bilayers in combination with leakage from liposomes it was found that peptide-induced membrane rupture to a great extent is related to peptide adsorption. The peptide activity and mechanism of action is highly dependent on peptide properties such as length, topology, charge, and hydrophobicity. Electrostatic interactions are crucial for peptide adsorption, whereas α-helix formation is of less importance, demonstrated by the dominating peptide conformation being random coil both in absence and presence of membranes, as investigated by circular dichroism. Comparable effects were observed in both mono- and bilayer systems, showing that formation of transmembrane structures is no prerequisite for membrane rupture by complement-derived peptides. Electrochemical studies on these peptides further demonstrated that hydrophobic interactions facilitate peptide penetration into the membrane, causing defects in close proximity to the peptides, while strong electrostatic interactions arrest the peptide in the headgroup region. Increasing the peptide hydrophobicity, by e.g., tryptophan end-tagging, also increases salt resistance. Good correlations were found between model membrane investigations and antibacterial activity towards both Gram-negative and Gram-positive bacteria, showing that membrane rupture is a key mechanism of action for the peptides investigated. In addition, for all peptides investigated cell toxicity is low.

Los biotensioactivos son compuestos producidos por microorganismos que poseen actividad superficial, baja toxicidad, carácter biodegradable y efectividad a temperaturas y valores de pH extremos. Los ramnolípidos, son un tipo de biotensioactivos con interesantes actividades biológicas, que parecen estar relacionadas con su interacción con las membranas. En esta tesis doctoral se estudia la interacción de la fracción dirramnolipídica purificada de cultivos de Pseudomonas aeruginosa con membranas modelo y membranas biológicas, la interacción con albúmina de suero bovino, utilizada como proteína modelo, y por último, se evalúa el papel de este biotensioactivo en la estabilización de liposomas sensibles a pH y su aplicación en la introducción de compuestos en células animales. Para el desarrollo de este trabajo de investigación se han utilizado técnicas como calorimetría, FT-IR, difracción de rayos X, dispersión de luz dinámica y microscopía electrónica. / Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. This thesis presents a detailed molecular level study of the dirhamnolipid interactions with artificial phospholipids membranes and biological membranas, and the dirhamnolipid interactions with bovine serum albumin, used as a general globular protein model. Furthemore, this work includes the characterization of phosphatidylethanolamine/diRL pH-sensitive liposomes described as efficient systems for cytoplasmic delivery of foreign compounds into living cells. It has been employed a number of physical techniques such as calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering.

Recent work has demonstrated that vast amounts of soil organic carbon (SOC) are redistributed and buried within Canadian croplands; however, the effects of redistribution on SOC dynamics and biological properties of the soil environment remain unknown. Because soil microorganisms are drivers of carbon (C) turnover in soil, the effects of such processes on microbial community dynamics are important in assessing the overall effects of redistribution and the stability of displaced C. This is particularly important in the face of future climate change scenarios and potential disturbances. The objectives of this study were to examine microbial community dynamics with depth and among landscape positions in an eroded landscape, and to assess C mineralization response between surface and subsurface soil layers in a depositional position. Microbial abundance was highly influenced by SOC redistribution. This was most evident in the buried backslope position where substantial soil and SOC deposition had occurred, creating a very thick A horizon (ca. 80 cm). Phospholipid fatty acid (PLFA) analysis revealed substantial concentrations of microbial biomass located at depth (30-60 cm), which was greater than PLFA concentration at the soil surface and correlated with SOC concentration. Community structure analysis demonstrated the strong influence of landscape position and depth in structuring microbial communities near the soil surface (0-20 cm). Communities in positions that were predominantly erosional were the most different from those in the depositional position, accounting for the largest amount of variation (60%) in the overall analysis. The existence of distinct microbial communities found in depositional material (0-25 cm) and within the buried A horizon (30-80 cm) in the buried backslope position indicate a strong influence of depth and redistribution in structuring microbial communities. The existence of significant viable biomass in the buried A horizon of the depositional position leads to question the persistence of highly concentrated, buried SOC over many decades. When soils from surface (0-5 and 20-25 cm) and subsurface (40-45 and 65-70 cm) depths were incubated in surface-like conditions, greater mineralization response in surface relative to subsurface soils, despite relatively similar SOC concentration, suggests that redistribution protects buried C from decomposition. Distinct microbial communities found at the onset and completion of the mineralization study between surface and subsurface soil layers may indicate the influence of microbial community structure on mineralization response. Depth was the largest source of variation in microbial community structure, and although a shift occurred after exposure to incubation conditions, the effect of depth remained the strongest influence. This work indicates that SOC redistribution strongly influences microbial abundance and community structure development, primarily driven by altered substrate gradients occurring with depth, and suggests that C is less susceptible to decomposition once buried in depositional positions.

The ATP-binding cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport, which is the elimination of excess sterols from peripheral cells and their transport to the liver for elimination. Early studies failed to detect significant ATPase activity, prompting the suggestion that ABCA1 was an ATP-regulated receptor, rather than an active transporter. We have provided evidence that ABCA1 can bind ATP and trap its hydrolysis product, ADP, in the presence of either ortho-vanadate or beryllium fluoride and Mg2+ or Mn2+. We have also shown that both nucleotide-binding domains (NBDs) trap nucleotide comparably, suggesting that ABCA1 is a functional ATPase. In addition, we have shown that ABCA1 can directly transport 25-hydroxycholesterol (25-OHC) in an ATP-dependent manner using a membrane vesicle uptake assay, and can do so when the physiological substrate acceptor apoA-I is replaced with BSA as a non-specific binding protein. Although more than 50 naturally occurring missense mutations and polymorphisms in ABCA1 have been identified in individuals with HDL-C levels within the lowest 5th percentile of the general population, the extent to which many of these mutations affect ABCA1 function is not known and cannot be predicted. Naturally occurring extracellular loop (ECL) mutations W590S and C1477R have both been shown to effectively eliminate the ability to mediate lipid efflux, despite the fact that the W590S mutant protein retains the ability to bind apoA-I. We show that neither mutant can transport nor efflux 25-OHC, whether in the presence of apoA-I or BSA, despite apparently full retention of the ability to bind and trap nucleotide. This suggests that these two ECL mutations inhibit transport by a mechanism that is independent of their effect on apoA-I binding. By introduction of naturally occurring mutations in the NBDs, we show that although some mutations associated with Tangier Disease, such as N935S, essentially eliminate nucleotide trapping and substrate translocation, other polymorphisms such as L1026P and T2073A associated with low HDL-C, appear to be fully functional. Lastly, we observed differences in the behaviour of both wild-type and mutant forms of ABCA1-GFP depending on whether they were expressed in insect or mammalian cell lines. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-02-12 11:14:11.381

Climate change is likely to alter plant species composition and interactions between plants and soil microbes that together dictate the quantity and quality of forage produced in pastures, the base of animal production in central Kentucky. This study assessed the seasonal dynamics of soil microbes and their response to increased temperature (+3oC) and growing season precipitation (+30% of the mean annual). Total soil microbial biomass, community composition, enzyme activities, potential carbon mineralization, and catabolic responses to selected substrates were measured seasonally in the different climate treatments. In this system, seasonal variability was a dominant driving factor for all the soil microbial characteristics that I investigated. Summer maxima and winter minima were identified in the active microbial biomass, while soil microbial community structure differed between each season. Extracellular enzyme activities were generally highest in either the spring or summer, while seasonal patterns for each substrate were unique across catabolic response profiles. Climate treatments produced few significant main or interactive effects on the soil microbial biomass and function. This resiliency, coupled with evidence of functional redundancy, suggests that central Kentucky pasture ecosystems may be well-equipped to handle future environmental stress associated with climate change and to maintain critical ecosystem services.

Effect of lipids on binding characteristics of opioid receptors in membranes prepared from rat brain were studied. Lipid concentrations causing changes in specific binding of [3H]Endomorphin-1 (ProE1), an opioid agonist highly specific to mu-type opioid, [3H]Ile5,6deltorphin II (DIDI), an agonist ligand highly specific to delta type receptor and [3H]Naloxone (Nlx), a universal opioid receptor antagonist were determined. Inhibition of [3H]ProE1, [3H]DIDI and [3H]Nlx specific binding was also examined by homologous displacement experiments in the presence and absence of lipids. In order to understand whether the changes occurring in the specific binding is due to changes in equilibrium dissociation constant (KD) or maximum number of binding sites (Bmax), the equilibrium binding experiments were performed. Arachidonic acid (AA) inhibited binding of both agonist and antagonist ligand in a dose dependent manner with IC50 values of 0.15, 0.1, and 0.6 mM for [3H]ProE1, [3H]DIDI and [3H]Nlx, respectively. Kd values were not affected while Bmax values decreased 38 % and 76 % for mu, and delta receptor subtypes, respectively. For [3H]Nlx, Bmax values decreased 20 and 56 % in the absence and presence of 100 mM NaCl, respectively. Cholesteryl hemisuccinate (CHS) enhances (100 % of control) ligand binding at mu-sites however no effect was encountered at delta sites. Furthermore, CHS also enhances (50 % of control) the binding of antagonist ligand in the absence of NaCl. Bmax values were increased by 70 % for mu sites and 40% for antagonist ligand binding site. Under similar conditions Kd values were not affected. Phosphatidic acid (PA) and phosphatidylcholine (PC) exhibited negligible effect on ligand binding. PA decreased specific binding of ProE1 and DIDI by 16 and 10 %, respectively. Specific binding of antagonist ligand Nlx decreased 11 % in the presence of NaCl whereas in the absence of NaCl specific binding is very close to control. In the presence of PC specific binding of both agonist and antagonist ligands were around control values. In this study modulatory effect of lysophospholipids, lysophosphatidic acid and lysophosphatidylcholine on opioid binding sites were evaluated for the first time. Both lysophospholipids exhibited similar effects: decreasing specific binding in receptor subtype independent manner between 0.1 to 1 mM range. Kd values were not significantly affected, while remarkable decrease (45-75 %) in Bmax values were observed.

Neste trabalho foram estudadas as interações entre os corantes ciânicos com dois cromóforos (BCD) e monocamadas de Langmuir de fosfolipídio (DPPC). Devido ao alto coeficiente de absorção molar da luz na região ? > 600 nm, ao alto rendimento quântico do estado tripleto, à alta fotocitotoxicidade e à alta afinidade com estruturas celulares os BCD são promissores para serem utilizados em terapia fotodinâmica. Os estudos das características da interação de BCD com a monocamada são importantes tanto para esclarecer os efeitos estruturais na interação de monocamadas com outras moléculas quanto para suas aplicações em terapia fotodinâmica e em outras áreas tecnológicas tais como nanoeletrônica, fotônica etc. As isotermas de pressão superficial (?-A) obtidas mostram que os BCD e as monocamadas interagem, pois as isotermas (?-A) se expandem conforme é aumentado à porcentagem de BCD. Esta interação é dependente tanto da estrutura do BCD quanto da força iônica da subfase. A partir das isotermas (?-A) foi obtido o módulo de compressibilidade (Cs-1 = -A (??/?A) das monocamadas, do qual foram analisadas, de maneira mais detalhada, as modificações da monocamada de DPPC devido à presença dos BCD. Estas modificações compreendem o aparecimento da fase líquida expandida (LE) e formação dos domínios antecipada em comparação com a monocamada de DPPC puro. Foi observado que a área mínima por molécula e a elasticidade da monocamada na fase condensada (C) aumentam juntos com a porcentagem dos BCD. Na presença de BCD 180º as isotermas de potencial superficial (?V-A) sofrem uma expansão, no entanto, o valor máximo do potencial permanece nalterado. Isto indica que os BCD estimulam (antecipam) a orientação das moléculas de DPPC na interface ar-água e, consequentemente, a co-orientação das cabeças polares do DPPC, mas não contribuem no potencial superficial. Foi observado que a presença de NaCl na subfase aumenta o efeito dos BCD na formação da monocamada DPPC. Acreditamos que este efeito sinérgico é devido às interações mútuas dos ânions Cl- com a carga positiva do grupo trimetilamônio da cabeça polar do DPPC e das cargas positivas do BCD com a carga negativa do grupo fosfato do DPPC. As imagens obtidas pela microscopia de ângulo de Brewster mostram, em concordância com a análise das isotermas de pressão (?-A) e de potencial (?V- A), que a formação dos domínios, e conseqüentemente da monocamada, é antecipada na presença de BCD. Entretanto, a presença do BCD não altera o formato dos domínios, mas diminui seu tamanho. A análise realizada sobre os espectros de absorção óptica mostrou que os BCD podem formar agregados tipos H na monocamada e não foram observados agregados tipo J. Baseando-se na análise do efeito da estrutura dos BCD, na interação com a monocamada, e dos espectros de absorção propusemos dois modelos de interação dos BCD com a monocamada e acreditamos que o seguinte modelo é a melhor modelagem do sistema: - BCD se inseri na monocamada, paralelamente ao eixo da molécula de DPPC, interagindo tanto com a cauda hidrofóbica quanto com a cabeça polar. Entretanto, os resultados obtidos não permitem excluir completamente o segundo modelo proposto, no qual o BCD se localiza na parte polar da monocamada, perpendicular ao eixo da molécula de DPPC. / In this work the interaction of cyanine dyes with two chromophores with the DPPC phospholipid Langmuir monolayers was studied. Due to their high optical absorption in the region ? > 600 nm, high triplet state quantum yields, high photocytotoxicity and high affinity with cell structures the BCD are promising for application in photodynamic therapy. The study of the interaction of BCD with monolayers is important to clarify the structural effects on the monolayer interaction with other molecules as well as for application in photodynamic therapy and in other fields of technology, such as nanoelectronics, photonics, etc. The superficial pressure isotherms (?-A) demonstrate that BCD do interact with monolayers, the (?-A) isotherms being expanded when the BCD relative content increases. This interaction depends on the BCD structure and the subphase ionic strength, as well. The curves of the compressibility module (Cs-1 = -A (??/?A) of the monolayers were obtained from the (?-A) isotherms, which were used for detailed analysis of the DPPC monolayer formation in the BCD presence. The analysis demonstrated that the monolayer expanded liquid phase and the domain were formed earlier in the BCD presence than for pure DPPC. It was observed that both the minimum area per molecule and the condensed phase elasticity increased when the BCD relative content increased. In the presence of BCD 1800 the superficial potential isotherms (?V-A) were expanded, but the maximum potential value was unchanged. This indicates that BCD stimulated the DPPC molecule orientation on the air-water interphase and, consequently, the co-orientation of the DPPC polar heads, but did not contribute itself in the surface potential. It was observed that NaCl in the subphase increased the BCD effects on the DPPC monolayer formation. We suppose that this synergetic effect is due to the mutual interaction of Cl- anions with positive charges of the trimethylammonium group of the DPPC polar head and that of positive BCD charges with its phosphate group negative charges. Images obtained with the Brewster microscopy confirmed that BCD stimulated the domain and, consequently, the monolayer formation in accordance with the (?-A) and (?V-A) analysis. At the same time the BCD did not change the domain geometry, but reduced their sizes. The analysis of the BCD optical absorption spectra demonstrate that the BCD molecules in the monolayer form H aggregates and no J aggregates were observed. Basing on the analysis of the BCD structure effects on the monolayer formation we can propose two models of the BCD - monolayer interaction and we believe that the follow model is better: - the BCD molecule is inserted in the monolayer interior being parallel with the DPPC molecule axis and interacting with both hydrophobic and polar DPPC parts; However, our data are not sufficient to exclude completely the possibility for the BCD molecule to be localized just in the polar head monolayer part being perpendicular to the DPPC molecule axis.

Les fonctions physiologiques spécifiques de la voie de N-méthylation de la phosphatidyléthanolamine (PE), une des deux voies de biosynthèse de la phosphatidylcholine (PC), restent relativement énigmatiques. Il a été démontré chez les poissons euryhalins qu’un stress hyperosmotique induisait une activation de cette voie métabolique au niveau hépatique. L’objectif de notre travail était de vérifier si ce phénomène se produit aussi chez d’autres animaux euryhalins. Les études réalisées in vivo sur deux espèces de crâbes, Eriocheir sinensis et Carcinus maenas, nous ont permis de montrer que l’acclimatation en eau de mer de ces animaux active la synthèse de PC par N-méthylation de la PE dans l’hépatopancréas. Les marquages radioisotopiques montrent aussi que cette PC est échangée avec le plasma et que ce phénomène est amplifié chez les animaux en eau de mer. Ce pool de PC est utilisé comme précurseur de la bétaïne, un osmoeffecteur organique important chez ces animaux. Nous avons ensuite caractérisé la voie de N-méthylation de la PE chez un animal osmoconformeur, la moule Mytilus galloprovincialis. Les résultats, obtenus in vivo et in vitro sur les tissus isolés, démontrent qu’une activité de N-méthylation de la PE en PC est exprimée dans la glande digestive et les hémocytes circulant de M. galloprovincialis. La PC ainsi synthétisée dans ces tissus est échangée avec l’hémolymphe de l’animal. De l’ensemble de ces observations, nous pouvons conclure que la synthèse de PC par N-méthylation est largement exprimée chez les animaux marins euryhalins et qu’une des fonctions physiologiques de cette voie métabolique est de synthétisée des osmolytes organiques comme la bétaïne / The specific physiological functions of the N-methylation of phosphatidylethanolamine (PE), one of the two biosynthetic pathways of phosphatidylcholine (PC), remain relatively mysterious. It has been demonstrated in euryhaline fish that hyperosmotic stress induced activation of this pathway in the liver. The aim of our work was to verify whether this phenomenon also occurs in other euryhaline animals. In vivo studies on two species of crabs, Eriocheir sinensis and Carcinus maenas, showed that seawater acclimation activates PC synthesis by N-methylation of PE in the hepatopancreas. Radioisotopic labelling also showed that PC is exchanged with the plasma and that this phenomenon is amplified in animals in seawater. This pool of PC is used as a precursor of betaine, an important organic osmoeffector in these animals. We then characterized the process of PE N-methylation in an osmoconforming animal, the mussel Mytilus galloprovincialis. The results, obtained in vivo and in vitro on isolated tissues, show that N-methylation of PE to PC is expressed in the digestive gland and circulating haemocytes in M. galloprovincialis. The PC synthesized in these tissues is exchanged with hemolymph of the animal. From all these observations, we conclude that the synthesis of PC by N-methylation is widely expressed in marine euryhaline animals and that a physiological function of this pathway is to provide organic osmolytes such as betaine

Le déclin de la production de pétrole, lié avec la diminution des matières premières carbonées pour la synthèse chimique ont mené les scientifiques à chercher de nouvelles sources de carbone pour l'industrie chimique. L'utilisation du dioxyde de carbone aiderait à réduire les émissions de gaz à effet de serre tout en fournissant une matière première renouvelable à base de bloc moléculaire en C1. En renversant les équilibres biologiques de trois déshydrogénases, nous avons effectué la biosynthèse multienzymatique en cascade du méthanol à partir de CO2 en utilisant la formiate déshydrogénase de Candida boidinii, la formaldéhyde déshydrogénase de Pseudomonas putida et l'alcool déshydrogénase de Saccacharomyces cerevisiae. Nous avons optimisé le système en ajustant les conditions catalytiques et la quantité relative de chaque déshydrogénase. La phosphite déshydrogénase de Pseudomonas stutzeri a été également choisi comme système de régénération du cofacteur nicotinamide adénine dinucléotide réduit (NADH) parmi 4 systèmes de régénération étudiés. L'ensemble du système a été encapsulé dans des nanocapsules poreuses de silice qui a permis d'augmenter 15 fois les productivités en méthanol. Nous avons montré que les dernières limitations rencontrées, comme la disponibilité du CO2 et l'accumulation du méthanol, peuvent être dépassées en mettant en place un système catalytique en flux continu en phase gaz. / The decline of oil production, linked with the decrease of carbon feedstock for chemical synthesis leads scientist to find new sources of carbon for the chemical industry. Use of carbon dioxide would help to reduce the greenhouse gas emissions while providing a renewable feedstock of C1 molecular building blocks. By reversing the biological metabolic reaction pathway of three dehydrogenase, we carried out multistep multienzyme biosynthesis of methanol from CO2 using formate dehydrogenase from Candida Boidinii, formaldehyde dehydrogenase from Pseudomonas Putida and alcohol dehydrogenase from Saccacharomyces cerevisiae. We improved the system active by adjusting the catalytic conditions and the relative quantity of each dehydrogenase. Phosphite dehydrogenase from Pseudomonas stutzeri was also chosen among 4 different studied systems to be introduced into the catalysis as a cofactor regenerating system for reduced nicotinamide adenine dinucleotide. The enzymatic system was then immobilized by encapsulation into novel phospholipid templated silica nanocapsules, allowing an increase of the methanol productivity by a factor 15. We show that the last limitation of the process as substrate availability and product accumulation can be overcome by running continuous enzymatic flow conversion in a gas phase.

This thesis is focused on investigation of phospholipid-hyaluronan system. First, appropriate method for preparation of bulk solution of phospholipid/lipid and suitable fluorescence probe were chosen. Sonification was selected as a method for preparation of bulk solution and pyrene was chosen as a fluorescence probe. From the group of phospholipids lecithin was selected. Next to phospholipid, lipid with no phosphate group (DPTAP) was utilized for comparison, alternatively a mixture of lipid (DPTAP) and phospholipid (DPPC). Instead of hyaluronan another polyelectrolytes (sodium polystyrene sulfonate, sodium alginate) were used too. Measurements were performed in water environment and in phosphate buffer saline (PBS). All investigation was accomplished by fluorescence spectroscopy and dynamic light scattering.