No description available.

No description available.

No description available.

La famille des lipases pancréatiques est constituée de trois sous-familles : la lipase pancréatique classique (PL) et les lipases pancréatiques apparentées de type 1 et 2 (PLRP1 et PLRP2). Contrairement à la PL, qui est active seulement sur les triglycérides, la PLRP2 possède trois types d’activités enzymatiques : lipase, phospholipase de type A1 et surtout galactolipase. Cette dernière activité est particulièrement intéressante car les galactolipides, principalement les monogalactosyldiacylglycérols (MGDG) et les digalactosyldiacylglycérols (DGDG) sont les lipides les plus abondants dans la nature. Un test continu, spécifique et fiable de dosage de l’activité galactolipase à été mis au point en utilisant la technique du pH-stat et un galactolipide synthétique à chaîne moyenne comme substrat, le MGDG en C8. Ce nouveau test de dosage nous a permis de mesurer pour la première fois des activités spécifiques importantes avec les PLRP2 humaine (HPLRP2) et du cobaye (GPLRP2) (1786±100 et 5420±85 U/mg, respectivement). Nous avons par la suite utilisé cette même technique pour étudier l’hydrolyse par les PLRP2 des galactolipides naturels à chaînes longues purifiés à partir de feuilles d’épinard. Les activités spécifiques mesurées sont du même ordre de grandeur que celles mesurées avec le MGDG en C8 mais aussi le DGDG en C8 synthétique, montrant ainsi que l’activité galactolipase n’est affectée ni par la longueur de la chaîne acyle ni par la présence d’un ou de deux résidus galactoses. Grâce à ce test nous avons pu réaliser un criblage de lipases microbiennes de différentes origines à la recherche d’activités galactolipase. Dans une deuxième partie, nous avons étudié les propriétés physico-chimiques du bis-monoacylglycérol-phopshate (BMP). Nous avons montré que non seulement ce phospholipide rare est hydrolysé par la PLRP2, mais le BMP et la PLRP2 humaine peuvent être trouvés dans la même cellule, le monocyte, suggérant un nouveau rôle physiologique pour la PLRP2. / The pancreatic lipase family consists of three sub-families : the classical pancreatic lipase (PL) and the pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). On the contrary to PL, which is active only on triglycerides, the PLRP2 possesses three types of enzymatic activities: lipase activity, phospholipase A1 activity and especially galactolipase activity. This latter activity is particularly interesting because monogalactosyl-diacylglycerol (MGDG) and digalactosyldiacyl-glycerol (DGDG) are the most abundant lipids in nature. A specific, continuous and robust galactolipase assay using the pH-stat technique and a synthetic medium chain MGDG as substrate was developed, and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related proteins 2 were tested as model enzymes. This new assay allowed us to measure for the first time high specific activities with both PLRP2 (1786±100 and 5420±85 U/mg, respectively). We then used this technique to study the hydrolysis by PLRP2 of natural long chain galactolipids purified from spinach leaves. The length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain and long chain galactolipids. We also used the pH-stat technique and medium chain MGDG and DGDG for screening several microbial lipases for their galactolipase activity.In a separate study, we investigated the physico-chemical properties of bis-monoacylglycorol-phopshate (BMP) and found that this rare phospholipid was hydrolyzed by PLRP2. Moreover, BMP and human PLRP2 could be found in the same cell, the monocyte, suggesting a new physiological role for PLRP2.

No description available.

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

The main objective of this study was the formulation of food-grade water-in-oil (W/O) nano-dispersions based mainly on medium or long-chain triglycerides. Two types of dispersions were formulated and structurally compared, namely emulsions and microemulsions. The systems were used as matrices for encapsulating targeted bioactive molecules with specific characteristics such as antioxidants or peptides. The structural characterization of the formulated systems was investigated using techniques such as Electron Paramagnetic Resonance (EPR) spectroscopy, Dynamic Light Scattering (DLS), Cryogenic Transmission Electron Microscopy (Cryo-TEM) and Small Angle Xray Scattering (SAXS). The existence of swollen inverse micelles was revealed for the case of microemulsions whereas larger droplets still at the nano-scale were observed for the case of emulsions. Structural differences in the presence of the bioactive molecules or induced by the alteration of components were also observed. In order to study the efficacy of the formulations, the proposed loaded systems were assessed either using EPR spectroscopy or Well Diffusion Assay (WDA) depending on the bioactive molecule. It was found that the encapsulated molecules retained their claimed characteristics when encapsulated to the proposed matrices. Finally, some of the formulated dispersions were investigated for their behavior under gastrointestinal (GI) conditions. A two-step digestion model using recombinant Dog Gastric Lipase (rDGL) and Porcine Pancreatic Lipase (PPL) was proposed to simulate lipid hydrolysis in humans. The studies revealed significant decrease of the rDGL specific activity in the presence of the microemulsion while in the presence of lower percent of surfactants (case of emulsion) no alterations were observed.

Abstract ACCURATE METHODOLOGY FOR MONITORING BIOMEMBRANE EVENTS By Christine A. Winschel, Ph.D. A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in Chemistry at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Director: Dr. Vladimir A. Sidorov ASSOCIATE PROFESSOR, DEPARTMENT OF CHEMISTRY This study describes the synthesis and characterization of a new receptor (cyclen 1) capable of strong selective binding of pyrene-based anionic dyes under near-physiological conditions. This receptor comprises four naphthylthiourea groups tethered to a cyclen core via an ester linkage. The most important finding was the ability of cyclen 1 to bind efficiently to a pH-sensitive pyranine dye, a dye that is commonly used in various biomembrane assays. The high affinity of cyclen 1 to pyranine, its impermeability to the lipid bilayer membrane, fast kinetics of binding, and ability to quench pyranine’s fluorescence were used as a basis for a new membrane leakage assay. This membrane leakage assay is fully compatible with the commonly applied pH-stat transport assay, and therefore it allows for differentiation of ion transport and nonselective leakage mechanisms within a single set of experiments. In the second part of this study a new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of a newly synthesized cascade-blue-labeled lipid through complex formation with cyclen 1. This receptor-dye complexation also has high affinity for binding at micromolar concentrations and can be reversed by either competitive displacement of the lipid probe or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. This new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments, and is less invasive to the model and cell membranes than the commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Lastly, new pH-sensitive lipids were synthesized and utilized in the formulation of liposomes suitable for controlled drug release. These liposomes contain various amounts of internal NaCl and undergo internal acidification upon the exogenous addition of an HCl co-transporter in a physiologically relevant NaCl solution. Therefore, acidification ultimately leads to the hydrolysis of the pH-sensitive lipids and subsequent contents release. These liposomes were found to be insensitive to physiological concentrations of human serum albumin and to be non-toxic to cells at concentrations exceeding pharmacological relevance. These results render this new drug release model potentially suitable for in vivo applications.