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Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic SwineHodges, William Anderson 28 February 2001 (has links)
Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity. / Master of Science
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Modification de domaines de liaison à la choline en vue de leur utilisation comme étiquette de purification de protéines recombinantes.DE SCHREVEL, Nathalie 21 October 2005 (has links)
Le but de ce travail consiste à créer un nouveau tag de purification d'affinité permettant la purification de protéines recombinantes sur une matrice DEAESépharose Fast Flow.
Dans la nature, certaines protéines de surface de Streptococcus pneumoniae sont liées à la paroi bactérienne par des interactions non convalentes faisant intervenir des molécules de choline présentes sur les acides téichoiques et lipotéichoiques. Ces protéines de surface présentent une organisation modulaire avec le domaine catalytique et le domaine de liaison fonctionnant indépendamment l'un de l'autre. La choline étant un analogue structural du DEAE, l'étude des domaines de liaison à la choline constitue une approche de choix pour concevoir un tag de purification présentant une affinité pour le DEAE-Sépharose.
Nous avons plus particulièrement travaillé sur la N-acétyl-L-alanine amidase (LytA) qui dégrade spécifiquement certaines liaisons du peptidoglycan de la paroi de Streptococcus pneumoniae. Son domaine de liaison à la choline C-terminal (ClytA) se compose de six motifs répétés imparfaits, constitué chacun d'une vingtaine de résidus.
Deux stratégies ont été développées pour concevoir le tag de purification. D'une part, 126 motifs répétés de 19 domaines de liaison à la choline ont été alignés pour définir une séquence consensus. Cette approche a permis de mettre en évidence les résidus importants conservés parmi les motifs répétés. D'autre part, nous avons construit des protéines de fusion portant des fragments du domaine de liaison ClytA de longueur variable. Des expériences de chromatographies sur matrice DEAESépharose nous ont permis d'isoler un petit fragment de ClytA(L234), présentant toujours une affinité spécifique pour le DEAE Sépharose. Cette affinité est maintenue lorsque le fragment L234 est fusionné à l'extrémité C-terminale d'une autre protéine reporter. Cependant, nos résultats suggèrent que le candidat tag L234 est instable et qu'il conduit à l'insolubilisation de la protéine de fusion lors de la production de celle-ci dans Escherichia coli. Afin d'améliorer la solubilité/stabilité du fragment L234, nous avons développé trois approches bioinformatiques. Cellesci ont permis de définir trois groupes de mutations permettant d'améliorer potentiellement la solubilité et/ou la stabilité du fragment L234. Les tags mutants ont été construits et fusionnés à l'extrémité C terminale de la thiorédoxine. Le premier tag mutant, EDE-L234, est plus soluble que la version non mutante mais présente une perte d'affinité pour le DEAE Sépharose. Le second mutant, NG-L234, ne montre pas d'augmentation de solubilité et perd également une partie de son affinité pour la matrice. Le troisième tag mutant, V1V2V3-L234, présente une augmentation d'affinité pour le DEAE-Sépharose bien que sa solubilité reste inchangée.
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Novel biomimetic polymeric nanoconjugates as drug delivery carriers for poorly soluble drugsKola-Mustapha, Adeola Tawakalitu January 2013 (has links)
Active Pharmaceutical Ingredients with poor solubility have presented significant difficulties in drug product design and development including slow and ineffective absorption leading to inadequate and variable bioavailability. Therefore it has become increasingly desirable to overcome the low aqueous solubility of drug candidates and develop more novel and innovative formulation approaches to increase the dissolution rate of the poorly soluble drugs. This work focuses on the formulation of novel amorphous ibuprofen-polymer nanoconjugates based on the polymer-drug complexation in order to improve its physical and dissolution characteristics without the use of toxic organic solvents. Plain and ibuprofen-loaded binary and ternary nanoconjugates were prepared using four modified co-precipitation techniques including melt solubilization; alkaline solubilization; surfactant solubilization and hydrotropic complexation techniques. A remarkably high loading capacity was achieved ranging from 89.05 to 99.49% across the four techniques and polymer-polymer ratio of 50:50 was found to be most efficient. All the four techniques reduced the size of ibuprofen (2.87 μm) significantly in the presence of 2.0 x10-3 mM of Diethylaminoethyl Dextran (DEAE-Dextran) in the order melt solubilization (203.25 nm) > alkaline solubilization (185.68 nm) > surfactant (Tween 80) solubilization (122.17 nm) > hydrotropic complexation (77.92 nm). 5.0 x 10-4 mM of chitosan also reduced the size of ibuprofen from 2872.12 to 10.70 nm (268-fold reduction). The FTIR spectroscopic analysis revealed electrostatic, hydrophobic and hydrogen bonding interaction between solubilized ibuprofen and the cationic polymers (DEAE-Dextran and chitosan) to form a new product (an amide). Polymer-polymer complexation also occurred between DEAE-Dextran and gellan as well as chitosan and gellan to a different extent depending on the mixing ratios. 1H and 13C NMR analysis confirmed the conjugation between ibuprofen and each of the cationic polymers as well as the formation of a new amide product. DSC thermal analysis showed that the nanoconjugates exhibited new broad and diffuse peaks confirming that they did exist in amorphous state as multiple complexes. The TGA thermograms of the binary nanoconjugates exhibited one step degradation profile compared with the physical mixture which exhibited two steps. However the ternary nanoconjugates exhibited two steps degradation profile confirming the formation of multiple complexes. Marked enhancement of drug release was achieved by the four techniques compared with the ibuprofen control. All the DG (DEAE-Dextran - Gellan) complexes exhibited a higher release profile than ibuprofen control. Fickian and non-Fickian anomalous mechanisms were deduced for the drug release of ibuprofen from the binary conjugates. The ternary nanoconjugates exhibited non-Fickian (anomalous) diffusion, Fickian diffusion and Super Case II transport release mechanisms. The ternary nanoconjugate hydrogels exhibited complete release (100%) within 48 h. The lowest concentration of DEAE-Dextran, Gellan - Ibuprofen - DEAE-Dextran (GIbDD) 2:0.125, increased the release of ibuprofen by 13.4% however higher concentrations of DEAE-Dextran decreased the release profile steadily. It was concluded that DEAE-Dextran has potentials in the formulation of modified (extended) release of ibuprofen. The most prominent mechanism of release of ibuprofen from the nanoconjugate hydrogel was Super Case II transport. SEM and AFM micrographs of the drug loaded composite pharmaceutical films exhibited concentric spheres with two and three layers for the binary and ternary films respectively. This supports the evidence of internalization of ibuprofen by the polyelectrolyte complex. The FTIR and DSC results confirmed electrostatic and hydrophobic interactions between ibuprofen and DEAE-Dextran as well as between gellan and DEAE-Dextran. Thermal analysis revealed that plain bilayer films were thermally more stable than composite films. The addition of ibuprofen significantly increased (p < 0.05, n = 4) the swelling ratio of the films compared with films without the drug. The drug loaded bilayer films exhibited Fickian diffusion mechanism while the dominating mechanism for composite films was anomalous (Non-Fickian) transport. From the foregoing, it was evident that ibuprofen-polymer nanoconjugate present a novel tool for the delivery of ibuprofen with potential application for transdermal delivery.
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Hydrogely na bázi kladně nabitých poylelektrolytů / Hydrogels based on cationic polyelectrolytesJarábková, Sabína January 2016 (has links)
This diploma thesis deals with the study of physical hydrogels based on positively-charged polyelectrolyte. The study is to investigate the interaction of selected positively-charged polyelectrolytes with oppositely charged surfactants in water and in physiologicla saline solution (0.15 M NaCl). The influence of the process for preparing hydrogels. Hydrogels were prepared by dry or wet. Were tested solubilization abilities hydrogels prepared using the hydrophobic dye oil red O were also measured rheological properties of the hydrogels prepared using frequency oscillatory tests and flow tests, depending on the concentration of the polymer or surfactant. For the rheological experiments suggest that chitosan is capable of forming rigid hydrogels with better mechanical properties than the dextran in the presence of both surfactants. Selected samples were also carried out experiments using fluorescent sold prodan, nile red and -naphthol as fluorescent probes. And in selected experiments was determined by solids content in percentage depending on the concentration used polyelectrolytes or surfactants.
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Studium interakce záporně nabitých vezikulárních systémů s polykationty / Study of interaction of negatively charged vesicular systems with polycationsRepová, Romana January 2020 (has links)
This diploma thesis deals with the preparation and characterization of negatively charged catanionic vesicular systems and their combination with selected polycations. The catanionic vesicular system was prepared by mixing of two oppositely charged surfactants SDS and CTAB. The negative charge as well as the stability of the vesicular system was provided by the incorporation of phosphatidic acid. Polycations, DEAE and TMC, have been selected for use in a pharmaceutical applications. Characterization of the prepared systems was performed by measuring DLS and ELS. The results indicate that we were able to prepare stable negatively charged vesicles that were eligible to non-covalently interact with selected polycations.
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Decoding protein networks during porcine epidemic diarrhea virus (PEDV) infection through proteomicsValle-Tejada, Camila Andrea 07 1900 (has links)
No description available.
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Biosurfaktanty a jejich využití pro inkorporaci hydrofobních domén do moderních nosičových systémů / Utilization of biosurfactants for incorporation of hydrophobic domains into modern controlled-release systemsNešpor, Tomáš January 2021 (has links)
This work deals with the current topic of carrier systems. Since the biggest problem is the passage of hydrophobic drugs through the bloodstream, or through universal body barriers (eg blood-brain), it is necessary to chemically modify these carriers in order to be able to administer hydrophobic substances effectively. Based on a literature search, several systems are designed and subsequently studied, in which there is a presumption of possible use for carrier systems and at the same time they have biosurfactants incorporated in them due to their ability to solubilize hydrophobic molecules. The theoretical part of this work will describe the individual biosurfactants, the process of their production, their physicochemical properties, and the possibility of their use in carrier systems. At the same time, the individual carrier systems, the procedure of their preparation, the possibilities of their use are described, and their advantages and disadvantages are also compared. In the practical part, the screening of both individual substances and their mutual interactions, as well as methods used to study the emerging structures is then performed. The study of molecular interactions is primarily performed using the technique of dynamic light scattering. The next part describes the optimization of hydrogel formation with incorporated biosurfactants in their structure and then the formed gels are subjected to rheological and solubilization tests. The study of the internal structure of these gels is performed using a scanning electron microscope.
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