Dissertations / Theses on the topic 'Protein functionalization'
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Buck, Chelsea. "Characterization and Functionalization of Suckerin-12 Protein Hydrogels." University of Dayton / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1542729200115677.
Takeda, Shigeo. "Functionalization of Glucan Dendrimers and Bio-applications." Kyoto University, 2020. http://hdl.handle.net/2433/253505.
Tabe, Hiroyasu. "Studies on Functionalization of Porous Protein Crystals by Immobilizing Organometallic Complexes." 京都大学 (Kyoto University), 2015. http://hdl.handle.net/2433/200445.
Yildirim, Eda Didem Sun Wei Guceri S. I. "Plasma and protein surface functionalization for three-dimensional polycaprolactone tissue scaffolds /." Philadelphia, Pa. : Drexel University, 2010. http://hdl.handle.net/1860/3326.
Takaoka, Yosuke. "Development of New Methods for Chemical Labeling, Functionalization and Detection of Proteins by Ligand-tethered Probes." 京都大学 (Kyoto University), 2010. http://hdl.handle.net/2433/120896.
Ahmad, Asad Ali. "Surface Functionalization and Analysis Thereof for an Ovarian Cancer Diagnostic Biosensor." Scholar Commons, 2011. http://scholarcommons.usf.edu/etd/2977.
Cai, Yixiao. "Bio-Nano Interactions : Synthesis, Functionalization and Characterization of Biomaterial Interfaces." Doctoral thesis, Uppsala universitet, Tillämpad materialvetenskap, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-277121.
Darwish, Amina M. "Silica Surface Modifications for Protein Separation." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1416231191.
Schumacher, Dominik. "Site-specific functionalization of antigen binding proteins for cellular delivery, imaging and target modulation." Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18547.
Antibodies and antigen binding proteins conjugated to fluorophores, tracers and drugs are powerful molecules that enabled the development of valuable diagnostic and therapeutic tools. However, the conjugation itself is highly challenging and despite intense research efforts remains a severe bottleneck. In addition to that, antibodies and antigen binding proteins are often not functional within cellular environments and unable to penetrate the cellular membrane. Therefore, their use is limited to extracellular targets leaving out a vast number of important antigens. Both limitations are core aspects of the presented thesis. With Tub-tag labeling, a novel and versatile method for the site-specific functionalization of biomolecules and antigen binding proteins was developed expanding the toolbox of protein functionalization. The method is based on the microtubule enzyme tubulin tyrosine ligase. Tub-tag labeling was successfully applied for the site-specific functionalization of different proteins including antigen binding nanobodies which enabled confocal microscopy, protein enrichment and super-resolution microscopy. In addition to that, cell permeable antigen binding nanobodies have been generated constituting a long thought goal of tracking and manipulating intracellular targets by in vitro functionalized antigen binding proteins. To achieve this goal, two different nanobodies were functionalized at their C-terminus with linear and cyclic cell-penetrating peptides using expressed protein ligation. These peptides triggered the endocytosis independent uptake of the nanobodies with immediate bioavailability. Taken together, Tub-tag labeling and the generation of cell-permeable antigen binding nanobodies strongly add to the functionalization of antibodies and their use in biochemistry, cell biology and beyond.
Lella, Divya Jyothi. "Functionalization and Modification of Naphthaquinone Analogs as HER2 Kinase Inhibitors." TopSCHOLAR®, 2014. http://digitalcommons.wku.edu/theses/1325.
Wan, Fan. "Biomimetic Surface Coatings from Modular Amphiphilic Proteins." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31639.
ARTUSIO, FIORA. "Novel crystallization platforms for drugs and biomolecules: self-assembled surface functionalization and gels." Doctoral thesis, Politecnico di Torino, 2021. http://hdl.handle.net/11583/2903508.
Deng, Lingquan. "Photochemical Surface Functionalization : Synthesis, Nanochemistry and Glycobiological Studies." Doctoral thesis, KTH, Organisk kemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-41973.
QC 20111004
Zou, Zhi Verfasser], Ulrich [Akademischer Betreuer] [Schwaneberg, and Andrij [Akademischer Betreuer] Pich. "Directed sortase evolution for site-specific protein engineering and surface functionalization / Zhi Zou ; Ulrich Schwaneberg, Andrij Pich." Aachen : Universitätsbibliothek der RWTH Aachen, 2019. http://d-nb.info/1192308719/34.
Zou, Zhi [Verfasser], Ulrich [Akademischer Betreuer] Schwaneberg, and Andrij [Akademischer Betreuer] Pich. "Directed sortase evolution for site-specific protein engineering and surface functionalization / Zhi Zou ; Ulrich Schwaneberg, Andrij Pich." Aachen : Universitätsbibliothek der RWTH Aachen, 2019. http://d-nb.info/1192308719/34.
António, Maria de Almeida Vasconcelos. "Detection of C-reactive protein using functionalized gold nanoparticles." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22538.
Este projeto teve como objetivo desenvolver novas estratégias para funcionalizar nanopartículas de ouro (Au NPs) de forma a detetar a proteína C-reativa (CRP). Assim, foram sintetizadas nanopartículas de ouro esféricas com tamanhos médios de aproximadamente 10 e 40 nm. Posteriormente, a superfície das Au NPs foi modificada utilizando duas abordagens diferentes. Na primeira abordagem as Au NPs estabilizadas com citrato foram funcionalizadas com grupos ácidos carboxílicos pela modificação da superfície com os ligandos: ácido 11-mercaptoundecanóico (MUDA) e ácido mercaptopropiónico (MPA). Posteriormente a citidina difosfocolina (CDP) foi acoplada covalentemente à superfície das Au NPs utilizando 1-etil-3-(3’- dimetilaminopropil)carbodiimida (EDC) para promover a reação. Na segunda abordagem as Au NPs estabilizadas com citrato foram modificadas com um aptâmero específico para a CRP. Estes materiais foram caracterizados utilizando diversas técnicas de análise nomeadamente espectroscopia de UVvis e de infravermelho (FTIR), medidas de potencial Zeta e de diâmetro hidrodinâmico e microscopia eletrónica de transmissão (TEM). Amostras selecionadas de Au NPs modificadas com aptâmero (Au NPs@ssDNA+NaCl+KCl) e de Au NPs bioconjugadas com CDP (Au NPs@MUDA@CDP_40nm e Au NPs@MPA@CDP_10nm) foram utilizadas para o estudo da deteção da proteína: CRP. As Au NPs foram adicionadas a soluções de concentração conhecida (10 - 100 nM). A deteção da CRP foi investigada através da aquisição de espectros de UV-vis. O aumento da razão de agregação, isto é entre a absorvância a 620 nm e a absorvância da banda de ressonância de plasmão de superfície localizada (A620/ALSPR), foi monitorizada ao longo do tempo, indiciando a deteção da CRP para todas as amostras testadas. Para os sistemas Au NPs@MUDA@CDP_40nm e Au NPs@ssDNA+NaCl+KCl foi possível correlacionar a razão A620/ALSPR com a concentração da CRP através de uma relação linear nas gamas de concentração de 20 - 50 nM (R2=0.9425) e de, 20 - 45 nM (R2=0.9382) respetivamente. Os resultados obtidos, embora preliminares, são promissores sendo necessário avaliar em estudos futuros aspetos tais como reprodutibilidade e interferência de outras proteínas em estudos futuros, tendo em vista o desenvolvimento de biossensores válidos para a deteção de CRP
The main goal of this project was to develop novel strategies for the functionalization of gold nanoparticles (Au NPs) aiming the detection of Creactive protein (CRP). Au NPs with an average size of 10 and 40 nm were synthesized. Then, the surface of Au NPs was modified following two different approaches. The first approach consisted on the surface functionalization of citrate stabilized Au NPs with carboxylic acid groups, by surface modification using 11-mercaptoundecanoic acid (MUDA) and mercaptopropionic acid (MPA). The cytidine diphosphocholine (CDP) was covalently attached to the surface of functionalized Au NPs using 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC) to promote the coupling reaction. In the second approach, citrate stabilized Au NPs were modified with a CRP specific aptamer. These materials were characterized using several analysis techniques namely UV-vis spectroscopy, infra-red spectroscopy (F-T IR), zeta potential and hydrodynamic diameter measurements and transmission electron microscopy (TEM). Selected samples of Au NPs modified with aptamer (Au NPs@ssDNA+NaCl+KCl) and CDP bioconjugated Au NPs (Au NPs@MUDA@CDP_40nm e Au NPS@MPA@CDP_10nm) were used for studying CRP detection. Au NPs samples were added to CRP solutions of known concentration (10-100 nM). The detection was measured through acquisition of the UV-vis spectra. The increase of aggregation ratio between absorbance at 620 nm and the absorbance of localized surface plasmon resonance band (A620/ALSPR) was monitored along time and, indicated that CRP was detected for all the Au NPs samples tested. For the systems Au NPs@MUDA@CDP_40nm and Au NPs@ssDNA+NaCl+KCl it was possible to find a linear correlation between the ratio A620/ALSPR and the CRP concentration, within the concentration range 20 - 50 nM (R2=0.9425) and, 20 - 45 nM (R2=0.9382), respectively. The results obtained are very promising but still preliminary. Further studies are needed to evaluate key aspects such as reproducibility and interference of other proteins, keeping as main objective the development of valid biosensors for CRP detection.
Hsin-Yi, Chiu [Verfasser], and Thomas [Akademischer Betreuer] Bein. "Synthesis and functionalization of multifunctional large-pore mesoporous silica nanoparticles for intracellular protein delivery / Chiu Hsin-Yi ; Betreuer: Thomas Bein." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1165503964/34.
Schlipf, Daniel M. "BIOMOLECULE LOCALIZATION AND SURFACE ENGINEERING WITHIN SIZE TUNABLE NANOPOROUS SILICA PARTICLES." UKnowledge, 2015. http://uknowledge.uky.edu/cme_etds/44.
Christmann, Sarah [Verfasser]. "Synthesis of functional amphiphilic block copolymers as stabilizer for various nanocarriers and further surface functionalization to change protein interactions / Sarah Christmann." Mainz : Universitätsbibliothek Mainz, 2017. http://d-nb.info/1131115260/34.
Herranz, Díez Carolina. "Functionalization of a Ti-based alloy with synthesized recombinant fibronectin fragments to improve cellular response." Doctoral thesis, Universitat Politècnica de Catalunya, 2014. http://hdl.handle.net/10803/277537.
De acuerdo con un estudio de la Comisión Europea, aproximadamente un millón de caderas son remplazadas por prótesis en el mundo anualmente. La interacción del cuerpo humano con materiales externos sujetos a una carga mecánica alternante en un medio altamente corrosivo todavía presenta ciertos desafíos. Los factores que contribuyen principalmente al fallo de una prótesis son el apantallamiento de cargas y la pobre osteointegracion. En la presente tesis el problema de la fallida de prótesis ha sido abordado desde el punto de vista del material y de la osteointegracion en un intento de dar una solución global al problema. El niobio y el hafnio, cuya total biocompatibilidad ha sido demostrada, se han utilizado para diseñar una aleación de titanio. El efecto de dichos aleantes respecto a la microestructura y el módulo elástico ha sido estudiado y la mejor composición ha sido profundamente caracterizada en términos de microestructura, módulo elástico, resistencia a la corrosión y energía superficial. Fragmentos recombinados de fibronectina han sido sintetizados abarcando la zona de adhesión celular y la unión de heparina, las cuales son esenciales para la viabilidad celular. Dichos motivos han sido utilizados para funcionalizar la superficie de la aleación TiNbHf. Dos métodos de unión diferentes han sido estudiados: fisisorción y silanización. La silanización es un método que no se ha utilizado hasta el momento para inmovilizar fragmentos de fibronectina sobre superficies metálicas y en la presente tesis su idoneidad ha sido demostrada. Finalmente, estudios celulares in vitro se han llevado a cabo con cada fragmento y con diferentes combinaciones de ambos, lo cual ha mostrado la importancia de la zona de unión de heparina para obtener una respuesta celular equivalente a la obtenida con la molécula de fibronectina en cuanto a adhesión celular, proliferación y diferenciación.
DiLillo, Ana M. ""Noncovalent Complexation of Single-Wall Carbon Nanotubes with Biopolymers: Dispersion, Purification, and Protein Interactions"." Cleveland State University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=csu1624461866858216.
Fromell, Karin. "Nanoscale Reaction Systems." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8249.
Baladi, Tom. "Autour du noyau imidazo[4,5-b]pyridine : inhibiteurs potentiels de la protéine kinase Tyro3 et fonctionnalisation directe de liaisons C – H." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS386/document.
Bladder cancer is a major medical issue, being the fourth most frequent cancer in men and treatable only with heavy surgery and/or broad-spectrum chemotherapy. This thesis project deals with the discovery of new targeted therapies of bladder cancer by blocking specifically, at a molecular scale in cancer cells, the signaling pathways in which protein kinase Tyro3 is involved. Indeed, its overexpression in most bladder cancers and the major part it plays in cancer cells survival have led to the validation of protein kinase Tyro3 as a therapeutic target for the treatment of bladder cancer. This thesis project can be divided into three main parts: the development of new synthetic methods around the imidazo[4,5-b]pyridine scaffold, the synthesis of a library of compounds using these methods and eventually the study of structure-activity relationships of these compounds versus Tyro3
Balderrama, Fanor Alberto. "Incorporation of recombinant fibronectin into genetically engineered elastin-based polymers." Thesis, Atlanta, Ga. : Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/31640.
Committee Chair: Chaikof, Elliot; Committee Member: Conticello, Vincent; Committee Member: Jo, Hanjoong. Part of the SMARTech Electronic Thesis and Dissertation Collection.
Joguet, Nicolas. "Utilisation et fonctionnalisation de protéines pour la conception de nouvelles microsphères permettant la protection et le relargage contrôlé de vitamine A." Thesis, La Rochelle, 2014. http://www.theses.fr/2014LAROS037.
The main objective of this thesis was to study the influence of functionalization of proteins by sugars or grape polyphenols in the vitamin A microsphere formulation and behavior. The formulation of different conjugated stemming either from the Maillard reaction or from the complexation of polyphenols on proteins was made on three proteins : pea proteins, sodium caseinate and type A gelatin. In a first part, the characteristics and the emulsifying power of the combined were studied, and confirmed the potential of stabilization of oil in the time. A second part was on the observations with scanning electron microscope of microspheres and on the methodology of specific observation in this kind of sample. The third part studied the influence of functionalization on vitamin A stability in the time, liberation on gastric or enteric digestion media, and liberation of co-encapsulated geraniol. The last study concerned the muco-adhesive potential of microspheres by using an original analysis
Sharan, Siddharth. "Compréhension des mécanismes à l'origine des propriétés fonctionnelles et de la flaveur d’ingrédients riches en protéines issus de féveroles." Electronic Thesis or Diss., université Paris-Saclay, 2021. http://www.theses.fr/2021UPASB061.
The growing population is demanding new healthy, sustainable solutions for foods and beverages. Fava bean (Vicia faba L.) is a promising plant source that can provide nutritional and functional ingredients for different food applications. Fava bean is processed to form ingredients and they can be further modified to render them fit for food applications. This PhD work aimed to understand the role of processing conditions on functional and flavor properties, and apply this understanding to produce and use fava bean protein-rich ingredients. It investigated the effects of certain industrially relevant process conditions using a cross-dimensional approach to find the right kind of compromise between different ingredients properties. To be precise, a very gently processed fava bean protein rich concentrate was industrially procured, which was then modified by process conditions such as pH (2, 4, 6.4 and 11), temperature (55, 75 and 95 °C) and treatment duration (30 and 360 min) to produce 36 different ingredients. These were further utilized at two pH (4 and 7) in systems close to beverage applications. During ingredient utilization, beverage functionalities (foam and emulsion) along with odor perception and non-volatile compounds were investigated for all ingredients as a function of process conditions.Results showed that process conditions were able to drive functional and flavor properties of the fava bean concentrate, strengthened by different statistical models. Foam and emulsion properties were predominantly governed by the pH during ingredient utilization. In general, utilization pH around the isoelectric point of fava proteins (pH 4) was not suitable for foam stability, emulsion capacity nor emulsion stability. Strong correlations between functional and physico-chemical properties were identified and explained by protein properties. In addition, flavor was heavily driven by the modification and utilization conditions, especially the pH.From gentler to vigorous process conditions, perception can be modified from more green to more cooked flavors, whereas different conditions of application (e.g. pH) can modulate between “sweet” and rancid perceptions. Considering volatiles composition, aldehyde signals were primarily detected in ingredient suspensions head-space. But furanoids, terpenoids, alcohols and ketones signals had the next higher contribution for modifications at pH2, 4, 6.4 and 11 respectively. Lipid oxidation was deemed important in generating volatiles, along with other reactions including proteins, sugars and carotenoids degradation. Going deeper into understanding of physico-chemical and sensory properties, determinants of antioxidant potential, taste (bitterness and astringency), color and even anti-nutritional effects were also investigated. Phenolic compounds (flavan-3-ols, flavones, flavonols, hydroxycinnamic acids) and saponins were significantly impacted by process conditions during ingredient modification, especially by pH. For phenolic compounds, acidic and alkaline conditions (pH 2, 4 and 11) were highly distinct compared to the non-pH adjusted process (pH 6.4) in changing the phenolic and saponin profiles of the ingredients. When looked closely at non-pH adjusted processes, their variability due to increasing degree of processing seemed to be either a function of their variable extractability and/ or their reactions involving their structural rearrangement.Thus, process conditions played an important role in fava bean ingredient properties, and this work opens up new arena for inter-disciplinary study based on nutritional (anti-oxidant and anti-nutritional aspects), sustainability (life cycle assessment), functionality (gelation) and sensory (texture, sweetness, bitterness) considerations of fava bean as potential ingredients for industrial food applications
Platt, Virginia M. "Surface functionalization of liposomes with proteins and carbohydrates for use in anti-cancer applications." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2010. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3390073.
Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Francis C. Szoka.
Sharpe, Kathleen Angela. "Functionalization of single walled carbon nanotubes with proteins : a comparison of methods and efficiency." Thesis, University of Surrey, 2010. http://epubs.surrey.ac.uk/843398/.
Périé, Lucie. "Formulation et alvéolation de pâtes céréalières à l’aide d’ingrédients biosourcés." Electronic Thesis or Diss., Bordeaux, 2024. http://www.theses.fr/2024BORD0090.
The replacement of controversial additives in cereal matrices represents a major challenge to meet consumers’ expectations. Leavening agents are functional ingredients that are required to obtain porous biscuit products according to industrial manufacturing methods. Their incorporation into biscuit dough determines the expansion of dough pieces during the baking stage. In this work, we considered two cereal doughs with different hydration levels that determine the gas incorporation pathways, aiming to completely suppress the need for leavening agents. In a low-hydration laminated biscuit dough, the study considered the use of baker's yeast as a substitute for leavening agents. The configuration of the gluten network conditions the dough elasticity and its ability to stretch to allow the biscuits to expand during baking. In a sponge drop (whipped) dough, air incorporation relies on the formation of a stable foam simultaneously with the gas release induced by the leavening agents. The removal of leavening agents from this matrix was enabled by using functionalized plant proteins through various treatments (physical or enzymatic). A design of experiments approach was implemented to optimize functionalities and thus, ensure the obtention of biscuits with a uniform crumb structure. During this process, the interfacial properties of the dough proteins determine their ability to stabilize the air bubbles in the matrix. These were studied using tensiometry and interfacial rheology
Issa, Sabin. "Fonctionnalisation de la surface du titane pour les implants dentaires." Thesis, Paris Est, 2014. http://www.theses.fr/2014PEST1075/document.
The objective of this thesis is to create new nanostructured surfaces with bioactive coatings and to study theirs physicochemical properties in order to develop better dental implants designs and promote their osseointegration. This functionalization was performed in two steps; starting by the nanostructuration of TiO2 surface by anodisation to create reactive sites on the edges of titanium nanotubes which acts as points of “attachment" to bioactive coatings. The second step was the surface chemical modification by coating the nanostructured surface with bioactive coatings of calcium phosphate (CaP) and strontium doped calcium phosphate (Sr.CaP). This coating was performed by pulsed electrodeposition. The physicochemical characterization by XPS, SEM and IR showed that doping with Sr promotes a non-apatitic compound similar to DCPD or DCPA (Dicalcium Phosphate Dihydrate or Anhydrous), while undoped CaP coating looks like an amorphous apatite-like compound ACP. The addition of strontium has the double advantage of optimizing the cellular multiplication and of giving an inorganic phase with bio-performance better than apatitic compounds. We also evaluated the adsorption proprieties of these functionalized surfaces by investigating the adsorption of proteins (BSA). This adsorption was performed onto tblank nanotubes, nanotubes coated with CaP and Sr doped CaP and evaluated according to deposition time and to the pH value of the solution that affect both protein and surface charge. The kinetic and structural evaluation reveals different adsorption geometries according to pH and adsorption time and also according to the chemical nature of surface. Such results of protein adsorption and conformation may form a database to understand and control protein activities and reactions with living body when used for dental implants system
Gutmann, Marcus [Verfasser], Tessa [Gutachter] Lühmann, Jürgen [Gutachter] Seibel, and Lorenz [Gutachter] Meinel. "Functionalization of cells, extracellular matrix components and proteins for therapeutic application / Marcus Gutmann ; Gutachter: Tessa Lühmann, Jürgen Seibel, Lorenz Meinel." Würzburg : Universität Würzburg, 2018. http://d-nb.info/1171132972/34.
Šibíková, Anna. "Syntéza kvantových teček pro detekci proteinů." Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2015. http://www.nusl.cz/ntk/nusl-221376.
Anaya, Castro Maria Antonieta. "Optimisation de la pH-sensibilité de protéines végétales en vue d'améliorer leurs capacités d'encapsulation de principes actifs destinés à la voie orale." Thesis, Toulouse, INPT, 2018. http://www.theses.fr/2018INPT0017/document.
In the pharmaceutical field, the oral route remains the preferred route of administration because it is simpler and better accepted by patients. However, this mode of administration is problematic for many active pharmaceutical ingredients (API) with low solubility, low permeability and/or instability in the gastrointestinal environment. Their microencapsulation in polymeric matrices can make them able to respond to these factors, especially if the microparticles generated resist the environments encountered during the gastrointestinal tract and then play a protective role, both for the API and for the mucous membranes encountered. The search for new excipients, from agroresources such as natural polymers, is booming. Vegetable proteins, thanks to their functional properties such as good solubility, relatively low viscosity, and emulsifying and film-forming properties, are preferred candidates. In addition, the great diversity of their functional groups makes it possible to envisage various chemical or enzymatic modifications. The aim of this work was to study the interest of soy protein as a coating material for API intended for the oral route, and more particularly as a candidate for the development of gastro-resistant forms. A soy protein isolate (SPI) was used as a coating material and the atomization as a process. Ibuprofen, a nonsteroidal anti-inflammatory drug, was chosen as a model molecule because of its low solubility requiring an improvement in its bioavailability, and its gastric side effects requiring an enteric shaping. Two chemical modifications of proteins (acylation and succinylation) have been studied in order to modify the solubility of the soy protein. These modifications were carried out in accordance with the principles of Green Chemistry, especially in the absence of organic solvent. The microcapsules obtained by spray-drying were characterized in terms of rate and encapsulation efficiency, morphology and size distribution of the particles, physical state of the encapsulated API and capacity of release in simulated gastric and intestinal medium. The results obtained validated the interest of the chemical modifications of the soy protein to modulate the release kinetics of API. The chemical modifications appeared particularly suitable for the encapsulation of hydrophobic active ingredients, and allowed to obtain ibuprofen release kinetics decreased to acidic pH (gastric). The last part of this work allowed to validate this last hypothesis by the realization of gastro-resistant forms on the model of MUPS tablets (multiple unit pellet system). The results of this exploratory work demonstrate that soy protein, combined with a multiparticle shaping process coupled with direct compression, can be a biosourced, environmentally friendly alternative (aqueous solvent handling, drying and compression steps reduced) and confident to the coating used in traditional gastroresistant forms
Schumacher, Dominik [Verfasser], Christian P. R. [Gutachter] Hackenberger, Dorothea [Gutachter] Fiedler, and Heinreich [Gutachter] Leonhardt. "Site-specific functionalization of antigen binding proteins for cellular delivery, imaging and target modulation / Dominik Schumacher ; Gutachter: Christian P. R. Hackenberger, Dorothea Fiedler, Heinreich Leonhardt." Berlin : Humboldt-Universität zu Berlin, 2017. http://d-nb.info/1185578390/34.
Schwenen, Lando Lantbert Gregor. "Untersuchung einzelner SNARE-vermittelter Membranfusionsereignisse auf planaren porenüberspannenden Membranen." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://hdl.handle.net/11858/00-1735-0000-0023-9650-F.
Bogner, Pamela [Verfasser]. "Generation of recombinant antibody fragments specific formurine mesangial cells : functionalization of highly specific fusion proteins for diagnostic approaches and the development of a murine mesangioproliferative glomerulonephritis disease models / Pamela Bogner." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2015. http://d-nb.info/1066813574/34.
Nesterenko, Alla. "Etude et fonctionnalisation de protéines végétales en vue de leur application en microencapsulation." Thesis, Toulouse, INPT, 2012. http://www.theses.fr/2012INPT0148/document.
Proteins extracted from vegetables are relatively low-cost, non-toxic, biocompatible and biodegradable raw materials. They represent a good alternative to animal-based proteins and petroleum-extracted polymers. In this study, proteins derived from soybean and sunflower seeds were used as wall materials for microencapsulation of hydrophobic (-tocopherol) or hydrophilic (ascorbic acid) active material by spray-drying technique. Soybean proteins are widely used in food and non-food applications, especially in microencapsulation. They were studied in this work as wall material of reference. Sunflower proteins are not actually used in industrial application, but only in the form of oil-cake for animal feeding. That’s why new ways of valorization of this agricultural by-product should be investigated. Several proteins’ modifications such as enzymatic hydrolysis, acylation, cross-linking and cationization were studied in order to improve encapsulating properties of wall material. In the context of green chemistry, all the modifications and preparations were performed without use of organic solvents and chemical catalysts. The effect of protein chemical and enzymatic modifications, and process parameters (homogenization pressure, wall/core ratio and protein concentration) on different characteristics of liquid preparations and microparticles (viscosity, emulsion droplet size, microparticle size and morphology) and on parameters related to the spray-drying process (yield and efficiency of microencapsulation) was particularly investigated in this study. The obtained results confirmed that sunflower proteins are quite suitable as encapsulating agent and provide the microencapsulation efficiencies significantly higher compared to those obtained with soy proteins
Bellat, Vanessa. "Ingénierie d'un nouveau nanobiohybride à base de nanorubans de titanates pour la médecine régénérative." Thesis, Dijon, 2012. http://www.theses.fr/2012DIJOS056/document.
This research work is devoted to new nanohybrid engineering composed of titanate nanoribbons for regenerative medicine. Over a first phase, nanoribbons were synthesized by hydrothermal treatment and their morphological, structural and chemical features were defined. A fine characterization by means of different techniques of transmission electron microscopy mainly enabled to determine their thickness; dimension which had never been measured so far. Subsequently, titanate nanoribbons were functionalized by different home-made heterobifunctional PEG. Those polymers present at one of their extremities specific functional groups being able to couple with numerous biological molecules. Some collagen type peptides containing cellular recognition sites were grafted onto those extremities. The so-formed nanobiohybrid will permit cellular adhesion and proliferation favouring in fine tissue healing and regeneration. To evaluate new nanohybrid biological properties, titanate nanoribbons cytoxicity and aggregating power were determined by MTT tests, performed on two cell populations (fibroblasts and cardiomyocytes) and platelet aggregation tests (human blood). Finally, when used to promote healing process, the new nanobiohybrid was formulated in the form of sodium alginate hydrogel permitting a direct application on damaged tissues. To confirm the interest of this galenic form, initial in vivo tests were realized
Abou-Dalle-Messaikeh, Hana. "Polymères insolubles fonctionnels : affinité spécifique pour les anticorps anti VIIIc." Paris 13, 1989. http://www.theses.fr/1989PA132006.
Peigneguy, Fanny. "Synthèses et Caractérisations de Glucides Électrostimulables pour des Applications Antifouling." Electronic Thesis or Diss., Angers, 2020. http://www.theses.fr/2020ANGE0073.
Marine biofouling represents the undesirable accumulation of biological organisms on the surfaces of structures submerged in seawater. Unfortunately, this natural phenomenon has serious economic, environmental and material consequences. Since the ban of some biocides in antifouling paints (TBT in January 2008) because of their toxicity on the nontargeted marine species and their accumulation in the marine environment, research has focused on the development of new efficient, durable and environmentally friendly antifouling coatings without releasing toxic species. Thus, the work of this thesis deal with the functionalization of glassy carbon surface by carbohydrates linked to an electrostimulable conjugated system via a triazole link in order to develop surfaces with antifouling activity. Indeed, this kind of coating was designed to intervene in the first steps of biofouling. First, the carbohydrate, which is very hydrophilic, should fight against the formation of the conditioning film by surronding itself with an aqueous barrier resistant to proteins. On the other hand, the continuous modification of charge state by applying an electric current to the electroactive conjugated system is expected to disrupt the bacterial colonization delaying the installation of marine biofilm. Our study is therefore based on the synthesis and the immobilization of electrostimulable carbohydrates on a glassy carbon surface by aromatic amine oxidation in organic and aqueous media. A microbiological test was carried out on one of the carbohydrate coatings in the presence of the TC8 bacterial strain in the wells of a microplate containing electrochemical cells connected to a potentiostat. Electrical stimulation of this coating allowed to improve its antibacterial properties
Costa, André Manuel Abreu da. "Functionalization of protein-based polymers for advanced biomaterials." Doctoral thesis, 2016. http://hdl.handle.net/1822/42525.
Recombinant DNA technology has enabled us to create and produce new genetic (re)combinations otherwise not found in natural proteins or peptides. Recombinant protein-based polymers (rPBPs) arise from this technology as a class of biopolymers based on repetitive blocks of amino acid residues commonly found in structural proteins, such as the VPGVG block from mammalian elastin and the GAGAGS block from silk fibroin. The main objective of this thesis was focused on developing, production and processing of rPBPs, targeting its biomedical application. Antimicrobial Peptides (AMPs) are natural alternatives to common antibiotics which can act as antimicrobial domain when fused with a rPBP sequence. In this work genetic sequences of rPBPs were recombined with different AMPs sequences originating a set of materials in whose physicochemical and biological properties were analysed. Chapter I presents a comprehensive review on the state of the art of AMPs classification, structure and mode of action. A review on the use of AMPs for the creation of antimicrobial polymers is also presented. With respect to the rPBPs, the literature review was mainly focused on elastin-like recombinamers (ELRs) and silk-elastin-like proteins (SELPs). The experimental approaches on the functionalization of ELRs using different AMPs, as well as the material´s characterization are described in chapters II to VI. Chapters VII to IX are focused on the production of the recombinant co-polymer SELP-59-A and the material´s characterization by exploiting distinct processing methods. Chapter II describes the creation, recombinant protein production and nonchromatographic purification of a functional polymer based on the ELR A200 and the ABP-CM4 peptide, originating the new polymer termed CM4-A200. The purified material when processed into free-standing films displayed high antimicrobial activity against bacteria and fungi. The films demonstrated non-cytotoxic effects against human skin cell lines. Furthermore, they remained stable in dry and wet conditions for a wide range of temperatures, without the use of any crosslinking agent. The developed ex vivo protocol using pig skin for antimicrobial testing confirmed the antimicrobial capability of CM4-A200 films for skin applications. In chapter III is presented the work performed with CM4-A200 polymer processed by electrospinning. The fibre mats were characterized and compared to the cast films described in chapter II. Similarly to the films, the fibres retained the antimicrobial activity and revealed no cytotoxic effects. The stability of the material in wet conditions was only maintained at temperatures above 30 ºC, however the fibre mats were more resistant to thermal degradation than the cast films. Structure prediction of the ABP-CM4 peptide and genetic sequence modifications of CM4-A200 construction are presented in chapter IV. In this regard, the antimicrobial activity of the new peptides and polymers suffered no improvements. The processing procedure of the CM4-A200 polymer was modified originating nanotube patterned films, a modification that increases the contact surface area with the microbial cells. As a result, the antimicrobial activity of the CM4-A200 polymer against Staphylococcus aureus was improved. In chapter V the results on the modification of the peptide BMAP-28 sequence and further functionalization of A200 polymer are presented. When processed in cast films the BMAP-18A200 polymer presented high antimicrobial activity against bacteria, yeasts and filamentous fungi. The deletion of the last 10 amino acid residues of BMAP- 28 sequence was essential to achieve the production of the functional polymer based on this peptide. In chapter VI the peptide Hepcidin was utilized to functionalize the A200 polymer creating a highly antibacterial material in the soluble form, a feature not found in the materials described in preceding chapters. Chapter VII describes the production optimization of the recombinant polymer SELP- 59-A in Escherichia coli which resulted in the volumetric productivity of 0.5 g/L. The one-factor-a-time optimization allowed a 2.5-fold improvement over the previous described values. In the two following chapters, distinct techniques were explored to process rPBPs. In chapter VIII, SELP-59-A and SELP-1020-A were processed by electrospinning. The electrospun mats presented size-dependence on the solvent type and polymer concentration and good compatibility for skin cell line proliferation. This study was further extended to solvent-cast films of SELP-59-A, as presented in chapter IX. The films were stable at temperatures until 220 ºC and displayed electric insulating properties. Methanol treatment induced improvement of the mechanical properties and introduction of glycerol as plasticizer improved flexibility of the material. In the final chapter (X) a general discussion of the results described in the precedent chapters is presented as well as the insights on future perspectives. Overall, this thesis provides new insights on the functionalization of rPBPs, the improvement of the biological production and purification procedures, as well as on the application of distinct processing methods. The rPBPs that were created will certainly contribute to fulfil the demand of new antimicrobial materials in particular for advanced biomaterials.
A utilização de tecnologia de DNA recombinante permite a criação e produção de novas (re)combinações genéticas, que de outro modo não se encontram em proteínas ou péptidos naturais. Desta tecnologia surgiram os polímeros recombinantes de origem proteica (rPBPs), uma classe de biopolímeros baseada na repetição de blocos de aminoácidos normalmente encontrados em proteínas estruturais, tais como o bloco VPGVG da elastina de mamífero ou o bloco GAGAGS da fibroína da seda. Esta tese focou-se no desenvolvimento, produção e processamento de rPBPs, tendo como objetivo a sua aplicação biomédica. Os péptidos antimicrobianos (AMPs) são alternativas naturais aos antibióticos de origem química, podendo ser utilizados como domínio funcional aquando da fusão com rPBPs. Neste trabalho, procedeu-se à recombinação de sequências genéticas de rPBPs com diferentes AMPs, originando uma pletora de novos materiais cujas propriedades físico-químicas e biológicas foram analisadas. No capitulo I é apresentada uma revisão do estado de arte sobre a função, modo de ação e estrutura dos AMPs, bem como a sua utilização para a criação de polímeros com atividade antimicrobiana. Relativamente aos rPBPs, a revisão da literatura focou-se maioritariamente nas famílias dos polímeros recombinantes semelhantes à elastina (ELRs) e dos co-polímeros com base na seda e na elastina (SELP). Os capítulos II a VI focam-se na funcionalização de ELRs utilizando diferentes AMPs, bem como na caracterização e processamento dos polímeros obtidos. Os capítulos VII a IX descrevem os trabalhos desenvolvidos com o co-polímero SELP-59-A, mais especificamente na otimização da sua produção e na exploração de diferentes técnicas de processamento. O capitulo II descreve a criação, produção e purificação por meios não cromatográficos de um polímero funcional baseado no ELR A200 e no péptido ABP-CM4, originando o polímero CM4-A200. O material purificado foi processado na forma de filme apresentando alta atividade antimicrobiana contra bactérias e fungos. Os filmes não apresentaram efeitos citotóxicos em linhas celulares humanas de pele. Além disso, mantiveram-se estáveis em condições secas e húmidas, sem o uso de qualquer agente de reticulação. O ensaio ex vivo com pele de porco que foi desenvolvido demonstrou o potencial dos filmes de CM4-A200 para aplicação na pele. O trabalho com o polímero CM4-A200 processado por eletrofiação é apresentado no capítulo III. As fibras criadas foram caracterizadas e as suas propriedades comparadas com os filmes mencionados no capítulo anterior. Tal como nos filmes, as fibras apresentaram atividade antimicrobiana e ausência de efeitos citotóxicos em linhas celulares humanas. As fibras apenas se mantêm estáveis a temperaturas superiores a 30 °C, quando em condições húmidas, contudo, são mais resistentes à degradação térmica do que os filmes. No capítulo IV descreve-se a previsão de estrutura do péptido ABP-CM4 e a introdução de modificações na construção CM4-A200. No entanto, estas modificações foram ineficazes no melhoramento da atividade antimicrobiana. O processamento dos filmes de CM4-A200 foi modificado originando-se um padrão em “floresta de nanotubos”, resultando no aumento da superfície em contacto com as células de microrganismos. Esta técnica resultou numa maior atividade contra a bactéria Staphylococcus aureus. No capítulo V está descrita a modificação da sequência do péptido BMAP-28 e a sua utilização na funcionalização do polímero A200. Quando processado na forma de filme, o polímero BMAP-18A200 apresentou alta atividade antimicrobiana contra bactérias, leveduras e fungos filamentosos. A remoção dos últimos 10 aminoácidos do péptido BMAP-28 resultou na produção do polímero funcionalizado. No capítulo VI, o péptido hepcidina foi utilizado para a funcionalização do polímero A200. Este revelou propriedades antibacterianas na forma solúvel, contrariamente ao observado nos polímeros mencionados nos capítulos anteriores. No Capítulo VII descreve-se a otimização da produção do polímero recombinante SELP-59-A em Escherichia coli, obtendo-se uma produtividade volumétrica de 0,5 g/L. A otimização do método resultou num aumento de 2,5x relativamente ao descrito na literatura. No capítulo VIII, os polímeros SELP-59-A e SELP-1020-A foram processados por eletrofiação. O diâmetro das fibras foi dependente do solvente e da concentração da solução utilizados. O material demonstrou ausência de citotoxicidade em culturas de linhas celulares de pele. No capítulo IX estudou-se o processamento e caracterização de filmes de SELP-59-A. Este material mostrou-se estável até 220 °C, e apresentou propriedades de isolamento elétrico. O tratamento com metanol induziu alterações nas propriedades mecânicas e a composição com glicerol resultou num aumento da flexibilidade do material. No último capítulo (X) procedeu-se à discussão geral dos resultados apresentados e apresentam-se as perspetivas futuras. De um modo geral, esta tese providencia novas soluções para a funcionalização, produção, purificação e processamento de rPBPs. Com este trabalho pretende-se que os rPBPs aqui criados contribuam para responder à procura de novos materiais com propriedades antimicrobianas, e particularmente, na área dos biomateriais avançados.
The work presented in this dissertation was performed in the Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho and in the BioForge institute, University of Valladolid, Spain. The financial support was given by Fundação para a Ciência e Tecnologia (FCT) by means of a grant, SFRH/BD/75882/2011. Financial support for part of the work was supported by FCT I.P. through the strategic funding UID/BIA/04050/2013
Fernandes, Margarida M. "Protein disulfide isomerase-assisted functionalization of proteinaceous substrates." Doctoral thesis, 2011. http://hdl.handle.net/1822/19687.
The formation of intramolecular disulphide bonds is critical in the process of protein folding and on the stabilization of protein tertiary structure. Their formation involves the oxidation of two thiol groups that should be correctly paired. The incorrect paring inhibit the protein folding into its native conformation. The rearrangement of incorrectly disulfide bonds on proteins is catalysed in vivo by the protein disulphide isomerase (PDI). This versatile enzyme is able to catalyse the oxidation, reduction and isomerisation of disulfide bonds in a broad range of protein substrates. This dissertation successfully presents the use of PDI for functionalization of cysteine-containing (CC) proteinaceous substrates such as keratin fibres and RNase A microspheres. These approaches take advantage of the presence of thiol moieties and disulphide bonds in these substrates. It was shown that the type of reaction catalysed by PDI can be predicted, by controlling the redox environment. When the active site of PDI is in its oxidized state due to is characteristic potential redox (Eº = -180 mV) an oxidation reaction is catalyzed. When the active site of PDI is transformed to its reduced state (ΔE = - 260 mV), the isomerisation of disulfide bonds is promoted. PDI was able to incorporate CC functional molecules on wool and hair trough disulfide bonds, as suggested by matrix-assisted laser desorption and ionization time-offlight results (MALDI-TOF) analysis. Similarly, PDI increased the affinity of a synthesised keratin-based peptide (KP) towards hair and facilitated the penetration into its cortex. Targeting a biomedical approach, Ribonuclease A (RNase A) was oxidatively attached to the wool surface through disulphide bonds, and PDI was shown to induce its release. Aiming a cosmetic application, KP and other synthesized surfactant-based peptide (SPB) were applied in over-bleached, damaged hair. Both peptides induced an improvement on its mechanical properties and thermal stability. Thus, recovering of the fibre integrity loss during the hair bleaching process was achieved. In the presence of PDI, peptides were linked by disulphide bonds and the thermal stability increased at higher levels. Due to the great properties developed on over-bleached hair after KP application, in the absence of PDI, other type of hair (relaxed hair) was treated with this peptide and the same properties were measured. The relaxing treatment, commonly applied on excessively curly hair, often result on the weakening of the fibre. KP was then applied to this weakened hair and its ability to recover its mechanical and thermal properties was proved. Two different peptide formulations were evaluated. In one formulation KP was dissolved in aqueous solution (WF) while in the other KP was dissolved in organic solvent solution (OF). The last imparted better mechanical and thermal properties to the hair, however, the safety assessment showed that OF is potentially cytotoxic, inhibit cell growth, and genotoxic. The KP itself did not inhibit the cell growth and was found to be non-cytotoxic and non-genotoxic, hence suitable for the application on cosmetic formulations at concentrations up to 0.5 g/L. Showing its ability to act on a broad range of cysteine-containing compounds, PDI was also able to oxidize the thiol groups found in sphere-like particles, recovering their biological function, at the conditions that lead PDI‟s active site on its oxidized state. Using a reducing environment, PDI promoted the released of native RNase A from protein-based microspheres. The research presented in this thesis shows the versatility of PDI to promote diverse functionalization on proteinaceous substrates, resulting in a wide applicability in different areas such as cosmetic, textile and biotechnology. The work was carried out partially in collaboration with a cosmetic company; hence the research included promising biotoolsbased strategies for hair-care product development, especially for the application on several types of damaged hair.
As pontes dissulfídicas são uma característica muito importante para a estabilização da estrutura terciária de proteínas. A formação das mesmas envolve a oxidação de dois grupos tiol, os quais devem estar correctamente ligados. Caso contrário, a proteína perde a sua actividade biológica característica, devido à formação de pontes dissulfídicas nãonativas, e desnatura. In vivo, aquando da formação das proteínas na célula, uma enzima tem uma função muito importante: promover o rearranjo das pontes dissulfídicas nãonativas e prevenir a agregação das proteínas desnaturadas. Esta enzima é a Protein Disulfide Isomerase (PDI), uma isomerase de pontes dissulfídicas que catalisa a oxidação, redução ou isomerisação de uma vasta gama de substratos contendo cisteína na sua constituição. Esta dissertação apresenta com sucesso a aplicação desta enzima na funcionalização substratos proteicos, tais como as fibras queratinosas ou microesferas de Ribonuclease A (RNase A). As pontes dissulfídicas dos substratos, acima mencionados, são as ligações-alvo nesta abordagem. Foi demonstrado que o potencial de redução do centro activo da PDI é uma ferramenta importante na pré-determinação das reacções que esta enzima pode catalisar. Quando o centro activo da PDI está no seu estado oxidado, devido ao seu característico potencial redox (Eº = -180 mV), uma reacção de oxidação é catalisada. Quando o centro activo da PDI é transformado para o seu estado reduzido (ΔE = - 260 mV), a isomerisação das pontes dissulfídicas é promovida. A PDI catalisou a incorporação de moléculas contendo cisteína (CC) em lã e cabelo através de pontes dissulfídicas, sugerido pela análise de espectroscopia de massa (MALDITOF). A PDI facilitou também, a penetração de um péptido de queratina (KP) no córtex da fibra de cabelo e induziu a libertação de uma proteína modelo (Ribonuclease A) da superfície da lã. Com o objectivo de uma aplicação cosmética, o KP e outro péptido, derivado de um surfactante humano (SPB) foram aplicados em cabelo danificado, previamente sujeito a vários ciclos de branqueamento oxidativo. Ambos os péptidos melhoraram as propriedades mecânicas e a estabilidade térmica do cabelo danificado, provando a sua capacidade para recuperar a integridade da fibra. Quando a PDI foi aplicada, os péptidos ligaram-se ao cabelo através de pontes dissulfídicas e a estabilidade térmica aumentou para valores ainda mais elevados. Devido ao efeito renovador que o KP teve sobre o cabelo branqueado, outro tipo de cabelo (cabelo relaxado) foi tratado com este péptido e as mesmas propriedades foram medidas. Os tratamentos de relaxamento em cabelo extremamente encaracolado resultam em enfraquecimento do mesmo. O KP foi por isso aplicado neste tipo de cabelo e a sua capacidade para o melhorar foi provado. Dois tipos de formulações peptídicas foram também testados. Numa formulação o KP foi diluído numa solução aquosa (WF), enquanto na outra o KP foi diluído numa solução contendo solventes orgânicos (OF). Esta última promoveu melhores resultados, contudo, revelou-se potencialmente cytotoxica, genotoxica e inibidora do crescimento celular. Demonstrou-se, todavia, que o péptido em solução aquosa pode ser aplicado em formulações cosméticas até à concentração de 0.5 g/L não manifestando citotóxicidade, genotóxicidade ou inibição do crescimento celular. Demonstrando a sua aptidão para actuar em compostos contendo cisteína, a PDI foi também capaz de oxidar os grupos tiol presentes nas microesferas, recuperando a sua função biológica nas condições que promovem o estado oxidado do seu centro activo. Usando um ambiente mais redutor, promoveu a libertação da proteína nativa das microesferas proteicas. Os resultados apresentados nesta tese demonstram a versatilidade da PDI para promover a funcionalização de substratos proteicos, resultando numa ampla aplicabilidade em áreas distintas como cosmética, têxtil e biotecnologia. O trabalho foi desenvolvido em parceria com uma empresa de cosmética, o que fomentou a procura de estratégias biológicas para o desenvolvimento de novos produtos para cabelo, especialmente para a aplicação em vários tipos de cabelo danificado.
Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/38363/2007
Bhagawati, Maniraj. "Photolithographic surface functionalization for spatio-temporally controlled protein immobilization." Doctoral thesis, 2012. https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2012012710014.
"Developing Novel Protein-based Materials using Ultrabithorax: Production, Characterization, and Functionalization." Thesis, 2011. http://hdl.handle.net/1911/70269.
Nayak, Nayan Nagesh. "Design and Functionalization of Alumina Monoliths for Protein Purification by Chromatography." Doctoral thesis, 2019. http://hdl.handle.net/10362/91584.
Hsieh, Feng-Jen, and 謝豐任. "Surface Modification and Functionalization of Fluorescent Nanodiamonds for Membrane Protein Targeting and Imaging." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/j53esv.
國立臺灣大學
生化科學研究所
107
Fluorescent nanodiamonds (FNDs) containing nitrogen-vacancy (NV) defects as light emitters have been extensively used as contrast agents for bioimaging due to their superior optical properties, such as high photostability. Forming aggregates in biofluids and lacking specific targeting ability, however, have significantly impeded their applications in specific protein labelling and imaging. In this thesis, two strategies were developed to modify and functionalize FNDs to overcome existing limitations. The first approach, lipid encapsulation method has the advantages of simple manipulation and time-effective. Desired functional groups can be added onto FND surface through minor changes in the lipid composition. Alkyne-modified hyperbranched polyglycerol (alkyne-HPG) grafting by ring opening reaction is the other approach for functionalization of FNDs. Although this approach has longer processing time, the coating layer is much more stable because of the formation of covalent bonds between the coating layer and particles. Both coatings endow FNDs with not only high dispersity in physiological medium but also specific targeting ability of cell membrane proteins. By combining the exclusive optical features (e.g., chemical inert), biotinylated lipid coated FNDs (bL-FNDs) successfully simplified the complicated protocol to localize CD44 antigens on cell surface by correlative light electron microscopy (CLEM). A thorough literature search reveal that FND is to date, the only carbon nanomaterial having the ability to act as a dual contrast agent for CLEM. Moreover, taking advantage of magneto-optical property of NV- centers, highly sensitive and accurate quantification of CD44 antigens on cell surface with 35 nm of bL-FNDs was accomplished. Finally, high temporal and spatial resolution of continuous long-term observation of integrins α5 was achieved with alkyne-HPGFNDs. The superb photostability (no photo-bleaching and -blinking) of FNDs allows for the detailed transportation route of integrins α5 to be studied through short- and long-term observation, which cannot be viewed by any other dye molecules or quantum dots. To sum up, two reliable surface functionalization methods for FNDs was successfully demonstrated in this thesis. These novel FNDs shorten the gap between light and electron microscopy and serve as a platform for continuous long-term imaging of membrane protein tracking with high temporal and spatial resolution. In the future, the applicability of other kinds of biohybrid FNDs (e.g., antibody modified HPGFNDs or L-FNDs) may be conducted to further simplify the protocol of FNDs for biolabeling.
Kulkarni, Chethana. "Selective Functionalization of the Protein N-Terminus with N-Myristoyl Transferase in Bacteria." Thesis, 2013. https://thesis.library.caltech.edu/7167/1/Kulkarni-2013_PhD-Thesis_Ch0.pdf.
Proteins are involved in myriad processes in all organisms. They provide structural support in the membrane and scaffolding of each cell; they aid in the transmission of biochemical signals within and between cells; and they play central roles in combating various disease states. The development of techniques enabling selective and site-specific functionalization of proteins is an active area of investigation, as such modifications are critical to many studies and uses of proteins. For instance, with the addition of a unique reactive handle, a protein may be conjugated to a polymer for the production of protein-based therapeutics exhibiting improved bioavailability. Alternatively, proteins may be attached to slides to prepare diagnostic microarrays, reacted with hydrogels to create functional biomaterials, or decorated with fluorophores for in vivo imaging. Site-specific protein tagging techniques have already contributed greatly to biomedical research and will continue to advance the state of the field.
The focus of my thesis research has been the development of a novel site-specific protein labeling method centered on the eukaryotic enzyme N-myristoyl transferase (NMT). In a process called myristoylation, NMT appends a fatty acid to the N-terminus of numerous substrate proteins. Previous work demonstrated that NMT tolerates a wide range of chemically functionalized analogs of its natural fatty acid substrate. Here, we describe efforts that exploit various features of NMT: its ability to bind and utilize reactive fatty acid analogs, its exquisite selectivity toward its protein substrates, and its orthogonality toward those proteins naturally present in bacteria.
First, in Chapter II, we discuss the development of a model system for NMT-mediated protein labeling in the bacterium Escherichia coli. We synthesized an azide fatty acid analog that can participate in bioorthogonal chemistries, and we prepared two GFP-based substrate proteins, each displaying a recognition sequence derived from a known substrate of NMT. Our experiments indicate that labeling by NMT is site-specific, quantitative, and highly selective for each engineered substrate within the bacterial milieu.
As summarized in Chapter III, the model system was extended to the N-terminal labeling of two neuronal proteins, calcineurin (CaN) and calmodulin (CaM). While CaN is naturally myristoylated, CaM was engineered to achieve labeling by NMT. Experiments with CaN and CaM confirmed that our NMT-based system is quantitative and selective in its labeling of both natural and engineered substrate proteins. Extensive characterization of each protein allowed us to identify constructs that retain wild-type levels of activity even after labeling with the azide fatty acid.
Three of the protein constructs reported in Chapters II and III were utilized for microarray studies, as described in Chapter IV. We achieved rapid surface immobilization of each azide-labeled protein directly from lysate, a significant advantage when considering the time and resources normally required to purify proteins for downstream applications. The experiments and methods summarized in this chapter will be adapted for high-throughput biochemical research with protein microarrays.
Finally, the orthogonality of NMT toward bacterial systems was probed further for the purpose of selective labeling of individual bacterial proteins for live-cell imaging. In addition to identifying an azide fatty acid suitable for such studies, we also selected two bacterial proteins that exhibit interesting functions and localization patterns, and we developed corresponding protein constructs for NMT-mediated labeling. Progress toward the use of NMT for in vivo imaging applications in bacteria is described in Chapter V.
Ultimately, our objective throughout the design and execution of these projects was to create and validate a new technique to achieve site-specific protein labeling. The particular advantages of NMT include its tolerance of reactive fatty acid analogs and engineered substrate proteins, and its lack of interaction with proteins present in the widely used E. coli expression host. We believe that the ideas and results presented in this thesis establish NMT-mediated protein labeling as a valuable tool for addition to the existing set of site-specific protein labeling methods. Development of such methods represents an important and exciting area within the field of modern chemical biology.
Su, Jing. "Laccase: a green catalyst for biosynthesis of poly-phenols." Doctoral thesis, 2020. http://hdl.handle.net/1822/76121.
Laccase is one of the multi-copper oxidases which can catalyze the oxidation of phenols, aromatic amines and other compounds using oxygen as the terminal electron acceptor. As an environment protecting biocatalyst, laccase shows great potential applications in various fields and industries, including textile industry, pulp and paper industry, environmental pollutant conversion and others In the field of synthesis, laccase catalyzed reactions have been widely investigated. However, the mechanism of laccase catalyzed aromatic compounds still needs to be further explored. The research and development of laccase on the polymer making study meet with requirement of the green chemistry, which will promote the application of the synthetic polymer materials. This PhD thesis intends to explore new strategies and mechanisms for the synthesis of aromatic polymers using different laccase forms and different reactors. Aromatic polymers were prepared using laccase from ascomycete Myceliophthora Thermophila. Different modifications of laccase were designed to improve its performance, including PEGylation and/or immobilization. Molecular modeling simulations were used to predict the geometry and structural changes of laccase. During the polymerization process, three reactors with different energy environment were applied, and the function of high-energy environment was investigated with the assistance of homology modelling and molecular simulation. The polymers obtained were characterized and applied onto different fabrics to achieve multi-functional fabrics. This study will provide fundamental knowledge for the polymer synthesis by laccase, and offer the theoretical and technical support for the establishment of high efficiency laccase-catalyzed synthesis system. Firstly, the polymerization of catechol was conducted using laccase as catalyst. Polyethylene glycol (PEG) was used in both non-covalent and covalent modification of laccase, then the performance of modified laccase was evaluated. The catalytic performance of different laccases was compared in both aqueous and gel phases. The results show that both non-covalent and covalent modification of laccase using PEG (PEGylated laccase) could promote the polymerization and improve the polymerization yield, as well as the degree of polymerization. However, these events were only detected in aqueous state. Molecular simulation shows that the presence of PEG slows down the inactivation of laccase. Later, the immobilization of laccase and PEGylated laccase was performed with epoxy resins as the carrier, using PEG also as linker compound to connect laccase and epoxy resin. The performance of immobilized laccase in aqueous solution for polymerization was explored and the structure of the polymer formed was proposed. After immobilization, the half-life time of laccase was improved, as well as the stability. The MALDI-TOF MS analysis showed that, when epoxy-native laccase, epoxy-PEGylated laccase and epoxy-PEG-laccase were used, the degree of polymerization was enhanced with the presence of immobilized laccase. Secondly, different reactors namely water bath, ultrasonic bath and high-pressure homogenizer were applied to perform the polymerization of catechol. The activity and stability of laccase in those reactors were discussed. The pathway and mechanism of laccase synthesis in high-energy environment were investigated. The polymers were characterized to speculate their structures. The results showed that high-energy environment promote the interaction between enzyme and substrate, incrementing the polymerization yield. The conversion yield when using the ultrasonic bath and high-pressure homogenizer was higher than when water bath was applied. Laccase under ultrasound and high-pressure homogenization showed less stability compared with under normal water bath, however, the polymerization proceeds earlier than this inactivation, thus no obvious negative effect on the synthesis was detected. The performance of native, PEGylated and Epoxy-PEGylated laccases was studied under the high-pressure homogenizer. Their activity and stability were compared and the corresponding polymers produced were evaluated. Both PEGylated laccase and Epoxy-PEGylated laccase showed the greatest catalytic properties and stability. Afterwards, catechol and p-phenylenediamine were polymerized using polyester (PET), cotton and wool as enzyme container in a high-pressure homogenizer using laccase as catalyst. The functionalization of fabrics was achieved via in-situ polymerization of aromatic substrate onto fabric containers. Both polymers, poly(catechol) and poly(p-phenylenediamine), present good thermal stability and resistance to thermal degradation, as well as free radical scavenging ability. Colored polymers were generated which conferred color to the fabrics. The scanning electron microscopy (SEM) observation shows uniform distribution of the polymer on the surface of cotton, wool and PET. All the fabrics reveal color fastness to washing, sunlight and rubbing. The conductivity of fabrics was determined after treatment with poly(p-phenylenediamine) and all the fabrics showed good conductivity. Both polymers are able to confer antimicrobial activity to all the coated fabrics against Gram positive (S. aureus) and Gram-negative (E.coli). The cytotoxicity tests performed on functionalized fabrics revealed that both polymer diffusion and porous fabric structure may affect cell viability, which could be avoided by the adjustment of the polymer concentration. This project aims to study the impact of the enzyme modification and processing conditions on the structure of the multi-functional reaction products, and aims to set up a high efficiency reaction system for laccasecatalyzed synthesis of a wide range of phenolic compounds. The oligomers/polymers obtained are supposed to show different functions to be applied on textile, medical and other areas.
A lacase é uma das oxidases multicobre que é capaz de oxidar compostos fenólicos, aminas aromáticas e outros compostos usando o oxigénio como aceitador de electrões. Sendo um biocatalisador ambientalmente aceite, a lacase mostra elevado potencial em diversas áreas de investigação assim como ao nível industrial, incluindo a Industria Têxtil, a Industria do papel, e na área de conversão de agentes poluentes, entre outros. No campo da síntese, as reações catalisadas pela lacase têm sido intensamente investigadas, no entanto o mecanismo de catálise de compostos aromáticos necessita de ser estudado em maior profundidade. A pesquisa e desenvolvimento da lacase na produção de polímeros atendem aos requisitos da química verde, o que promoverá uma vasta aplicação dos materiais poliméricos sintéticos. Esta tese de doutoramento pretende explorar novas estratégias e mecanismos para a síntese de polímeros usando lacase em diferentes formas e diferentes tipos de reatores. Os polímeros aromáticos foram produzidos usando a lacase do fungo ascomicete Myceliophthora Thermophila. Foram desenhadas diferentes modificações para a lacase de modo a incrementar a sua performance, que incluíram a PEGilação e/ou imobilização. Ao mesmo tempo foram realizadas simulações de modelação molecular de forma a prever a geometria e as modificações estruturais da enzima. Durante o processo de polimerização, foram estudados três tipos de reatores com distintos níveis de energia associados. Os polímeros obtidos foram caracterizados e aplicados em diferentes substratos de forma a obter tecidos multi-funcionais. Este estudo visa fornecer conhecimento fundamental sobre a síntese de polímeros pela lacase promovendo ao mesmo tempo suporte teórico e técnico para o estabelecimento de sistemas catalíticos eficientes. Primeiramente, a polimerização do catecol foi feita usando a lacase como catalisador. O polietilenoglicol (PEG) foi usado na modificação não covalente e covalente da lacase sendo a sua performance posteriormente avaliada. A performance catalítica das diferentes lacases PEGiladas foi comparada em fase aquosa e em gel. Os resultados demonstram que a lacase modificada, quer de forma não-covalente ou covalente (lacase PEGilada), é capaz de promover a polimerização do catecol, aumentando quer o grau de conversão quer o grau de polimerização. No entanto, estes eventos foram unicamente detetados em fase aquosa. Os estudos de modelação molecular demonstram que a presença do PEG desacelera a desativação da enzima em fase aquosa. Posteriormente, foi efetuada a imobilização da lacase nativa e da lacase PEGilada em resina epoxy, usando polietilenoglicol como agente de ligação entre a enzima e o suporte. A performance de polimerização em fase aquosa das enzimas imobilizadas foi explorada e a estrutura dos polímeros obtidos foi proposta. Depois de imobilizada, o tempo de meia-vida da lacase foi incrementado assim como a sua estabilidade. A análise de MALDI-TOF MS revelou um aumento do grau de polimerização quando as enzimas imobilizadas, epoxy-lacase nativa, epoxy-PEGilada e epoxy-PEG-lacase, foram usadas como catalisador. Posteriormente, foram estudados diferentes reatores para a polimerização do catecol, nomeadamente banho termostatizado com agitação orbital, banho de ultrassons e homogeneizador de alta pressão. A atividade e estabilidade da lacase nesses reatores foram primeiramente avaliadas assim como o mecanismo de síntese do catecol em presença de ambientes de elevada energia. Os polímeros obtidos foram caracterizados sendo proposta a sua estrutura final. Os resultados demonstraram que ambientes de maior energia fornecem uma maior interação entre a enzima e o substrato, incrementando assim o rendimento de polimerização. O grau de conversão aquando da utilização quer do banho de ultrassons quer do homogeneizador de alta-pressão foi superior ao obtido quando a polimerização foi efetuada no banho termostatizado com agitação orbital. A lacase sob efeito de ultrassons ou do homogeneizador de alta pressão revelou menor estabilidade que quando sob o efeito do banho termostatizado. No entanto, dado que a polimerização ocorre antes dessa inativação, os ambientes de maior energia não provocam efeito negativo sobre a síntese do polímero. A performance catalítica das lacases, nativa, PEGilada e epóxi-PEGilada, foi avaliada quando sob o efeito do homogeneizador de alta pressão. Foi comparada a atividade e estabilidade assim como os correspondentes polímeros produzidos. As formas da lacase, PEGilada e Epoxi-PEGilada, revelaram a maior atividade catalítica assim como maior estabilidade. Em seguida, o catecol e a p-fenilenodiamina foram polimerizados no homogeneizador de alta-pressão pela lacase usando sacos feitos de poliéster (PET), algodão e lã que serviram de recipientes da enzima. A funcionalização dos tecidos foi conseguida através da polimerização in situ dos substratos aromáticos sobre os tecidos contendo a enzima. Ambos os polímeros, poli(catecol) e poli(p-fenilenodiamina), apresentam aceitável estabilidade dimensional e resistência à degradação térmica, assim como capacidade de eliminação de radicais livres. Foram obtidos polímeros com cor com capacidade de coloração dos tecidos usados. As observações efetuadas por microscópio eletrónico de varredura mostram uma distribuição uniforme do polímero na superfície dos tecidos de poliéster, algodão e lã, tendo os mesmos revelado alguma resistência à lavagem, à luz solar e à fricção. Os tecidos após funcionalização com poli(p-fenilenodiamina) revelaram condutividade elétrica. Ambos os polímeros produzidos foram capazes de conferir capacidade antimicrobiana aos tecidos com eles funcionalizados contra bactérias Gram positiva (S. aureus) e Gram negativa (E.coli). Ensaios de toxicidade efetuados nos tecidos funcionalizados revelaram que a difusão dos polímeros, depende da porosidade dos tecidos, e afeta diretamente a viabilidade celular, que pode ser aumentada ajustando a concentração do polímero. Este projeto visa o estudo do impacto da modificação da enzima e das condições de processamentos na estrutura dos produtos de reação com funções múltiplas, tendo ao mesmo tempo o objetivo de estabelecer um sistema reacional de catálise de uma panóplia de compostos fenólicos pela lacase. Os oligomeros e/ou polímeros produzidos poderão ter diferente aplicações em áreas como têxtil, médica, entre outras.
The research presented in this document was done in the Bioprocess and Bionanotechnology Research Group, Centre of Biological Engineering, University of Minho, Braga, Portugal. Part of this work was funded by the Portuguese Foundation for Science and Technology (FCT) through the grant CEB-BI-24-2017 and CEB-BI-06-2018 (strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01- 0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte). Part of this work was also funded by the Chinese Government Scholarship under China Scholarship Council (No. 201606790036), Chinese Foundation Key projects of governmental cooperation in international scientific and technological innovation (No. 2016 YFE0115700).
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