Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the meta­bolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification. The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small mole­cules purification. Specifically, this investigation aims to see if it is achievable to make a bio­tinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis. Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermi­dine, histamine and nicotinamide have been selected. The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control non­biotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.

In this thesis, a novel single molecule technique will be presented that will, for the first time, give direct access to the interaction energy landscapes of small molecules. The technique relies on the interpretation of thermal position fluctuations of a colloidal probe particle tethered to the molecular complex of interest and a geometrical amplification effect that converts Ångstrom scale fluctuations of the ligand in the binding pocket of the receptor to tens of nanometer fluctuation of the bead. The position of the bead is measured with 0.5 MHz bandwidth and 2 nm spatial resolution. The surface characteristic of the substrate was found to be critical for this new technique and various surface effects were observed. Methods were developed to block nonspecific interaction between the surfaces. The mobility of specifically bound particles was found to depend strongly on the density of specific bonds and the length of the molecular complex; low concentration and short linker lead to slow ligand-receptor mediated surface diffusion, high concentration and/or long linkers to an immobilization of the particle. Transient bond formation was observed for the intermediate range. Details of the interaction energy landscape were not resolved. However, a systematic change in the linker length from 22 Å to 29 Å led to a corresponding change in the lateral position fluctuations from 12.9 nm to 13.2 nm in excellent agreement with our theoretical calculations, confirming the geometrical amplification effect. Also, a new phenomenon of nanometer scale friction in the gap between the bead and the surface was discovered. In summary, the results underline that the novel technique might be able to measure details of the interaction energy landscape of a specific ligand-receptor bond and thus test theoretical predictions for its shape. / text

Cancer vaccines represent a promising treatment modality for a world-wide health problem. Whether as an adjuvant or as a stand-alone therapy, cancer vaccines represent a tumor-specific and systemic treatment potentially capable of eliminating metastatic lesions without the severe side-effects often associated with chemotherapy. Specifically, whole cell tumor vaccines have shown promise in preclinical and clinical settings and the studies presented here represent the beginnings of an approach to improve the antitumor potency of these vaccines. This project demonstrates as "proof of concept" the feasibility of manufacturing tumor cell-particle hybrids. The coupled use of these two components, whole tumor cells and cargo-carrying biodegradable particles, as one entity in a cancer vaccine system is a new line of research. Stable cell-particle hybrids were assembled using avidin-biotin chemistry where cargo-carrying PLGA particles (500 nm diameter) were coated with streptavidin and allowed to bind to tumor cells that had been indirectly labeled with biotin (using an integrin-specific biotinylated antibody). That successful cell-particle hybrids were assembled was determined by multiple means, including flow cytometry, laser scanning confocal microscopy and scanning electron microscopy. Two murine tumor cell lines (representing melanoma and prostate cancer) were investigated in this study and successfully demonstrated the general applicability of the assembly method. Particles appeared to be localized on the cell surface (rather than endocytosed) as determined by microscopic imaging. The cell-particle hybrid was shown to be stable to irradiation, an important consideration since whole tumor cells need to be treated with ionizing radiation prior to being used as vaccines in order to render them nonproliferative and immunogenic. We also characterized loading and release profiles of CpG, a prospective vaccine adjuvant, into PLGA particles. We conclude that we have developed a method for manufacturing cell-particle hybrids comprising PLGA nanoparticles and irradiated tumor cells. The next step would be to use CpG-loaded particles in the assembled hybrid and test the anti-tumor immune efficiency of this cancer vaccine formulation in either a melanoma or prostate cancer model.

No description available.

Despite recent advances in stem cell biology, immunotherapy and transplantation, substantial barriers still exist in the large-scale specific separation of a discrete population of human therapeutic cells from a cell suspension. The ideal purification technique should combine high cell purity, yield and function, with fast processing and affordability. Currently, fluorescence-activated cell sorting with flow cytometry (FACS) and magnetic activated cell sorting (MACS®) are the most used methods for cell separation and purification and have been employed extensively in molecular biology, diagnostic and cell sorting applications, because they are considered to be gentle, fast and scalable. However, these methods have several key disadvantages; they are invariably expensive, yield low log cell reduction (LCR) rates, and suffer from drawbacks when applied to niche cell populations, such as those requiring multiple tandem separation steps and/or involving combined positive and negative cell selection steps. To address this challenge, a new cell affinity selection system was developed. The selectivity is based on the reversible monomeric avidin biotin interaction and it is primary designed for positive selection. The initial studies were performed on flat, nonporous, glass coverslips and the technology was then successfully transferred on high grade smooth non-porous glass beads (with a diameter of 79.12 to 118.59 μm). The multi-step layer-by-layer deposition procedure culminating in dextran-coated supports bearing monomeric avidin was rigorously characterized and subsequently employed in packed bed chromatography experiments with human erythrocytes isolated from cord blood and B lymphocytes from cell lines. The developed affinity selection platform was highly selective, efficient and, most importantly, resulted in high yields, cell purity and viability comparable with MACS® technology. Additionally scale up is possible and could be easily transferred to another chromatographic matrix with the appropriate structure.

Antimicrobial proteins contained in the albumen represent maternal effects, including the non- genetic component allocated into the egg during its oogenesis. Especially for species, whose broods are exposed to environmental influences until completation, these proteins play a crucial role in the viability of embryos due to their potential to influence the risk of microbial infection, which is considered one of the main causes of reduced hatchability. Also, it is assumed that these proteins, beacause of their specific traits, may influence phenotype of chicks, especially its size and immunity in the early postembryonal stage. In my thesis I focused on three antimicrobial proteins of avian egg white - avidin, lysozyme and ovotransferrin, which vary in their antimicrobial activity. For a better understanding of causal relationships between the concentrations of these proteins in the albumen and their effect on hatching success or offspring phenotype, a series of manipulation experiments and correlative measurements were performed. These experiments were held on the eggs of two precocial species - Japanese Quail (Coturnix japonica) and Mallard (Anas platyrhynchos). Our results indicate a crucial role of antimicrobial proteins in reducing the risk of bacterial infection and their natural concentration...

Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their reproducible identification and quantitation. However, carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allow for targeting carbonyl PTMs by enriching proteins and peptides carrying these modifications. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma from a healthy donor were derivatized with ARP, digested with trypsin, and enriched using biotin-avidin affinity chromatography prior to nano reversed-phase chromatography coupled online to electrospray ionization tandem mass spectrometry with travelling wave ion mobility spectrometry (nRPC-ESI-MS/MS-TWIMS). The presented workflow addresses several analytical challenges by using ARP-specific fragment ions to reliably identify ARP peptides. Furthermore, the reproducible recovery and relative quantitation of ARP peptides were validated. Human serum albumin (HSA) in plasma was heavily modified by a variety of direct amino acid oxidation products and adducts from reactive carbonyl species (RCS), with most RCS modifications being detected in six hotspots, i.e., Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525 of mature HSA.

Les liquides ioniques connaissent depuis quelques décennies un essor particulier en raison de leurs nombreuses propriétés physico-chimiques intéressantes, telles qu’une faible pression de vapeur saturante, une viscosité limitée, une faible miscibilité avec la plupart des solvants communs, ou encore des propriétés d’agencement supramoléculaire, qui en font des outils puissants dans de nombreux domaines de la chimie. Les sels d’imidazolium représentent la plus grande famille de liquides ioniques à ce jour. Leur modulabilité leur permet d’être dérivés pour de nombreuses applications spécifiques, notamment en synthèse organique, où ils sont utilisés majoritairement comme solvants, et plus récemment comme catalyseurs. Les travaux présentés dans cette thèse se concentrent sur leur utilisation en synthèse organique, à la fois comme solvants et principalement comme catalyseurs chiraux, catalyseurs pour lesquels l’anion du sel est l’espèce catalytique, permettant d’ajouter de la flexibilité et de la mobilité au système. En tirant parti de la tolérance des liquides ioniques envers la majorité des macromolécules naturelles, l’objectif principal des travaux présentés dans cette thèse est le développement d’un nouveau type de catalyseur bio-hybride reposant sur l’encapsulation d’un sel d’imidazolium dans une protéine. Par le biais de la technologie biotine-avidine, l’inclusion supramoléculaire de sels d’imidazolium biotinylés portant des contre-anions catalytiques dans l’avidine a été réalisée et exploitée en catalyse. Dans un premier temps, le développement et l’étude de deux sels de 1-butyl-3-méthylimidazolium possédant des anions chiraux dérivés de la trans-4-hydroxy-L-proline sont rapportés, ainsi que leur comportement dans des réactions énantiosélectives d’aldol et d’addition de Michael. Ces types de composés se sont révélés actifs et performants en milieu liquide ionique. Dans un second temps, la préparation de sels d’imidazolium dont le cation est biotinylé et portant un contre-anion achiral, a été réalisée. Le comportement de l’avidine en milieu liquide ionique et son apport en termes de chiralité sur le système bio-hybride ont été étudiés. Les résultats montrent le rôle crucial des liquides ioniques sur la conformation de la protéine et l’efficacité du catalyseur pour des réactions d’aldol. Dans un dernier temps, l’influence de la structure du cation et de l’anion sur le système a été étudiée. Différents espaceurs ont été introduits successivement dans les squelettes cationiques et anioniques des sels d’imidazolium biotinylés. Dans le cas du cation, les résultats ne révèlent aucune influence majeure sur l’efficacité du catalyseur. La structure de l’anion se montre cependant beaucoup plus importante : la préparation de différents catalyseurs bio-hybrides possédant des anions aux propriétés physico-chimiques différentes a permis d’obtenir de plus amples informations sur le mode de fonctionnement du système bio-hybride et de la coopérativité entre l’avidine et l’anion du sel d’imidazolium.La nature ionique de la liaison cation-anion offrant une liberté de mouvement accrue à l’anion dans la protéine, la tolérance à différents substrats a également été abordée après optimisation du système. / Ionic liquids have gained a growing interest due to many interesting properties, such as low vapor pressure, reasonably low viscosity, poor miscibility with common organic solvents, and also exhibit supramolecular organization in solution, which make them interesting tools for several fields of applications in chemistry. As of today, imidazolium salts make up the largest family of ionic liquids. Their modulability allows them to be used for a wide range of applications, notably in organic chemistry, where they are mainly used as solvents, but also more recently as actual catalysts. The work presented in this thesis focuses on their use as solvents and chiral catalysts, in which the catalytic species is the anion of the imidazolium salts, adding more flexibility and mobility to the whole system. Taking advantage from the tolerance of ionic liquids toward biological macromolecules, the main goal of this work is the design and development of a new type of bio-hybrid catalyst based on the encapsulation of an imidazolium salt inside the cavity of a host protein. Based on the biotin-avidin technology, the supramolecular ligation of biotinylated imidazolium salts inside avidin, bearing catalytic counter-anion, is discussed. As a first step, the development and studies of two 1-butyl-3-methylimidazolium-based salts, bearing trans-4-hydroxy-L-proline-derived anions are reported. Their use for asymmetric catalysis in ionic liquids media is disclosed, both for the aldol and Michael additions. Results show that these compounds are viable and efficient organocatalysts in ionic liquids. Subsequently, the preparation of biotinylated imidazolium salts, bearing a racemic pyrrolidine-based counter-anion is reported. Avidin behaviour in ionic liquid media, as well as its contribution for the stereocontrol for the whole bio-hybrid system, is assessed. Results highlight the critical role of the ionic liquid reaction medium on the protein’s conformation, and thus the efficiency of the bio-hybrid catalyst towards aldol reactions. Finally, the influence of the structure of the cation and anion on the catalytic properties of the biohybrid system were investigated. Several spacers were inserted successively both in the cation and anion structures of the biotinylated imidazolium salts. Regarding the cation modifications, results show no major influence on the bio-hybrid catalyst behaviour. However, modifying the anion structure revealed the much more important role of the anion towards catalysis. Preparation of different anions, each bearing a different spacer, granting them different physico-chemical properties, gives rise to further information regarding the behaviour of the bio-hybrid catalyst, and possible cooperativity between avidin and the imidazolium salt. The ionic character of the interaction between the anion and the cation, allowing a greater freedom of movement of the anion inside the avidin’s cavity, and the tolerance of the bio-hybrid system to different substrates were studied.

Tumor diseases are severe problem worldwide with increasing number of patients suffering from various types of malignancies. Many of approved therapeutics cause serious side toxicities. Therefore, there are intensive efforts to improve cancer treatment protocols. The aim of this study was to deplete regulatory T (Treg) cells without affecting other immunocompetent cells playing a positive role in tumor eradication. Treg cells were reported to hamper anti-tumor immunity and promote tumor growth and survival. Thus, their selective elimination could lead to induction of anti-tumor responses and tumor rejection if combined with chemotherapy with selected N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer-bound drug conjugates. Original approach was to deplete of Treg cells without the use of anti-CD25 mAb that has been widely exploited for Treg cell elimination; however, its long-term persistence in circulation together with inhibitory effect on activated effector cells (CD25+ ) are its main disadvantages. Thus, Treg cells were sensitized to cell cycle-specific cytostatic drugs via application of IL-2/anti-IL-2 JES6.1 mAb immunocomplexes that induce vigorous selective proliferation of this cell population. Subsequent application of cell cycle-specific cytostatics showed steep decrease of Treg cell...