No description available.

We report the assembly of three-dimensional (3D) liver sinusoidal mimics comprised of primary rat hepatocytes, human or rat liver sinusoidal endothelial cells denoted as hLSECs and rLSECs respectively, and an intermediate chitosan-hyaluronic acid (HA) polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30-55nm and exhibited a shear modulus of ~ 100kPa. Primary rat hepatocytes coated with 5 and 15 PE layers exhibited stable urea and albumin production over a seven day period and these values were either comparable or superior to that in a collagen sandwich (CS). Hepatocyte-PEM-hLSEC liver mimics exhibited stable urea production and increasing albumin secretion over the culture period in comparison to hepatocyte-LSEC samples. In the 3D liver mimics, hLSEC phenotype was maintained and verified by the uptake of acetylated low-density lipoprotein (AcLDL). A sixteen-fold increase in CYP1A1/2 activity was observed for hepatocyte-PEM-10,000 hLSEC samples, thereby, suggesting that interactions between hepatocytes and hLSECs play a key role in enhancing hepatic phenotypes in in vitro cultures. As the first step towards elucidating key signaling pathways involved in cell-cell communications, global genome-wide transcriptional profiles of primary hepatocytes cultured in CS and hepatocyte monolayers (HMs) were performed over an eight-day period using DNA microarray measurements and Gene Set Enrichment Analysis (GSEA) in order to derive biologically meaningful information at the level of gene sets. The gene expression in CS cultures steadily diverged from that in HMs. Gene sets up-regulated in CS are those linked to liver metabolic and synthetic functions, such as lipid, fatty acid, alcohol and carbohydrate metabolism, urea production, and synthesis of bile acids. Monooxygenases such as CYP enzymes were significantly up-regulated starting on day 3 in CS cultures. These results serve as a baseline for further investigation into the systems biology of engineered liver tissues. 3D hepatic constructs were also assembled with primary rat hepatocytes and rLSECs, and a chitosan-HA PEM. In these hepatic models, the phenotype of hepatocytes and rLSECs were maintained. rLSEC phenotype was verified over a twelve-day period through immunostaining with the sinusoidal endothelial-1 (SE-1) antibody. In contrast, rLSECs cultured as monolayers lost their phenotype within 3 days. A two-fold increase in albumin production was observed only in the 3D liver models. rLSEC-PEM-hepatocyte cultures exhibited three- to six-fold increased CYP1A1/2 and CYP3A enzymatic activity. Well-defined bile canaliculi were observed in only 3D hepatic constructs. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte constructs can be used as liver models for future studies. / Ph. D.

Reactive oxygen species (ROS) such as superoxide radical anion (O2•¯), hydroxylradical (OH•), hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) that are produced during the metabolism of oxygen under oxidative stress in aerobic organisms destroy several key biomolecules and lead to a number of disease states. Mammalian systems possess several effective defense mechanisms including antioxidant enzymes to detoxify these ROS. The selenocysteine-containing Glutathione peroxidase (GPx) is particularly an efficient enzyme in the detoxification of H2O2 and other hydroperoxides by using glutathione (GSH) as cofactor. The chemistry at the active siteof GPx has been extensively investigated with the help of synthetic selenium compounds. Although the anti-inflammatory compound ebselen(2-phenyl-1,2-benzoisoselenazol-3(2H)-one) is undergoing phase III clinical trial as antioxidant, the chemistry of ebselen is still not understood. The present study on a number of ebselen derivatives with various N-substitutions reveals that the substitution at the N atom is important for the antioxidant activity. This study also suggests that the nature for thiol cofactor has a dramatic effect on the GPx activity of ebselen derivatives. It has been shown that ebselen exhibits very poor catalytic activity in the presence of aromatic thiols mainly due to strong Se….O nonbonded interactions that lead to extensive thiol exchange reactions in the selenenyl sulfide intermediate. To prevent the se….O interactions, a series of tertiary amide-based diselenides have been synthesized along with their secondary amide counterparts. Detailed structure-activity correlation studies reveal that the GPx-like activity of the sec-amide-based compounds can be significantly enhanced by the substitution at the free-NH group of sec-amide functionality. The N,N-dialkylbenzylamine-based diselenides exhibit their catalytic activities via the generation of selenols which was confirmed by the reaction with anti-arthritic gold(I) compounds. Interestingly, the replacement of the hydrogen atom at the 6th position of the benzene ring of N,N-dialkylbenzylamine-based diselenides by a methoxy group prevents the thiol exchange reactions mainly be weakening the Se…N interactions and thus enhances the GPx activity. On the other hand, the catalytic activity of the tert-amine-based diselenides can also be increased by replacing the tert-amino groups with the corresponding sec-amine moieties. It has been observed that the basic amino group in the amine-based diselenides deprotonates the selenol and also the thiol cofactor, which is crucial for the higher catalytic activities of the amine-based compounds. Peroxynitrite (PN, ONOO), a strong nitrating agent, is known to inactivate a number of proteins, enzymes and other biomolecules by nitration of tyrosine residues. In this study, we have shown that the commonly used antithyroid drugs and their analogues inhibit protein tyrosine nitration. This study reveals that antithyroid agents having PN scavenging activity may be beneficial of hyperthyroidism as these compounds may protect the thyroid gland from nitrative or nitrosative stress.

Off-label use of traditional Oropharyngeal Airway (OPA) as a bite-block, and the subsequential procedure of force exertion of the device by physician has caused many cases of patient’s teeth damage and monetary loss, as the patient’s incisors were damaged while clenching on the OPA during an adverse scenario called “Emergency Clenching”. To remedy this harmful situation, Bardo OPA was developed by Dr. Theodore Burdumy. The Bardo airway has unique design to transfer the clenching force from incisor to the molar. However, the Bardo OPA is one-sized, and cannot fit most of the patients like the commonly-used OPAs, such as the Berman and Gudel OPA, which have a spectrum of sizes to ensure fit. In this project, a Computer Assisted Design (CAD) simulation platform was developed to simulate the scenario where OPA is placed in a patient’s oral cavity. CAD – related technique and tools, such as 3D scanner (ScanStudio HD), RapidWorks, SolidWorks and Mimics were utilized to create the models used to construct the platform. The purpose of this platform is to generate data to support the development of additional sizes and other modification to improve the current design of the Bardo OPA. MRI sets of nine (9) patients were obtained and converted into STL mesh models. Berman and Guedel OPA were used as the standard for comparison against the Bardo OPA. It was found that the Bardo OPA was able to fit into all sample patients’ models, while these models were fitted with Berman and Guedel OPA of 70-90mm (Small to medium adult) sizes. It can only be concluded that the Bardo is compatible with these OPA sizes and there was not enough evidence to show the need for additional sizes. Nevertheless, some functional features of the Bardo OPA were found potentially harmful to the patients or ineffective. Three approaches were suggested to improve the design of the Bardo to achieve better safety and efficacy.

Living amidst a milieu of pathogenic organisms, vertebrates are in constant threat of contracting one or the other disease. As a mechanism of protection against such ‘invasions’, the vertebrate immune system has evolved to serve two main functions. One, to generate a specific immune response against the invading pathogen (in the from of specific antibodies and cell mediated immune responses). And two, to ‘remember’ the pathogen after the first exposure and mount a heightened and quicker immune response upon subsequent encounters. This phenomenon is called immunological memory, or anamnestic response and is achieved by the generation of memory B and T cells. The generation of specific Immunological memory is indeed the most important requirement/purpose of prophylactic vaccination Though different mechanisms are known to operate to maintain memory B and T cells, some aspects are still debatable. The ‘relay hypothesis’ (Nayak etal., Immunology.102(4)(2001); Nayak R etal., Microbes. Infect.(2005)), addresses some of those key issues. It describes that antigen specific memory B cells can be maintained by the interaction of membrane bound idiotypic (Id, Ab1) and anti-idiotypic (α-Id, Ab2) antibodies on B cells. Anti-Ids binding to idiotopes on Abs (Ab1) are known to be potential regulators of immunity in a variety of diseases, such as autoimmunity, cancer as well as viral, bacterial or parasitic infections. The relay hypothesis outlines the mechanism of persistence of memory cells in the absence of persisting antigen. This is achieved through the ‘internal image’ of the antigen on the Ab2 variable region, which serves as surrogate antigen thus helping in maintenance of immunological memory even in the absence of persisting antigen. It also explains that all antigens, protein or nonprotein can be converted to the common “coinage” of internal image peptides, otherwise called peptido-mimics. Peptido-mimics that have similar binding properties to MHC as the antigenic epitope, will ensure that the antigen specific memory T cells are also maintained. Hence T cell activation could also occur in the absence of nominal antigen, a potentially important process in T-B cooperation and immune regulation. Scope and objectives of this work: To demonstrate the presence of idiotypic and cognate anti-idiotypic antibody for the given antigen  To examine the likelihood of the three dimensional structural similarity between antigen and Ab2 variable region  To demonstrate the presence of peptido-mimics of the antigenic epitope in the Ab2 variable region; and if those peptido-mimics have structural and functional similarity with antigenic peptides when bound to MHC-I To examine the (immunological) memory associated phenotype of thediotypic anti-idiotypic B cells. The antigen of choice for the current study is Heamagglutinin-Neuraminidase (HN) protein of peste des petits ruminants virus (PPRV). Idiotypic (Id, Ab1), antiidiotypic (α-Id, Ab2) hybridoma against a deletion mutant of PPRV HN were generated and characterized. These hybridoma served as surrogate B cells for the study of Id α-Id B cell interactions. Anti-anti-idiotypic (Ab3) lymphocytes were also generated by immunizing syngenic BALB/c mice with Ab2 hybridoma. Results not only indicated the interplay of idiotypic and anti-idiotypic B and T cells in this cascade but also the mimicry of the antigen by Ab2. Ab2 Mab could recognize idiotopes of anti-PPRV HN Ab1 raised in diff species of animals, thus demonstrating that Ab2 was indeed an antigen mimic that interacts with Ab1 paratope irrespective of which species the Ab1 originates from. Ab2 Mab also mimicked the antigen (Hemagglutinin-neuraminidase) in functional assays by bringing about hemagglutination. Similarly, Polyclonal Ab3 which reacts with Ab2 Mab and with antigen, inhibits hemagglutination, just as Ab1 does, albeit to a lesser extent. This suggests Ab3 has functional similarity with Ab1. It is imperative that T cells be involved in this network of B cells for the maintenance of antigen specific immunological memory. This is because B cells require T cell help in the form of cytokines for proliferation and Cytotoxic T Lymphocytes (CTLs) are needed to control the specific population of Id and anti-Id B cells to maintain homeostasis. The Ab2 hybridoma as well as soluble Ab2 stimulated the proliferation of antigen specific T cells. Similarly, Ab3 splenocytes were stimulated to proliferate by the Ab2 as well as the antigen. Peptides generated from monoclonal Ab2 heavy and light chain variable regions (VH and VL) showed structural and functional similarity to the antigenic peptides in terms of p-MHC binding. These peptides stimulated the proliferation of antigen and Ab2 specific T cells, and also triggered 4-5 times higher CTL targeted cell lysis of peptide pulsed RMAS-Kd cells, as compared to a control peptide. VH, VL and antigenic peptides stabilized MHC-I on the cell surface of the TAP deficient, RMAS-Kd cell line for upto six hrs as compared to the ‘empty’ MHC-I, which remained on the surface only for one hr. The presence of peptido-mimics in the Ab2 variable region, which have structural similarity with antigenic peptide (when bound to MHC I), was also established using insilico software tools. Antigenic peptides and VH and VL peptides were modeled onto MHC-I crystal structures using the molecular modeling software InsightII and the minimization program, CNS. Putative MHC-I binding peptides from these sequences were generated using the p-MHC-I binding prediction algorithm, BIMAS. By replacing these peptides in the respective crystal structure of MHC I and superimposing the two structures, we have tried to establish that through structural similarity in binding to MHC-I, peptidomimics have a role in the maintenance of antigen-specific CTL memory. Consequently CTL memory specific to antigenic epitope can be preserved even in the absence of antigen by its peptidomimic. Following long-term immunizations, as expected of a secondary immune response, the serum Ab1 titre was found to be higher than the titer during primary response. It was also noted that though the number of Ab1 and Ab2 cell number was comparable in the total splenocyte population, Ab1 titre in the serum was higher than Ab2, immaterial of Ag/Ab2 booster. The same trend was noticed in prolifertion assay and CTL assays when the splenocytes were stimulated by Ag/Ab2 pulsed bone-marrow derived dendritic cells (BMDCs) as APCs. That is, irrespective of immunization and boost with Ag/Ab2, Ag pulsed BMDCs stimulated the proliferation and CTL lysis of long term immunized splenocytes to a greater extent than Ab2 pulsed BMDCs. Memory markers present on B and T cell surface might help maintain their close interactions in the idiotypic network. CD27/CD70 (CD27L) might play a role in maintaining these cells in a memory state. The Id α-Id B cells in addition to being triggered through the membrane bound Id, α-Id antibodies, can also be activated through CD27/CD70 to differentiate into plasma cells upon activation by antigen. Id and α-Id B cells were demonstrated to possess the CD27 memory marker on their surface in addition to the membrane bound IgM. Antigen specific IgM and CD27 double positive cells were detected in the range of 1-3% in the total splenocyte population. In conclusion: PPRV HN immunization triggered the generation of Ab1, Ab2, Ab3 (Id, α-Id, α-α-Id) cascade through the interaction of membrane bound immunoglobulin of the corresponding B cells. Ab2 was demonstrated to be a significant structural and functional mimic of the antigen. Peptidomimics of the antigenic epitope, present in the Ab2 variable region, can serve the purpose of maintaining antigen specific T cell memory response. These findings re-confirm the importance of anti-id antibodies in the regulation of immune responses. Ever since the concept of antigen mimicry by anti-Id antibody has been confirmed by several laboratories, the utility of anti-Ids as surrogate antigens for the purpose of prophylactic vaccination has received great attention. The results of the current work are especially significant for the purpose of development of vaccines for diseases related to antigens that are very cumbersome to purify (for ex., in case of several cancers) or when it is too dangerous to immunize with the antigen itself (for ex., in case of some pathogenic organisms). The results also signify that immaterial of the nature of the antigen, their respective petidomimics can establish and maintain immunological memory.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-06-06T14:46:41Z No. of bitstreams: 1 arquivototal.pdf: 3774616 bytes, checksum: 0876fb49507ce01bcd8d4756cb29ba71 (MD5) / Made available in DSpace on 2017-06-06T14:46:41Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3774616 bytes, checksum: 0876fb49507ce01bcd8d4756cb29ba71 (MD5) Previous issue date: 2016-09-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The superoxide anion is among the reactive oxygen species closely related to physiopathological states associated with oxidative stress. The physiological superoxide levels are controlled in vivo by the superoxide dismutase enzymes (SOD). Pentacationic Mn porphyrins derived from 2-N-alkylpyridylporphyrins have been explored as potent SOD mimics and efficient redox modulators of oxidative stress. The in vivo efficiency of these compounds is related with their intrinsic catalytic activity, lipophilicity, bioavailability, and toxicity. The present study describes the synthesis of two A3B-type neutral porphyrins of low symmetry (A = 2-pyridyl, B = 3-methoxy-4-hydroxyphenyl or 3,4-dimethoxyphenyl), the methylation of these compounds to yield tricationic porphyrins and the preparation of the corresponding tetracationic Mn(III) porphyrins; overall, 8 new compounds are described: 5-(3-methoxy-4-hydroxyphenyl)-10,15,20-tris(2-pyridyl)porphyrin (H2VanTri-2-PyP), 5-(3,4-dimethoxyphenyl)-10,15,20-tris(2-pyridyl)porphyrin (H2MVanTri-2-PyP), 5-(3-methoxy-4-hydroxyphenyl)-10,15,20-tris(N-methylpiridinium-2-yl)porphyrin chloride (H2VanTriM-2-PyPCl3), 5-(3,4-dimethoxyphenyl)-10,15,20-tris(N-methylpiridinium-2-yl)porphyrin chloride (H2MVanTriM-2-PyPCl3), 5-(3-methoxy-4-hydroxyphenyl)-10,15,20-tris(2-pyridyl)porphinatomanganese(III) (MnVanTri-2-PyPCl), 5-(3,4-dimethoxyphenyl)-10,15,20-tris(2-pyridyl)porphinatomanganese(III) chloride (MnMVanTri-2-PyPCl), 5-(3-methoxy-4-hydroxyphenyl)-10,15,20-tris(N-methylpiridinium-2-yl)porphinatomanganese(III) chloride (MnVanTriM-2-PyPCl4) and 5-(3,4-dimethoxyphenyl)-10,15,20-tris(N-methylpiridinium-2-yl)porphinatomanganese(III) chloride (MnMVanTriM-2-PyPCl4). The spectroscopic, electrochemical properties and lipophilicity of all compounds were compared with A4-type 2-N-pyridyl porphyrin analogues. The monocationic compounds MnVanTri-2-PyP+ and MnMVanTri-2-PyP+ showed too low Mn(III)/Mn(II) reduction potential incompatible with superoxide dismuting activity, whereas the tetracationic complexes MnVanTriM-2-PyP4+ and MnMVanTriM-2-PyP4+ showed potential values suitable for the development these compounds as SOD mimics based on structure-activity relationships reported for analogous systems. The lipophilicity of MnVanTriM-2-PyP4+ and da MnMVanTriM-2-PyP4+ suggests promising bioavailability for in vitro and in vivo testing. / O ânion radical superóxido constitui uma das espécies reativas de oxigênio intimamente relacionada a estados fisiopatológicos associados ao estresse oxidativo. Os níveis fisiológicos do superóxido são controlados in vivo pelas enzimas Superóxido Dismutases (SOD). Mn-porfirinas pentacatiônicas derivadas das 2-N-alquilpiridilporfirinas têm se destacado como mímicos potentes das enzimas SOD e, por sua vez, potentes moduladores redox de estresse oxidativo. A eficiência in vivo destes compostos está relacionada à atividade catalítica intrínseca, à lipofilicidade, à biodisponibilidade e à toxicidade. No presente estudo, descreve-se a síntese de 2 porfirinas neutras de baixa simetria do tipo A3B (A = 2-piridil, B = 3-metoxi-4-hidroxifenil ou 3,4-dimetoxifenil), a metilação destes compostos para obtenção de porfirinas tricatiônicas e a preparação das Mn(III) porfirinas tetracatiônicos correspondentes; ao todo são descritos 8 compostos inéditos: 5-(3-metoxi-4-hidroxifenil)-10,15,20-tris(2-piridil)porfirina (H2VanTri-2-PyP), 5-(3,4-dimetoxifenil)-10,15,20-tris(2-piridil)porfirina (H2MVanTri-2-PyP), cloreto de 5-(3-metoxi-4-hidroxifenil)-10,15,20-tris(N-metilpiridinio-2-il)porfirina (H2VanTriM-2-PyPCl3), cloreto de 5-(3,4-dimetoxifenil)-10,15,20-tris(N-metilpiridinio-2-il)porfirina (H2MVanTriM-2-PyPCl3), cloreto de 5-(3-metoxi-4-hidroxifenil)-10,15,20-tris(2-piridil)porfirinatomanganês(III) (MnVanTri-2-PyPCl), cloreto de 5-(3,4-dimetoxifenil)-10,15,20-tris(2-piridil)porfirinatomanganês(III) (MnMVanTri-2-PyPCl), cloreto de 5-(3-metoxi-4-hidroxifenil)-10,15,20-tris(N-metilpiridinio-2-il)porfirinatomanganês(III) (MnVanTriM-2-PyPCl4) e cloreto de 5-(3,4-dimetoxifenil)-10,15,20-tris(N-metilpiridinio-2-il)porfirinatomanganês(III) (MnMVanTriM-2-PyPCl4). As propriedades espectroscópicas, eletroquímicas e de lipofilicidade de todos os compostos foram comparadas com os derivados das 2-N-piridilporfirinas análogas do tipo A4. Os complexos monocatiônicos MnVanTri-2-PyP+ e MnMVanTri-2-PyP+ apresentam potenciais de redução Mn(III)/Mn(II) muito baixos para catalisarem a dismutação do superóxido, enquanto os complexos tetracatiônicos MnVanTriM-2-PyP4+ e MnMVanTriM-2-PyP4+ apresentaram potenciais apropriados para o desenvolvimento de mímicos SOD com base nas relações estrutura-atividade reportadas para sistemas correlatos. A lipofilicidade da MnVanTriM-2-PyP4+ e da MnMVanTriM-2-PyP4+ sugere que os compostos têm biodisponibilidade promissora para testes in vitro e in vivo.

Blood vessel mimics (BVMs) are simple tissue engineered blood vessel constructs intended for preclinical testing of vascular devices. This thesis developed and implemented methods to characterize two of these components. The first aim of this thesis investigated the effect of cell culture duration and flow conditions on endothelial cell gene expression, especially regarding endothelial-to-mesenchymal transition (EndMT). A trend of decreased endothelial marker gene expression and increased mesenchymal marker gene expression would indicate EndMT. qPCR analysis revealed that increased cell culture duration did not result in EndMT, and in fact increased endothelial marker expression as cell culture duration increased. Disturbed flow conditions decreased endothelial marker and increased mesenchymal marker expression relative to static culture. The second aim of this thesis developed methods to determine cytotoxicity of, and endothelial cell adhesion to, novel BTEAC salt scaffolds. Immunostaining was used to visualize these scaffold effects. The cytotoxicity elution assay showed that BTEAC salt scaffolds were not more cytotoxic than the standard PLGA scaffold. Direct contact assays spanning several timepoints also found that BTEAC salt scaffolds were not more cytotoxic than standard scaffolds but had higher endothelial cell adhesion and coverage than standard scaffolds. Overall, this thesis developed and implemented methods to characterize the endothelial cells used in the BVM model.

Engineering artificial cells is currently an emerging area of research that involves constructing mimics of biological cells. These biomimetic cellular systems hold tremendous promise for the different biomedical applications (diagnostics, therapy, tissue engineering, gene transfection, bioactive coatings) as well as aspects of synthetic biology. A key architectural principle of the cell is a multicompartmentalized assembly, which is one of the features of biological cells that enable the performance of multiple complex biochemical reactions within confined environments. For this purpose, this study demonstrates novel artificial cells, not only presenting organelle mimics but also incorporating various stimuli for regulating enzymatic cascade reactions within the artificial cell and for controlled simultaneous and/or subsequent release of the encapsulated (therapeutic) molecules. To successfully fabricate the multifunctional polymeric multicompartmentalized systems as artificial cells aimed for, in the first step a hollow capsule as biomimetic cellular membrane was developed to simulate a key characteristic of functional artificial cells for the selective uptake and release of (bio)molecules and particles for intra- and intercellular signaling processes. Herein using LbL technique which involved alternate deposition of oppositely charged polyelectrolytes on silica template via electrostatic interaction, the pH and temperature dual-responsive and photo-crosslinked hollow capsule was fabricated and they can be used for the subsequent post-encapsulation process of protein-like macromolecules (≤ 11 nm) and their controllable release triggered by external stimuli for mimicking the controllable bio-inspired functions of cell membranes. The reversible temperature and pH dual-response ability of the hollow capsules has been analyzed. The uptake and release properties of the resulting hollow capsules with different degree of photo-crosslinking for cargos have been further investigated at various temperatures (25, 37 or 45°C) and pH (5.5 or 7.4) of the solution. Next, the design of the polymersomal subcompartmens as organelle mimics, which divide the interior of the multicompartmentalized systems into subcompartments and can stably encapsulate fragile hydrophobic and hydrophilic cargo, e.g., enzymes in order to conduct encapsulated catalysis-resembling cell organelles, was also an important subject. The fabrication of these subcompartments was starting with the synthesis of suitably end-group block copolymers to realize the enzyme-loaded, multifunctional, pH-responsive, photo-crosslinked and post-labelled polymersomes decorated with adamantane groups. The pH sensitivity and various enzymatic reactions of the established multifunctional Ada-polymersomes have been investigated. Based on the above concepts, a bottom-up approach was developed to assemble a structural and functional eukaryotic cell mimics, including “membrane-associated” multicompartmentalized system (MS1) and “free-floating” multicompartmentalized system (MS2), by loading pH-sensitive Ada-polymersomes inside the multifunctional cell membrane. The creation of these multicompartmentalized systems was based on the assembly of enzyme-loaded Ada-polymersomes as organelle mimics onto sacrificial particle templates by host-guest interaction, followed by the LbL deposition of temperature-responsive and photo-crosslinkable PMA(β-CD)/[PAH/PNMD]3 multilayers and outer protective capping PAH/PMA(PEG) bilayer as biomimetic cellular membrane. Upon photo-crosslinking the polymer biomimetic membrane and dissolution of the particle templates, multicompartmentalized systems were obtained. Spatial position of the subcompartments can be controlled using non-covalent host-guest concept, which yielded multicompartmentalized systems containing “membrane-associated” and “free-floating” subunits. Moreover, the metabolism mimicry of multicompartmentalized systems by performing multiple successive two-enzyme cascade reactions in the cells and the multiple parallel reactions by using a third enzyme for deactivating the reaction product and interfering the cascade reaction have been investigated. In conclusion, these multicompartmentalized systems, combining the advantages of both pH-responsive Ada-polymersomes as organelle mimics and multifunctional hollow capsule as biomimetic cellular membrane, present new opportunities for the development of functional cell mimics. The presented studies highlight crucial aspects for the successful applications of such cell mimics for diagnostics, tissue engineering, as nanoreactors, as carriers for multiple drug delivery with controlled release profiles, or as therapeutic artificial cells.

Les polymersomes, obtenus par auto-assemblage en solution aqueuse de copolymères à blocs amphiphiles en structure vésiculaire, sont présentés comme d’excellent mimes synthétiques des virus, dont les propriétés membranaires – principalement élasticité, perméabilité, fonctionnalité- peuvent être très proches. Il y a ainsi un fort engouement quant à leur utilisation en biotechnologie et surtout en vectorisation d’actifs pharmaceutiques ou cosmétiques. Afin d’aller encore plus loin dans le biomimétisme ou la bio-inspiration, une étape devait être franchie : encapsuler ces polymersomes les uns dans les autres. Ce cloisonnement ou multi-compartimentalisation permet de mimer cette fois la structure d’une cellule dite eukaryote, elle-même constituée de compartiments internes (organelles) et d’un cytoplasme (lui conférant entre autres une certaine stabilité mécanique) contenues dans le compartiment externe représenté par la membrane cellulaire. Toutefois, l’obtention d’un simple mime structural d’une structure si complexe représente déjà un challenge en soi, nécessitant maîtrise de la physico-chimie des systèmes, de la stabilisation des interfaces et des outils de formulation. Une méthode d’émulsion-centrifugation a été développée et a permis d’obtenir de telles structures compartimentalisées (mimes d’organelles) à cavité gélifiée (mime de cytoplasme). Finalement, différentes voies d’exploitation de ces systèmes sont présentées, allant de l’encapsulation multiple, la libération contrôlée jusqu’au développement de réactions enzymatiques en cascade confinées, mimant ainsi le métabolisme cellulaire. / Amphiphilic block copolymers self-assemble in water into vesicles, coined “polymersomes”; these vesicles are described as excellent synthetic mimics of viral capsids due to the resemblance of their respective membrane properties (in terms of elasticity, permeability, and functionality). As a result, they were massively investigated over the last years regarding applications in biotechnology and more particularly for the targeted delivery of pharmaceutical or cosmetic actives.In order to go further towards bio-inspiration and cell biomimicry, the next step required the encapsulation of polymersomes in other polymersomes. This multicompartmentalization indeed enables to mimic the structure of an eukaryotic cell; an outer cellular membrane compartment encloses internal compartments (organelles) and a cytoplasm responsible amongst others for a certain mechanic stability. However, alone the controlled formation of a system mimicking such a complex structure represents a technological challenge in terms of control over the physical chemistry of these systems, the stabilization of their interfaces and their formulation. A formation method based upon an emulsion-centrifugation has been developed and enabled the formation of such multicompartmentalized structures (organelle mimics) with a gelified lumen (cytoplasm mimic). Finally, various potential applications of these systems are presented: from multiple encapsulation, controlled drug release, to the development of enzymatic and confined cascade reactions that mimick the cellular metabolism.

Les glycosylamines sont des précurseurs très utiles pour préparer une grande diversité de produits naturels et leurs analogues d’intérêts biologiques. Elles ont été particulièrement utilisées comme imines latentes dans des réactions avec différents nucléophiles principalement organométalliques pour former des imino-C-glycosides. Considérant les avantages des t-butanesulfinyl imines d’Ellman en tant qu’électrophiles pour l’addition stéréocontrôlée de différents réactifs pour former des amines, nous avons synthétisé puis exploré la réactivité des t-butanesulfinyl glycosylamines encore peu connues. Nous rapportons ici nos résultats sur la préparation de plusieurs glycosylamines et leur réactivité vis-à-vis de dérivés de magnésium et de lithium, y compris des réactifs fluorés, pour former des amines sous forme de chaîne ouverte. Des détails sont donnés sur les effets de stéréochimie à l’oeuvre dans cette méthode. Nous présentons également la transformation des amines intermédiaires en imino-C-glycosides, incluant des composés importants comme des mimes de glycosyl phosphates portant une aglycone fluorée. L’évaluation biologique de plusieurs de ces composés a été réalisée sur la GlfT2, une cible enzymatique ayant fort un potentiel pour le traitement de la tuberculose. / Glycosylamines are convenient precursors of a diversity of natural products and analogs of biological interest. In particular they have been used as latent imine equivalents in reactions with various organometallic nucleophiles to prepare imino-C-glycosyl compounds. Considering the advantages of Ellman t-butanesulfinyl imines as convenient electrophiles for the stereocontrolled addition of various groups to form amines, we explored the synthesis and reactivity of yet little known t-butanesulfinyl glycosylamines. We report herein our results on the preparation of various glycosylamines and their reactivity towards magnesium and lithium derivatives, including fluorinated reagents, to give open-chain amines. Details are given on the stereochemical effects involved in this process. We also present the transformation of the intermediate amines into imino-C-glycosyl products, including such significant compounds as glycosyl phosphate mimics bearing a fluorinated aglycone. Biological evaluation of number of compounds has been performed on GlfT2, an enzyme of importance regarding to its potential as target for the treatment of tuberculosis disease.