Quitosana é um polissacarídeo usado em diversas aplicações biológicas, por exemplo, em liberação controlada de drogas, transfecção, aceleração da cicatrização de feridas e como agente bactericida, entre outras. Em todas essas aplicações, o polímero interage com tecidos e células. Entretanto, embora sua ação seja comprovada, os mecanismos de ação e a interação do polímero com células e biomembranas no nível molecular ainda não são conhecidos. Nesta tese de doutorado, filmes de Langmuir e Langmuir-Blodgett (LB) de lipídios foram usados como modelos de membrana celular para estudar em nanoescala a interação e os efeitos causados pela quitosana. Primeiramente, observou-se que a quitosana, um polieletrólito solúvel em pH ácidos, possui atividade superficial induzida na presença de um filme interfacial de lipídio, demonstrando que o polímero possui interação favorável com membranas. Após adsorver sobre as monocamadas, a quitosana expande as mesmas, o que ocorre apenas até uma determinada concentração de polímero, denominada concentração de saturação. A magnitude dessa expansão é menor para filmes compactos, o que sugere que a quitosana é parcialmente expulsa da interface, localizando-se na subsuperfície. Isso foi comprovado com o uso de filmes LB, que mostraram que filmes mistos com quitosana têm rugosidade cerca de 10 vezes a de filmes puros de ácido dimiristoil fosfatídico (DMPA). Foi possível confirmar que a quitosana penetra na monocamada, formando agregados com até 150 nm de altura. Além disso, a maior orientação das moléculas de fosfolipídios, sugerida por isotermas de potencial de superfície (V-A) para filmes de Langmuir, também foi comprovada para os filmes LB por medidas de espectroscopia de geração de soma de freqüências (SFG). Filmes mistos de DMPA e colesterol também foram estudados, sendo que o colesterol provoca condensação nos filmes de DMPA a baixas pressões, mas expande as monocamadas em altos estágios de compactação. Quando a quitosana interage com os filmes mistos, ela provoca a mesma expansão para todas as monocamadas independentemente da proporção de colesterol na mistura. Embora esse comportamento possa sugerir um papel inerte do colesterol, ele é explicado pela modulação da penetração da quitosana nos filmes pelo colesterol. Isso ocorre porque há um número fixo de pontos de interações eletrostáticas entre os grupos NH3+ da quitosana e PO2- do DMPA, o que foi comprovado por medidas de espectroscopia de reflexão-absorção na região do infravermelho com modulação da polarização (PM-IRRAS). Com esta técnica para filmes de Langmuir, e espectroscopia SFG para filmes LB, pôde ser traçado um panorama dos efeitos da inserção de colesterol na membrana de DMPA, seguido da interação da quitosana com a membrana mista. A adição do colesterol ao filme de fosfolipídio acarreta em diminuição da ordem das cadeias de DMPA, detectado por variações nas bandas de s(CH2) e ass(PO2-) do fosfolipídio no espectro de PM-IRRAS, e pela razão s(CH3)/s(CH2) nos espectros de SFG. Por outro lado, a interação da quitosana com esse filme misto causa recuperação da orientação das caudas polares do fosfolipídio, verificada pela análise das mesmas bandas de PM-IRRAS e pela razão s(CH3)/s(CH2), que diminui de 6,62 para 4,58 com a adição de colesterol, mas volta a 5,97 após a interação com o polímero. De forma geral, a ação da quitosana sobre biomembranas é governada principalmente por interações eletrostáticas com lipídios carregados negativamente, na superfície externa das mesmas. Dentre os principais efeitos causados pelo polímero, destaca-se a diminuição da elasticidade da membrana e o aumento da orientação das moléculas de lipídio, que podem ter importantes implicações biológicas. A observação de uma concentração de saturação dos efeitos, na maioria dos casos, sugere que a dosagem e a estrutura química da quitosana devem ser bem controladas para alcançar o efeito biológico desejado. / Chitosan is a polyssaccharide with many biological applications, as in drug delivery, transfection, wound healing and as bactericidal agent, for instance. In all these applications the polymer interacts with tissues and cells. The efficacy of chitosan has been proven, but the mechanisms of action and the interactions with cells and biomembranes are still unknown. In this thesis, Langmuir and Langmuir-Blodgett (LB) films made of lipids were employed as cell membrane models, in order to investigate the interactions and modulations caused by chitosan at the molecular level. Firstly, the soluble polyelectrolyte chitosan was found to induce surface activity when a lipid monolayer is at the air/water interface, demonstrating that the interaction of chitosan with membranes is favorable. Upon chitosan adsorption, the monolayers were increasingly expanded with increasing chitosan concentration in the subphase up to a saturation concentration. The extension of this expansion was lower for highly packed films, suggesting that chitosan was partially expelled from the interface after the compression, being located at the sub-monolayer region. This was confirmed by the 10-fold increase in film roughness observed for the areas without aggregates in LB films. Also, we could observe aggregates as high as 150 nm on the film surface, thus confirming chitosan penetration in the dimyristoyl phosphatidic acid (DMPA) monolayer. Mixed DMPA-cholesterol Langmuir monolayers were also produced, with cholesterol inducing condensation of the DMPA films at low pressures, and film expansion at high pressures. Regardless of the cholesterol proportion in the film, chitosan always induced the same degree of expansion on the DMPA mixed monolayers as for a neat DMPA monolayer. Although this behaviour may suggest an inert role for cholesterol, it can only be explained if the sterol is assumed to regulate the extension of chitosan penetration into the monolayer. This occurs because there is a fixed number of sites for electrostatic interactions between NH3+ groups from chitosan and PO2- from DMPA, probed by infrared reflection-absorption spectroscopy (PM-IRRAS) measurements. Indeed, with PM-IRRAS measurements for Langmuir monolayers and sum-frequency generation spectroscopy (SFG) measurements for LB films, we could establish an overview of the effects from cholesterol on DMPA films upon interaction with chitosan. The addition of cholesterol to the DMPA monolayer caused a decrease in the chain order, which was detected by changes in the s(CH2) and ass(PO2-) bands from the phospholipid in the PM-IRRAS spectrum, and by the s(CH3)/s(CH2) intensity ratio in SFG measurements. On the other hand, the interaction of chitosan with these mixed monolayers restored chain order, as observed from the analysis of PM-IRRAS bands and the s(CH3)/s(CH2) in SFG. The latter dropped from 6.62 to 4.58 with cholesterol addition, but further increased to 5.97 with the chitosan interaction. Overall, the chitosan action on biomembranes is mainly governed by electrostatic interactions with negatively charged lipids at the external leaflet of the membrane. The main effects from chitosan to the membrane models are the decrease in membrane elasticity and the increase in molecular ordering, which can lead to important biological implications. Moreover, the existence of the so-called concentration of saturation for most systems suggests that the dosage and chemical structure of chitosan must be well controlled to obtain the desired biological effect.

There is compelling evidence that smokers are less responsive to vaccination. We reported that both therapeutic and prophylactic vaccines fail to protect and cure animals from disease due to negative effects of nicotine on DCs’ ability to generate effector T cells. We have been investigating whether vaccine formulated with TLR agonist(s) could potentially overcome the immunosuppressive effects of nicotine on human DC-NK cross-talk essential for effector T cell generation. Monocyte-derived DCs and nicDCs were stimulated with individual and combined TLR agonists prior to co-culture with purified T cells. The phenotypes and cytokine profiles of T cell were assessed using Flow Cytometry and ELISA, respectively. We found nicDCs cultured with TLR-8/7 alone or in combination with TLR-3 produce quantitatively and qualitatively similar IFN-γ producing effector T cells when compared to control DCs. Our data suggest that the addition of appropriate TLR agonist to vaccine formulation could potentially overcome the immunosuppression seen in smokers, thereby containing the spread of infectious disease to vulnerable population / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Seto Tai Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 122-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.v / Abstract (in Chinese) --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xvi / List of Figures --- p.xv / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Tobacco seed as bioreactor --- p.4 / Chapter 1.2.1 --- Advantages of using tobacco seed to produce bioactive human lysosomal enzyme --- p.4 / Chapter 1.2.2 --- Disadvantages and potential problems of using tobacco seed to produce bioactive human lysosomal enzyme --- p.5 / Chapter 1.2.2.1 --- Difference of asparagine-linked N-glycosylation between plant and human protein --- p.8 / Chapter 1.2.2.2 --- Immunogenicity of recombinant protein with plant-derived N-glycan to human --- p.10 / Chapter 1.2.2.3 --- "Strategy to ""humanize"" plant-derived recombinant human lysosomal enzyme" --- p.10 / Chapter 1.2.2.4 --- Lack of specific glycan structure一mannose-6-phosphate (M6P) tag addition --- p.11 / Chapter 1.2.2.5 --- Strategy for M6P tag addition on plant-derived human lysosomal enzyme --- p.12 / Chapter 1.3 --- The plant secretory pathway --- p.13 / Chapter 1.3.1 --- Plant vacuole in tobacco seed --- p.16 / Chapter 1.3.2 --- Soluble protein trafficking in plant cell --- p.17 / Chapter 1.3.3 --- Integral membrane protein trafficking in plant cell --- p.17 / Chapter 1.3.4 --- Components involved in integral membrane protein trafficking to PSV crystalloid --- p.19 / Chapter 1.3.4.1 --- BP-80 (80-kDa binding protein) --- p.19 / Chapter 1.3.4.2 --- α-TIP (α-tonoplast intrinsic protein) --- p.20 / Chapter 1.3.5 --- Using specific integral membrane protein trafficking system to target recombinant human lysosomal enzyme to tobacco seed PSV --- p.21 / Chapter 1.4 --- Homo sapiens α-L-iduronidase (hIDUA) --- p.21 / Chapter 1.4.1 --- Global situation of lysosomal storage disease一hIDUA deficiency --- p.21 / Chapter 1.4.2 --- Physiological role --- p.22 / Chapter 1.4.3 --- Molecular property --- p.24 / Chapter 1.4.3.1 --- Mutation and polymorphism --- p.24 / Chapter 1.4.4 --- Lysosomal secretory pathway --- p.24 / Chapter 1.4.5 --- Biochemical property --- p.25 / Chapter 1.4.6 --- Clinical application --- p.27 / Chapter 1.4.6.1 --- Enzyme replacement therapy (ERT) --- p.27 / Chapter 1.4.6.2 --- Clinical trial --- p.28 / Chapter 1.4.6.3 --- Economic value --- p.29 / Chapter 1.4.7 --- Expression system --- p.29 / Chapter 1.4.7.1 --- Production (overexpression) of rhIDUA in CHO cell system --- p.30 / Chapter 1.4.7.2 --- Production of rhIDUA in tobacco plant leaf --- p.30 / Chapter 1.5 --- Project objective and long-term significance --- p.30 / Chapter 1.5.1 --- Project objective --- p.30 / Chapter 1.5.2 --- Long-term significance --- p.31 / Chapter Chapter 2 --- Generation and Characterization of Anti-IDUA Antibodies --- p.32 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Chemical --- p.33 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Generation of polyclonal anti-IDUA antibody --- p.35 / Chapter 2.3.1.1 --- Design of synthetic peptide --- p.35 / Chapter 2.3.1.2 --- Conjugation of synthetic peptide to carrier protein --- p.39 / Chapter 2.3.1.3 --- Immunization of rabbit --- p.39 / Chapter 2.3.2 --- Characterization of polyclonal anti-IDUA antibody in rabbit serum --- p.40 / Chapter 2.3.2.1 --- Dot-blot analysis --- p.40 / Chapter 2.3.3 --- Purification of polyclonal anti-IDUA antibody --- p.42 / Chapter 2.3.3.1 --- Construction of anti-IDUA antibody affinity column --- p.42 / Chapter 2.3.3.2 --- Affinity-purification of anti-IDUA antibody --- p.42 / Chapter 2.3.4 --- Western blot detection of denatured rhIDUA --- p.42 / Chapter 2.4 --- Results --- p.43 / Chapter 2.4.1 --- Characterization of polyclonal anti-IDUA antibody --- p.43 / Chapter 2.5 --- Discussion --- p.51 / Chapter 2.6 --- Conclusion --- p.51 / Chapter Chapter 3 --- Generation and Characterization of Transgenic Tobacco Plants Expressing rhIDUA Fusions --- p.52 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.1.1 --- Signal peptide of hIDUA (hIDUA SP) --- p.54 / Chapter 3.1.2 --- Signal peptide of proaleurain (Pro. SP) --- p.54 / Chapter 3.1.3 --- Hypothesis to be tested in this study --- p.54 / Chapter 3.2 --- Materials --- p.55 / Chapter 3.2.1 --- Chemical --- p.55 / Chapter 3.2.2 --- Primers --- p.55 / Chapter 3.2.3 --- Bacterial strain --- p.58 / Chapter 3.2.4 --- The insert-Homo sapiens α-L-iduronidase (hIDUA) cDNA used in this study --- p.58 / Chapter 3.2.5 --- The vector-pLJ526 used in this study --- p.59 / Chapter 3.3 --- Methods --- p.61 / Chapter 3.3.1 --- Construction of chimeric gene construct --- p.61 / Chapter 3.3.1.1 --- Restriction endonuclease´ؤPfIMIl --- p.61 / Chapter 3.3.1.2 --- Recombinant DNA and molecular cloning techniques used in this study --- p.61 / Chapter 3.3.1.3 --- Cloning of pSPIDUA-FLAG --- p.62 / Chapter 3.3.1.4 --- Cloning of pSPIDUA-control --- p.62 / Chapter 3.3.1.5 --- Cloning of a universal construct (pUniversal) --- p.62 / Chapter 3.3.1.6 --- Cloning of pSP-IDUA-T7 --- p.66 / Chapter 3.3.1.7 --- Cloning of pSP-IDUA-control --- p.66 / Chapter 3.3.1.8 --- Cloning of chimeric gene construct into Agrobacterium binary vector --- p.66 / Chapter 3.3.2 --- Expression of chimeric gene construct in tobacco plant --- p.73 / Chapter 3.3.2.1 --- Tobacco plant --- p.73 / Chapter 3.3.2.2 --- Electroporation of Agrobacterium --- p.73 / Chapter 3.3.2.3 --- Agrobacterium-mediated transformation of tobacco plant --- p.74 / Chapter 3.3.2.4 --- Selection and regeneration of tobacco transformant --- p.75 / Chapter 3.3.3 --- Characterization of transgenic tobacco plant expressing rhIDUA fusion --- p.75 / Chapter 3.3.3.1 --- Genomic DNA polymerase chain reaction (PCR) --- p.75 / Chapter 3.3.3.2 --- Southern blot analysis --- p.76 / Chapter 3.3.3.3 --- Total RNA reverse transcription-PCR (RT-PCR) --- p.77 / Chapter 3.3.3.4 --- Northern blot analysis of tobacco leaf --- p.78 / Chapter 3.3.3.5 --- Western blot analysis --- p.79 / Chapter 3.3.4 --- Purification of plant-derived rhIDUA fusion --- p.81 / Chapter 3.3.4.1 --- Construction of affinity column with anti-IDUA antibody --- p.81 / Chapter 3.3.4.2 --- Affinity-purification of rhIDUA fusion from tobacco mature seed --- p.81 / Chapter 3.3.5 --- Confocal immunoflorescence study --- p.82 / Chapter 3.3.5.1 --- Preparation of paraffin section --- p.82 / Chapter 3.3.5.2 --- Single immunocytochemical labeling --- p.82 / Chapter 3.3.5.3 --- Double labeling with one monoclonal and one polyclonal antibodies --- p.83 / Chapter 3.3.5.4 --- Double labeling with two polyclonal antibodies --- p.83 / Chapter 3.3.5.5 --- Image collection --- p.84 / Chapter 3.4 --- Results --- p.85 / Chapter 3.4.1 --- Chimeric gene construction and confirmation --- p.85 / Chapter 3.4.2 --- Selection and regeneration of tobacco transformant with kanamycin- resistance --- p.86 / Chapter 3.4.3 --- Genomic DNA PCR screening of tobacco transformant --- p.88 / Chapter 3.4.4 --- Southern blot analysis of tobacco transformant --- p.91 / Chapter 3.4.5 --- Total RNA RT-PCR screening of tobacco transformant --- p.93 / Chapter 3.4.6 --- Northern blot analysis of tobacco transformant --- p.93 / Chapter 3.4.7 --- Western blot analysis --- p.96 / Chapter 3.4.7.1 --- Western blot analysis of pSP-IDUA-T7-121 transformant leaf --- p.96 / Chapter 3.4.7.2 --- Western blot analysis of pSP-IDUA-T7-121 transformant mature seed --- p.98 / Chapter 3.4.8 --- Affinity-purification of rhIDUA fusion --- p.98 / Chapter 3.4.9 --- Expression level of rhIDUA fusion --- p.102 / Chapter 3.4.10 --- Subcellular localization of rhIDUA fusion --- p.102 / Chapter 3.5 --- Discussion --- p.111 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.117 / References --- p.122 / Appendix 1 --- p.139 / Appendix II (List of Abbreviations) --- p.141

Membrane rafts are highly dynamic heterogeneous sterol- and sphingolipid-rich micro-domains on cell surfaces. They are generally believed to provide residency for cell surface molecules (e.g., adhesion and signaling molecules) and scaffolding to facilitate the functions of these molecules such as membrane trafficking, receptor transport, cell signaling, and endocytosis. Using laser scanning confocal microscopy and reflection interference microscopy (RIM), we studied the spatial and temporal distributions of membrane rafts and surface receptors, signaling molecules, and cell organelles during the formation of phagocytic contact areas. K562 cells, which naturally express CD32A, a cell surface receptor for the Fc portion of Immuno-globulin g (IgG), was chosen as a model for neutrophils. An opsonized target was modeled using a glass supported lipid bilayer reconstituted with IgG. CD32A was found to cluster and co-localize with membrane rafts. Placing the K562 cells on the lipid bilayer triggered a process of contact area formation that includes binding between receptors and ligands, their recruitment to the contact area, a concurrent membrane raft movement to and concentration in the contact area, and transport of CD32A, IgG, and membrane rafts to the Golgi complex. Characterization of these processes was performed using agents known to disrupt detergent resistant membranes (DRMs), dissolve actin microfilaments, and inhibit myosin motor activity, which abolished the CD32A clusters and prevented the contact area formation. The relevance to phagocytosis of contact area formation between K562 cells and lipid bilayers was demonstrated using micro-beads coated with a lipid bilayer reconstituted with IgG as the opsonized target instead of the glass supported planar lipid bilayer. Disruption of membrane rafts, salvation of the actin cytoskeleton, and inhibition of myosin II activity were found to inhibit phagocytosis. Here we have provided evidence that membrane rafts serve as platforms that are used to pre-cluster CD32A and transport CD32A along the actin cytoskeleton to the site of phagocytic synapse formation, followed by internalization to the Golgi complex.

In this study, we introduce novel, truly non-ionic hydrogen-bonded layer-by-layer (LbL) coatings for cell surface engineering capable of long-term support of cell function. Utilizing the LbL technique imparts the ability to tailor membrane permeability, which is of particular importance for encapsulation of living cells as cell viability critically depends on the diffusion of nutrients through the artificial polymer membrane. Ultrathin, permeable polymer membranes are constructed on living cells without a cationic pre-layer, which is usually employed to increase the stability of LbL coatings. In the absence of the cytotoxic PEI pre-layer, viability of encapsulated cells drastically increases to 94%, as compared to 20-50% in electrostatically-bonded shells. Engineering surfaces of living cells with natural or synthetic compounds can mediate intercellular communication, render the cells less sensitive to environmental changes, and provide a protective barrier from hostile agents. Surface engineered cells show great potential for biomedical applications, including biomimetics, biosensing, enhancing biocompatibility of implantable materials, and may represent an important step toward construction of an artificial cell.

Bibliography : leaves 197-209 / 210 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Botany, 1991

Thesis (Ph.D.)--University of Western Sydney, Hawkesbury, 2000. / "A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy-Science". Bibliography : p. 191-233.

L’objectif de mon travail de thèse a été de caractériser l’adhésion de vésicules géantes lipidiques et de cellules vivantes. Dans le but d’obtenir des informations quantitatives sur l’adhésion, j’ai développé deux techniques de nanoscopie de fluorescence basées sur la microscopie TIRF (Total Internal Reflection Fluorescence). Cette technique repose sur création d’une onde évanescente à proximité d’une interface. J’ai développé pour cela un montage optique, qui permet de contrôler finement les caractéristiques de l’onde évanescente (longueur d’atténuation, état de polarisation, etc.). L’adhésion des vésicules a été étudiée par nTIRF (TIRF normalisé) : les images TIRF sont normalisées par des images en épi-fluorescence. J’ai pu ainsi caractériser l’adhésion non spécifique (interaction électrostatique) et spécifique (interaction biotine-streptavidine) de vésicules sur différentes surfaces fonctionnalisées. Pour quantifier l’adhésion des cellules, j’ai utilisé l’approche VA-TIRF (TIRF à angle variable). Cette dernière consiste à enregistrer une série d’images en régime évanescent à différents angles d’incidence. Ceci nous a permis d’établir une cartographie des distances entre la membrane ventrale d’une cellule et la surface pour différents comportements d’adhésion initiés sur divers substrats : chimiques ou protéiques. Ces deux techniques permettent de mesurer la distance membrane-surface avec une précision nanométrique, ≈20nm, ce qui est particulièrement adapté à l’étude du processus d’adhésion / The aim of my thesis was to characterize the adhesion of Giant Unilamellar Vesicles and living cells. In order to obtain a quantitative information about the state of the adhesion, I developed two fluorescence nanoscopy techniques based on microscopy TIRF (Total Internal Reflection Fluorescence). This technique consist of creating an evanescent wave in the vicinity of an interface. I developed the experimental setup, which allows an accurate control of characteristics of the evanescent wave (penetration depth, polarization state, etc.). The vesicles adhesion was studied by nTIRF (normalized TIRF). TIRF images are normalized by epi-fluorescence images. I was able to characterize the nonspecific adhesion (electrostatic interaction) and specific adhesion (biotin-streptavidin interaction) of vesicles on different functionalized surfaces. To quantify the adhesion of cells, I used the VA-TIRF approach (variable angle TIRF). The latter is to record a series of images at different angles of incidence in the evanescent regime. This allowed us to map the distances between the ventral membrane of a cell and the surface for different adhesion behaviors initiated on various substrates: chemical or protein. These two techniques permit to measure the membrane surface-distance with the nanometer precision ≈20nm, which is particularly suitable for the study of the adhesion process

Au cours de cette thèse nous avons étudié les propriétés anticancéreuses du pigment majoritaire de la betterave rouge : la bétanine, ainsi que son mode d’action. Nos travaux reposent sur une approche pluridisciplinaire. Nous avons tout d’abord mis au point un protocole d’extraction et de purification de la bétanine à partir de betteraves rouges fraîches. Plusieurs étapes de purification se terminant par la séparation des molécules d’intérêt sur HPLC semi-préparative sont nécessaires à l’obtention de la bétanine à un degré de pureté de 90 %, une qualité d’extrait jusqu’à présent inégalé. Nous avons ensuite évalué l’effet cytotoxique de notre extrait sur cellules cancéreuses. Nous avons pu démontrer son innocuité sur cellules non cancéreuses et identifier les voies de signalisation pouvant être impliquées. Nous avons ainsi pu avancer des pistes concernant le mode d’action de la bétanine sur les cellules, mais également soumettre pour la première fois l’idée d’une implication de l’autophagie dans la mort cellulaire induite par la bétanine. Enfin, nous avons montré, par des techniques d’analyse biophysique aux interfaces appliquées aux membranes cellulaires et biomimétiques, qu’indépendamment de son insertion jusqu’au cœur hydrophobe des membranes, la bétanine n’influait pas sur la fluidité et la perméabilité membranaire. Ce travail exploratoire confirme l’intérêt à porter à la bétanine qui, compte tenu de sa haute biodisponibilité, présente de nombreuses applications thérapeutiques potentielles. / During this thesis we studied the anticancer properties of the major beetroot’s pigment: betanin. Our work is based on a multidisciplinary approach.First we developed a protocol for the extraction and the purification of betanin from fresh beetroots. Several purification steps ended by separation in semi-preparative HPLC are required to obtain a betanin at 90 % pure, which is the highest purity ever recorded. Then we assessed the cytotoxic effect of our extract on cancer cells and its safety on non-cancer cells. By identifying the signaling pathways that might be involved in these effects, we were thus able to suggest ways concerning the mode of action of betanin on cells, but also propose, for the first time, the idea of an involvement of autophagy in cell death induced by betanin. Finally, we have shown by interfacial biophysical techniques applied on cell and biomimetic membranes that, regarless to its deep insertion in the hydrophobic core of the lipid bilayer, betanin did not affect the physical properties of the membrane such as its fluidity or its permability.This scoping study confirms the interest to bring to betanin which, given its high bioavailability, has many potential therapeutic applications.

Em S. cerevisiae, as proteínas de membrana são responsáveis pelo transporte de açúcares através da membrana celular e, portanto, são importantes para os processos fermentativos. Visando melhorar a compreensão do metabolismo de açúcares, estudamos o transporte ativo de açúcares mediado pela permease Agt1p e o processo de inativação catabólica, promovido pela glicose. Para isso, mutantes em resíduos específicos do Agt1p foram gerados por mutagênese e expressados em uma linhagem agt1<font face=\"Symbol\">D. Os resultados obtidos indicam que os aminoácidos Glu-120, Asp-123, Glu-167, Arg-504 e Ile-505 estão envolvidos com o simporte açúcar-H+ realizado pelo Agt1p. Em relação aos resíduos e/ou domínios envolvidos com o processo de inativação catabólica, os resultados demonstram que a região N-terminal do Agt1p, bem como a alça citoplasmática presente entre os TMs 6 e 7, são essenciais para a resposta celular frente a presença de glicose. Finalmente, a fusão do Agt1p com GST permitiu purificar uma proteína de ~67 kDa, condizente com a massa molecular prevista para este transportador. / In S. cerevisiae, membrane proteins are responsible for the transport of different sugars across the cellular membrane and, therefore, are important for fermentation processes. In order to improve our understanding of sugar metabolism, we studied the active sugar transport mediated by Agt1p permease and the catabolite inactivation induced by glucose. Thus, mutants in specific residues of the Agt1p were generated by site direct mutagenesis and expressed in a strain agt1<font face=\"Symbol\">D. The results indicate that the Glu-120, Asp-123, Glu-167, Arg-504 and Ile-505 residues are involved in the sugar-H+ symport mediated by the Agt1p permease. Regarding residues and/or domains involved in the process of catabolite inactivation promoted by glucose, the results indicate that the N-terminal region of Agt1p, and the intracellular loop between TMs 6 and 7, are essential for the cellular response to the presence of glucose. Finally, the fusion of the Agt1p to GST allowed the purification of a ~67 kDa protein, consistent with the predicted molecular weight of this transporter.