Les cytochalasines, métabolites secondaires issus de PKS-NRPS fongiques, représentent une large famille de composés bioactifs intéressants de par leur système tricyclique. Ce document expose les stratégies de synthèse vers l’obtention de cytochalasines de petite taille, les périconiasines A-C, isolées de Periconia sp. F-31, qui partagent un système 9/6/5 et des cytotoxicités intéressantes envers l’adénocarcinome du colon, et la synthèse totale de la périconiasine G qui possède le plus petit macrocycle rencontré jusqu’à présent dans cette famille avec un système 7/6/5. Plusieurs analogues du produit naturel dont l’bis-iso-périconiasine C qui se caractérise par une isomérisation des alcènes initiaux sont décrits, ainsi que leurs tests sur des cellules HeLa et MDA ce qui a permis l’identification d’un composé hautement cytotoxique. D’autre part, la synthèse totale de la périconiasine G a conduit à réviser la structure initialement attribuée par Dai et al. et réaliser des tests biologiques pertinents sur les bactéries gram-négatives Micrococcus luteus et Staphylococcus aureus ainsi que sur le phytopathogène Botrytis cinerea responsable de la « pourriture grise » des cultures à travers le monde. Une spécificité de la périconiasine G contre le phytopathogène a pu être découverte ce qui amène à considérer le rôle protecteur des endophytes dans le mutualisme plante-champignon. / Cytochalasins are secondary metabolites born from fungal PKS-NRPS which possess a characteristic tricyclic core and display a large variety of biological properties. This document exposes the strategies elaborated toward total syntheses of small cytochalasins periconiasins A-C, isolated from Periconia sp. F-31 and sharing a 9/6/5 backbone, and periconiasin G which possesses the smallest macrocyclic ring so far in this family with a 7/6/5 tricyclic ring. Several analogues of natural product periconiasin C, including isomer bis-iso-périconiasin C, are described, as well as their bioassays on HeLa and MDA cell lines which allowed identification of a highly toxic lead. Moreover, the total synthesis of periconiasin G led us to the revise the structure published by Dai et al. and carry out pertinent bioassays on gram-positives bacteria Micrococcus luteus and Staphylococcus aureus, and on the phytopathogen Botrytis cinerea responsible of the gray mold disease throughout the world. A specificity of periconiasin G against the phytopathogen was discovered, leading us to consider the protective role of endophytes in the plant-fungus mutualism.

Electric cell-substrate impedance sensing (ECIS) pioneered by Giaever and Keese is suitable for continuous, automatic and real-time cell attachment analysis. ECIS is a novel electrical method to study, in real time, many of the activities of animal cells when grown in tissue culture. These include morphological changes, cell locomotion, and other behaviors directed by the cell's cytoskeleton. One of the most direct ECIS measurements is that of the attachment and spreading behaviors of cells. These measurements allow one to study and quantify the interaction of cultured cells with extracellular matrix (ECM) proteins and other macromolecules continuously and in real time. Traditionally, cell attachment and spreading measurements are labor intensive, requiring many manipulations of the cultures for microscopic evaluation of cell behavior. With ECIS, these same measurements can be made in an automated approach without opening the door of the incubator. The ECIS core technology is based on a technique of measuring the change in impedance of a small electrode to AC current flow. The heart of the measurement is a specialized slide that has 8 individual wells for cell culturing. The base of the device has an array of gold film electrodes that connect to the ECIS electronics to each of the 8 wells. In our work we used ECIS to study the attachment and spread of HUVEC and 3T3 cells. The curve of HUVEC showed higher resistances than that of 3T3 cells. This was due to the fact we used gelatin to aid in attachment of HUVECs which accounted for the high resistances. 3T3 cells attached easily without help of gelatin. We also studied the cytotoxicity of HUVEC and 3T3 cells. The drugs that we used were CB, H7 and CdCl2. We found that the best drug was CB since it affected the cells even at low concentrations. H7 effects were mild while CdCl2 only worked at high concentrations. HUVEC cells make loose contact on electrodes and are easily detached by drugs. 3T3 makes firm at tachment to the electrodes and are not easily detached from the electrodes. Electrical impedance measurements on multiple electrodes are highly attractive in this application because of the potential for direct computer control.

The investigations conducted within this dissertation centers around the use of electric cell-substrate impedance sensing (ECIS). This system is able to characterize in real-time analysis, the adhesion of cells to their substrate and neighboring cells. With this, valuable information can be gathered with in-vitro experiments regarding a tissue culture's response to physiological stimulation. This dissertation has taken advantage of ECIS' ability to analyze toxicology, barrier function, and cancer invasion on a tissue culture. With proper analysis modifications, trans-epethelial resistance (TER) can be used as a cytotoxicity assay with higher sensitivity than previously thought. In vitro assessment of cytotoxicity based on TER needs more quantitative methods to analyze the alteration of cell morphology and motility. Here, we applied ECIS to evaluate dose-dependent responses of human umbilical vein endothelial cells (HUVEC) and mouse embryonic fibroblasts (NIH 3T3) exposed to cytochalasin B and protein kinase inhibitor H7. To detect subtle changes in cell morphology, the frequency-dependent impedance data of the cell monolayer were measured and analyzed with a theoretical cell-electrode model. To detect the alternation of cell micromotion in response to cytochalasin B and H7 challenge, time-series impedance fluctuations of cell-covered electrodes were monitored and the values of power spectrum, variance, and variance of the increment were calculated to verify the difference. While a dose-dependent relationship was generally observed from the overall resistance of the cell monolayer, the analysis of frequency-dependent impedance and impedance fluctuations distinguished cytochalasin B levels as low as 0.1µM and H7 levels as low as 10 µM for HUVEC and 3T3 layers. Even though overall resistance values are relatively small for 3T3 layers, and frequency scan measurements are negligible, impedance fluctuation analysis reveals significant micromotion for cytotoxic detection. Our results show that cytochalasin B and H7 causes a decrease of junctional resistance between cells and an increase of membrane capacitance. Cigarette smoke is cytotoxic and tumorigenic. Initial studies were conducted to evaluate the cytotoxicity of cigarette smoke condensate (CSC) on HUVEC layers. The focus was then turned to investigations involving in vitro cancer invasion assays with CSC on HUVEC layers. ECIS is an excellent investigative device that can be utilized to observe cancer invasion on normal tissue cultures due to the significantly higher impedance signature of cancer cells. The investigation in this dissertation focused on cigarette smoke's influence on cellular mechanics of endothelial cells and the invasive potential of two ovarian cancer cell lines (ALST and OVCA429) against a fully active endothelium. The HUVEC cultures responded to CSC with an increase in junctional binding, where as ALST and OVCA429 relieved adhesion thereby providing an improved motility when evaluated in wound healing assays. Transmigration of the HUVEC layer by ALST cells exhibit a pre-CSC exposure time-dependence affecting the effectiveness of ALST transmigration. The HUVEC layer's decreased tight junction binding that resulted from CSC exposure, allowed for a more aggressive ALST layer formation that occurred during simulated intravasation. Increased HUVEC layer tight junction binding that occurred in the first five hours in response to CSC during extravasation contributes to impeding ALST transmigration at high concentrations of CSC. Overall, CSC has an impeding effect on ALST transmigration during extravasation while causing aggressive transmigration during intravasation.

Submitted by Rafael Vieira (vieira.rafa1986@gmail.com) on 2016-01-06T18:57:51Z No. of bitstreams: 1 dissertacao final rafael.pdf: 11211535 bytes, checksum: e982b9dd12cb7bf43a451db3f5e30076 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-01-06T19:24:55Z (GMT) No. of bitstreams: 1 vieira_r_me_araiq_par.pdf: 1125273 bytes, checksum: 201f41df3c870e0118ce7d37d6eca396 (MD5) / Made available in DSpace on 2016-01-06T19:24:55Z (GMT). No. of bitstreams: 1 vieira_r_me_araiq_par.pdf: 1125273 bytes, checksum: 201f41df3c870e0118ce7d37d6eca396 (MD5) Previous issue date: 2015-12-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este trabalho de mestrado tem, como foco principal, analisar o comportamento do fungo Xylaria sp. perante variações físico-químicas, através da abordagem OSMAC (One Strain, Many Compounds). Para realizar tais induções e explorar a variabilidade da rede metabólica deste micro-organismo, um planejamento fatorial foi desenhado visando induzir a variabilidade (ou a potencialização) de metabólitos. Diante de respaldos quimiométricos, induções planejadas foram impostas ao micro-organismo e observou-se variações no perfil químico nos extratos brutos. Somado a isso, técnicas de desreplicação foram usadas a fim de estudar quimicamente as matrizes complexas produzidas por este fungo visando a produção de metabólitos de alto valor agregado e determinou-se condições específicas para produção de moléculas de citocalasina D e de griseofulvina. / This work has as its main focus, analyze the behavior of the fungus Xylaria sp. before physicochemical variations via the OSMAC approach (One Strain, Compounds Many). To achieve such induction and exploit the variability of the metabolic network of this micro-organism, a factorial design was designed to induce variability (or potentiating) metabolites. Before chemometric backrests, planned inductions were imposed on the micro-organism and it was observed variations in the chemical profile in crude extracts. Added to this, dereplication techniques were used to study the chemically complex matrices produced by this fungus aimed at producing high added value and metabolites was determined specific conditions for the production of molecules cytochalasin D and griseofulvin.

Carrier mediated nutrient import is vital for cell and tissue homeostasis. Structural insights of carrier mediated transport, particularly the human glucose transporter GLUT1, are essential for understanding the mechanisms of human metabolic disease, and provide model systems for cellular processes as a whole. GLUT1 function and expression is characterized by a complexity unexplained by the current hypotheses for carrier-mediated sugar transport (9). It is possible that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae(RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1) to characterize its functional properties. Identical protein sequences generated different kinetic parameters when expressed in RE700A yeast, erythrocytes, and HEK293 cells. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific. Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 sugar export site. Paradoxically, very low concentrations of these inhibitors produce a modest stimulation of sugar transport (16). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, intracellular binding sites for e1 ligands CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of exit site ligands determine binding site affinity and cooperativity. Our findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of GLUT1 quaternary structure and evaluate the major determinants of ligand binding affinity and cooperativity. Cytochalasin B (CB) inhibits GLUT1 substrate transport at or near the endofacial sugar binding site. N-bromosuccinamide analysis combined with 3H-CB photolabeling implicates the region between Trp388 and Trp412 in ligand binding. Although its structure has been modeled(5), the specific residues comprising the sugar binding site are unknown. A series of alanine point mutants were made, and mutant protein 2-deoxy glucose transport was tested in the presence of increasing [CB]. Arg126Ala and Cys421Ala GLUT1 mutations altered CB affinity but were determined not to be in the e1 site. The Arg400Ala mutation decreased binding affinity for CB, and may comprise part of the e1 binding site. Because point mutations were individually insufficient to abrogate CB binding, Trp388 to Trp412 chimeras were made. GLUT1/GLUT4388-412/GLUT1 and GLUT1/GLUT5388-412/GLUT1 chimeras showed moderately less sensitivity to CB inhibition of transport; these amino acids likely comprise regions determinant of CB binding affinity. Furthermore GLUT1/GLUT5388-412/GLUT1 shows enhancement of 2-DG uptake at 50nM CB, but an overall dose response indistinguishable from WT GLUT1. A multisite fit of the data suggested GLUT1/GLUT5388-412/GLUT1 chimera possesses strong first site affinity for CB but slight negative second-site cooperativity. We conclude that point mutants were insufficient to abrogate CB binding and that the Trp388 to Trp412 sequence is necessary for CB binding affinity but is not the sole determinant of inhibition of 2 deoxyglucose uptake by CB. We discuss these results with their implications for structure-function sequence localization of the CB binding site, and by extension, the e1 sugar binding site.

No description available.

<p>The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein.</p><p>The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization.</p><p>In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.</p>

The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein. The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization. In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.

<p>Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. </p><p>Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state <i>in vitro</i> and <i>in vivo</i>. </p><p>Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.</p><p>An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented. </p>

<p>The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.</p><p>Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.</p>