Cette étude vise à définir l'effet des lipoprotéines de basses densité natives (LDL) et oxydées (ox-LDL) sur les fonctions des cellules endothéliales en relation avec les processus physiopathologiques de l'athérosclérose. Le microscope à force atomique (AFM) fut utilisé en combinaison avec les méthodes biochimiques traditionnelles afin d'acquérir de l'information sur les propriétés biomécaniques des cellules endothéliales. L'AFM est un outil permettant l'acquisition d'images et de mesures de forces quantitatives concernant les propriétés viscoélastiques des cellules vivantes selon leur exposition aux LDL ou ox-LDL. L'AFM rassemble localement des informations sur la membrane cellulaire et le cytosquelette des cellules et ce, de manière non invasive. Il est ensuite possible de corréler les résultats obtenus avec les marquages immunohistochimiques afin d'évaluer la réponse cellulaire suite à une exposition à des LDL ou ox-LDL. Ces données recueillies, les protocoles étant au point, il ne restera plus qu'à effectuer les tests avec les antioxydants afin de déterminer les agents et les dosages appropriés permettant une protection salutaire de l'endothélium. Ce travail amène donc de nouvelles connaissances sur les mécanismes moléculaires fondamentaux de la dysfonction endothéliale en vue éventuellement de développer de nouvelles thérapies cytoprotectrices efficaces. Une méthode d'imagerie des LDL a également été mise au point en utilisant l'AFM. Il est maintenant possible d'obtenir des images de bonne qualité permettant aussi de mesurer les dimensions de LDL individuelles. Cette technique pourrait entre autre servir à évaluer des pathologies touchant les LDL comme le diabète.

Moléculas de origem vegetal foram há muito testadas como fonte de drogas antineoplásicas com sucesso promissor. Este trabalho trata dos efeitos antiangiogênicos do Metil Jasmonato. Este derivado hidrofóbico do ácido jasmônico foi demonstrado anteriormente como um agente de dano seletivo para a mitocôndria de células neoplásicas. In vitro, o Metil Jasmonato 1-10 mM promoveu a morte celular de células endoteliais humanas de cordão umbilical (HUVEC) e de melanoma murino (B16 -F10), enquanto concentrações micromolares foram inócuas. A inclusão do Metil Jasmonato em liposomos de fosfatidilcolina e em um nanocarreador hidrofílico baseado em açúcar mostrou efeitos diferenciais sobre a citotoxicidade. A interrupção do ciclo celular foi observada em concentrações citotóxicas, enquanto a diminuição na produção de VEGF e algum grau de autofagia foram sugeridos em concentrações micromolares. In vivo, Metil Jasmonato 1-10mM foi francamente tóxico, e reduziu a densidade de vasos em membranas corioalantóicas de embrião de galinha (CAM). Entretanto, concentrações entre 1-10 ?M produziram um efeito complexo. Ocorreu aumento no brotamento capilar, mas os novos vasos apresentaram-se frágeis e menos organizados que os controles correspondentes. Sugere-se que, além da toxicidade direta, a ação do Metil Jasmonato sobre a angiogênese seja relevante para seu efeito antineoplásico. / Molecular plant components have long been tested as sources for antineoplasic drugs with promising success. The present work deals with the anti-angiogenic effects of Methyl Jasmonate. This hydrophobic Jasmonate derivative was previously demonstrated to selectively damage the mitochondria of cancer cells. In vitro, 1-10 mM Methyl Jasmonate induced the cell death of the human umbilical vein endothelial cells (HUVEC) and the Murine melanoma cells (B16-F10), while micromolar concentrations were ineffective. Methyl Jasmonate inclusion in phosphatidylcholine liposomes and in an hydrophilic sugar based nanocarrier presented differential effects upon citotoxicity. Cell cycle arrest was observed in citotoxic concentrations, while VEGF withdrawn and some autophagy was suggested in the micromolar range. In vivo, 1-10mM concentrations were explicitly toxic and reduced the vessel density of the Chorioallantoic Membrane of the Chicken Embryo (CAM). However, 1-10 ?M concentrations produced a complex effect. There was increased capillary budding, but the new vessels were leakier and less organized than corresponding controls. It is suggested that not only direct toxicity, but also the drug effects upon angiogenesis are relevant to the antineoplasic effects of Methyl Jasmonate.

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.

Syphilis is a global public health concern with 36.4 million cases worldwide and 11 million new infections per year. It is a chronic multistage disease caused by the spirochete bacterium Treponema pallidum and is transmitted by sexual contact, direct contact with lesions or vertically from an infected mother to her fetus. T. pallidum is a highly invasive pathogen that rapidly penetrates tight junctions of endothelial cells and disseminates rapidly via the bloodstream to establish widespread infection. Previous investigations conducted in our laboratory identified the surface-exposed adhesin, pallilysin, as a metalloprotease that degrades the host components laminin (major component of the basement membrane lining blood vessels) and fibrinogen (primary component of the coagulation cascade), as well as fibrin clots (function to entrap bacteria and prevent disseminated infection). Furthermore, pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis conferred upon this bacterium the ability to degrade fibrin clots. It was hypothesized that pallilysin is integral to the process of T. pallidum dissemination, and interference with its functioning will prevent spread throughout the host and establishment of chronic infection. To test this hypothesis, a two-pronged approach was undertaken during my thesis research. Bioinformatics analyses were used to trace the evolutionary history of pallilysin in an attempt to gain further insight into its role in the pathogenesis of T. pallidum. The sequence conservation of pallilysin was analyzed in the context of its homologues. The bioinformatics analyses revealed homologues in three spirochete genera, namely Treponema, Spirochaeta, and Borrelia, presented in decreasing order of the degree of sequence conservation. The HEXXH motif, part of the active site of the pallilysin metalloprotease, was fully conserved only in T. pallidum and T. paraluiscuniculi, both of which are systemic pathogens. However, the flanking sequences showed a high degree of conservation, especially in the Treponema and Spirochaeta genera. The minimum laminin-binding region of pallilysin identified previously was partially conserved among the treponema and spirochaeta homologues with the highest degree of conservation observed with the homologues from T. paraluiscuniculi and T. phagedenis, as well as among the homologues from the human oral pathogens. In vitro dissemination studies were performed to investigate the dissemination capacity of T. phagedenis heterologously expressing pallilysin. Human Umbilical Vein Endothelial Cells were seeded and grown to confluence on permeable inserts coated with growth factor-reduced Matrigel to create an artificial endothelial barrier. Wild type T. phagedenis, and T. phagedenis transformed either with the pallilysin open reading frame or its empty shuttle vector, were incubated with the barriers under anaerobic conditions. Dissemination across the barrier was assessed as percent traversal by both dark-field microscopic counts of treponemes and real-time quantitative PCR of genomic DNA extracted from the treponemes. The results were inconclusive. However, a traversal trend suggested heterologous expression of pallilysin may facilitate traversal of T. phagedenis across the artificial endothelial barrier. This study presented the first step towards elucidating the role of pallilysin in endothelial monolayer traversal and provided supporting evidence for the role of pallilysin in the widespread dissemination of T. pallidum in vivo. / Graduate

Moléculas de origem vegetal foram há muito testadas como fonte de drogas antineoplásicas com sucesso promissor. Este trabalho trata dos efeitos antiangiogênicos do Metil Jasmonato. Este derivado hidrofóbico do ácido jasmônico foi demonstrado anteriormente como um agente de dano seletivo para a mitocôndria de células neoplásicas. In vitro, o Metil Jasmonato 1-10 mM promoveu a morte celular de células endoteliais humanas de cordão umbilical (HUVEC) e de melanoma murino (B16 -F10), enquanto concentrações micromolares foram inócuas. A inclusão do Metil Jasmonato em liposomos de fosfatidilcolina e em um nanocarreador hidrofílico baseado em açúcar mostrou efeitos diferenciais sobre a citotoxicidade. A interrupção do ciclo celular foi observada em concentrações citotóxicas, enquanto a diminuição na produção de VEGF e algum grau de autofagia foram sugeridos em concentrações micromolares. In vivo, Metil Jasmonato 1-10mM foi francamente tóxico, e reduziu a densidade de vasos em membranas corioalantóicas de embrião de galinha (CAM). Entretanto, concentrações entre 1-10 ?M produziram um efeito complexo. Ocorreu aumento no brotamento capilar, mas os novos vasos apresentaram-se frágeis e menos organizados que os controles correspondentes. Sugere-se que, além da toxicidade direta, a ação do Metil Jasmonato sobre a angiogênese seja relevante para seu efeito antineoplásico. / Molecular plant components have long been tested as sources for antineoplasic drugs with promising success. The present work deals with the anti-angiogenic effects of Methyl Jasmonate. This hydrophobic Jasmonate derivative was previously demonstrated to selectively damage the mitochondria of cancer cells. In vitro, 1-10 mM Methyl Jasmonate induced the cell death of the human umbilical vein endothelial cells (HUVEC) and the Murine melanoma cells (B16-F10), while micromolar concentrations were ineffective. Methyl Jasmonate inclusion in phosphatidylcholine liposomes and in an hydrophilic sugar based nanocarrier presented differential effects upon citotoxicity. Cell cycle arrest was observed in citotoxic concentrations, while VEGF withdrawn and some autophagy was suggested in the micromolar range. In vivo, 1-10mM concentrations were explicitly toxic and reduced the vessel density of the Chorioallantoic Membrane of the Chicken Embryo (CAM). However, 1-10 ?M concentrations produced a complex effect. There was increased capillary budding, but the new vessels were leakier and less organized than corresponding controls. It is suggested that not only direct toxicity, but also the drug effects upon angiogenesis are relevant to the antineoplasic effects of Methyl Jasmonate.

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