The invasion of cancer cells is an important aspect of cancer progression. Single tumor cells exhibit at least two types of invasion in 3D environment, mesenchymal and amoeboid invasion. Tumor cells can switch between these two modes of movement depending on cellular status and surrounding environment. Amoeboid-mesenchymal transition (AMT) is less explored then mesenchymal-amoeboid transition (MAT). We performed a proteomic analysis of amoeboid-mesenchymal transition of human melanoma cell line A375M2. We have induced amoeboid-mesenchymal transition by treatment with a ROCK inhibitor Y27632 in 3D matrigel matrices and in 2D environment. Induction of the amoeboid-mesenchymal transition has changed a level of expression of 92 proteins and a level of phosphorylation of 15 proteins. Expression of only 17 proteins and phosphorylation of 8 proteins was identically changed in both of these environments. We found that PKCα regulates amoeboid migration and that treatment of cells with a PKCα inhibitor Gö6976 induces amoeboid-mesenchymal transition. Analysis of the proteomics data have further shown that induction of AMT by the ROCK inhibitor Y27632 leads to activation of antiapoptotic signals and activation of signaling pathways involved in regulation of actin cytoskeleton especially regulation of focal...

The catfish industry suffers losses primarily due to enteric septicemia of catfish and columnaris disease caused by Edwardsiella ictaluri and Flavobacterium columnare, respectively. Understanding the host-pathogen interactions is vital for prevention and eradication of these diseases. Hence, the overall objective of this study was to analyze whole cell proteomes of these two bacteria, and to determine the changes in E. ictaluri protein expression against in vitro iron-restriction and host serum treatment. High-throughput proteomic analysis of these bacteria was conducted using two-dimensional liquid chromatography followed by electrospray ionization tandem mass spectrometry (2-D LC ESI MS/MS) and two-dimentional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-oflight mass spectrometry (2-DE MALDI TOF/TOF). Identified proteins were clustered into functional groups using clusters of orthologous groups, and subcellular locations as well as possible functional relationships were determined. A total of 788 unique E. ictaluri and 621 unique F. columnare proteins were identified, which represented 12 and 28 pathways, respectively. Vertebrate hosts tend to chelate free iron of their body and make the environment hostile for bacteria. Hence, reduced availability of iron may cause significant stress for pathogens and is considered a signal that leads to alteration in virulent gene expression. Similarly, E. ictaluri might use the catfish blood stream effectively for quick systemic invasion. Hence, exposure to catfish serum components might reveal the ability of E. ictaluri to protect against host defense mechanisms. Using two-dimensional difference gel electrophoresis, responses of E. ictaluri due to in vitro iron-restriction and host serum treatment were determined. A total of 50 and 19 proteins were identified to be differentially expressed due to in vitro iron-restriction and catfish serum treatment, respectively. Among the differentially expressed proteins, several putative virulent determinants, immunogenic proteins, chaperones, and housekeeping genes were noted. To initiate functional studies, four differentially expressed E. ictaluri genes (lamB, glyS, malE, and sdhA) were mutated by inrame deletion. Results from this study provided experimental evidence for many predicted proteins. In addition, identification of differentially expressed proteins provided targets for further functional analysis, which could help elucidate pathogenic mechanisms of E. ictaluri.

Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation, the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) comparing a wild type and a Δtda1 deletion mutant. To additionally address possible consequences of glucose repression/derepression, both were grown at 2% and 0.1% (w/v) glucose. A total of eight protein spots exhibiting a minimum twofold enhanced or reduced fluorescence upon TDA1 deficiency was detected and identified by mass spectrometry. Among the spot identities are—besides the expected Hxk2—two proteoforms of hexokinase 1 (Hxk1). Targeted proteomics analyses in conjunction with 2D-DIGE demonstrated that TDA1 is indispensable for Hxk2 and Hxk1 phosphorylation at serine 15. Thirty-six glucose-concentration-dependent protein spots were identified. A simple method to improve spot quantification, approximating spots as rotationally symmetric solids, is presented along with new data on the quantities of Hxk1 and Hxk2 and their serine 15 phosphorylated forms at high and low glucose growth conditions. The Δtda1 deletion mutant exhibited no altered growth under high or low glucose conditions or on alternative carbon sources. Also, invertase activity, serving as a reporter for glucose derepression, was not significantly altered. Instead, an involvement of Tda1 in oxidative stress response is suggested.

Microcystine sind die wohl bekanntesten cyanobakteriellen Toxine. Sie werden im Wesentlichen durch die im Süßwasser weltweit verbreitete, koloniebildende Gattung Microcystis synthetisiert. Die biologische Funktion dieser Peptide ist jedoch ungeklärt. In dieser Studie wurde die Fragestellung erstmals über einen globalen Ansatz auf molekularer Ebene analysiert. Die proteomischen Analysen zwischen M. aeruginosa PCC 7806/ Wildtyp und einigen Microcystin-freien Mutanten deuten auf eine physiologische Rolle der Microcystine. Microcystine beeinflussen die Abundanz zahlreicher Proteine. Prominentester Vertreter ist RubisCO – Schlüsselenzym des Calvin Zyklus. RubisCO und andere im 2D selektierte Proteine konnten außerdem als mögliche zelluläre Bindepartner des Microcystins identifiziert werden. Möglicherweise bindet MC an bestimmte Cysteinreste dieser Proteine. Mit dem Knockout der mcy-Gene geht außerdem eine Überexpression eines Morphotyp-spezifischen Proteins einher, das MrpC genannt wurde. Dieses Protein vermittelt möglicherweise Zell-Zell-Interaktionen in Microcystis. / Microcystins are the most common cyanobacterial toxins found in freshwater lakes and reservoirs throughout the world. They are frequently produced by the unicellular, colonial cyanobacterium Microcystis; however, the role of the peptide for the producing organismen is poorly understood. In this study we describe the first global approach to investigate this topic on a molecular level. Proteomic studies with M. aeruginosa PCC 7806 wild-type and several microcystin-deficient mutants indicated a physiological function for microcystin. Microcystin was shown to influence the abundance of several proteins which have an intra- or extracellular function. A prominent candidate is RubisCO, the key enzyme of the calvin cycle. RubisCO and other proteins, initially selected by 2D analysis, are putative cellular binding partners of microcystin. A potentially interaction mechanismen is the kovalent binding of microcystin to cysteine residues of the protein. Moreover, several knockouts of microcystin biosynthesis genes result in an overexpression of a putative morpho-type specific factor, named MrpC. This protein possibly mediates cell-cell interactions in Microcystis.

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est un pathogène d’importance dans l’industrie porcine et est responsable d’importantes pertes économiques. Il n’existe pas d’antiviral efficace contre celui-ci. Il a récemment été mis en évidence que le surnageant de culture d’Actinobacillus pleuropneumoniae, l’agent étiologique de la pleuropneumonie porcine, possédait une activité antivirale in vitro contre le VSRRP dans la lignée cellulaire SJPL. Les objectifs de mon projet sont (i) d’étudier les mécanismes cellulaires menant à l’activité antivirale causée par le surnageant de culture d’A. pleuropneumoniae, et (ii) de caractériser les molécules actives présentes dans le surnageant de culture d’A. pleuropneumoniae. Dans un premier temps, des analyses de protéome ont été effectuées et ont permis d’observer que le surnageant de culture modulait la régulation du cycle cellulaire. Dans le but d’analyser le cycle cellulaire des cellules SJPL, la cytométrie en flux a été utilisée et a permis de démontrer que le surnageant de culture induisait un arrêt du cycle cellulaire en phase G2/M. Deux inhibiteurs de la phase G2/M ont alors été utilisé. Il s'est avéré que ces inhibiteurs avaient la capacité d’inhiber le VSRRP dans les cellules SJPL. Enfin, la spectrométrie de masse a été utilisée dans le but de caractériser les molécules actives présentes dans le surnageant de culture d’A. pleuropneumoniae et d’identifier deux molécules. Ce projet a permis de démontrer pour la première fois qu’A. pleuropneumoniae est capable de perturber le cycle cellulaire et que ce dernier était un élément important dans l’effet antiviral contre le VSRRP. / Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the swine industry and causes important economic losses. No effective antiviral drugs against it exist. It was recently reported that the culture supernatant of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, possesses an antiviral activity in vitro against PRRSV in SJPL cells. The objectives of this project were (i) to identify the mechanism behind the antiviral activity displayed by A. pleuropneumoniae, and (ii) to characterize the active molecules present in the culture supernatant. Proteomic analyses were first conducted and demonstrated the culture supernatant ability to induce modulations of the cell cycle regulation. In order to determine the SJPL cell cycle, flow cytometry analyses were performed and demonstrated that the culture supernatant induced a G2/M-phase cell cycle arrest. Two G2/M-phase cell cycle inhibitors were then used and their ability to inhibit PRRSV infection in SJPL cells was demonstrated. Finally, in order to characterize the active molecules present in the A. pleuropneumoniae culture supernatant, mass spectrometry was used and two molecules were identified. This is the first study demonstrating the A. pleuropneumoniae ability to disrupt cell cycle and the cell cycle importance to the inhibitory activity against PRRSV.

No description available.

L'hépatite chronique virale C est associée à une défaillance du système immunitaire. Nous nous sommes intéressés aux cellules NK et aux lymphocytes Treg, partenaires de la réponse immunitaire innée. Le nombre des NK, particulièrement les CD56dim, est significativement diminué chez les patients infectés, dans le foie plus que dans le sang, et s'accentue avec la fibrogenèse. Le nombre de CD3-CD56+brightNKG2A+ circulantes est corrélé à la sévérité de l'inflammation et de la fibrose et celui des CD3-CD56+dimNKG2A+ inversement corrélé à la charge virale. Les NK sont fonctionnelles, en capacité de produire de l'IFN-γ et d'engager un processus de cytolyse. L'expression de CD158 est significativement diminuée à la surface des NK hépatiques mais conservée dans les NK circulantes. L'expression de NKG2A,C,D dans les NK circulantes et hépatiques est identique à celles de patients non infectés. Les Treg intrahépatiques FoxP3+ sont quasi-exclusivement de phénotype CD4+. En analyse multivariée, le nombre de FoxP3+ est indépendamment associé à celui de CD8+, surtout dans les lésions nécrotico-inflammatoires et une corrélation forte est observée entre les transcrits CD8, FoxP3, IL-10 et TGF-β, suggérant que les Treg bloquent l'expansion et la cytotoxicité des TCD8 par contact cellulaire ou par le biais de cytokines immunosuppressives. L'équilibre entre FoxP3 et CD8 est rompu dans les grades et stades Métavir A>2 et F>3, avec un effondrement du rapport FoxP3/CD8. L'inflammation hépatique chronique s'accompagne de fibrose, aboutissant à la cirrhose, principale cause de CHC. Dans les cirrhoses virales C avec ou sans CHC, les lymphocytes CD3+, CD4+, CD8+, CD20+, CD56+, TCRγδ +, FoxP3+ sont plus nombreux dans la fibrose que dans le parenchyme. Le nombre de CD20+, CD3+, CD4+, CD8+ et l'expression d'IFN-γ et RANTES sont plus élevés dans les cirrhoses qui développent un CHC. En analyse multivariée, CD8 est le seul facteur indépendament associé à la récidive tumorale et à une diminution de la survie sans récidive à 5 ans. Les CD20+, CD3+, CD4+, CD8+, CD56+, TCRγδ+, FoxP3+ sont significativement moins nombreux dans le CHC que dans la cirrhose. Mais les FoxP3+ sont significativement plus nombreux et les CD56+ moins nombreux dans le CHC que dans le nodule parenchymateux, sans modification des LT, conduisant à une augmentation du rapport FoxP3/CD8 dans la tumeur. Les CD56+ diminuent de la cirrhose au CHC. Aucune corrélation n'est observée entre la densité intra-tumorale des lymphocytes étudiés et la récidive carcinomateuse. Conclusion. Un infiltrat inflammatoire dense au sein de la cirrhose C, particulièrement riche en CD8, favorise le développement et/ou la récidive du CHC. / Chronic hepatitis C is associated with the failure of the immune system. We were interested to NK cells and Treg cells, partners in the innate immune response. The number of NK, particularly the CD56+dim, is significantly reduced in infected patients, in the liver more than in the blood, and increases during the process of fibrogenesis. The number of circulating CD3- CD56+brightNKG2A+ correlates with the severity of inflammation and fibrosis and that of CD3- CD56+dimNKG2A+ inversely correlates with viral load. The NK functional capacity to produce IFN-γ and initiate a process of cytolysis is maintened. The CD158 expression is significantly reduced on the surface of intrahepatic NK, whereas NKG2A,C,D expression in circulating and hepatic NK is identical to that of non-infected patients. The intrahepatic Treg FoxP3+ are almost exclusively CD4+ phenotype. In multivariate analysis, the number of FoxP3+ is independently associated with that of CD8+, especially in necroinflammatory lesions and a strong correlation is observed between CD8, FoxP3, IL-10 and TGF-β, suggesting that Treg could inhibit CD8 expansion and cytotoxicity by cell contact or through immunosuppressive cytokines. The balance between FoxP3 and CD8 is broken in the most severe stages of the disease (METAVIR A>2 and F>3), which results in a drop in the FoxP3/CD8 ratio. Chronic inflammation is accompanied by liver fibrosis, leading to cirrhosis, the main cause of HCC. In viral C cirrhosis with or without HCC, CD3+, CD4+, CD8+, CD20+, CD56+, TCR γδ+, FoxP3+ lymphocytes are more numerous in fibrosis than in parenchyma. The number of CD3+, CD4+, CD8+, CD20+ and the expression of IFN-γ and RANTES were higher in cirrhosis developing HCC. In multivariate analysis, CD8 is the only independent predictor of tumor recurrence and is associated with a significant decrease in the 5 years disease free survival. The CD3+, CD4+, CD8+, CD20+, CD56+, TCR γδ+, FoxP3+ tumor infiltrating lymphocytes were significantly lower than in distant cirrhosis. However, FoxP3+ are significantly higher and CD56+ significantly lower in HCC than in parenchymatous nodules, without LT changes, leading to an increase in the FoxP3/CD8 ratio into the tumor. The number of CD56+ decreases from cirrhosis to HCC. No correlation was found between the density of studied tumor infiltrating lymphocytes and HCC recurrence. Conclusion. A dense inflammatory infiltrate in viral C cirrhosis, particularly rich in CD8, promotes HCC development and/or recurrence.

GONÇALVES, Nidyedja Goyanna Gomes. Ação da lectina de Dioclea altissima sobre células tumorais: Citotoxidade e Perfil Proteômico da Linhagem PC3M. 2012. 106 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-06-27T12:21:35Z No. of bitstreams: 1 2012_dis_ngggoncalves.pdf: 5791111 bytes, checksum: 5bb7d007364d7c5364b8faa3b419bf9c (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:07:30Z (GMT) No. of bitstreams: 1 2012_dis_ngggoncalves.pdf: 5791111 bytes, checksum: 5bb7d007364d7c5364b8faa3b419bf9c (MD5) / Made available in DSpace on 2016-08-02T20:07:30Z (GMT). No. of bitstreams: 1 2012_dis_ngggoncalves.pdf: 5791111 bytes, checksum: 5bb7d007364d7c5364b8faa3b419bf9c (MD5) Previous issue date: 2012 / Recently, plant lectins have attracted great interest due to their several biological activities of which stands out the antitumoral action in vivo and in vitro that in general result in inhibition of cell growth and induction of cell death by apoptosis. In the present study, it was investigated the effect of the Dioclea altissima (DAL) lectin, a legume alfa-D-mannose ligand lectin on A549 (lung cancer), OVCAR-8 (ovarian cancer) and PC3M (prostate cancer) and normal line PBMC (cell blood tissue). DAL was isolated and purified by affinity chromatography on a Sephadex G-50 column and its cytotoxicity was evaluated by MTT assay. DAL was selectively cytotoxic to cancer cells A549, PC3M after 48 and 72 hours of incubation, and OVCAR-8 after 72 hours of treatment with DAL (CI50 values between 23.0 e 55.7 μg/mL). Moreover, it was observed cell agglutination from 24 hours of incubation. Comet assay revealed DAL does not cause direct DNA damage. The line PC3M was selected for proteomic analysis by mass spectrometry (nanoUPLC® nanoESI-MSE) to present the best evidence of sensitivity to DAL. PC3M line was treated with various concentrations of DAL during 24, 48 e 72 hours, it was identified a total of 837 proteins, 140 (24h), 321 (48h) e 376 (72h). The study of differential protein expression of the DAL-treated PC3M cells compared to control demonstrated apoptotic effect generated, mainly, via ER stressed-dependent. / Nas últimas décadas, as lectinas vegetais têm atraído grande interesse devido às suas diversas atividades biológicas das quais se destaca a ação antitumoral in vivo e in vitro que, em geral, causa a inibição do crescimento celular e a indução da morte celular por apoptose. No presente estudo, foi investigado o efeito da lectina de Dioclea altissima (DAL), uma lectina de leguminosa, alfa-D-manose ligante, sobre as linhagens tumorais A549 (carcinoma pulmonar), OVCAR-8 (carcinoma de ovário) e PC3M (carcinoma de próstata) e linhagem normal CMSP (células do tecido sanguíneo). DAL foi isolada e purificada por cromatografia de afinidade em coluna de Sephadex G-50 e sua citotoxicidade foi avaliada através do ensaio do MTT. DAL foi seletivamente citotóxica para as células cancerígenas A549, PC3M, após 48 e 72 horas de incubação, e para OVCAR-8, após 72 horas de tratamento apresentando valores de CI50 entre 23,0 e 55,7 μg/mL, promovendo aglutinação celular a partir de 24 horas de incubação. DAL não se mostrou citotóxica para células normais. O teste do cometa revelou que DAL não causa dano direto ao DNA. A linhagem PC3M foi selecionada para análise proteômica por espectrometria de massas (nanoUPLC® nanoESI-MSE) por apresentar maior sensibilidade à DAL. Após tratamento das células com diversas concentrações de DAL, por 24, 48 e 72 horas, foi identificado um total de 837 proteínas válidas, 140 (24h), 321 (48h) e 376 (72h). O estudo das proteínas diferencialmente expressas das células tratadas com a lectina em relação ao controle definiu o efeito citotóxico de DAL em PC3M como apoptótico gerado, principalmente, via estresse do retículo endoplasmático.

Notre équipe vient de découvrir une méthode originale d’obtention d’extraits de Bacteroides thetaiotaomicron (E) qui préserve sa viabilité. Après culture anaérobie de ce commensal intestinal en milieu gélosé pauvre en facteurs de croissance, puis exposition à l’air, la bactérie semble posséder et générer dans E tout l’équipement de détoxication des espèces réactives de l’oxygène in vitro. Il laisse alors augurer d’un pouvoir thérapeutique à visée anti-inflammatoire.Objectifs et méthodes : Le but est d’abord de caractériser E, aux plans glucidique, lipidique et protéique. Dans ce dernier cas, il s’agit de séparer les protéines produites par la bactérie vivante et contenues dans E par électrophorèse bidimensionnelle et de les identifier par la technique des cartes peptidiques massiques. Les gels (n≥6) sont traités statistiquement (PDQuest®, Bio-Rad). Pour mieux localiser ces protéines dans la bactérie, elles sont comparées avec celles obtenues par destruction de B. thetaiotaomicron et identifiées dans la fraction cellulaire relative à la membrane bactérienne externe. Un travail de microscopie électronique est aussi entrepris pour visualiser les éventuels évènements intervenant pendant l’extraction.Le but est alors de vérifier, in vitro, l’effet antioxydant de l’extrait bactérien standardisé et d’en contrôler l’innocuité en modèles cellulaires utilisant le granulocyte neutrophile. L’effet thérapeutique anti-inflammatoire est ensuite recherché chez l’animal. L’action de E est d’abord évaluée en modèle murin d’inflammation cutanée auriculaire induite par dépôt de chlorure de benzalkonium, sous anesthésie générale. Des témoins positifs et négatifs de traitement et d’autres ne subissant pas d’irritation sont testés en parallèle. L’épaisseur des oreilles est mesurée toutes les heures pendant 5 h et des coupes histologiques d’oreilles, effectuées au bout de 2 h chez certains animaux. Deux colorations différentes permettent alors d’évaluer la quantité de mastocytes dégranulant localement.L’action de E, administré par voie intra-rectale (IR), est ensuite testée chez des souris subissant les premières phases d’un processus inflammatoire, en modèle de colite aiguë. Celle-ci est induite per os par du dextran sulfate sodium (DSS) ; elle évolue sur 8 jours. Sont considérés en parallèle des témoins positifs et négatifs de traitement et d’autres ne subissant pas de colite. Des scores cliniques et des scores histologiques de sévérité sont établis tous les jours de l’expérience. Des marqueurs de l’inflammation sont suivis dans les tissus murins après autopsie des animaux. [...] / Our team had discovered a new method to obtain extracts of Bacteroides thetaiotaomicron (E) which preserved its viability. This intestinal symbiont was anaerobically grown on an agar medium poorly supplemented in growth factors. After exposure to air, the bacterium seemed to possess and generate in E all the equipment able in vitro to detoxify reactive oxygen species. It let us expect a therapeutic power referred to anti-inflammatory properties.Objectives and methods: The aim was first to characterize E, in terms of carbohydrates, lipids and proteins. To achieve this last-mentioned goal, proteins contained in E coming from living bacteria were separated by two-dimensional electrophoresis and identified by the peptide mass fingerprinting technique. The gels (n ≥ 6) were statistically analyzed (PDQuest®, Bio-Rad). To find the origin of these proteins in bacteria, they were compared with those obtained by destruction of B. thetaiotaomicron (BT) and identified in the cell fraction containing the bacterial outer membrane proteins. Electron microscopy work was also undertaken to visualize any event occurring during extraction.The antioxidative effect of standardized E extracts was checked in vitro. E safety was also controlled in cell models using polymorphonuclear neutrophils. An E anti-inflammatory effect was then searched in animal models. E was first evaluated using a skin irritation mouse model. Inflammation was induced by benzalkonium chloride on ears of anesthetized mice. Positive and negative controls were treated in parallel. The ear thickness was measured every hour for 5 h and histological ear sections were performed after 2h for some animals. Two different staining methods enabled the enumeration of degranulating mast cells in ear sections.The effect of the bacterial extract was next tested locally by intrarectal (IR) instillations in mice undergoing the early stages of inflammation in a dextran sodium sulfate (DSS)-induced colitis. This acute model evolved over 8 days. In parallel, positive and negative animal controls underwent or not the colitis and were treated or not. Clinical and colonic histological severity scores were daily determined. Inflammation markers were measured in mouse colonic tissues after animal autopsy. [...]

The aim of this Ph.D. thesis was to analyze the morphine-induced changes of frontal brain cortex protein composition in rats exposed to increasing doses of morphine (10-50 mg/kg) for prolonged period of time (10 days). The first part of this work was oriented to the analysis of the phenomenon of hypersensitization/superactivation of adenylyl cyclase (AC), which is regarded as one of the crucial molecular mechanisms causing drastic pathological consequences of drug addiction. The increase of AC activity represents a "compensatory" response and is functionally related to the desensitization of G protein response to prolonged morphine exposure of target cells. The clear desensitization of µ-OR- and δ-OR-stimulated G protein response by morphine was demonstrated in our laboratory by analysis of the dose-response curves of DAMGO and DADLE-stimulated, high-affinity [35 S] GTPγS binding in plasma membranes isolated from frontal brain cortex of rats exposed to morphine according to the same protocol as that used in my Ph.D. thesis (10-50 mg/kg, 10 days). The κ-OR-stimulated [35 S] GTPγS binding was unchanged. It has been determined the amount of all AC isoforms (AC I-IX) in plasma membranes (PM) isolated from control and morphine-treated rats which were sacrificed 24 hours since the last dose of morphine....