Iraq is a country of great cultural significance as it is where civilization first began. As a result of its lengthy occupation, it is virtually one large archaeological site. In spite of numerous warnings to the governments of both the United States and the United Kingdom, no efforts were made to protect the Iraqi National Museum in Baghdad when the American-led coalition unlawfully invaded Iraq. Indeed, orders were given not to interfere with the looting. During the occupation that followed, the United States failed to take steps to protect Iraqi cultural property. In terms of international law, it was obliged to protect Iraq’s cultural property. The United States also chose to exacerbate its unlawful conduct by occupying archaeological sites and damaging them further by illegal construction. As a result many significant sites have been irreparably damaged or destroyed. Their conduct was the result of complete indifference to the Iraqi cultural heritage. Although their actions render them iin breach of international law, it is unlikely that the United States will ever be prosecuted for its actions. / Biblical and Ancient Studies / M.A. (Ancient Near Eastern Studies)

This dissertation focuses on utopian versus dystopian perceptions of rural indigenous African societies with regard to the modern metropolis. Since the evolution of the modern metropolis, rural African societies have undergone significant and complex cultural changes that have dislodged rural cultures from being perceived in terms of the traditional notion of fixity. This has lead to the modern city being seen as either utopian or dystopian by rural African societies. The dissertation questions the “utopianess” of the modern metropolis with a special focus on its central idea of “progress”. Special attention is given to artists who explore this cultural phenomenon in the utopian–dystopian paradigm. The dissertation goes further to address the cultural impact of recent technological developments on rural and urban societies, the researcher’s perceptions of this impact and how this has contributed to the dynamics that characterise the cultures of contemporary rural and urban migrants / Art History, Visual Arts and Musicology / M.A. (Visual Arts)

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Escherichia coli enteroagregativa (EAEC) é um patotipo emergente e heterogêneo que causa a diarréia aguda ou persistente em indivíduos de diferentes faixas etárias e em pacientes imunocomprometidos. Além disso, EAEC é um dos principais agentes etiológicos da diarréia dos viajantes. O padrão de aderência agregativa de EAEC está associado ao plasmídeo de aderência agregativa (pAA). Genes presentes no plasmídeo e no cromossomo codificam proteínas envolvidas na secreção extracelular de fatores de virulência na superfície ou diretamente na célula hospedeira. A capacidade de produção de muco e biofilme, elaboração de toxinas, aderência e indução de inflamação intensa na mucosa intestinal são importantes características da patogenicidade de EAEC. Nesse estudo, determinamos o perfil genotípico de genes do sistema de secreção Tipo V (SST5) e sistema de secreção Tipo VI (SST6) em cepas de EAEC. Os genes do SST5 ocorreram com mais frequência que os genes do SST6. A presença de pelo menos um gene do SST5 foi detectada em 79% das cepas, enquanto que os genes relacionados ao SST6 foram detectados em apenas 42% das cepas analisadas. A produção de biofilme foi observada em teste quantitativo e verificamos que 67% das cepas produziram biofilme. No teste qualitativo, o tipo de biofilme que predomina é o biofilme moderado (11 cepas), seguido do biofilme forte (9 cepas) e do biofilme discreto (4 cepas). A presença ou ausência de genes do SST5 e SST6 não parece interferir com a capacidade de produção de biofilme, nem com o tipo de biofilme formado. Em ensaios de citotoxicidade, apenas 25% das cepas EAEC (sobrenadante) causaram redução significativa na viabilidade de células T84 avaliada pelo teste de redução com MTT. Nossos resultados mostram que as cepas EAEC isoladas de crianças com diarréia aguda ou de grupo controle são invasoras para células T84. Ao compararmos a capacidade invasora das cepas clinicas e controle, observamos que a média do índice de internalização obtido nas 15 cepas do grupo clinico foi de 5,7% 1,7 e para as 9 cepas do grupo controle foi de 2.4 % 0,7; entretanto essa diferença observada não foi estatisticamente significativa. Não foi possível correlacionar o perfil genotípico dos genes do SST5 e SST6 com o perfil fenotípico analisado (formação de biofilme, citotoxicidade e invasão).O que pode ser atribuído a heterogeneidade genotípica e fenotípica, uma característica relevante de cepas EAEC. / Enteroaggregative Escherichia coli (EAEC) is an emerging and heterogeneous pathotype that causes acute or persistent diarrhea in individuals of different age groups and HIV-positive patients. In addition, EAEC is a major etiologic agent of travelers' diarrhea. The pattern of aggregative adherence of EAEC is associated with the aggregative adherence plasmid (pAA). Genes present in the plasmid and chromosome encode proteins involved in extracellular secretion of virulence factors on the surface or directly into the host cell. The production of mucus, biofilm and toxins; and induction of intense inflammation in the intestinal mucosa and mucosal adherence are major features of EAEC pathogenesis. In this study, we determined the genotypic profile of type V secretion system (T5SS) and type VI secretion system (T6SS) genes in EAEC strains. T5SS genes occurred more frequently than T6SS genes. The presence of at least one T5SS gene was detected in 79% of the strains, while genes related with T6SS were detected in only 42% of the strains tested. The biofilm production was observed in quantitative test, we have found that 67% of strains produced biofilm. In the qualitative test, we detected three distinctive patterns of biofilm formation: moderate (11 strains), strong (9 strains) and discrete biofilm (4 strains). The presence or absence of T5SS and T6SS genes do not interfere with the ability to produce biofilm, neither with biofilm pattern. In cytotoxicity assays, only 25% of EAEC strains (supernatant)resulted in significant reduction of T84 cells activity as evaluated by MTT reduction test. Our results show that EAEC strains isolated from children with acute diarrhea or the control group are able to invade T84 cells. When comparing the invasive ability of clinical and control strains, we observed that the average rate of internalization obtained in 15 clinical strains was 5.7% 1.7, for the nine strains of control group was 2.4% 0. 7, however this difference was not statistically significant. It was not possible to correlate the genotypic profile of T5SS and T6SS genes with the phenotypic profile analysed (biofilm formation, cytotoxicity and invasion), which can be attributed to genotypic and phenotypic heterogeneity, an important characteristic of EAEC strains

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Escherichia coli enteroagregativa (EAEC) é um patotipo emergente e heterogêneo que causa a diarréia aguda ou persistente em indivíduos de diferentes faixas etárias e em pacientes imunocomprometidos. Além disso, EAEC é um dos principais agentes etiológicos da diarréia dos viajantes. O padrão de aderência agregativa de EAEC está associado ao plasmídeo de aderência agregativa (pAA). Genes presentes no plasmídeo e no cromossomo codificam proteínas envolvidas na secreção extracelular de fatores de virulência na superfície ou diretamente na célula hospedeira. A capacidade de produção de muco e biofilme, elaboração de toxinas, aderência e indução de inflamação intensa na mucosa intestinal são importantes características da patogenicidade de EAEC. Nesse estudo, determinamos o perfil genotípico de genes do sistema de secreção Tipo V (SST5) e sistema de secreção Tipo VI (SST6) em cepas de EAEC. Os genes do SST5 ocorreram com mais frequência que os genes do SST6. A presença de pelo menos um gene do SST5 foi detectada em 79% das cepas, enquanto que os genes relacionados ao SST6 foram detectados em apenas 42% das cepas analisadas. A produção de biofilme foi observada em teste quantitativo e verificamos que 67% das cepas produziram biofilme. No teste qualitativo, o tipo de biofilme que predomina é o biofilme moderado (11 cepas), seguido do biofilme forte (9 cepas) e do biofilme discreto (4 cepas). A presença ou ausência de genes do SST5 e SST6 não parece interferir com a capacidade de produção de biofilme, nem com o tipo de biofilme formado. Em ensaios de citotoxicidade, apenas 25% das cepas EAEC (sobrenadante) causaram redução significativa na viabilidade de células T84 avaliada pelo teste de redução com MTT. Nossos resultados mostram que as cepas EAEC isoladas de crianças com diarréia aguda ou de grupo controle são invasoras para células T84. Ao compararmos a capacidade invasora das cepas clinicas e controle, observamos que a média do índice de internalização obtido nas 15 cepas do grupo clinico foi de 5,7% 1,7 e para as 9 cepas do grupo controle foi de 2.4 % 0,7; entretanto essa diferença observada não foi estatisticamente significativa. Não foi possível correlacionar o perfil genotípico dos genes do SST5 e SST6 com o perfil fenotípico analisado (formação de biofilme, citotoxicidade e invasão).O que pode ser atribuído a heterogeneidade genotípica e fenotípica, uma característica relevante de cepas EAEC. / Enteroaggregative Escherichia coli (EAEC) is an emerging and heterogeneous pathotype that causes acute or persistent diarrhea in individuals of different age groups and HIV-positive patients. In addition, EAEC is a major etiologic agent of travelers' diarrhea. The pattern of aggregative adherence of EAEC is associated with the aggregative adherence plasmid (pAA). Genes present in the plasmid and chromosome encode proteins involved in extracellular secretion of virulence factors on the surface or directly into the host cell. The production of mucus, biofilm and toxins; and induction of intense inflammation in the intestinal mucosa and mucosal adherence are major features of EAEC pathogenesis. In this study, we determined the genotypic profile of type V secretion system (T5SS) and type VI secretion system (T6SS) genes in EAEC strains. T5SS genes occurred more frequently than T6SS genes. The presence of at least one T5SS gene was detected in 79% of the strains, while genes related with T6SS were detected in only 42% of the strains tested. The biofilm production was observed in quantitative test, we have found that 67% of strains produced biofilm. In the qualitative test, we detected three distinctive patterns of biofilm formation: moderate (11 strains), strong (9 strains) and discrete biofilm (4 strains). The presence or absence of T5SS and T6SS genes do not interfere with the ability to produce biofilm, neither with biofilm pattern. In cytotoxicity assays, only 25% of EAEC strains (supernatant)resulted in significant reduction of T84 cells activity as evaluated by MTT reduction test. Our results show that EAEC strains isolated from children with acute diarrhea or the control group are able to invade T84 cells. When comparing the invasive ability of clinical and control strains, we observed that the average rate of internalization obtained in 15 clinical strains was 5.7% 1.7, for the nine strains of control group was 2.4% 0. 7, however this difference was not statistically significant. It was not possible to correlate the genotypic profile of T5SS and T6SS genes with the phenotypic profile analysed (biofilm formation, cytotoxicity and invasion), which can be attributed to genotypic and phenotypic heterogeneity, an important characteristic of EAEC strains

Invasive species represent a major challenge through their consequences on<p>biodiversity, human health and economy. Their effects are especially important on fragile and<p>unique insular biotas such as the Galápagos Islands. Ants in particular are keystone species<p>implicated in ecosystem functioning and biodiversity and they can be dramatic invaders. In<p>the Galápagos Islands, the tropical fire ant Solenopsis geminata is considered a high-impact<p>invasive species, though it remains surprisingly poorly studied. The objectives of this work<p>are to document the invasion of S. geminata in the Galápagos Islands by 1) updating its<p>distribution; 2) determining its reproduction and dispersal strategies and reconstruct its<p>invasion history throughout the archipelago and 3) evaluating its impact on the native fauna<p>(focusing on ants and arthropods communities and endemic land tortoises).<p>First, we added 66 new records of S. geminata in the Galápagos since 2008. It has<p>now been recorded on seven islands and 11 islets in a wide range of habitats, including<p>nesting sites of 24 endemic and/or endangered vertebrate species, for which it constitutes a<p>potential threat.<p>Secondly, by combining Bayesian clustering methods, coalescent-based scenario<p>testing using microsatellite data and historical records, we determined that genetic diversity<p>of populations of S. geminata collected in Galápagos Islands is significantly lower than the<p>genetic diversity of populations from native areas (Costa Rica). The Galápagos populations<p>form three clusters corresponding to an island or groups of islands. They appear to be the<p>result of a single introduction in the first half of the 19th century, probably from mainland<p>Ecuador, which acted as a bridgehead population to two subsequent introductions within the<p>archipelago, corresponding human colonization fluxes in the archipelago.<p>We sampled ants in all main habitats of Santa Cruz Island. Introduced ant species<p>were largely prevalent, and S. geminata was the dominant species and was associated with<p>low evenness of ant communties and lower abundance of native ants. We found that<p>Galápagos’ ant communities are determined by the vegetation type and altitude, but found<p>only little evidence for competitively structured assemblages, except in disturbed areas.<p>The arthropod diversity was investigated in two agricultural sites of Santa Cruz Island<p>by combining three complementary sampling techniques. More than half of the species were<p>either endemic or native, but introduced species constituted the majority of the catches.<p>Solenopsis geminata was by far the most abundant and common species.<p>Finally, we investigated the mortality of Cheloidis land tortoise’s eggs and hatchlings<p>in an area infested by S. geminata on Santa Cruz Island with regard to the abundance of fire<p>ants and the duration of incubation. Egg survival was negatively associated with longer incubation times but we found no direct relation between ant density and tortoise mortality<p>despite a high abundances of fire ants in the vicinity of the majority of the tortoise burrows.<p>Our work allows addressing ecological and genetical aspects of the invasion of S.<p>geminata in the Galápagos Islands. We analyzed our results in the light of an ecoevolutionary<p>framework presenting different invasion scenarios and discussed S. geminata<p>as an invasive ant. This provided us with information useful for the study and management of<p>this invasive species in the Galápagos Islands.<p>/<p>Les espèces invasives constituent un défi majeur à cause de leur impact sur la<p>biodiversité, la santé humaine et l’économie. Leurs effets sont particulièrement importants<p>sur les environnements insulaires fragiles et uniques comme les île Galápagos. Les fourmis<p>en particulièr sont des espèces clé de voûte du fonctionnement des écosystèmes et de la<p>biodiversité. Elles peuvent de ce fait devenir des envahisseurs spectaculaires. Dans les îles<p>Galápagos, la fourmi de feu tropicale Solenopsis geminata fait partie des espèces invasives<p>à haut impact et cependant elle a été étonnamment peu étudiée. Les objectifs de ce travail<p>consistent à documenter l’invasion de S. geminata dans l’archipel des Galápagos: 1) en<p>mettant à jour sa distribution; 2) en déterminant ses stratégies de reproduction et de<p>dispersion et en reconstruisant l’histoire de son invasion dans l’archipel et 3) en évaluant son<p>impact sur la faune native (particulièrement sur les communautés de fourmis et<p>d’arthropodes et sur les tortues terrestres endémiques).<p>Tout d’abord, nous avons ajouté 66 nouveaux relevés de S.geminata aux Galápagos<p>depuis 2008. À ce jour, la fourmi a été observée sur 7 îles et 11 îlots, et ce dans une grande<p>variété d’habitats. On la trouve également sur les sites de ponte de 24 espèces de vertébrés<p>endémiques ou en voie de disparition, qu’elle menace ainsi potentiellement.<p>Ensuite, en combinant des méthodes bayésiennes de regroupement et des<p>comparaisons de scénarios en se basant sur des séquences microsatellites et des données<p>historiques, nous avons montré que la diversité génétique des populations de S. geminata<p>des Galápagos est significativement inférieure à celle des populations des zones d’indigénat<p>(Costa Rica). Les populations des Galápagos - réparties en 3 groupes correspondant à une<p>île ou un groupe d’îles – sont le résultat d’une introduction unique ayant eu lieu dans la<p>première moitié du 19ème siècle. Elles proviennent vraisemblablement de l’Equateur<p>continental et constituent une population “tête de pont” pour deux introductions ultérieures au<p>sein de l’archipel. Ces mouvements correspondent aux flux de populations humaines.<p>Nous avons échantillonné les fourmis dans tous les principaux habitats de l’île de<p>Santa Cruz. Les fourmis introduites sont largement prévalentes. Solenopsis geminata<p>constitue l’espèce dominante et se trouve associée avec une faible équitabilité des<p>communautés de fourmis ainsi qu’avec une diminution de l’abondance des fourmis natives.<p>Nos résultats indiquent que les communautés de fourmis des Galápagos sont structurées<p>par le type de végétation et l’altitude, alors que les assemblages de fourmis ne sont pas<p>structurés compétitivement, à l’exception des zones perturbées.<p>Nous avons investigué la diversité arthropodienne de deux sites agricoles de l’île de<p>Santa Cruz en combinant trois méthodes d’échantillonnage complémentaires. Plus de la moitié des espèces collectées étaient soit endémiques, soit natives. Les espèces introduites<p>ont toutefois constitué la majorité des individus collectés. Solenopsis geminata était de loin la<p>plus abondante et la plus commune des espèces récoltées.<p>Enfin, dans une zones infestées par S. geminata sur l’île de Santa Cruz, nous avons<p>mis en relation la mortalité des oeufs et juvéniles de tortues terrestres Chelonoidis avec<p>l’abondance des fourmis de feu et la durée d’incubation des oeufs. Le taux de survie des<p>oeufs est négativement corrélé à leur durée d’incubation. Cependant, malgré de très hautes<p>abondances de fourmis de feu à proximité des nids de tortues, nous n’avons pas trouvé de<p>relation directe avec leur mortalité.<p>Pour conclure, ce travail aborde les aspects génétiques et écologiques de l’invasion<p>de la fourmi de feu tropicale dans les îles Galápagos. Nos résultats sont analysés au sein<p>d’un cadre éco-évolutif présentant différents scénarios d’invasion. Nous discutons également<p>de S. geminata en tant qu’espèce invasive. Nous espérons apporter des informations utiles<p>dans le cadre de l’étude et du contrôle de cette espèce invasive aux Galápagos. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Vegetation changes in Czech lowland forests over the past decades Martin Kopecký ABSTRACT To explore decadal changes in plant species diversity and composition of typical lowland forests in the Czech Republic, I compared vegetation on plots sampled decades ago with vegetation on the same plots sampled recently. First I evaluated the robustness of the approach used in my thesis. In Chapter 1, we provided the first direct test of the effect of uncertainty in original plot location on results from vegetation resurvey. We found that temporal trends in vegetation diversity and composition were comparable between exactly relocated permanent and only approximately relocated semi-permanent plots. Therefore, we conclude that the resurvey of semi-permanent plots is robust to the uncertainty in original plot location. Then, we showed that vegetation in lowland oak forest shifted from species-rich communities of thermophilous forest toward species poorer communities of mesic forest (Chapter 2). The species typical for thermophilous oak forests and nationally endangered species suffered the most significant decline. We identified as the main driver behind these changes shift from traditional coppicing toward high forest management after WWII. Further, we explored the processes behind temporal shifts in species diversity...

Dentre as 18 espécies do gênero Yersinia, as espécies Y. enterocolitica, Y. pseudotuberculosis e Y. pestis foram extensivamente caracterizadas em diversos aspectos como ecologia, epidemiologia e mecanismos de patogenicidade. Sete das 15 espécies restantes (Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii e Y. rohdei), usualmente conhecidas como Y. enterocolitica-like, até o momento, não tiveram seu potencial patogênico caracterizado e são, geralmente, consideradas não-patogênicas. Entretanto, dados da literatura sugerem que algumas dessas espécies possam causar doença. Esses dados estimularam o surgimento de questões sobre os mecanismos pelos quais as espécies de Y. enterocolitica-like possam interagir com as células do hospedeiros e causar doenças. Esse projeto teve como principal objetivo caracterizar o potencial patogênico de linhagens de Y. enterocolitica-like, especificamente das espécies Y. frederiksenii, Y. kristensenii e Y. intermedia. No presente trabalho, o potencial patogênico de 118 linhagens de Y. enterocolitica-like (50 Y. frederiksenii, 55 Y. intermedia e 13 Y. kristensenii) foi avaliado pela pesquisa da presença dos genes relacionados à virulência ail, fepA, fepD, fes, hreP, myfA, tccC, ystA, ystB e virF por PCR. Além disso, a habilidade de algumas linhagens de Yersinia de aderir e invadir células Caco-2 e HEp-2 após diferentes períodos de incubação, bem como, de sobreviver no interior de macrófagos humanos U937 foi testada. Aspectos morfológicos da adesão bacteriana foram visualizados por microscopia eletrônica. Finalmente, a presença de possíveis novos mecanismos de virulência foi avaliada a partir do sequenciamento de RNA de uma linhagem de Y. enterocolitica-like. As linhagens estudadas apresentaram os seguintes genes: Y. frederiksenii, fepA (44%), fes (44%) e ystB (18%); Y. intermedia, ail (53%), fepA (35%), fepD (2%), fes (97%), hreP (2%), ystB (2%) e tccC (35%); e Y. kristensenii, ail (62%), ystB (23%), fepA (77%), fepD (54%), fes (54%) e hreP (54%). De modo geral, as linhagens de Y. enterocolitica-like tiveram a habilidade de aderir e invadir células Caco-2 e HEp-2 inferior à da linhagem altamente patogênica Y. enterocolitica 8081. Contudo, Y. kristensenii FCF 410 e Y. frederiksenii FCF 461 apresentaram elevado potencial de invasão a células Caco-2 após cinco dias de pré-incubação, os quais foram 45 e 7,2 vezes maiores do que o controle Y. enterocolitica 8081, respectivamente, porém, o gene ail não foi detectado nessas linhagens. O ensaio de sobrevivência em macrófagos humanos U937 ii mostrou que as linhagens de Y. frederiksenii FCF 461 (40,0%) e Y. frederiksenii FCF 379 (24,6%) tiveram porcentagens de sobrevivência superior à de Y. enterocolitica 8081 (13,4%). Todavia, linhagens de Y. intermedia e Y. kristensenii apresentaram uma capacidade reduzida de sobreviver em macrófagos. A microscopia eletrônica de varredura mostrou as bactérias em contato com a filipódia celular. As bactérias foram distribuídas tanto individualmente quanto em pequenos aglomerados. Portanto, podemos concluir que a presença dos genes relacionados à virulência encontrados nas Y. enterocolitica-like estudadas indicou o possível potencial patogênico de algumas dessas linhagens. Os ensaios de adesão e invasão a células de mamíferos sugerem que a patogenicidade de Y. kristensenii e Y. frederiksenii possa ser linhagem-dependente. O ensaio de sobrevivência em macrófagos humanos U937 evidenciou o potencial patogênico de algumas linhagens de Y. frederiksenii. Em conjunto, os resultados obtidos sugerem a existência de mecanismos de virulência alternativos aos mecanismos clássicos descritos para Y. enterocolitica patogênica. Contudo, a presença de possíveis novos mecanismos de virulência não pode ser verificada, uma vez que a plataforma 454 GS Junior (Roche) não se mostrou adequada para a realização de sequenciamento de RNA de amostras provenientes de interações com células devido à baixa cobertura obtida. / Among the 18 species of the Yersinia genus, Y. enterocolitica, Y. pseudotuberculosis and Y. pestis were extensively characterized in different subjects as ecology, epidemiology and pathogenicity mechanisms. Seven among the remaining 15 species (Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii and Y. rohdei), often called Y. enterocolitica-like have not their pathogenic potential characterized and are usually considered to be nonpathogenic. However, literature data suggest that some of these species can cause diseases. These data stimulate the upsurge of questions about the mechanisms of which Y. enterocolitica-like species may interact with host cells and cause diseases. The main objective of this preject was to characterize the pathogenic potential of Y. enterocolitica-like strains, specifically of the species Y. frederiksenii, Y. kristensenii and Y. intermedia. This work evaluated the pathogenic potential of 118 Y. enterocolitica-like strains (50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii) searching for the presence of the virulence-related genes ail, fepA, fepD, fes, hreP, myfA, tccC, ystA, ystB and virF by PCR. Besides, Yersinia strains ability of adhesion and invasion to Caco-2 and HEp-2 cells after different pre-incubation periods, and its survival within human macrophages U937 were tested. Morphologic aspects of bacterial adhesion were observed by scanning electronic microscopy. Finally, the presence of new possible virulence mechanisms was evaluated through RNA sequencing of one Y. enterocolitica-like strain. The studied strains showed the following genes: Y. frederiksenii, fepA (44%), fes (44%) and ystB (18%); Y. intermedia, ail (53%), fepA (35%), fepD (2%), fes (97%), hreP (2%), ystB (2%) and tccC (35%); and Y. kristensenii, ail (62%), ystB (23%), fepA (77%), fepD (54%), fes (54%) and hreP (54%). Usually Y. enterocolitica-like strains presented less ability of adhere and invade Caco-2 and HEp-2 cells than the highly pathogenic strain Y. enterocolitica 8081. On the other hand, Y. kristensenii FCF 410 and Y. frederiksenii FCF 461 showed high potential of invasion in Caco-2 cells after 5 days of pre-incubation, which were 45 and 7.2 times higher than the control Y. enterocolitica 8081 respectively, but ail gene was not found in these strains. Survival assay in human macrophages U937 showed that Y. frederiksenii FCF 461 (40.0%) and Y. frederiksenii FCF 379 (24.6%) strains presented survival percentages higher than Y. enterocolitica 8081 (13.4%). However, Y. intermedia and Y. iv kristensenii strains showed a reduced capability of surviving in macrophages. Scanning electron microscopy showed bacteria at the surface in contact with the cellular filopodia. The bacteria were distributed either individually or in small clumps. Therefore, it may be concluded that the presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Mammal cells adhesion and invasion assays suggest that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent. Human macrophages U937 surviving assay highlighted the pathogenic potential of some Y. frederiksenii strains. Together, the results suggest the existence of alternative virulence mechanisms other than the classical mechanisms described for pathogenic Y. enterocolitica. However, we could not verify the presence of possible new virulence mechanisms because 454 GS Junior (Roche) platform was not suitable for RNA sequencing of strains from cells interaction due its low coverage obtained.

O câncer de mama é o tipo de câncer mais comumente detectado em mulheres de todo o mundo. Na maioria das pacientes, a causa de morte se deve, principalmente, à doença metastática que pode se desenvolver a partir do tumor primário. O processo metastático envolve uma complexa cascata de eventos, incluindo a quebra organizada dos componentes da matriz extracelular por metaloproteinases de matriz (MMPs). A atividade das MMPs é precisamente regulada por inibidores específicos, os inibidores teciduais das MMPs (TIMPs). Dado seu papel na progressão tumoral, níveis elevados de MMPs têm sido associados com prognóstico desfavorável para pacientes com câncer. Por outro lado, sendo os TIMPs proteínas multifuncionais, níveis elevados de TlMP-1 e de TIMP-2 correlacionam com agressividade do tumor e prognóstico ruim em diferentes tipos de câncer, incluindo o câncer de mama. O gene supressor de metástase RECK codifica uma glicoproteína de membrana capaz de inibir a invasão e a metástase tumoral através da regulação negativa da atividade de MMPs envolvidas em carcinogênese: MMP-2, MMP-9 e MMP-14 (MT1-MMP). A fim de analisar o papel das MMPs e de seus inibidores (TIMPs e RECK) na progressão tumoral do câncer de mama, o perfil de expressão destes genes foi detectado, através de ensaios de Real-Time PCR, em um painel de cinco linhagens celulares de carcinoma de mama humano com diferentes potenciais invasivos e metastáticos e em 72 amostras teciduais de tumores primários de mama e 30 amostras teciduais de borda normal adjacente ao tumor. O perfil de expressão protéica de RECK foi avaliado em 236 amostras de tumores primários de mama através de ensaios de Tissue Microarray. Além disso, a atividade proteolítica das MMPs foi detectada em ensaios de Zimografia. Os resultados obtidos indicam que a progressão do câncer de mama humano está relacionada com um aumento dos níveis de expressão das MMPs e de seus inibidores específicos. O aumento dos níveis de expressão dos TIMPs parece estar relacionado ao seu papel como proteína multifuncional que pode estar funcionando de maneira a promover, mais do que suprimir, a progressão tumoral. Níveis elevados da expressão protéica de RECK estão associados com pior prognóstico. No entanto, para pacientes em estádios clínicos avançados, altos níveis de expressão de RECK podem estar correlacionados com melhor prognóstico, dependendo do balanço MMP/inibidor. Os níveis de expressão das MMPs apresentaram correlação positiva em relação aos níveis de expressão de seus inibidores específicos, sugerindo a existência de fatores e vias de sinalização comuns envolvidas na regulação coordenada destes genes. Além disso, a síntese do inibidor pode estar relacionada a uma resposta celular ao aumento da expressão e atividade de proteases. O balanço transcricional enzima/inibidor favorece a enzima nas amostras tumorais e, de modo contrário, o inibidor específico nas amostras de borda normal, sugerindo o balanço como o principal fator na determinação da degradação da MEC em processos invasivos e metastáticos. Os resultados obtidos podem contribuir para um melhor entendimento da complexidade dos mecanismos envolvidos na metástase do câncer de mama. / Breast cancer is among the most common tumors affecting women. Like most solid tumors, metastatic disease rather than the primary tumor itself is responsible for death. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix by matrix metalloproteinases (MMPs). The activity of these proteases is tightly regulated by specific inhibitors, known as tissue inhibitors of MMPs (TIMPs). Consistent with their role in tumor progression, high levels of a number of MMPs have been shown to correlate with poor prognosis in human cancers. On the other hand, TIMPs are multifunctional molecules with high levels of TIMP-1 and TIMP-2 having been shown to predict adverse prognosis and correlate with tumor aggressiveness in several different human cancers, including breast cancer. The RECK metastasis suppressor gene encodes a membrane-associated MMP regulator protein that is able to suppress tumor invasion and metastasis by negatively regulating MMPs involved in carcinogenesis, namely: MMP-2, MMP-9 and MMP-14 (MT1-MMP). In order to analyse the role of these genes in breast cancer progression, the expression levels of MMPs and theirs inhibitors were detected by Real Time PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential and in 72 primary breast cancer and 30 adjacent normal tissue specimens. The RECK protein expression profile was also examined in 236 primary breast cancer tissue specimens by Tissue Microarray technology. The proteolytic activity of MMPs was examined by Zymography. The results suggest that high expression levels of MMPs and their inhibitors are correlated with breast cancer progression. High levels of TIMP transcript may be involved in tumor-promoting activity as a result of their multifunctional role. Increased levels of the RECK protein are correlated with poor prognosis for the patient. However, high levels of RECK would be expected to confer a favorable prognosis to patients with advanced disease. The expression levels of MMPs significantly correlated with the levels of TIMPs and may be explained by coordinate correlation of these molecules or, alternatively, the synthesis of an inhibitor may be a cellular reaction to the presence of the protease. The enzyme/inhibitor balance at the transcriptional level favors the enzyme in tumor tissue and the inhibitor in adjacent normal tissue. It is probably the parameter that will determine the matrix degradation at invasion and metastatic process. Our results are likely to contribute for better understanding of the complex mechanisms involved in breast cancer metastasis.

Abstract Between October 1999 and October 2003, 30 adult and 48 young (&amp;lt; 1 year) raccoon dogs (Nyctereutes procyonoides) were monitored using radio-telemetry in an area of Germany which has been occupied by this invasive alien species since the early 1990s. Additionally, three pairs of raccoon dogs were observed by continuous radio-tracking during the first six weeks after parturition in 2003. Furthermore 136 raccoon dog pubs were ear-tagged between June 1999 and August 2006. No adult animals dispersed from the area during the study period and home ranges tended to be used for several years, probably for life. The average annual home range size, calculated using 95% fixed kernel, was 382.2 ha ± 297.4 SD for females (n = 30 seasonal home ranges) and 352.4 ha ± 313.3 SD for males (n = 32 seasonal home ranges). Paired raccoon dogs had home ranges of similar size, with pair sharing the same area all year round. Raccoon dogs occupied large core areas (85% kernel) covering 81.2% of their home ranges. The home ranges were at their smallest during the mating season. The slightly larger size of home ranges in winter suggests that, due to the temperate climate, raccoon dogs do not hibernate in Germany. Males and females formed a long-term (probably lifelong) pair bond. Same-sex neighbours ignored each other and even adjacent males/females showed neither preference nor avoidance. Thus, it can be assumed that the raccoon dog in Central Europe is monogamous without exclusive territories, based on the results of home range overlap analysis and interaction estimations. Habitat composition within home ranges and within the whole study area was almost equal. Although, percentage shares of farmland and meadow was 16.35% smaller and 12.06% higher within the home ranges, respectively. All nine habitat types (farmland, forest, settlement, water, meadows, maize fields, small woods, reeds and hedges) were used opportunistically by raccoon dogs. No significant, recognisable difference for habitat preferences between seasons was detected. Male and female raccoon dog showed equal habitat preference pattern. A comparison of active and inactive locations in different habitats found no remarkable differences. Habitat composition of individual home ranges was used to classify animals. If the percentage of forest within a home range exceeded 50% the individual was classified as a ‘forest type’ raccoon dog. If the percentage of forest habitats within a home range was less than 5%, the share of pastureland was mean 81.82% ± 16.92 SD. Consequently the individual was classified as a ‘agrarian type’ raccoon dog. Neither habitat preference nor habitat selection process differed between the two ‘types’. Habitat use and preference is discussed with relation to the ability of the raccoon dog to expand its range towards Western Europe. Males spent noticeably more time (40.5% of the time ±11.7 SD) alone with the pups than females (16.4% of the time ±8.5 SD). Females had noticeably larger 95% kernel home ranges (98.24 ha ±51.71 SD) than males (14.73 ha ±8.16 SD) and moved much longer daily distances (7,368 m ±2,015 SD) than males (4,094 m ±2,886 SD) in six weeks postpartum. The raccoon dogs being studied left the breeding den in the 6th week after the birth of the pups. In situ video observation showed that the male carried prey to the den to provide the female and the litter with food. A clear division of labour took place among parents during the period in which the pups were nursed: males guarded the litter in the den or in close vicinity of it, while the females foraged to satisfy their increased energy requirements. There were relocations of 59 (43.4%) ear-tagged young racoon dogs and mean distance from marking point was 13.5 km ±20.1 SD. Dispersal mortality rate was 69.5% among young raccoon dogs. Most animals (55.9%) were recovered nearer than 5 km from the marking point, whereas only 8.5% relocations were recorded further than 50 km from the marking point. There was no difference in the distances of relocations between sexes. Most (53.7%) relocations of ear-tagged young raccoon dogs were in August and September and, only 34.1% were recorded from October to April. Hunting (55 %) and traffic (27 %) were the major mortality factors. Radio-collared young raccoon dogs generally dispersed between July and September. The mean natal home range size (MCP 100%) with and without excursions was 502.6 ha ±66.4 SD (n = 9) and 92.1 ha ±66.4 SD (n = 17), respectively. There were no differences between sexes in the month of dispersal. The direction of travel for dispersing animals appeared to be random, with distances from 0.5 km to 91.2 km. A highly flexible dispersing behaviour is certainly one of the reasons which contribute to the high expansion success of the species.

Between October 1999 and October 2003, 30 adult and 48 young (< 1 year) raccoon dogs (Nyctereutes procyonoides) were monitored using radio-telemetry in an area of North-East Germany which has been occupied by this invasive alien species since the early 1990s. Additionally, three pairs of raccoon dogs were observed by continuous radio-tracking during the first six weeks after parturition in 2003. Furthermore 136 raccoon dog pubs were ear-tagged between June 1999 and August 2006. No adult animals dispersed from the area during the study period and home ranges tended to be used for several years, probably for life. The average annual home range size, calculated using 95% fixed kernel, was 382.2 ha ± 297.4 SD for females (n = 30 seasonal home ranges) and 352.4 ha ± 313.3 SD for males (n = 32 seasonal home ranges). Paired raccoon dogs had home ranges of similar size, with pair mates sharing the same area all year round. Raccoon dogs occupied large core areas (85% kernel) covering 81.2% of their home ranges. The home ranges were at their smallest during the mating season. The slightly larger size of home ranges in winter suggests that, due to the temperate climate, raccoon dogs do not hibernate in Germany. Males and females formed a long-term (probably lifelong) pair bond. Same-sex neighbours ignored each other and even adjacent males/females showed neither preference nor avoidance. Thus, it can be assumed that the raccoon dog in Central Europe is monogamous without exclusive territories, based on the results of home range overlap analysis and interaction estimations. Habitat composition within home ranges and within the whole study area was almost equal. Although, percentage shares of farmland and meadow was 16.35% smaller and 12.06% higher within the home ranges, respectively. All nine habitat types (farmland, forest, settlement, water, meadows, maize fields, small woods, reeds and hedges) were used opportunistically by raccoon dogs. No significant, recognisable difference for habitat preferences between seasons was detected. Male and female raccoon dog showed equal habitat preference pattern. A comparison of active and inactive locations in different habitats found no remarkable differences. Habitat composition of individual home ranges was used to classify animals. If the percentage of forest within a home range exceeded 50% the individual was classified as a ‘forest type’ raccoon dog. If the percentage of forest habitats within a home range was less than 5%, the share of pastureland was mean 81.82% ± 16.92 SD. Consequently the individual was classified as a ‘agrarian type’ raccoon dog. Neither habitat preference nor habitat selection process differed between the two ‘types’. Habitat use and preference is discussed with relation to the ability of the raccoon dog to expand its range towards Western Europe. Males spent noticeably more time (40.5% of the time ±11.7 SD) alone with the pups than females (16.4% of the time ±8.5 SD). Females had noticeably larger 95% kernel home ranges (98.24 ha ±51.71 SD) than males (14.73 ha ±8.16 SD) and moved much longer daily distances (7,368 m ±2,015 SD) than males (4,094 m ±2,886 SD) in six weeks postpartum. The raccoon dogs being studied left the breeding den in the 6th week after the birth of the pups. In situ video observation showed that the male carried prey to the den to provide the female and the litter with food. A clear division of labour took place among parents during the period in which the pups were nursed: males guarded the litter in the den or in close vicinity of it, while the females foraged to satisfy their increased energy requirements. There were relocations of 59 (43.4%) ear-tagged young raccoon dogs and mean distance from marking point was 13.5 km ±20.1 SD. Dispersal mortality rate was 69.5% among young raccoon dogs. Most animals (55.9%) were recovered nearer than 5 km from the marking point, whereas only 8.5% relocations were recorded further than 50 km from the marking point. There was no difference in the distances of relocations between sexes. Most (53.7%) relocations of ear-tagged young raccoon dogs were in August and September and, only 34.1% were recorded from October to April. Hunting (55 %) and traffic (27 %) were the major mortality factors. Radiocollared young raccoon dogs generally dispersed between July and September. The mean natal home range size (MCP 100%) with and without excursions was 502.6 ha ±66.4 SD (n = 9) and 92.1 ha ±66.4 SD (n = 17), respectively. There were no differences between sexes in the month of dispersal. The direction of travel for dispersing animals appeared to be random, with distances from 0.5 km to 91.2 km. A highly flexible dispersing behaviour is certainly one of the reasons which contribute to the high expansion success of the species.