Detecção sorológica de riquétsias do grupo da febre maculosa e levantamento acarológico em equinos no Distrito Federal, Brasil

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Veterinária, 2014.A febre maculosa brasileira (FMB) é uma zoonose causada pela bactéria Rickettsia rickettsii e transmitida a humanos por carrapatos. Procurou-se detectar a presença do principal agente etiológico da FMB circulando em equinos apreendidos no Distrito Federal, Brasil. Durante um ano foram coletadas amostras de sangue e ectoparasitos de 122 equinos. Após teste por reação de imunofluorescência indireta (RIFI), para anticorpos IgG anti R. rickettsii 21 amostras foram positivas em titulações variáveis de 1:64 a 1:512. Dos 618 carrapatos coletados, 449 pertenciam ao gênero Anocentor, 113 ao Boophilus e 36 do gênero Amblyomma. Como conclusão foi possível identificar a circulação de Rickettsia em equinos no DF, Brasil. ______________________________________________________________________________________ ABSTRACTThe Brazilian spotted fever (BSF) is a zoonosis caused by the bacteria Rickettsia rickettsii and transmitted to humans by ticks. In this study we tried to detect the BSF ethiological agent in horses apprehended by animal control in Distrito Federal, Brazil. Over one year the blood and ectoparasites were sampled on 122 horses. The samples were tested by indirect imunnofluorescence antibody assay (IFA) for R. rickettsii anti-IgG antibody, 21 samples were positive for titer ranging from 1:64 to 1:512. Among the 618 ticks collected, 449 belonged to the Anocentor genus, 113 to Boophilus and 36 were Amblyomma. The bacteria were detected in horses in Distrito Federal

Subcellular localization and dynamics of transmembrane and membrane associated proteins in Corynebacterium glutamicum

The cytoplasmic membrane of Corynebacterium glutamicum comprises 660 predicted membrane integral proteins of various functional categories, including transport, signal transduction, protein translocation, proteolysis and energy metabolism. Being an important industrial producer of amino acids, studying the membrane proteome of this organism is relevant for a better understanding of its fundamental processes and possible targeted optimizations. Here, we analyzed the subcellular localization of different transmembrane and membrane-associated proteins of Corynebacterium glutamicum, including the fructose specific PtsF and the glucose specific PtsG transporters, the respiratory chain complex II component SdhA, the small-conductance mechanosensitive channel YggB, and the polar/septa scaffold DivIVA. Using widefield and single molecule localization microscopy, we discovered that PtsG and PtsF form membrane embedded clusters with no defined positions. A similar pattern was observed for SdhA, which curiously, seem to colocalize with PtsF in our widefield microscopy experiments. Size, number and fluorescence of the observed PTS clusters change upon presence or absence of the transported substrate. In presence of the transport substrate, clusters significantly increased in size. Photo-activated localization microscopy (PALM) data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas. This mechanism allows for efficient use of the limited membrane space under varying growth conditions without need of protein removal and re-synthesis. Clustering was also observed in YggB, that localized in a punctate pattern of different intensities at the membrane. By changing the NaCl concentration in medium, ranging from 0 mM to 170 mM, we could observe C. glutamicum cells under different degrees of osmotic stress, ranging from hypoosmotic to low hyperosmotic. The extreme studied treatments resulted in impaired growth, which suggests that the cells were indeed under constant osmotic stress. Under such conditions, no changes in YggB-mNeonGreen foci number or fluorescence were observed, hinting that yggB expression is constitutive and that prolonged exposure to osmotic stress does not result in the recruitment of more channels to the membrane. Finally, we studied the dynamics of DivIVA in cells of various shapes, including L-form cells with different morphologies by performing widefield microscopy and fluorescence recovery after photobleaching (FRAP) experiments with a strain expressing DivIVA-mCherry. DivIVA is a membrane-associated protein that serves as a scaffold for different proteins related to cell division and cell growth. Cell division in bacterial L-forms is unlike the coordinated and precise division observed in cell-walled bacteria. It takes place randomly, not necessarily linked to the divisome and is highly influenced by perturbations in the surrounding environment. Due to this, it is not uncommon to find L-form cells with multiple, parts of, or no chromosomes. Still, in these cells, DivIVA continues to localize in regions of negative curvatures, characterized as bumps in the cytoplasmic membrane. In completely spherical cells, with no regions of higher negative curvature, DivIVA localize as larger foci. Our FRAP results demonstrated that in regions of bumps, the diffusion rate of DivIVA-mCherry is similar to the one observed for poles of rod-shaped cells, but with a significant lower half-time recovery. Furthermore, the diffusion rate on smooth surfaces was more than double, and the half-time recovery less half as the one observed in bumps. This suggests that DivIVA is significantly more mobile in such regions. Moreover, using novel microscopy techniques, the work presented on this thesis shed a light on unexplored facets of membrane protein compartmentalization in Corynebacterium glutamicum.Die Zytoplasmamembran von Corynebacterium glutamicum beherbergt 660 annotierte Membranintegralproteine, die verschiedene Funktionen in Transport, Signaltransduktion, Proteintranslokation, Proteolyse und Energiestoffwechsel wahrnehmen. Da C. glutamicum eine wichtige Rolle in der industriellen Aminosäureproduktion spielt, ist die Untersuchung des Membran-Proteoms wichtig für ein besseres Verständnis grundlegender Prozesse und gezielter Optimierung der Produktionsprozesse. In dieser Arbeit wurde die subzelluläre Lokalisation verschiedener Transmembran- und membranassoziierter Proteine von C. glutamicum analysiert. Dazu gehörten der Fructose-spezifische PtsF- und der Glucose-spezifische PtsG-Transporter, die Komponente SdhA des Komplexes II der Atmungskette, der mechanosensitive Kanal YggB und das am Zellpol lokalisierte Gerüstprotein DivIVA. Unter Verwendung von Weitfeld- und Einzelmolekül-Lokalisationsmikroskopie konnte gezeigt werden, dass PtsG und PtsF in Membranen eingebettete Cluster ohne definierte Positionen bilden. Ein ähnliches Muster wurde für SdhA beobachtet, das in Weitfeldmikroskopie-Experimenten interessanterweise mit PtsF zu kolokalisieren scheint. Größe, Anzahl und Fluoreszenz der beobachteten PTS-Cluster änderten sich bei Anwesenheit oder Abwesenheit des transportierten Substrats. In Gegenwart des transportierten Substrats nahm die Größe der Cluster signifikant zu. Daten der photoaktivierbaren Lokalisationsmikroskopie (PALM) ergaben, dass in Gegenwart verschiedener Kohlenstoffquellen die Anzahl der EII-Proteine pro Cluster konstant bleibt, die Dichte dieser Cluster jedoch abnimmt. Diese Arbeit zeigt einen einfachen Mechanismus zur effizienten Regulierung der Membranbelegung. Cluster von PTS-EII-Transportern sind in Abwesenheit eines geeigneten Substrats dicht gepackt. In Gegenwart eines transportierten Substrats besetzen die EII-Proteine in einzelnen Clustern größere Membranflächen. Dieser Mechanismus ermöglicht die effiziente Nutzung des begrenzten Membranraums unter verschiedenen Wachstumsbedingungen, ohne dass Proteine entfernt und erneut synthetisiert werden müssen. Clusterbildung wurde auch bei YggB beobachtet, das in einem punktförmigen Muster unterschiedlicher Intensität an der Membran lokalisiert war. Durch Änderung der NaCl-Konzentration im Medium im Bereich von 0 mM bis 170 mM konnten wir C. glutamicum-Zellen unter osmotischem Stress beobachten, der von hypo- bis leicht hyperosmotisch reichte. Unter Extrembedingungen ließ sich ein negativer Effekt auf das Wachstum beobachten, was darauf hindeutet, dass die Zellen tatsächlich einem konstanten osmotischen Stress ausgesetzt waren. Auch unter solchen Bedingungen wurden keine Änderungen der Anzahl an YggB-mNeonGreen-Foci oder der Fluoreszenz beobachtet, was darauf hindeutet, dass die yggB-Expression konstitutiv ist und dass eine längere Exposition gegenüber osmotischem Stress nicht zur Rekrutierung weiterer YggB-Kanäle an die Membran führt. Schließlich wurde die Dynamik von DivIVA in Zellen verschiedener Formen, einschließlich L-Formen mit unterschiedlichen Morphologien, untersucht. Hierfür wurde ein DivIVA-mCherry-exprimierender Stamm sowohl mittels Weitfeldmikroskopie, als auch mittels Fluorescence Recovery After Photobleaching (FRAP) analysiert. DivIVA ist ein membranassoziiertes Protein, das als Gerüst für verschiedene Proteine dient, die mit der Zellteilung und dem Zellwachstum zusammenhängen. Die Zellteilung in bakteriellen L-Formen unterscheidet sich von der koordinierten und präzisen Teilung, die bei Bakterien mit Zellwänden beobachtet wird. Sie findet zufällig statt, ist nicht notwendigerweise mit dem Divisom verbunden und wird stark von Störungen in der Umgebung beeinflusst. Aus diesem Grund ist es nicht ungewöhnlich, L-förmige Zellen mit mehreren, oder auch ganz ohne Chromosomen zu finden. Dennoch lokalisiert DivIVA in diesen Zellen weiterhin in Regionen mit negativen Krümmungen, die als „Ausbuchtungen“ in der Zytoplasmamembran zu sehen sind. In vollständig kugelförmigen Zellen ohne Bereiche mit höherer negativer Krümmung lokalisiert DivIVA in größeren Foci. Unsere FRAP-Experimente zeigten, dass die Diffusionsrate von DivIVA-mCherry in Regionen mit hohes Membrankrümmung der von Polen stäbchenförmiger Bakterien ähnelt, jedoch mit signifikant geringerer Halbwertszeit in der Erholungsphase. Unsere Daten zeigen, dass die DivIVA Dynamik von der Zellgeometrie beeinflusst wird. Da DivIVA über die Rekrutierung des Zellwand-Synthese Apparates an der Ausbildung der Zellmorphologie beteiligt ist, stellt dieses System ein einfaches aber effektives Konzept der bakteriellen Selbstorganisation dar. Darüber hinaus war die Diffusionsrate auf wenig gekrümmten Oberflächen mehr als doppelt so hoch und die Halbwertszeit halb so hoch wie die Diffusionsrate in Regionen mit hohes Membrankrümmung. Dies deutet darauf hin, dass DivIVA in solchen Regionen wesentlich mobiler ist. zusammenfassend klären die in dieser Arbeit vorgestellten Daten mithilfe neuartiger Mikroskopietechniken noch nicht erforschte Facetten der Kompartimentierung von Membranproteinen in Corynebacterium glutamicum auf

COMMUNITY STRUCTURE AND DYNAMICS OF THE AZOREAN ROCKY INTERTIDAL: EXPLOITATION OF KEYSTONE SPECIES

Experimental work has shown the importance of grazing by patellid limpets in structuring intertidal assemblages. Little is known, however, about the effects of a largescale and chronic removal of limpets. Here I investigate the ecology of Patella candei, a seldom-studied limpet endemic to Macaronesia, and how its long-term fishery impacts the Azorean rocky intertidal. The specific aims of this thesis are to: examine the processes that affect the distribution of limpets in the Azores at a range of spatial scales; investigate the role of grazing by P. candei in structuring the Azorean rocky intertidal and if its harvesting has impacted the dynamics and functioning of this ecosystem. The distribution of limpets was variable at a range of spatial scales. At the scale of islands, inter-island variation in harvesting intensity affected the abundance and size structure of populations of limpets as well as the balance between grazers, algae and barnacles. Stocks of limpets showed clear signs of exploitation and there was evidence that current legislation, including limpet protected zones, have been largely ineffective in protecting these populations. At smaller spatial scales, substratum micro-topography influenced the distribution and sui-vival of limpets. I also showed that the experimental provision of microhabitats could be used as a measure to mitigate the effects of coastal urbanisation, whilst promoting a local enhancement of the stocks of limpets. Overall my results provide evidence for the population and community level effects of limpet harvesting and show that limpet harvesting has a strong impact on the structure and fiinctioning of the Azorean rocky intertidal.University of the Azores. Marine Biological Association of the U

Impact of chromossomal structure on the evolution of Schizosaccharomyces pombe undergoing Mutation Accumulation

Large chromosomal rearrangements are common in natural populations and thought to be involved in speciation events. In this project, we used experimental evolution to determine how the speed of evolution and the type of accumulated mutations depend on the ancestral chromosomal structure and genotype. We utilized two Wild Type strains and a set of genetically engineered Schizosaccharomyces pombe strains, different solely in the presence of a certain type of chromosomal variant (inversions or translocations), along with respective controls. Previous research has shown that these chromosomal variants have different fitness levels in several environments, probably due to changes in the gene expression along the genome. These strains were propagated in the laboratory at very low population sizes, in which we expect natural selection to be less efficient at purging deleterious mutations. We then measured these strains’ changes in fitness throughout this accumulation of deleterious mutations, comparing the evolutionary trajectories in the different rearrangements to understand if the chromosomal structure affected the speed of evolution. We also tested these mutations for possible epistatic effects and estimated their parameters: the number of arising deleterious mutations per generation (Ud) and each one’s mean effect (sd)

Lapas : pequenas Ilhas de diversidade

A secção UAciência é coordenada pelo Professor Universitário Armindo Rodrigues.[…]. Um estudo recente feito por investigadores do Centro de Investigação em Recursos Naturais da Universidade dos Açores examinou a diversidade de organismos que vivem sob as conchas da lapa Patella aspera. As lapas desempenham um papel importante nos ecossistemas uma vez que controlam a abundancia de algas prevenindo desta forma que estas monopolizem o espaço, permitindo assim a fixação de outro tipo de organismos como por exemplo a pequena craca Chthamalus stellatus. No entanto, o presente estudo mostrou que para alem do papel importante na ecologia das nossas costas, as lapas desempenham ainda um outro papel ao suportarem em si mesmas uma extraordinária diversidade. Foram observados um total de 707 conchas de lapa recolhidas em todas as ilhas do arquipélago nas quais se registaram uns surpreendentes 190 registos de espécies diferentes. […].info:eu-repo/semantics/publishedVersio

A urbanização nos ecossistemas costeiros : implicações para a biodiversidade

A secção UAciência é coordenada pelo Professor Universitário Armindo Rodrigues.[…]. A urbanização dos habitats costeiros, incluindo a construção de estruturas de defesa costeiras, como paredões, quebra-mares ou espigões, tem vindo a aumentar como resposta às previsões para o aumento do nível do mar, maior ocorrência de tempestades e aumento do transporte marítimo. O efeito destes habitats artificiais numa variedade de organismos marinhos tem recebido pouca atenção, apesar de ser fundamental para estabelecer diretrizes para a correta gestão das áreas costeiras urbanizadas. Existe, por tanto, um claro interesse em compreender as consequências ambientais da urbanização, razão que esteve na base do desenvolvimento dum projeto de investigação levado a cabo pelo grupo de Investigação Aquática Macaronésia (IAM) entre os anos 2013 e 2015. O IAM, integrado no CIIMAR é composto por elementos do Departamento de Biologia da Universidade dos Açores, coordenou o projeto “Estruturas urbanas: um fator de mudança na biodiversidade dos ecossistemas costeiros”. O projeto foi financiado pela Fundação para a Ciência e a Tecnologia e contou com a Prof. Ana I. Neto como investigadora responsável e com a Faculty of Science and Environment da Universidade de Plymouth como entidade colaboradora externa. […].info:eu-repo/semantics/publishedVersio

Evaluation of tone-mapping algorithms for focal-plane implementation

Scenes in the real world may simultaneously contain very bright and very dark regions, caused by different illumination conditions. These scenes contain a wide range of different light intensity values. Attempting to exhibit a picture of such scene on a conventional display device, such as a computer monitor, leads to (a possibly large) loss of details in the displayed scene, since conventional display devices can only represent a limited amount of different light intensity values, which span a smaller range. To mitigate the loss of details, before it is shown on the display device, the picture of the scene must be processed by a tone-mapping algorithm, which maps the original light intensities into the light intensities representable by the display, thereby accommodating the input high dynamic range of values into a smaller range. In this work, a comparison between different tone-mapping algorithms is presented. More specifically, the performances (regarding processing time and overall quality of the processed image) from a digital version of the tone-mapping operator originally proposed by Fern´andez-Berni et al. [11] that is implemented in the focal plane of the camera and from different tone-mapping operators that are originally implemented in software are compared. Furthermore, a second digital version of the focal-plane operator, which simulates a modified version of the original hardware implementation, is considered and its performance is analyzed. The modified hardware implementation is less complex and requires less space than the original implementation and, subjectively, keeps the overall image quality approximately equal to that achieved by digital operators. Issues regarding colors of the tone-mapped images are also addressed, especially the required processing that must be performed by the focal-plane operator after the tone mapping, in order to yield images without color distortions.Cenas no mundo real podem conter uma ampla faixa de valores de diferentes intensidades luminosas. Mostrar a cena original em um aparelho de exibição convencional, tal como um monitor de computador, leva a uma (possivelmente grande) perda de detalhes na cena exibida, uma vez que esses aparelhos são capazes de representar somente uma quantidade limitada de diferentes intensidades luminosas, as quais ocupam uma faixa de valores menor. Para diminuir a perda de detalhes, antes de ser exibida em tais aparelhos, a cena deve ser processada por um algoritmo de tone mapping, o qual mapeia os valores originais de intensidade luminosa em valores que são representáveis pelo aparelho de exibição, acomodando, com isso, a alta faixa dinâmica dos valores de entrada em uma faixa de valores menor. Neste trabalho, uma comparação entre diferentes algoritmos de tone-mapping é apresentada. Mais especificamente, são comparados entre si os desempenhos (referentes a tempos de execução e qualidade geral da imagem processada) da versão digital do operador de tone mapping originalmente proposto por Fernández-Berni et al. [11] que ´e implementado no plano focal da câmera e de diferentes operadores de tone mapping que são originalmente implementados em software. Além disso, uma segunda versão digital do operador no plano focal, a qual simula uma versão modificada da implementação original em hardware, é considerada e seu desempenho é analisado. Essa versão modificada requer um hardware que é menos complexo e ocupa menos espaço que o hardware da implementação original, além de, subjetivamente, manter a qualidade geral da imagem próxima daquela alcançada por operadores digitais. Questões referentes às cores das imagens processadas também são tratadas, especialmente os processamentos que são requeridos pelo operador do plano focal após o tone mapping, de modo a gerar imagens sem distorções de cor

A computational framework for sound segregation in music signals

Tese de doutoramento. Engenharia Electrotécnica e de Computadores. Faculdade de Engenharia. Universidade do Porto. 200