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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
31

Morfofisiologia do intestino médio de adultos do mosquito não hematófago Toxorhynchites (Lynchiella) theobaldi (Diptera, Culicidae) / Midgut of the non-hematophagous mosquito Toxorhynchites theobaldi (Diptera, Culicidae)

Godoy, Raquel Soares Maia 29 July 2015 (has links)
Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-16T10:37:27Z No. of bitstreams: 1 texto completo.pdf: 1515494 bytes, checksum: bd156721affe3babff6b245f7cc1e560 (MD5) / Made available in DSpace on 2015-11-16T10:37:27Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1515494 bytes, checksum: bd156721affe3babff6b245f7cc1e560 (MD5) Previous issue date: 2015-07-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Na maioria das espécies de mosquitos, as fêmeas precisam se alimentar do sangue de vertebrados para completar o desenvolvimento de seus ovos. No entanto, em mosquitos do gênero Toxorhynchites, os ovos se desenvolvem sem a dieta de sangue, e ambos, fêmeas e machos desse gênero se alimentam exclusivamente de substâncias açucaradas. O intestino médio é um órgão bem conhecido nos mosquitos que se alimentam de sangue, mas pouco se sabe a seu respeito em mosquitos não-hematófagos. No presente trabalho, foi investigada a morfologia detalhada do intestino médio de Toxorhynchites theolbadi utilizando-se métodos histoquímicos e ultraestruturais. O intestino médio de fêmeas e machos adultos de T. theobaldi consiste em um longo e fino do intestino médio anterior (AMG), e um curto e dilatado intestino médio posterior (PMG). O AMG é subdividido em AMG1 (curto, com pregas) e AMG2 (longo, sem pregas). Ramificações neuronais e células enteroendócrinas estão presentes no AMG e PMG, respectivamente. Em comparação com o PMG de mosquitos-fêmeas que se alimentam de sangue, o PMG de T. theobaldi é menor; no entanto, em ambos os mosquitos, o PMG parece ser a principal região de digestão e absorção de alimentos, e secreção de proteínas. As pregas presentes no AMG de T. theobaldi não foram relatadas em outros mosquitos; no entanto, a organização muscular do intestino médio e o controle endócrino do processo digestivo são conservados em ambos T. theobaldi e mosquitos hematófagos. / In most mosquito species, the females require a blood-feeding for complete egg development. However, in Toxorhynchites mosquitoes, the eggs develop without blood- feeding, and both females and males exclusively feed on sugary diets. The midgut is a well-understood organ in blood-feeding mosquitoes, but little is known about it in non- blood-feeding ones. In the present study, the detailed morphology of the midgut of Toxorhynchites theobaldi was investigated using histochemical and ultrastructural methods. The midgut of female and male T. theobaldi adults consists of a long, slender anterior midgut (AMG), and a short, dilated posterior midgut (PMG). The AMG is subdivided into AMG1 (short, with folds) and AMG2 (long, without folds). Nerve branches and enteroendocrine cells are present in AMG and PMG, respectively. Compared with the PMG of blood-feeding female mosquitoes, the PMG of T. theobaldi is smaller; however, in both mosquitoes, PMG seems be the main region of food digestion and absorption, and protein secretion. The epithelial folds present in the AMG of T. theobaldi have not been reported in other mosquitoes; however, the midgut muscle organization and endocrine control of the digestion process are conserved in both T. theobaldi and blood-feeding mosquitoes.
32

Brassica oleracea lectin: Isolation, characterization, and functional assessment of the first lectin with MATH domains / Lectina de Brassica oleracea: Isolamento, caracterização e avaliação funcional da primeira lectina com domínios MATH

Duarte, Christiane Eliza Motta 16 September 2016 (has links)
Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-01-23T12:20:24Z No. of bitstreams: 1 texto completo.pdf: 2708123 bytes, checksum: 9ac52444154e208b7e0074a15fb94bbd (MD5) / Made available in DSpace on 2017-01-23T12:20:24Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2708123 bytes, checksum: 9ac52444154e208b7e0074a15fb94bbd (MD5) Previous issue date: 2016-09-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Lectinas estão envolvidas em uma ampla variedade de processos biológicos, inclusive atuando como agentes imunomoduladores capazes de ativar a imunidade inata. Nós purificamos e caracterizamos uma nova lectina de couve-flor (Brassica oleracea ssp. botrytis – BOL) através de três etapas sequenciais de cromatografia, cuja pureza foi confirmada por SDS-PAGE. Além disso, avaliamos o papel dessa lectina na imunidade inata por meio de um ensaio de fagocitose, produção de H 2 O 2 e NO. BOL foi caracterizada como uma proteína não glicosilada com uma massa molecular de ~ 34 kDa em SDS-PAGE. Para otimizar o processo de obtenção da lectina e possibilitar um estudo mais aprofundado de sua estrutura e função, realizou-se a clonagem molecular e expressão heteróloga de BOL. Para isso utilizamos RNA total extraído de plântulas de couve-flor e isolamos a sequência de 1053 pb do cDNA codificante. Ferramentas de bioinformática foram utilizadas para determinar a sequência promotora de 1000 pb de Bol, a qual revelou vários elementos cis-regulatórios conhecidos por estarem envolvidos em várias condições de estresse na planta. A análise comparativa tecido-específica da expressão de Bol demonstrou níveis mais elevados do transcrito nas folhas, em comparação aos tecidos do caule e da raiz. A análise da sequência de aminoácidos e o alinhamento com proteínas homólogas deduzidas nos permitiu determinar que a proteína madura é constituída por 301 aminoácidos. A estrutura tridimensional predita confirmou que esta lectina apresenta uma estrutura em forma de cúpula com dois domínios MATH. Este é o primeiro relato do isolamento, clonagem e expressão bacteriana de uma lectina com domínios MATH e pode ser de interesse significativo para compreender o papel regulador desta proteína como agente imunoestimulante bem como para à fisiologia da própria planta. / Lectins are involved in a wide range of biological mechanisms, including act as immunomodulatory agent able to activate the innate immunity. We purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H 2 O 2 and NO. BOL was characterized as a non-glycosylated protein with a molecular mass of ~34 kDa in SDS-PAGE. To optimize the process of the lectin obtaining and allow further study of their structure and function, the molecular cloning and heterologous expression of BOL were carried out. Using total RNA extracted from cauliflower seedlings a Bol coding cDNA sequence of 1053 bp was isolated. Bioinformatics tools were used to determine a promoter sequence of 1000 bp of Bol which revealed several key cis-regulatory elements known to be involved in various plant stresses. Comparative expression analysis of tissue specific Bol demonstrated the highest transcript levels in leaves, as compared to stem and root tissues. Analysis of amino acid sequence and alignment with deduced homologous proteins allowed us to determine that the mature protein comprises 301 amino acids. Predicted three-dimensional structure confirmed that this lectin had an overall dome-like structure with two MATH-domains. This is the first report of isolation, cloning and bacterial expression of a lectin with MATH-domains and may be of significant interest to understand the regulatory role of this protein as immunostimulatory agent as well as to the physiology of the plant itself.
33

Dinámica biogeoquímica de nutrientes y metales pesados en ambientes intermareales de la laguna costera Mar Chiquita : potenciales efectos ecotoxicológicos sobre especies claves del ecosistema

Beltrame, María Ornela 23 March 2009 (has links)
Las lagunas costeras son ambientes estuariales con una alta dinámica y con características ambientales particulares, pues conectan el límite continental con la zona costera. Estos ecosistemas altamente productivos son relevantes para la conservación de la biodiversidad, ya que son refugio de aves migratorias y de la biota acuática. Sin embargo, son muy sensibles a las actividades antrópicas, puesto que están ubicados al final de las cuencas de drenaje. Se caracterizan por ser sitio de conjunción entre dos flujos hidrológicos: la descarga de agua continental proveniente de los tributarios y las intrusiones marinas. Mar Chiquita es la única laguna costera del tipo semiobturada de canal largo en la Argentina. Está situada en el partido de Mar Chiquita, al sudeste de la Provincia de Buenos Aires, entre los 3733 3743 S y los 5715 5730 O. Esta laguna fue declarada Reserva Mundial de Biosfera por el Programa Man and Biosphere de la UNESCO. Posee una cuenca de drenaje que supera los 10.000 km2 y recibe el aporte de gran cantidad de arroyos y canales artificiales, los cuales forman una extensa cuenca tributaria, que incluye áreas afectadas por numerosas actividades humanas, principalmente agrícolo-ganaderas. Teniendo en cuenta que en esta zona se realiza un uso intensivo de fertilizantes, plaguicidas y otros agroquímicos, la cuenca de drenaje constituye una fuente potencial de contaminantes cuyo destino es esta laguna. Por otras parte, una de las especies claves de este ambiente es el cangrejo cavador Neohelice granulata. En este estudio se analizó la distribución espacio temporal de parámetros hidrográficos (temperatura, salinidad, turbidez, oxígeno disuelto, pH), nutrientes (nitritos, nitratos, amonio, fosfatos y silicatos) y pigmentos fotosintéticos (clorofila a y feopigmentos) en agua de columna e intersticial. Por otra parte, se determinaron las concentraciones de metales pesados, Cu, Zn, Cd, Pb, Cr, Fe y Mn en muestras de sedimentos superficiales, agua intersticial y agua de columna en planicies de marea de la laguna. Además estos parámetros también fueron analizados en los tributarios de la laguna con el objetivo de evaluar sus aportes hacia el sistema. También se analizaron muestras de tejido de N. granulata con el fin de evaluar la bioacumulación de metales en esta especie. Los resultados indican que los nutrientes estuvieron presentes durante todo el año, y en muchos casos sus concentraciones fueron elevadas. Los rangos de concentración para cada uno de ellos fueron: amonio (nd130,04 μmol.L-1), nitratos (0,99-149 μmol.L-1), nitritos (0,11-2,47 μmol.L-1), fosfatos (0,37-18,72 μmol.L-1) y silicatos (8,17-880,22 μmol.L-1) y presentaron un comportamiento temporal, con un pico máximo de concentración durante los meses de primavera y otro pico de concentración menor a fines de otoño-principios de invierno. Las concentraciones de pigmentos fotosintéticos mostraron un comportamiento similar al de los nutrientes (el rango de concentración de la clorofila a fue nd-76,47 μg.L-1 y el de los feopigmentos fue nd-47,34 μg.L-1). Los metales estudiados no presentaron un patrón de comportamiento definido en función al tiempo ni en función a las mareas, y en varias ocasiones presentaron niveles muy elevados. Los rangos presentes de cada metal disuelto en agua de columna (AC) fueron (μg.L-1): Cd nd (no detectable)- 4,3; Cu nd-1000,23; Pb nd-850; Zn nd-1224,38; Fe nd-227,15; Cr nd-35,25 y Ni nd-79,84; mientras que en agua intesticial (AI) fueron (μg.L-1) : Cd nd-1,14; Cu nd-690,28; Pb nd-430, Zn nd-1395, Fe nd-236,07, Cr nd-38,01 y Ni nd-95,65. En tanto, los rangos de los metales en sedimento superficial fueron (μg.g-1): Cd 0,86-3,01; Cu nd-21,98; Pb nd-30,49; Zn 5,10-47,68; Fe nd-47900; Cr 0,34-8,45; Mn 58,76-1116,06 y Ni 5,34-34,89. Las concentraciones de metales en material particulado en suspensión (MPS) fueron (μg.g-1): Cd nd-58,64; Cu nd-22921; Pb nd-345,84; Zn nd-16922; Fe 49-54680; Cr nd-59,36; Mn 2,87-2415,88 y Ni nd-16327. El comportamiento de cada uno de los tributarios con respecto a los parámetros hidrológicos, nutrientes y metales pesados fue diferente. Cada uno presentó diferentes concentraciones de los compuestos estudiados de acuerdo a su recorrido y a las fuentes por las cuales atraviesa. Si bien todos los afluentes transportan grandes concentraciones de nutrientes, el Arroyo Grande y el Canal 7 son los tributarios que aportan en mayor grado compuestos nitrogenados hacia la laguna, mientras que los metales pesados son transportados en diferentes magnitudes por cada uno de los tributarios. Finalmente se realizaron ensayos de toxicidad de Zn en el cangrejo N. granulata. Este metal fue seleccionado por considerarlo de potencial riesgo para el ambiente bajo estudio. En esta tesis se evaluaron los efectos que produce este metal, y se demostró que puede interferir tanto en el proceso de muda como en el desarrollo embrionario de esta especie. Desde un punto de vista ecotoxicológico, los efectos observados en los ensayos de toxicidad realizados representan una información de gran utilidad para evaluar el status ambiental de esta laguna costera, no sólo considerando los niveles presentes de este metal en este ecosistema, sino también los potenciales efectos de este contaminante sobre su biota. Los estudios de bioacumulación en N. granulata indicaron que esta especie puede considerarse un potencial biomonitor para esta laguna costera. El conjunto de los resultados obtenidos durante este trabajo permiten sostener que las actividades antrópicas desarrolladas en la cuenca de drenaje, especialmente las actividades agrícolo-ganaderas, tienen una gran influencia sobre la condición ambiental de esta laguna. / Coastal lagoons are estuarine environments with a high dynamic and particular environment characteristics, because they connect the continental limit with the coastal zone. These highly productive ecosystems are relevant for the biodiversity conservation, since they are refuge for some migratory birds and aquatic biota. However, they are very sensitive to anthropogenic activities, since they are located at the end of the drainage basin. They are sites where two hydrologic flows meet: the continental water discharge of the tributaries and the marine intrusions. Mar Chiquita is the unique semiobtured and long channel coastal lagoon within Argentine. It is located between 3733 3743 S and 5715 5730 W within the Atlantic coast of Buenos Aires Province. It has been declared a Biosphere Reserve under the UNESCO Man and Biosphere Programme. It has a tributary basin of 10.000 km2. It receives a great input from streams and artificial channels, which together conform an extensive drainage basin, that includes areas with several human activities, mainly agriculture. Considering that in this area an intensive use of fertilizers, pesticides and other agrochemicals is developed, this drainage basin constitutes a potential source of pollution for this lagoon. On the other hand, one of the key species of this environment is the burrowing crab Neohelice granulata. In this study the spatial and temporal distribution of several hydrographic parameters (temperature, salinity, turbidity, dissolved oxygen, pH), nutrients (nitrites, nitrates, phosphates, silicates), ammonium and photosynthetic pigments (chlorophyll a and phaeopigments) in column and interstitial water was analyzed. On the other hand, heavy metal concentrations, (Cu, Zn, Cd, Pb, Cr, Fe y Mn) in samples of superficial sediments, interstitial and column water in intertidal environments were determined. Moreover, these parameters were analysed in all the tributaries and was evaluated the contribution of each one to the lagoon. Also, N. granulata tissues samples were analyzed in order to evaluate the metal bioaccumulation within this species. The obtained results indicate that nutrients were present during all the year at high concentrations. Nutrient concentrations ranged nd130,04 μmol.L-1 for ammonium, 0,99-149 μmol.L-1 for nitrates, 0,11-2,47 μmol.L-1 for nitrites, 0,37-18,72 μmol.L-1 for phosphates, and 8,17-880,22 μmol.L-1 for silicates. These nutrients showed a seasonal trend, with maximum values during spring and other minor peak during the end of autumn and early winter. Photosynthetic pigments concentrations showed a trend similar to that of nutrients (chlorophyll a range was nd-76,47 μg.L-1 and phaeopigments range was nd- 47,34 μg.L-1). Heavy metals did not present any particular behaviour, neither temporal or seasonal, nor tidal influence, and occasionally were present in high levels. Concentrations within water column were (in μg.L-1 units): nd (no detectable)-4,36 for Cd, nd-1000,23 for Cu, nd-850 for Pb, nd-1224,38 for Zn, nd-227,15 for Fe, nd-35,25 for Cr and nd- 79,84 for Ni. Heavy metal concentrations within pore water were (in μg.L-1 units): Cd nd-1,14; Cu nd-690,28; Pb nd-430; Zn nd-1395; Fe nd-236,07; Cr nd-38,01 and Ni nd-95,65. Heavy metals concentrations within surface bottom sediments were (in μg.g-1 units) Cd 0,86-3,01; Cu nd- 21,98; Pb nd-30,49; Zn 5,10-47,68; Fe nd-47900; Cr 0,34-8,45; Mn 58,76-1116,06 y Ni 5,34-34,89. Heavy metals concentrations within suspended particulate matter (SPM) were (in μg.g- 1units): Cd nd-58,64; Cu nd-22921; Pb nd-345,84; Zn nd-16922; Fe 49-54680; Cr nd-59,36; Mn 2,87-2415,88 and Ni nd-16327. Tributaries behaviour regarding their hydrological parameters, nutrients and heavy metals was different among them, since they showed different concentrations according to their route and sources through which they run. All affluents transported great amounts of nutrients, but Arroyo Grande stream and Canal 7 channel are the main contributors of nitrogen compounds towards Mar Chiquita coastal lagoon, whereas heavy metals are transported in different amounts by each tributary. Finally, Zn toxicity bioassays on the crab N. granulata were developed. This metal was selected because constitutes potential risk for this environment. This study evaluated the effects of this metal on the embryonic development and on the molt process. It was demostrated that this metal can interfere in both processes and generate deletereous effects. From an ecotoxicological point of view, the observed effects represent information of great utility in order to evaluate the environmental status of this coastal lagoon, considering not only the present levels in this ecosystem, but also the potential effects of this pollutant on the biota. Bioaccumulation studies on N. granulata indicated that this species can be considered as a potential biomonitor within this coastal lagoon. Results as obtained within this study allows to sustain that anthropogenic activities developed within the drainage basin, specially agriculture activities, have a great influence on the environmental conditions of this coastal lagoon.
34

Biología y ultraestructura de codium spp. (bryopsidophyceae, chlorophyta) : morfologías vegetativa y reproductiva, ciclos de vida y epifitismo

Miravalles, Alicia Beatriz 17 April 2009 (has links)
Se estudiaron poblaciones de tres taxa del género Codium que crecen en las costas de las Provincias de Buenos Aires, Río Negro y Chubut: C. decorticatum, C. fragile subsp. novae-zelandiae y C. vermilara. Los talos de los tres taxa, erguidos y ramificados dicotómicamente, se fijaban al sustrato mediante un disco basal. El tamaño y forma de los utrículos y gametangios, presencia/ausencia de mucrón fueron las características principales que permitieron diferenciar los taxa. Ultraestructuralmente los utrículos mostraron una vacuola central rodeada por una fina capa de citoplasma parietal en la que se encontraban las organelas. La ultraestructura de los pelos y de los filamentos medulares fue similar a la de los utrículos. Los gametangios se formaban a los lados de los utrículos. Se observaron gametangios femeninos en los tres taxa y masculinos en C. fragile. Los masculinos eran más delgados que los femeninos. La liberación de ambos tipos de gametas inmersas en abundante mucílago se produjo a través de la ruptura del extremo apical. Este es el primer estudio ultraestructural del proceso de gametogénesis y de las gametas femeninas y masculinas en el género Codium. Las gametogénesis se iniciaban con la división de núcleos y cloroplastos. La delimitación de las gametas se producía por la actividad de vesículas dictiosómicas que rodeaban porciones de protoplasma uninucleadas. Las gametas femeninas eran piriformes; poseían una papila apical de la que emergían los flagelos. Anteriormente se encontraban el núcleo y una mitocondria grande con forma de V invertida. El resto de la gameta estaba ocupado por numerosos cloroplastos. Las gametas masculinas eran ovoides alargadas, de menor tamaño; poseían dos o tres cloroplastos escasamente desarrollados y una papila apical hialina de la que emergían los flagelos. Se comprobó la germinación partenogenética de las gametas femeninas de C. fragile en ausencia de gametas masculinas. También se observó la germinación intragametangial de las gametas femeninas. Se verificó la reproducción sexual en C. fragile en presencia de ambos tipos de gametas por la presencia de cigotos tetraflagelados y su posterior germinación. Para cada uno de los tres taxa se proporcionó información cariológica, con un número haploide de 10, como el obtenido por otros autores para otras especies del género. Se confirmó la partenogénesis para las tres especies. Se determinó que C. vermilara y C. decorticatum presentaban un ciclo de vida haplobionte haplonte y C. fragile un ciclo de vida haplobionte haplonte o diplonte, de acuerdo a la capacidad de formación de gametas masculinas. Se identificaron las distintas especies de epífitos sobre C. vermilara de la localidad de Las Grutas. Los géneros de epífitos más comunes fueron Ceramium, Polysiphonia, Callithamnion y Ectocarpus. De acuerdo al grado de invasión de las estructuras de fijación se diferenciaron cuatro modos de fijación de los epífitos al hospedante. Se estudió con microscopía óptica y electrónica la interfase entre los epífitos y el hospedante para determinar el grado de daño que ejercía el epífito al hospedante. Las distintas especies de epífitos que crecían sobre Codium y entre sus utrículos no causaban daño al talo. / The purpose of this thesis was to study populations of C. decorticatum, C. fragile subsp. novae-zelandiae and C. vermilara of the genus Codium growing on the coastal areas of the provinces of Buenos Aires, Río Negro and Chubut, Argentina. Their thalli were found to be erect and dichotomously branched and they were fixed to the substrate by a basal disk. The following characteristics were crucial for the differentiation of these taxa: i) size and shape of utricula and gametangia, and ii) presence and/or absence of mucron. At the ultrastructural level, it could be observed that utricula showed a central vacuole which was surrounded by a thin layer of parietal cytoplasm in which organella were located. The ultrastructure of hairs and medular filaments was similar to that of utricula. Gametangia were formed on the sides of utricula. Female gametangia were observed in the three taxa analyzed whereas male gametangia were found only in C. fragile and they were thinner than the female ones. The release of both types of gametes, which were immersed in abundant mucilage, occurred through a slit. This is the first ultrastructural study conducted to date on gametogenesis and on female and male gametes in the genus Codium. Findings from this thesis indicate that gametogenesis begins with the division of nuclei and chloroplasts and that gametes are individualized as a result of the activity of dyctiosomic vesicles which surround uninucleate portions of protoplasm. It could also be observed that female gametes are pyriform and that they have an apical papilla from which flagella emerge. The nucleus and a large inverted V-shaped mitochondrium were found to be located in the anterior region of gamete while the rest of the gamete was occupied by numerous chloroplasts. Male gametes were found to be ovoid and elongated, they were smaller than female gametes and they had two or three chloroplasts poorly developed and a hyaline apical papilla from which flagella emerged. Parthenogenetic germination of female gametes of C. fragile was observed in the absence of male gametes. Intragametangial germination of female gametes was also observed. On the other hand, the presence of tetraflagellate zygotes and their subsequent germination in C. fragile was indicative of sexual reproduction in this species. Karyological information was provided for each of the three taxa with a haploid number of 10. The latter agreed with that obtained by other authors for other species of the genus. Parthenogenesis was confirmed for the three species. Also, it could be concluded that C. vermilara and C. decorticatum have a haplobiont haplont life cycle and that C. fragile, depending on the presence or absence of male gametes, has either a haplobiont diplont or haplont life cycle. Different species of epiphytes on C. vermilara could also be identified in Las Grutas, Argentina, the most common epiphytic genera being Ceramium, Polysiphonia, Callithamnion and Ectocarpus. Taking into account the degree of invasion of fixing structures, four modes of attachment of epiphytes to the host were identified. The epiphyte-host interface was studied via optical and electron microscopy in order to determine the degree of damage exerted by the epiphyte to the host. It could be observed that the different species of epiphytes growing on Codium and among its utricula induce no damage to the thallus.
35

Identificação e análise de sequências codificantes com atributos conflitantes em genomas procariotos / Analysis and identification of prokaryotic coding sequences with confliting atributes eng

Guadalupe Del Rosario Quispe Saji 23 August 2010 (has links)
O advento de novas tecnologias de sequenciamento e o desenvolvimento de ferramentas computacionais que facilitam a análise dos genomas gerou o aumento exponencial dos bancos de dados genômicos. As abordagens in-silico da genômica comparativa usam esse tipo de dados nas suas comparações. Trabalhos recentes desenvolvidos sobre o genoma de Escherichia coli indicam que o estado atual das sequências codificantes (CoDing Sequences CDS) de genomas anotados nos bancos de dados contêm erros nas sequências que precisam ser verificados (Ochman e Davalos 2006). Portanto a correta descrição de uma CDS é importante para permitir futuras comparações genômicas. Atualmente existe uma nova proposta da comunidade científica de bancos de dados biológicos para estabelecer padrões para a submissão de sequências dos projetos de genoma na nova era de sequenciamento. Dentro desse contexto, destaca-se a identificação e/ou correção de frameshifts durante o processo de montagem de sequências genômicas. A finalidade deste trabalho foi desenvolver uma ferramenta com dois métodos comparativos para identificar CDSs com atributos conflitantes. Usa-se a descrição de conflito para descrever atributos como frameshifts, grandes inserções ou deleções, truncamentos, que são detectados a partir de uma CDS ou várias CDSs usadas como referência, dependendo do modelo. Finalmente, a ferramenta proposta permite visualizar os resultados graficamente e fornecer acesso a outras ferramentas, dando suporte para futuras análises genômicas de maneira amigável e rápida. Foi realizada a busca de CDSs com atributos conflitantes no genoma de E. coli estirpe CFT073 (NCBI) versão AE014075.1, (última data de atualização: 20 de abril do 2006), como referência foi usado o genoma da E.coli estirpe O157:H7 EDL933 versão AE005174.2 ( última data de atualização : 6 de junho do 2008). Através dessa análise foram identificadas e armazenadas 1.865 CDSs (incluem-se possíveis parálogos), por apresentarem alinhamentos únicos com cobertura superior a 30% da CDS de referência. Em uma análise mais fina destes resultados, sobressaltam 144 CDSs no genoma alvo que provavelmente apresentam frameshifts, dos quais 21 acontecem em regiões intergênicas. A ferramenta desenvolvida neste trabalho foi também aplicada para o caso de estudo de uma região genômica da bactéria Klebsiella pneumoniae estirpe KP13. O genoma dessa bactéria foi sequenciado na Unidade Genômica Computacional (UGC) Darcy Fontoura de Almeida do LNCC (dados ainda não publicados). A partir das análises destes genomas, pode se concluir a importância do uso da ferramenta nas fases de identificação, verificação e correção de erros de anotação e, portanto a necessidade da sua inclusão em projetos de sequenciamento que desejam atingir altos padrões na submissão de dados genômicos. / The advent of new sequencing technologies and the development of computational tools that facilitate the analysis of genomes, generated the exponential growth of genome databases. New approaches in-silico of the comparative genomics use such data in its comparisons. Nevertheless, recent work on the genome of Escherichia coli indicate that the current state of coding sequences (Coding Sequences - CDS) from annotated genomes contain several errors, which need to be verified (Ochman e Davalos 2006). Therefore the correct description of a CDS is important to allow future genomic comparisons. Currently, there is an innovated proposal of the scientific community of biological databases to establish standards for the submission of the draft genome sequences in the new era of sequencing. Within this context, it is highlighted the identification and/or correction of frameshifts during the assembly of genomic sequences. The goal of this work was developing a tool with two comparative methods to identify CDSs with conflicting attributes. It uses the description of conflict to describe attributes such as frameshifts, large insertions or deletions, truncations, etc.. that are detected from a CDS or several CDSs used as references, depending on model. Also, the proposed tool allows to user to view of the results graphically and provide access to other tools, providing support for future friendly and faster genomic analysis. As a model of study, it was used the analysis of CDSs with conflicting attributes of the genome of E. coli strain CFT073 (NCBI) version AE014075.1, (last update date: April 20 of 2006), with this purpose was used as a reference genome of E.coli strain O157: H7 EDL933 version AE005174.2 (last update date: 6 June of 2008). Through this analysis were identified and stored 1865 CDSs (Included possible paralogs) because they present only alignments with coverage exceeding 30% of the CSD of reference. In a more detailed analysis of these results, 144 CDSs startle in the target genome by probably present frameshifts, of which 21 occur in intergenic regions. The tool developed in this work, also was applied to the case study of a genomic region of the bacterium Klebsiella pneumoniae strain KP13. The genome of this bacterium was sequenced in Computational Genomics Unit (UGC) Darcy Fontoura de Almeida LNCC (unpublished data). From the analysis of these genomes, one can conclude the importance of using the tool in the stages of identification, verification and correction of errors in annotation,and thus the need for its inclusion in the sequencing projects that want to reach high standards in the submission of genomic data.
36

Estresse p?s-traum?tico e tratamento com cetamina em ratos Wistar : an?lise comportamental, histofisiol?gica, bioqu?mica e neurometab?lica

Saur, Lisiani 12 January 2017 (has links)
Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-01T15:01:05Z No. of bitstreams: 1 TES_LISIANI_SAUR_COMPLETO.pdf: 5532574 bytes, checksum: 990865e2748aa19880737e88991ec082 (MD5) / Made available in DSpace on 2017-06-01T15:01:05Z (GMT). No. of bitstreams: 1 TES_LISIANI_SAUR_COMPLETO.pdf: 5532574 bytes, checksum: 990865e2748aa19880737e88991ec082 (MD5) Previous issue date: 2017-01-12 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Funda??o de Amparo ? Pesquisa do Estado do Rio Grande do Sul (FAPERGS) / Post-traumatic stress disorder (PTSD) is a neuropsychiatric condition related to exposure to a traumatic event. It is clinically characterized by several debilitating symptoms including frequent re-experiencing of the traumatic event, avoidance bahavior, hypervigilance, and cognitive and mood changes among others. Not all individuals exposed to severe traumatic experiences develop PTSD. This psychopathology affects a vulnerable subpopulation of individuals confronted with a stressful experience that exceeds their capacity to cope. In this sense, it has been argued that PTSD can be considered a fear-related disorder. Thus, the brain regions most widely studied in this psychopathology are the prefrontal cortex, the hippocampus and the amygdala, because they are related to the storage and consolidation of new memories including emotional memories, such as fear and anger. Due to the complex clinical presentation of PTSD and its very variable symptoms, it is considered a condition to treat. Ketamine is an NMDA receptor antagonist that has shown promising effects in the treatment of depression. However, because it produces dissociative and psychotic effects, there is a concern this drug might be related with increased PTSD symptoms. One of the most widely used animal models to mimic the behavioral and neurochemical changes of PTSD is the inescapable footshock. In the first part of this study, we observed significant behavioral and histological changes caused by exposure to footshock. Animals with PTSD exhibited longer freezing bouts when re-exposed to the same aversive context, as well as increased pellet production. In addition, important changes were observed in astrocytes from the hippocampal CA1 region of these animals, such as decreased astrocytic density, ramification and fewer primary processes. Furthermore, the polarity of the astrocytes was also changed when compared to control animals. In the second part of this study, we analyzed the effects of ketamine on this animal model. Since not all animals have the same response during the re-exposure to aversive environment, that is, the animals have individual differences in their susceptibility to traumatic stress, we decided to separate the animals with PTSD into two groups: those with an extreme behavioral response (EBR) and those with a minimal behavioral response (MBR). Furthermore, the glucose metabolism and the BDNF levels in the frontal cortex, hippocampus and amygdala were analyzed 8 and 9 days after PTSD induction, respectively. BDNF levels were analyzed through biochemical assays and glucose metabolism was analyzed by 18F-FDG-microPET, which measures the glucose uptake in tissues, in other words, the metabolic demand. We observed that animals with PTSD classified as EBR showed an increase in freezing behavior and the treatment with ketamine worsened that behavioral response, that is, the ketamine worsened the PTSD symptoms. However, no changes were observed in BDNF levels or glucose metabolism. These results demonstrate that footshock as an animal model of PTSD induced significant behavioral and astrocytic alterations. We also observed that ketamine worsened the PTSD symptoms and that, as is the case with humans, not all individuals that are exposed to a traumatic stress develop PTSD. However, this animal model does not seem to be related to long-term changes in BDNF levels or glucose metabolism. / O transtorno do estresse p?s-traum?tico (Post-Traumatic Stress Disorder - PTSD) ? uma doen?a neuropsiqui?trica relacionada ? exposi??o a um acontecimento traum?tico. O PTSD ? caracterizado clinicamente por uma s?rie de sintomas debilitantes incluindo a reviv?ncia frequente do evento traum?tico, fuga de est?mulos associados ao trauma, hipervigil?ncia, mudan?as de cogni??o e humor entre outros. Nem todos os indiv?duos expostos a graves experi?ncias traum?ticas desenvolvem PTSD, esta psicopatologia afeta uma subpopula??o de indiv?duos vulner?veis cuja exposi??o a uma experi?ncia estressante excede sua capacidade de adapta??o. Neste sentido, foi argumentado que o PTSD pode, pelo menos em parte, ser um dist?rbio relacionado ao medo. Assim, as regi?es encef?licas mais amplamente estudadas nesta psicopatologia s?o o c?rtex pr?-frontal, o hipocampo e a am?gdala, pois s?o regi?es relacionadas com os processos de armazenamento e consolida??o de novas mem?rias incluindo mem?rias emocionais, tais como de medo e raiva. Devido ? complexa apresenta??o cl?nica do PTSD e a sua sintomatologia muito vari?vel esta ainda ? uma doen?a classificada como de dif?cil tratamento. A cetamina ? um f?rmaco antagonista de receptores NMDA que tem demonstrado promissores efeitos no tratamento da depress?o, entretanto, por ser uma droga que produz efeitos dissociativos e psic?ticos, existe a preocupa??o de que esta droga possa estar relacionada com o aumento dos sintomas do PTSD. Um dos modelos animais mais amplamente utilizados para mimetizar as altera??es comportamentais e neuroqu?micas do PTSD ? o choque inescap?vel ?nico. Na primeira parte do trabalho observamos importantes altera??es comportamentais e histol?gicas provocadas pela exposi??o ao choque nas patas. Os animais com PTSD apresentaram um maior comportamento de ?freezing? quando reexpostos ao mesmo contexto aversivo, bem como maior produ??o de bolos fecais. Al?m disso, importantes altera??es foram observadas nos str?citos da regi?o CA1 do hipocampo destes animais como: diminui??o na densidade, arboriza??o e quantidade de processos prim?rios. Ademais, a polaridade astrocit?ria tamb?m foi alterada em rela??o aos animais controle. Na segunda parte do trabalho analisamos os efeitos da cetamina sobre este modelo animal. Como nem todos os animais apresentam a mesma resposta durante a reexposi??o ao contexto aversivo, ou seja, os animais apresentam diferen?as individuais na sua susceptibilidade ao estresse traum?tico, n?s decidimos separar os animais com PTSD em dois grupos: aqueles que apresentaram uma resposta comportamental exagerada (Extreme Behavioral Response - EBR) e aqueles que apresentaram uma resposta comportamental m?nima (Minimal Bahavioral Response - MBR). Al?m disso, o metabolismo da glicose e os n?veis de BDNF no c?rtex frontal, hipocampo e am?gdala foram analisados oito e nove dias ap?s a indu??o do PTSD, respectivamente. Os n?veis de BDNF foram analisamos atrav?s ensaios bioqu?micos e o metabolismo da glicose foi analisado por meio do 18FFDG- microPET que avalia o consumo da glicose nos tecidos. Observamos que os animais com PTSD classificados como EBR apresentaram um aumento no comportamento de freezing e que o tratamento com cetamina piorou a resposta comportamental, ou seja, a cetamina agravou os sintomas do PTSD. Entretanto, nenhuma altera??o foi observada nos n?veis de BDNF e no metabolismo da glicose. Estes resultados demonstram que o choque nas patas, como modelos de PTSD em animais, induz importantes altera??es comportamentais e astrocit?rias. Observamos tamb?m que a cetamina piora os sintomas do PTSD e que, tal qual observado em humanos, nem todos os indiv?duos expostos a um estresse traum?tico desenvolvem os sintomas. Contudo, este modelo experimental de PTSD parece n?o estar relacionado com altera??es de longo-prazo nos n?veis de BDNF e metabolismo da glicose.
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Biologia de sistemas computacional aplicada ? via metab?lica do chiquimato : enfoque na enzima 3-desidroquinato desidratase (EC 4.2.1.10)

?vila, Maur?cio Boff de 20 March 2017 (has links)
Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-29T13:54:15Z No. of bitstreams: 1 DIS_MAURICIO_BOFF_DE_AVILA_COMPLETO.pdf: 2632464 bytes, checksum: 1a170d33bfad88ea8a7aba65cb0f4bea (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-29T13:54:25Z (GMT) No. of bitstreams: 1 DIS_MAURICIO_BOFF_DE_AVILA_COMPLETO.pdf: 2632464 bytes, checksum: 1a170d33bfad88ea8a7aba65cb0f4bea (MD5) / Made available in DSpace on 2017-06-29T13:54:34Z (GMT). No. of bitstreams: 1 DIS_MAURICIO_BOFF_DE_AVILA_COMPLETO.pdf: 2632464 bytes, checksum: 1a170d33bfad88ea8a7aba65cb0f4bea (MD5) Previous issue date: 2017-03-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Microorganisms, in general, are the major agents of disease in humans. Data from the Brazilian Ministry of Health show bacterial diseases as the main causes of death in the country. In the therapy of these organisms, antibiotics are considered the most successful chemotherapy methods of 21st century medicine, as they represent the first, and only, line of combat against bacterial diseases. The development of new antibiotic drugs is becoming increasingly necessary, as bacterial resistance rates become higher each year. At this point, shikimate pathway is attractive to this type of research, since it is considered an essential pathway for the maintenance of these organisms in the environment, besides being absent in animals. The pathway is responsible for the formation of chorismate, precursor of aromatic amino acids (Phe, Trp and Tyr), folic acid and ubiquinones in the groups of organisms that presents it. The third reaction of the shikimate biosynthetic pathway is performed by the enzyme DHQD. In this step the reversible dehydration of the DHQ molecule is performed aiming to transform into 3-dehydroshikimate, the focus reaction of this study. In the search for new DHQD inhibitors, docking simulations were performed against the three-dimensional structure of a target protein, since it is a process that seeks to find, among possible orientations/conformations of a ligand in the active site, the one that presents the lower binding energy and, consequently, greater affinity. In addition to the docking simulations, machine learning methods were used to formulate polynomial scoring functions, based on the MVD scoring functions, which were able to predict protein/binder affinity. At the end of all the simulations and tests carried out throughout the project, we conclude that the Polscore56 equation was the most skilled to predict the affinity between the active site of DHQD and tested compounds. For this polynomial, the results of test set (? = 0,900; p-value = 0,037), AUC (74,686%), EF1 (540) and EF2 (159,23) were, in most of the categories evaluated, the best, confirming the formulated hypotheses on the equation and indicating it for further studies with the enzyme. / Microrganismos, em geral, apresentam-se como os principais agentes de doen?as em seres humanos. Dados do Minist?rio da Sa?de Brasileiro demonstram doen?as bacterianas como as principais causas de morte no pa?s. Na terap?utica desses organismos, os antibi?ticos s?o considerados os m?todos quimioter?picos de maior sucesso da medicina do s?culo XXI, pois representam a primeira, e ?nica, linha de combate contra doen?as bacterianas. O desenvolvimento de novas drogas antibi?ticas torna-se cada vez mais necess?rio, uma vez que os ?ndices de resist?ncia bacteriana se tornam mais altos a cada ano. Nesse ponto, a rota metab?lica do chiquimato ? atraente a esse tipo de pesquisa, por ser considerada uma via essencial para a manuten??o desses organismos no ambiente, al?m de estar ausente em animais. A via ? respons?vel pela forma??o do corismato, precursor de amino?cidos arom?ticos (Phe, Trp e Tyr), ?cido f?lico e ubiquinonas nos grupos de seres vivos onde est? presente. A terceira rea??o da via biossint?tica do chiquimato ? realizada pela enzima DHQD. Nesse passo ? realizada a desidrata??o revers?vel da mol?cula DHQ visando transform?-la em 3-desidrochiquimato, rea??o foco desse estudo. Na busca por novos inibidores de DHQD foram realizadas simula??es de docking de pequenos ligantes contra a estrutura tridimensional de uma prote?na alvo, pois ? um processo onde se visa encontrar, entre as poss?veis orienta??es/conforma??es de um ligante no s?tio ativo, aquela que apresenta a menor energia de liga??o e, consequente, maior finidade. Al?m das simula??es de docking, foram realizados m?todos de Aprendizagem de M?quina na formula??o de fun??es escores polinomiais, a partir de fun??es escores presentes no MVD, que fossem capazes de prever a afinidade entre prote?na/ligante. Ao final de todas as simula??es e testes realizados ao longo do projeto, chegamos ? conclus?o de que a equa??o Polscore56 apresentou-se como a mais h?bil para prever a afinidade entre o s?tio ativo de DHQD com compostos testados. Para esse polin?mio os resultados de test set (? = 0,900; p-value = 0,037), AUC (74,686%), EF1 (540) e EF2 (159,23) foram, na maioria das categorias avaliadas, os melhores, confirmando as hip?teses formuladas sobre a equa??o e indicando-a para estudos posteriores com a enzima.
38

Investiga??o dos efeitos do pept?deo liberador de gastrina (GRP) e seu antagonista RC-3095 em c?lulas mieloides

Czepielewski, Rafael Sanguinetti 18 March 2016 (has links)
Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-24T18:23:29Z No. of bitstreams: 1 RAFAEL_SANGUINETTI_CZEPIELEWSKI_TES.pdf: 5487109 bytes, checksum: 88dbb99b9597e08253e2832cc01ccfce (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-25T17:50:46Z (GMT) No. of bitstreams: 1 RAFAEL_SANGUINETTI_CZEPIELEWSKI_TES.pdf: 5487109 bytes, checksum: 88dbb99b9597e08253e2832cc01ccfce (MD5) / Made available in DSpace on 2017-07-25T18:00:09Z (GMT). No. of bitstreams: 1 RAFAEL_SANGUINETTI_CZEPIELEWSKI_TES.pdf: 5487109 bytes, checksum: 88dbb99b9597e08253e2832cc01ccfce (MD5) Previous issue date: 2016-03-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Tumor microenvironment and inflammatory diseases promote alterations in our immune system along with their development. Several molecules are implicated in this modulation and are therefore considered therapeutic targets. Gastrin-releasing peptide (GRP) is produced in tumors where it promotes cellular proliferation. It is also correlated with chronic diseases, as in rheumatoid arthritis and asthma, and in the acute condition of sepsis. Recently, our group found a direct GRP action over neutrophils, promoting migration. This work aimed to study the interface between GRP-producing tumors and the recruitment of immune cells, as well as extend the cellular studies about neutrophil activation and migration processes promoted by the peptide. In tumors, we observed that a lung adenocarcinoma cell line does not proliferate in response to GRP. Yet, it is induced to migrate when exposed to the peptide, indicating a potential role for GRP in metastasis of this type of cancer. In our tumor immunology studies, we established a novel in vivo model by overexpressing GRP in a melanoma cell line (B16F10). We observed the augment of infiltrating inflammatory monocytes in the tumor microenvironment of these tumors. In parallel, we verified that reactive oxygen species production and migration in response to GRP is dependent of the NADPH oxidase complex. GRP stimulation promotes an intense activation, which culminates in neutrophil extracellular traps (NETs) release. In addition, the GRP receptor (GRPR) antagonist RC-3095 presented anti-inflammatory potential, inhibiting neutrophil migration towards IL-8 and reducing the extent of acetaminophen-induced liver damage. This effect was due to motility alterations in infiltrating neutrophils within the tissue and reduction of cell adhesion molecules. The results presented herein demonstrate the wide panorama of GRP?s interactions in tumor and immune biology. / O microambiente tumoral e as doen?as inflamat?rias promovem altera??es nas c?lulas do nosso sistema imune ? medida que progridem. Diversas mol?culas est?o envolvidas nessa modula??o, e por isso s?o alvos terap?uticos. O pept?deo liberador de gastrina (GRP) ? produzido por tumores, onde promove prolifera??o celular. Este tamb?m est? correlacionado com doen?as cr?nicas como a artrite reumatoide e asma, e em doen?as agudas, como a sepse. Recentemente, nosso grupo descobriu a??o direta do GRP em neutr?filos, promovendo indu??o de migra??o. O presente trabalho se prop?s a estudar a interface entre tumores produtores de GRP e o recrutamento celular, assim como aprofundar os estudos celulares sobre os processos de ativa??o e migra??o de neutr?filos promovidos pelo pept?deo. Em tumores, observamos que uma linhagem de adenocarcinoma pulmonar n?o prolifera quando exposto ao GRP, por?m ? induzida a migrar quando exposta ao pept?deo, estabelecendo um potencial papel deste na promo??o de met?stases para esse tipo tumoral. Na interface da imunologia tumoral, atrav?s do desenvolvimento de um modelo in vivo de superexpress?o de GRP em melanoma murino (B16F10), observamos que esse aumento do GRP induz a infiltra??o de mon?citos inflamat?rios no microambiente tumoral. Em paralelo, verificamos que a produ??o de esp?cies reativas de oxig?nio e a migra??o em dire??o ao GRP s?o dependentes do complexo NADPH oxidase. Esse est?mulo promove ativa??o intensa, culminando na produ??o de redes extracelulares de neutr?filos (NETs). J? o antagonista do seu receptor, GRPR, apresentou potencial antiinflamat?rio, sendo capaz de inibir a migra??o neutrof?lica via modula??o de IL-8 e reduzindo a extens?o da les?o hep?tica induzida por paracetamol (acetaminofeno), alterando a motilidade dos neutr?filos no tecido e a express?o de mol?culas de ades?o. Assim, os resultados aqui apresentados demonstram um panorama amplo da fun??o do GRP na biologia tumoral e no sistema imune.
39

Efeito da inje??o da prote?na b-amiloide 1-42 em diferentes formas no ventr?culo encef?lico : um modelo de aspectos celulares da doen?a de Alzheimer em zebrafish

Silva, Natalia Eltz 08 March 2017 (has links)
Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-11T14:38:07Z No. of bitstreams: 1 NATALIA_ELTZ_SILVA_DIS.pdf: 1036979 bytes, checksum: 23cdf77b8842f9e9ade0b13e6f7bc111 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-28T17:40:18Z (GMT) No. of bitstreams: 1 NATALIA_ELTZ_SILVA_DIS.pdf: 1036979 bytes, checksum: 23cdf77b8842f9e9ade0b13e6f7bc111 (MD5) / Made available in DSpace on 2017-07-28T17:47:24Z (GMT). No. of bitstreams: 1 NATALIA_ELTZ_SILVA_DIS.pdf: 1036979 bytes, checksum: 23cdf77b8842f9e9ade0b13e6f7bc111 (MD5) Previous issue date: 2017-03-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Aging-related diseases are becoming more common. Alzheimer's disease (AD), the most prevalent form of dementia, includes as initial symptoms cognitive deficits that are attributed to the toxic effects of amyloid? peptide (A?) that accumulates in senile plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The amyloid cascade initially proposed to explain the effects of A? pointed to the plaques as the most toxic form of the A? molecule responsible for neuronal dysfunction and death. Recently, several evidences point to the increased toxicity of the soluble forms of the peptide. A better understanding of the dynamics of amyloid aggregation, clearance and toxic potential of the soluble versions may foster significant advances in the understanding AD mechanisms and the identification of potential targets for AD therapies. In this study we used the zebrafish as a model. 24-hour embryos received intracerebroventricular injection of human A?1-42 prepared to have different aggregation potentials: monomeric, oligomeric and plaqueforming. At 5 days post-fertilization (dpf), quantification of A?1-42 levels demonstrated a remnant increase in peptide levels in the animals injected with the solution that favored plaque formation. After monitoring for embryotoxic and teratogenic effects, 5dpf the animals were also evaluated in relation to general physiological aspects and their cognitive ability. Although the injection did not significantly impact animal survival or exploratory ability, the oligomeric solution induced specific cognitive deficits in relation to the vehicleinjected control. Together these results support the revised version of the amyloid cascade in which, although the presence of plaques corresponds to a greater accumulation of A?1- oligomeric forms may induce significant neurotoxic effects and result in cognitive deficits specially at disease?s early stages. / Com o envelhecimento da popula??o, doen?as relacionadas com o envelhecimento v?m se tornando mais comuns. A Doen?a de Alzheimer (DA), a forma prevalente de dem?ncia, inclui como sintomas iniciais deficit cognitivos que s?o atribu?dos a efeitos t?xicos de pept?deo ?- amiloide (A?) que se acumula em placas senis e emaranhados neurofibrilares constitu?dos pela prote?na tau hiperfosforilada. A cascata amiloide inicialmente proposta para explicar os efeitos do A? apontava para as placas de dep?sito de A?1-42 como a forma t?xica da mol?cula respons?vel pela disfun??o e morte neuronal. Recentemente diversas evid?ncias apontam para a toxicidade das vers?es sol?veis do pept?deo antes da agrega??o em placas. O melhor entendimento da din?mica de agrega??o do amiloide, limpeza e potencial t?xico das vers?es sol?veis pode permitir significativos avan?os no conhecimento dos mecanismos da doen?a e a identifica??o de potenciais alvos para terapias da DA. Neste estudo utilizamos o tele?steo zebrafish como modelo para a caracteriza??o destes processos. Embri?es com 24 horas receberam inje??o intracerebroventricular de A?1-42 humano preparado de forma a ter diferentes potenciais de agrega??o: monom?rica, oligom?rica e formadora de placas. Ao atingirem 5 dias p?s-fertiliza??o (dpf), a quantifica??o dos n?veis de A?1-42 demonstrou um aumento remanescente dos n?veis do pept?deo nos animais injetados com a solu??o que favorecia a forma??o de placas. Ap?s monitorarmos eventuais efeitos embriot?xicos e teratog?nicos, ao atingirem 5dpf, os animais foram tamb?m avaliados em rela??o a aspectos fisiol?gicos gerais e sua capacidade cognitiva. Embora a inje??o n?o tenha impactado significativamente a sobreviv?ncia dos animais ou a capacidade explorat?ria, a inje??o da solu??o oligom?rica induziu deficit cognitivos espec?ficos em rela??o ao controle injetado com ve?culo. Juntos, estes resultados suportam a vers?o revisada da cascata amiloide na qual, embora a presen?a de placas corresponda a um maior ac?mulo de A?1-42, a presen?a de vers?es oligom?ricas pode induzir efeitos neurot?xicos significativos e resultar em deficit cognitivos, especialmente nos est?gios iniciais da doen?a.
40

Fenotipagem por DNA - variantes em genes humanos que regulam a pigmenta??o de olhos e de pele : an?lise fenot?pica e genot?pica de indiv?duos sulbrasileiros para fins forenses

Sawitzki, Fernanda Rosa 07 November 2017 (has links)
Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-02-26T11:20:29Z No. of bitstreams: 1 FERNANDA_ROSA_SAWITSKI_TES.pdf: 5519043 bytes, checksum: d8234f6f341fd9d5f2c4a39db88b714c (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-02-26T20:03:16Z (GMT) No. of bitstreams: 1 FERNANDA_ROSA_SAWITSKI_TES.pdf: 5519043 bytes, checksum: d8234f6f341fd9d5f2c4a39db88b714c (MD5) / Made available in DSpace on 2018-02-26T20:08:29Z (GMT). No. of bitstreams: 1 FERNANDA_ROSA_SAWITSKI_TES.pdf: 5519043 bytes, checksum: d8234f6f341fd9d5f2c4a39db88b714c (MD5) Previous issue date: 2017-11-07 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Genes related to externally visible characteristics (EVC) present a great importance for the prediction of skin and eye colors in humans. In this study, we tested the ability of a set of SNPs in pigment-related genes to predict skin and eye colors in an admixed south Brazilian population. First, we used the Cinderella - Tiana - Snow White model to analyze the allelic distribution of eight biallelic SNPs in pigment-related. Cinderella and Tiana patterns have respectively low and high melanin content in skin and eyes; Cinderella has white skin and blue eyes (low melanin content; LMC) and Tiana has dark skin and eyes (high melanin content; HMC). Comparative investigation between frequencies of genetic variants in Cinderella-Like versus Tiana-Like subjects may indicate which polymorphic variant is associated with melanin synthesis in skin and eyes. Coordinately, studies with Snow White-Like subjects may be informative to reveal any tissue-specific expression, since these individuals have both white skin (LMC) and dark eyes (HMC). Based on allele frequencies of different human popullations, allele ?L? was used for the alleles associated with low melanin content populations (Cinderela-like subjects), and allele ?H? was used for the alleles associated with high melanin content populations (Tiana-like subjects). Allelic distribution of eight SNPs showed that 100% of Cinderella- like subjects (N= 73) had less than eight H alleles, and 82% of Tiana-Like subjects (N= 61) had eight or more H alleles. The AUC (Area Under the Receiver Operating Characteristic Curve) value was 0.99, and the calculation of PGL (Pathway Genetic Load) and GP (Genetic Probability) showed that the SNP set presented 93% and 91% concordance between DNA genotype and phenotypes, respectively. Factorial discriminant analyses (FDA) performed in the Snow White group (light skin and dark eyes; N= 116) showed an association between SNPs rs16891982 (SLC45A2), rs8045560 (MC1R), rs1426654 (SLC24A5), rs2733832 (TYRP1), and rs1042602 (TYR) and the Cinderella cluster for skin phenotype, and an association between SNPS rs4778138 (OCA2), rs12913832 (HERC2), and rs916977 (HERC2) and the Tiana cluster for eye phenotype. Lastly, we studied 436 South Brazilian subjects with different skin and eye colors to construct a multinomial logistic regression model able to predict phenotype. The model was tested in 40 random subjects to measure the efficiency of the prediction. The data presented 93% concordance between the predicted and the observed phenotype, showing the successful phenotype prediction of the model in South Brazilian individuals. This is important since Brazil has an ethnically mixed population in which genomic structure may favor epistatic and pleiotropic effects not observed in populations that are more homogeneous. The analyses presented here are an important contribution to forensic DNA phenotyping scenario. / Os genes relacionados a caracter?sticas externamente vis?veis (EVC) apresentam grande import?ncia para a previs?o de cores de pele e olho em humanos. Neste estudo, testamos a capacidade de um conjunto de SNPs em genes relacionados ? s?ntese de pigmento melanina para prever cores de pele e olho em uma popula??o sul-brasileira. Inicialmente, utilizamos o modelo Cinderela - Tiana - Neve Branca para analisar a distribui??o al?lica de oito SNPs bial?licos associados ? produ??o da melanina. Os padr?es Cinderela e Tiana possuem respectivamente baixo e alto conte?do de melanina na pele e nos olhos; Cinderela tem pele branca e olhos azuis (baixo conte?do de melanina, LMC) e Tiana tem pele e olhos escuros (alto conte?do de melanina, HMC). A investiga??o comparativa entre frequ?ncias de variantes gen?ticas em indiv?duos com Cinderela-Like versus Tiana-Like pode indicar qual variante polim?rfica est? associada ? s?ntese de melanina na pele e nos olhos. De forma coordenada, os estudos com indiv?duos Branca de Neve-Like podem ser informativos para revelar a express?o tecido-espec?fica, uma vez que esses indiv?duos t?m pele branca (LMC) e olhos escuros (HMC). Com base em frequ?ncias de alelos de diferentes popula??es humanas, o nome alelo "L" foi utilizado para os alelos associados a popula??es de baixo conte?do de melanina (indiv?duos Cinderela-Like) e o nome alelo "H" foi utilizado para os alelos associados a popula??es de alto conte?do de melanina (Indiv?duos Tiana-Like). A distribui??o al?lica de oito SNPs mostrou que 100% de indiv?duos com Cinderela-Like (N= 73) tinham menos de oito alelos H e 82% de indiv?duos Tiana-Like (N= 61) tinham oito ou mais alelos H. O valor AUC (Area Under the Receiver Operating Characteristic Curve) foi de 0,99 e o c?lculo de PGL (Pathway Genetic Load) e GP (Probabilidade Gen?tica) mostrou que o conjunto de SNP apresentou concord?ncia de 93% e 91% entre gen?tipo e fen?tipo, respectivamente. As an?lises discriminantes fatoriais (FDA) realizadas no grupo Branca de Neve-Like (pele clara e olhos escuros; N= 116) mostraram associa??o positiva entre SNPs rs16891982 (SLC45A2), rs8045560 (MC1R), rs1426654 (SLC24A5), rs2733832 (TYRP1) e rs1042602 (TYR) e o cluster de Cinderela para o fen?tipo da pele, e associa??o positiva entre SNPS rs4778138 (OCA2), rs12913832 (HERC2) e rs916977 (HERC2) e o cluster Tiana para o fen?tipo dos olhos. Por fim, estudamos 436 sujeitos sul-brasileiros com diferentes cores de pele e de olhos para construir um modelo de regress?o log?stica multinomial capaz de prever o fen?tipo. O modelo foi testado em 40 indiv?duos aleat?rios para medir a efici?ncia da predi??o. Os dados apresentaram concord?ncia de 93% entre o fen?tipo previsto e observado, mostrando a previs?o fenot?pica bem-sucedida do modelo em indiv?duos sul-brasileiros. Isso ? importante, uma vez que o Brasil tem uma popula??o etnicamente mista, na qual a estrutura gen?mica pode favorecer os efeitos epistaticos e pleiotr?picos n?o observados em popula??es mais homog?neas. As an?lises aqui apresentadas s?o uma importante contribui??o para a ?rea da fenotipagem por DNA.

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