Light microscopy was used to characterize digestive cells in the gut of the hard tick Ixodes ricinus during feeding and different phases of digestion. Indirect immunofluorescent microscopy was used to localize three digestive peptidases - cathepsin B, cathepsin L, cathepsin D in the gut of the hard tick Ixodes ricinus.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 dissertacao_marcelo_gotze.pdf: 556489 bytes, checksum: cb0ceeb57d3c58da117f1a655a510d0d (MD5) Previous issue date: 2010-03-18 / Bovine babesiosis caused by Babesia bovis and Babesia bigemina, is the most important disease transmitted by Rhipicephalus (Boophilus) microplus in tropical and subtropical areas in South America. Definitive diagnosis can be made by detecting infected erythrocytes in blood smears. However, the parasitemia in peripheral blood is often too low for this method to be used for diagnostic purposes. For this reason, several serological tests, including complement fixation, indirect hemagglutination and indirect immunofluorescence (IIF) have been used to detect antibodies in infected cattle. Although these tests allow the detection of persistently infected animals, they have limitations in specificity and/or sensitivity. The IIF has been the most sensitive, but cross-reactivity between species, subjective interpretation, and low production has limited its usefulness. The enzyme linked immunosorbent assay (ELISA) have found wide application in the diagnosis of infectious diseases. The cELISA format (competitive) can provide an additional level of specificity, because the antibody is directed to a single epitope, specific for the organism to be detected. For these reasons, this study aimed to evaluate the sensitivity and specificity of cELISA compared to IIF and nested PCR (nPCR) for diagnosis babesiosis caused by B. bovis. Therefore, blood samples were collected from cattle in Brazil and Argentina, and processed for the diagnosis of B. bovis. The nPCR was used as the gold standard to validate the cELISA and the IIF as a comparative test. The cELISA for the diagnosis of B. bovis presented is easily processed with high levels of sensitivity and specificity. It is easily performed in a high number of samples, making it useful in cases of outbreaks of bovine babesiosis. / A babesiose bovina, causada por Babesia bovis e Babesia bigemina, é a doença mais importante transmitida por carrapatos Rhipicephalus (Boophilus) microplus em áreas tropicais e subtropicais da América do Sul. O diagnóstico definitivo pode ser feito através da detecção de eritrócitos infectados em esfregaços sanguineos, porém a parasitemia em sangue periférico é frequentemente muito baixa para que esse método seja utilizado de forma confiável para fins de diagnóstico. Por esse motivo, vários testes sorológicos, incluindo a fixação de complemento, hemaglutinação indireta e imunofluorescência indireta (IIF) têm sido usados para detectar anticorpos em bovinos infectados. Embora estes testes permitam a detecção de animais persistentemente infectados, eles têm limitações na especificidade e/ou sensibilidade. O IIF tem sido o mais sensível, mas a reatividade cruzada entre as espécies, interpretação subjetiva, e baixa produção tem limitado a sua utilidade. Os ELISA têm encontrado ampla aplicação no diagnóstico de doenças infecciosas. O formato cELISA (competitivo) pode fornecer um nível adicional de especificidade, pois o anticorpo é dirigido para um epitopo único específico para o organismo a ser detectado. Por estas razões, este estudo teve como objetivo avaliar a sensibilidade e especificidade do cELISA comparado com IIF e nested PCR (nPCR) para diagnóstico de babesiose causada por B. bovis. Para tanto, amostras de sangue bovino foram coletadas no Brasil e na Argentina e processadas para o diagnóstico de B. bovis. Utilizou-se o nPCR como teste padrão para a validação do cELISA, e a IIF como teste comparativo. O cELISA para diagnóstico de B. bovis apresentou-se de fácil processamento, com altos níveis sensibilidade e especificidade, além da rapidez no processamento de amostras em larga escala, sendo de grande utilidade para casos de surtos de babesiose bovina

Charles University Faculty of Pharmacy in Hradec Králové Department od Biochemical Sciences Candidate: Růžena Melicharová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of thesis: The detection of protein covalent complexes with DNA using fluorescent microscopy Anthracycline antibiotics are present one of the most potent antineoplastic drugs. The mechanism of their action is complex. They are reported to intercalate to DNA, form DNA adducts and interact with topoisomerase II (TopII) as its poisons. Catalytic cycle of TopII is interrupted when anthracyclines stabilize the covalent complex of DNA and TopII and that causes cell damage. However, using of anthracyclines is limited by several adverse effects e. g. myelotoxicity and cardiotoxicity. The mechanism of cardiotoxicity is still unclear but may be associated with poisoning of the TopIIβ isoform. Unlike the TopIIα, TopIIβ is present mostly in quiescent cells as cardiomyocytes. Furthermore, the only clinically approved cardioprotective drug dexrazoxane belongs to TopII catalytic inhibitors. Nevertheless, the details of the dexrazoxane-afforded protection are unclear. This thesis was aimed to optimize the TARDIS (trapped in agarose DNA immunostaining) assay to detect and quantify covalent cleavage complexes, compare different ways for analysis of...

The synthesis of iron-sulfur clusters is an essential cellular process, which depends on complex biosynthetic pathways. In model eukaryotes, these pathways are the ISC pathway in the mitochondria and the CIA pathway in the cytosol. A recent genome and transcriptome analysis showed, that an amitochondriate protist Monocercomonoides exilis lacks the canonical ISC pathway, which has been replaced by a bacterial SUF pathway. A close free-living relative of M. exilis, Paratrimastix pyriformis possesses a mitochondrion-related organelle, yet also possesses a SUF pathway instead of ISC. The acquisition of the SUF pathway has been suggested as the primordial cause for mitochondrial loss in M. exilis, which is the first documented eukaryotic organism without a mitochondrion. The SUF pathway has been the subject of numerous studies in bacteria, however, its role as the core provider of iron-sulfur clusters for eukaryotic cells has been reported in merely a handful of eukaryotes and was based predominantly on genomic data. This thesis focuses on the putative ATPase SufC and the putative scaffold protein SufB. Both proteins were successfully produced in recombinant forms. SufC has been found to possess ATPase activity in vitro, which was increased upon interaction with SufB. The conditions for theATPase...

Reproduction is an essential feature of all animals and a fundamental step to produce new generations. Study of sperm proteins is crucial for understanding of the sperm-egg recognition. We searched out sperm surface proteins involving in the zona pellucida (ZP) binding and studied whether these proteins are preserved throughout mammalian species. Indirect immunofluorescent technique was used to test a panel of monoclonal antibodies prepared against boar sperm surface proteins on spermatozoa of bull and mice. We found a cross-reactivity of some antibodies against boar sperm with bull ejaculated and mouse epididymal spermatozoa. Further, we isolated sperm proteins from different mammalian species, such as pig, bull, dog, cat, mouse and human. Proteins were separated by SDS- electrophoresis and protein/glycoprotein profiles from epididymal, ejaculated and in vitro capacitated sperm were compared. The interaction of sperm with ZP was studied on electrophoretically-separated sperm surface proteins from pig and bull with biotin-labeled ZP glycoproteins. Antibodies, which reacted with boar sperm surface proteins with ZP- binding activity, therefore could be potential egg-binding receptors, were used for monitoring of the sperm protein origin in reproductive fluids and tissues. (In Czech) Keywords: sperm...

Introdução: O remodelamento da matriz extracelular no líquen escleroso (LS) caracteriza-se histologicamente por uma faixa hialinizada situada predominantemente na derme superficial, semelhante ao que ocorre na lipoidoproteinose (LPE), uma genodermatose rara, na qual ocorre deficiência na produção da proteína 1 da matriz extracelular (ECM-1). Recentemente, em casos de LS foram descobertos auto-anticorpos contra a ECM-1 e um novo caminho foi proposto para desvendar a sua etiopatologia. O LS também é freqüentemente comparado com a Esclerodermia, visto que alguns autores consideram que são espectros de uma mesma doença. Na Esclerodermia e na LPE há aumento do colágeno tipo V (COL V), mas pouco se sabe sobre este tipo de colágeno no LS. Assim, o objetivo do presente trabalho foi demonstrar a localização e a quantidade de COL V e ECM-1 nas vulvas de pacientes com LS. Materiais e métodos: foram estudadas 21 biópsias de pacientes com LS vulvar e 21 biópsias de vulvas normais. A morfometria foi realizada nas imagens geradas através da imunofluorescência marcada com anticorpos contra os colágenos I (COL I), III (COL III) e V, e também nas de imunoistoquímica para ECM-1. Ademais, utilizou-se microscopia confocal a laser para visualizar o COL V e a ECM-1 na mesma lâmina. Resultados: Peles do grupo controle mostraram fraca e homogênea distribuição das fibras de COL I, III e V na imunofluorescência. Em contraste, peles com LS exibiram desarranjo da arquitetura da derme superficial e difuso aumento da fluorescência das fibras de COL I, III e V na faixa hialina. A fração de área das fibras de COL I, III, e V foram significativamente maiores nas peles de pacientes com LS em relação ao controle (p<0,05). Peles controles mostraram co-localização da ECM-1 e do COL V na parede de pequenos vasos e ao longo da membrana basal. Entretanto, no LS houve perda de expressão da ECM-1 na parede dos vasos sanguíneos (p<0,001) e difuso aumento da fluorescência verde do COL V (p<0,001). Conclusão: Houve inversa relação entre o COL V e a proteína ECe constatado pela histomorfometria, imunofluorescência, imunoistoquímica e reconstrução tridimensional, sugerindo que estratégias terapêuticas para prevenir o aumento da síntese de COL V e a diminuição da ECM-1 poderão ser promissoras no prognóstico e tratamento do LS / Background: The extracellular matrix remodeling in lichen sclerosus (LS) is characterized in routine light microscopy by a hyalinized band predominantly located in the superficial dermis. The same pattern occurs in the lipoid proteinosis (LPE), a rare genodermatosis, in which the production deficiency of extracellular matrix protein 1 (ECM-1) is responsible for triggering the disease. Recently, in cases of LS, autoantibodies were discovered against ECM-1 and a new way to unravel their aetiopathology was proposed. LS is also frequently compared with Scleroderma, since some authors consider that they are spectra of a similar disease. In Scleroderma and LPE there is an increase of type V collagen (COL V), but little is known about this type of collagen in LS. Thus, the objective of the current study was to demonstrate the location and quantity of COL V and ECM-1 on the vulvas of patients with LS. Materials and methods: Twenty one biopsies from patients with vulvar LS matched with 21 biopsies from normal vulvas were included in this study. Immunofluorescence against type I (COL I), (COL III) and V collagen and immunohistochemistry for ECM-1 was performed and the slides were analysed by morphometry. Furthermore, laser confocal microscopy was used to visualize the COL V fibers and the ECM-1 protein on the same slide. Results: Skins of the control group showed low and homogeneous distribution of fibers COL I, III and V in immunofluorescence. In contrast, skins with LS exhibited disruption of the architecture of the superficial dermis and diffuse increase in fluorescence fibers COL I, III and V in the range hyaline. The area fraction of fibers COL I, III, and V were significantly higher in the skin of patients with LS compared to control (p <0.05). Skins controls showed colocalization of ECM-1 and COL V the wall of small vessels and along the basement membrane. However, the LS had loss of expression of ECM-1 the wall of blood vessels (p <0.001) increase the fluorescence and diffuse green COL V (p <0.001). Conclusion: There was an inverse relationship between COL V and ECM-1 protein in LS as demonstrated by histomorphometry, immunofluorescence, immunohistochemistry and three-dimensional reconstruction, suggesting that therapeutic strategies to prevent the increased synthesis COL V and ECM-1 protein in LS as demonstrated by histomorphometry, immunofluorescence, immunohistochemistry and three-dimensional reconstruction, suggesting that therapeutic strategies to prevent the increased synthesis COL V and decreased ECM-1 may be promising in the prognosis and treatment of the LS

Introdução: O remodelamento da matriz extracelular no líquen escleroso (LS) caracteriza-se histologicamente por uma faixa hialinizada situada predominantemente na derme superficial, semelhante ao que ocorre na lipoidoproteinose (LPE), uma genodermatose rara, na qual ocorre deficiência na produção da proteína 1 da matriz extracelular (ECM-1). Recentemente, em casos de LS foram descobertos auto-anticorpos contra a ECM-1 e um novo caminho foi proposto para desvendar a sua etiopatologia. O LS também é freqüentemente comparado com a Esclerodermia, visto que alguns autores consideram que são espectros de uma mesma doença. Na Esclerodermia e na LPE há aumento do colágeno tipo V (COL V), mas pouco se sabe sobre este tipo de colágeno no LS. Assim, o objetivo do presente trabalho foi demonstrar a localização e a quantidade de COL V e ECM-1 nas vulvas de pacientes com LS. Materiais e métodos: foram estudadas 21 biópsias de pacientes com LS vulvar e 21 biópsias de vulvas normais. A morfometria foi realizada nas imagens geradas através da imunofluorescência marcada com anticorpos contra os colágenos I (COL I), III (COL III) e V, e também nas de imunoistoquímica para ECM-1. Ademais, utilizou-se microscopia confocal a laser para visualizar o COL V e a ECM-1 na mesma lâmina. Resultados: Peles do grupo controle mostraram fraca e homogênea distribuição das fibras de COL I, III e V na imunofluorescência. Em contraste, peles com LS exibiram desarranjo da arquitetura da derme superficial e difuso aumento da fluorescência das fibras de COL I, III e V na faixa hialina. A fração de área das fibras de COL I, III, e V foram significativamente maiores nas peles de pacientes com LS em relação ao controle (p<0,05). Peles controles mostraram co-localização da ECM-1 e do COL V na parede de pequenos vasos e ao longo da membrana basal. Entretanto, no LS houve perda de expressão da ECM-1 na parede dos vasos sanguíneos (p<0,001) e difuso aumento da fluorescência verde do COL V (p<0,001). Conclusão: Houve inversa relação entre o COL V e a proteína ECe constatado pela histomorfometria, imunofluorescência, imunoistoquímica e reconstrução tridimensional, sugerindo que estratégias terapêuticas para prevenir o aumento da síntese de COL V e a diminuição da ECM-1 poderão ser promissoras no prognóstico e tratamento do LS / Background: The extracellular matrix remodeling in lichen sclerosus (LS) is characterized in routine light microscopy by a hyalinized band predominantly located in the superficial dermis. The same pattern occurs in the lipoid proteinosis (LPE), a rare genodermatosis, in which the production deficiency of extracellular matrix protein 1 (ECM-1) is responsible for triggering the disease. Recently, in cases of LS, autoantibodies were discovered against ECM-1 and a new way to unravel their aetiopathology was proposed. LS is also frequently compared with Scleroderma, since some authors consider that they are spectra of a similar disease. In Scleroderma and LPE there is an increase of type V collagen (COL V), but little is known about this type of collagen in LS. Thus, the objective of the current study was to demonstrate the location and quantity of COL V and ECM-1 on the vulvas of patients with LS. Materials and methods: Twenty one biopsies from patients with vulvar LS matched with 21 biopsies from normal vulvas were included in this study. Immunofluorescence against type I (COL I), (COL III) and V collagen and immunohistochemistry for ECM-1 was performed and the slides were analysed by morphometry. Furthermore, laser confocal microscopy was used to visualize the COL V fibers and the ECM-1 protein on the same slide. Results: Skins of the control group showed low and homogeneous distribution of fibers COL I, III and V in immunofluorescence. In contrast, skins with LS exhibited disruption of the architecture of the superficial dermis and diffuse increase in fluorescence fibers COL I, III and V in the range hyaline. The area fraction of fibers COL I, III, and V were significantly higher in the skin of patients with LS compared to control (p <0.05). Skins controls showed colocalization of ECM-1 and COL V the wall of small vessels and along the basement membrane. However, the LS had loss of expression of ECM-1 the wall of blood vessels (p <0.001) increase the fluorescence and diffuse green COL V (p <0.001). Conclusion: There was an inverse relationship between COL V and ECM-1 protein in LS as demonstrated by histomorphometry, immunofluorescence, immunohistochemistry and three-dimensional reconstruction, suggesting that therapeutic strategies to prevent the increased synthesis COL V and ECM-1 protein in LS as demonstrated by histomorphometry, immunofluorescence, immunohistochemistry and three-dimensional reconstruction, suggesting that therapeutic strategies to prevent the increased synthesis COL V and decreased ECM-1 may be promising in the prognosis and treatment of the LS

Nuclear receptors constitute a large family of transcription factors that are powerful regulators of animal tissue metabolism, homeostasis, tissue maintenance and development. They are particularly attractive for their ability to respond to the binding of hormones, metabolites, xenobiotics and artificially prepared molecules and transmit the interaction with these small lipophylic molecules to specific regulatory potential. In search for nuclear receptors that are likely to be critical for neural tissues in invertebrates and conserved during the evolution of animals, we have identified a close homologue of vertebrate TLX in a planarian Schmidtea mediterranea. Planaria represent very promising biological model systems for studies on tissue maintenance and regeneration. Planaria are able to resorb their tissues and use them as sources of energy during fasting and they re-build their bodies from neoblasts when food is plentiful. Our search in Schmidtea mediterranea's publicly accessible genome sequencing data indicated that planarian genome contains at least one gene with a high degree of similarity to vertebrate TLX. We cloned full length CDS (coding DNA sequence of cDNA) and characterized the gene functionally. This showed that the planarian and vertebrate NR2E1 are highly similar...

Nuclear receptors constitute a large family of transcription factors that are powerful regulators of animal tissue metabolism, homeostasis, tissue maintenance and development. They are particularly attractive for their ability to respond to the binding of hormones, metabolites, xenobiotics and artificially prepared molecules and transmit the interaction with these small lipophylic molecules to specific regulatory potential. In search for nuclear receptors that are likely to be critical for neural tissues in invertebrates and conserved during the evolution of animals, we have identified a close homologue of vertebrate TLX in a planarian Schmidtea mediterranea. Planaria represent very promising biological model systems for studies on tissue maintenance and regeneration. Planaria are able to resorb their tissues and use them as sources of energy during fasting and they re-build their bodies from neoblasts when food is plentiful. Our search in Schmidtea mediterranea's publicly accessible genome sequencing data indicated that planarian genome contains at least one gene with a high degree of similarity to vertebrate TLX. We cloned full length CDS (coding DNA sequence of cDNA) and characterized the gene functionally. This showed that the planarian and vertebrate NR2E1 are highly similar...

Submitted by (edna.saturno@ufrpe.br) on 2016-07-25T13:02:29Z No. of bitstreams: 1 Neurisvan Ramos Guerra.pdf: 506781 bytes, checksum: 5f3717d4e83a80c6435609c31fc492be (MD5) / Made available in DSpace on 2016-07-25T13:02:29Z (GMT). No. of bitstreams: 1 Neurisvan Ramos Guerra.pdf: 506781 bytes, checksum: 5f3717d4e83a80c6435609c31fc492be (MD5) Previous issue date: 2013-02-20 / Trypanosoma vivax determines losses in production of ruminants related to morbidity, decline in production, reproductive problems and also mortality. The objective of this study was to determine the diagnosis of Trypanosoma (Dutonella) vivax in cattle of different regions of Pernambuco State, Brazil, through the techniques of Indirect Immunofluorescence Assay (IFA) and Polymerase Chain Reaction (PCR). Firstly, it was performed a serology of 2,053 serum samples from bovine herds from different counties in the state of Pernambuco. From these results the counties with the highest frequency by IFA test were selected and a total of 127 bovine blood samples were collected for molecular analysis by PCR. The results of IFAT showed 13.93% (286/2,053) of positive animals for IgG antibodies against Trypanosoma vivax. The amplification by PCR revealed positivity of 44.88% (57/127). These results suggest that Pernambuco state is an area endemic for Trypanosoma vivax being considered an enzootic instability area. On the other hand, PCR was effective in the diagnosis of Trypanosoma vivax in cattle blood, being an indispensable tool for studies epidemiological. / Trypanosoma vivax determina prejuízos à produção de ruminantes, relacionados com a morbidade, queda na produção, problemas reprodutivos além de mortalidade. O presente trabalho teve como objetivo realizar o diagnóstico de Trypanosoma (Dutonella) vivax em bovinos das diferentes Mesorregiões do Estado de Pernambuco, Brasil, através das técnicas de Reação de Imunofluorescência Indireta (RIFI) e da Reação em Cadeia da Polimerase (PCR). Iniciamente foi realizada sorologia de 2.053 amostras de soro sanguíneo de bovinos provenientes de rebanhos de municípios do estado de Pernambuco. A partir destes resultados foram selecionados os municípios com maiores frequências de anticorpos em cada mesorregião do Estado de Pernambuco e foram coletadas um total de 127 amostras de sangue bovino para análise molecular através da PCR. À sorologia, 13,93% (286/2.053) dos animais foram reagentes para anticorpos IgG anti-Trypanosoma vivax. Já na PCR foi obtida uma positividade de 44,88% (57/127). Esses resultados sugerem que o estado de Pernambuco é área endêmica para Trypanosoma vivax, apresentando-se como área de instabilidade enzoótica e que a PCR mostrou-se eficiente no diagnóstico da infecção por Trypanosoma vivax em sangue de bovinos, sendo uma ferramenta indispensável para estudos epidemiológicos.