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Amyloidosis is a group of diseases caused by proteins that have lost their correct three-dimensional conformation and instead assemble into insoluble fibrils in various tissues and organs. Today, more than 30 different proteins that can give rise to amyloid fibrils have been identified. Each protein that assembles into fibrils causes a specific disease. For clinical diagnosis of amyloid, antibodies are one of the most important tools. In this study, antibodies generated towards various amyloid-specific peptides were characterized and validated. This was assessed by immunohistochemistry, slot blot and SDS-PAGE and western blot. Congo red, an amyloid specific dye, was used for detection of amyloid. Immunohistochemical staining and slot blot analysis indicated that each antiserum used in this study was amyloid-specific. Antigen retrieval can facilitate staining by the techniques ability to break cross-linkages caused by fixation in formaldehyde. The results from the characterization of antisera in this study should be a great helpin clinical work on amyloid, and ensure correct diagnosis.

Babesia bovis is a causative agent of bovine babesiosis and is transmitted by vector ticks, Rhipicephalus (Boophilus) spp. The disease has a high mortality rate in susceptible cattle, causing serious economic loss. At present, the only commercial vaccine is culture-based with limited availability. No effective molecular vaccine has been developed to date. Generating a vaccine with specific critical epitopes responsible for protection against B. bovis is critically important. Immunity against B. bovis requires both innate and adaptive responses, with antigen-specific CD4+ T cells essential to the latter through production of IFN-γ. Fourteen B. bovis proteins were selected as putative vaccine candidates and their full-length genes cloned for recombinant protein production intended for evaluating peripheral blood mononuclear cell IFN-γ secretion level from experimentally infected animals in ELISPOT. All proteins expressed in insoluble form (inclusion bodies) and could not be purified. B. bovis genes were then truncated to exclude signal peptide and transmembrane regions, then cloned and expressed using pET101/D-TOPO in Escherichia coli to obtain soluble, useable proteins. Only recombinant B. bovis MSA1, MSA2b and MSA2a1 proteins were successfully expressed in soluble form. These proteins induce invasion-blocking antibodies in immunized cattle, are hypothesized to elicit protection in susceptible animals, but were previously studied by others. Due to failure to produce new candidates to assay, the animal experiments were not performed. Instead, sera from field-infected cattle were assayed for reactivity against the MSA proteins by indirect immunofluorescent antibody (IFA) and western blot (WB) analysis. Field sera from South Texas (#41) and the Mexican Yucatan (#6, #9 and #11) along with positive and negative controls were tested. In IFA test, cattle #6, #9 and #41 were positive while #11 was negative. In WB, #41 and #6 reacted with the recombinant MSA proteins and with control B. bovis whole parasite lysate. However, both #11 and #9 had no signal in WB, although the latter was positive in IFA. Several theories may explain this phenomenon, such as the different preparation process of the antigen in the two tests, strain differences between sera and test antigens, or the different design and nature of each test.

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Thesis(M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

Charles University, Faculty of pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucie Ženklová Supervisors: Prof. Fabio Altieri and PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Involvement of PDIA3 in oxidative stress response PDIA3 is a member of the protein disulfide isomerase family (PDI) and it is a stress- responsive protein. It is also involved in various cellular signalling pathways and has various functions in the cell. The best-known location is in the endoplasmic reticulum where it plays a major role mainly in the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class I. However, its existence has also been described in many other cell compartments, such as nucleus, mitochondria, cell surface or cytosol, where it interferes in various processes. While in some instances these roles need to be confirmed by further studies, a lot of observations confirmed its involvement in the signal transduction (for example releated with STAT protein) from the cell surface and the regulatory processes in the nucleus. Recent studies have also confirmed its increased expression in various pathological states. The aim of our work was to find out what is its role in the exposure of the MDA-MB 468...

Submitted by VICTOR AUGUSTO DOMINGOS DIAS null (victordiaszootecnista@gmail.com) on 2016-08-22T15:46:32Z No. of bitstreams: 1 TESE - VICTOR AUGUSTO DOMINGOS DIAS.pdf: 1882002 bytes, checksum: 9487fafaeb94e582e81be00a1501e16f (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-23T18:12:58Z (GMT) No. of bitstreams: 1 dias_vad_dr_jabo.pdf: 1882002 bytes, checksum: 9487fafaeb94e582e81be00a1501e16f (MD5) / Made available in DSpace on 2016-08-23T18:12:58Z (GMT). No. of bitstreams: 1 dias_vad_dr_jabo.pdf: 1882002 bytes, checksum: 9487fafaeb94e582e81be00a1501e16f (MD5) Previous issue date: 2016-08-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A atuação das enzimas proteolíticas durante o post-mortem é um dos principais fatores que determinam a qualidade da carne, sendo as enzimas calpaína e calpastatina uma das principais atuantes neste processo. O objetivo deste estudo foi avaliar a relação entre a expressão gênica dos genes CAPN1, CAPN2 e CAST, a quantificação das enzimas µ-calpaína, m-calpaína e calpastatina, dentro de grupos contrastantes para maciez e crescimento animal. Foram utilizados dados de 90 animais machos não castrados da raça Nelore, com idade aproximada de 24 meses, permanecendo em confinamento por 95 dias. Após o abate foram colhidas amostras do músculo Longissimus thoracis entre a 9ª e 13ª costelas, da meia-carcaça esquerda. As amostras coletadas foram utilizadas para medir a área de olho de lombo (AOL), espessura de gordura subcutânea (EGS), índice de marmorização (IM), perdas por cozimento, coloração instrumental da carne e força de cisalhamento (FC). A partir do restante da carne, foram coletadas alíquotas para a realização das análises de índice de fragmentação miofibrilar (MFI) e lipídeos totais (LT). Foram coletadas alíquotas para a análise da expressão gênica e quantificação das enzimas. Os dados de força de cisalhamento foram utilizados como critério para a separação em carne macia e dura. Também foram utilizadas o ganho de peso para a separação quanto ao crescimento animal. Os seguintes grupos (n=10) foram formados: Leve, Pesado, Duro e Macio. As análises estatísticas foram realizadas utilizando-se o procedimento GLM e MIXED do programa SAS. No presente trabalho não foi encontrada diferença nas expressões dos genes CAPN1 e CAPN2 entre os grupos. Para o grupo contrastante para maciez também não foi encontrada diferença significativa (p<0,05 para a quantidade das enzimas µ e m-calpaína, e da calpastatina. Não foi encontrada correlação da quantidade das enzimas µ e m-calpaína, e calpastatina com as características PF e GP. Não foi encontrada diferença significativa (p<0,05) para o grupo contrastante para maciez da quantidade das enzimas µ e m-calpaína. Porém foi encontrada diferença significativa (p<0,05) para a quantidade da enzima calpastatina, onde os animais do grupo de carne dura apresentaram maiores valores em relação aos de carne macia. Além de apresentarem uma alta correlação entre a quantidade de calpastatina e as características FC (0) e FC (7) (r= 0,81 e 0,74, respectivamente). A diferença entre os grupos contrastantes e a alta correlação entre os níveis de calpastatina com as características de maciez demonstra a importância do sistema das calpainas na proteólise das miofibrilas, sendo os níveis de calpastatina um alvo potencial para a seleção de animais, com o objetivo de melhorar as características da qualidade da carne dos animais da raça Nelore. / The role of proteolytic enzymes during the post-mortem is one of the main factors that determine the quality of the meat, and the enzymes calpain and calpastatin are one of the main factors in this process. The aim of this study was to evaluate the gene expression of the genes CAPN1, CAPN2 and CAST, and quantify the enzymes μ-calpain, m-calpain and calpastatin within contrasting groups animal growth, and thougness. 90 animals data were used uncastrated male Nellore, aged approximately 24 months remaining in confinement for 95 days. After slaughter were harvested Longissimus muscle samples thoracis between the 9th and 13th ribs, the left half-carcase. The samples were used to measure the ribeye area (REA), subcutaneous fat thickness (SFT), marbling index (MI), cooking loss, instrumental color of the meat and shear force (SF). From the rest of the meat, aliquots were collected to perform the myofibrillar fragmentation index analysis (MFI) and total lipids (TL). Were collected aliquots for the analysis of gene expression and quantification of enzymes. The data of shear force were used as criteria for the separation of tender and tough meat. The characteristics of weight gain for the separation on the animal growth were also used. The following groups (n=10) were formed: lightweight, Heavy, tough meat and tender meat. Statistical analyzes were performed using the GLM and MIXED procedure of Statistical Analysis System program. In this study there was no difference in the expression of CAPN1 and CAPN2 genes between the groups. In this study there was no difference in the expressions of CAPN1 and CAPN2 genes between the groups. For the contrasting group for tenderness meat was also no significant difference (p<0.05) for the amount of enzymes μ and m-calpain, and calpastatin. There was no correlation between the amount of μ and m-calpain enzymes, and calpastatin with PF and GP. There was no significant difference (p<0.05) for the contrasting group for meat tenderness of the amount of enzyme μ and mcalpain. However, a significant difference (p<0.05) for the amount of enzyme calpastatin where animals of tough meat group had higher values on the tender meat. In addition to having a high correlation between the amount of characteristics calpastatin and SF (0) and SF (7) (r = 0.81 and 0.74, respectively). The difference between the contrasting groups and the high correlation between the calpastatin levels with tenderness characteristics demonstrates the importance of the system of calpains proteolysis of myofibrils, and the calpastatin levels of a potential target for the selection of animals for the purpose of improve the quality characteristics of the meat of Nellore.

Resumo: A caracterização da estrutura genética de populações clonais de patógenos bacterianos tem sido feita, entre outros métodos, por "Southern blot" empregando-se como sondas seqüências de inserção ou genes avr. O presente trabalho objetivou o desenvolvimento de oligonucleotídeos iniciadores e sondas de DNA baseadas em genes de patogenicidade para estudo da diversidade genética de isolados de Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolii e Xanthomonas alfalfae subsp. citrumelonis. Observou-se a presença dos genes avrXacE1, avrXacE2 e lrp em todos isolados das subsp. citri e aurantifolii e a ausência do gene avrXacE2 no isolado da subsp. citrumelonis. Os perfis de restrição gerados por PCR-RFLP não revelaram polimorfismo nos genes amplificados e o uso de genes avr como sondas para "Southern blot" mostrou-se efetiva na diferenciação das espécies de Xanthomonas patogênicas a citros e na identificação de relativo polimorfismo entre isolados da mesma espécie. A diversidade haplotípica foi maior com o gene avrXacE1. Com exceção à sonda correspondente ao gene lrp, o número de haplótipos identificados (17) variou de acordo com a endonuclease utilizada em "Southern blot". Os isolados da subsp. citri apresentaram um maior número de cópias dos genes avr em comparação com isolados das subsp. aurantifolii e citrumelonis. O estado de São Paulo, que adota uma campanha de erradicação do cancro cítrico, apresentou a menor diversidade haplotípica, provavelmente em decorrência do curto período em que o patógeno interage com o hospedeiro. / Abstract: The genetic structure of clonal populations of bacterial pathogens has been characterized, among other methods, by Southern blot using insertion sequences or avr genes as probes. The present work aimed the development of DNA primers and probes based on pathogenicity genes to study the genetic diversity of Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolii, and Xanthomonas alfalfae subsp. citrumelonis strains. The presence of avrXacE1, avrXacE2 and leucine rich protein (lrp) genes was observed in all citri and aurantifolii strains, as well as the absence of the avrXacE2 gene in citrumelonis isolate. The restriction profiles generated by PCR-RFLP did not show polymorphism in the amplified genes and the use of avr genes as Southern blot probes showed to be effective to differentiate Xanthomonas species pathogenic to citrus and to identify a relative polymorphism between strains belonging to the same species. The haplotype diversity was higher with the avrXacE1 gene. Excepting the probe corresponding to lrp gene, the number of haplotypes identified (17) varied according to the endonuclease used on Southern blot. The strains of the citri species presented a higher number of copies of the avr genes compared to aurantifolii and citrumelonis. The state of São Paulo, that adopts a campaign of eradication of the citrus canker, presented the smaller haplotype diversity, probably as result of the short period where the pathogen interacts with the host. / Orientadora: Maria Inês Tiraboschi Ferro / Coorientador: José Belasque Júnior / Banca: João Martins Pizauro Junior / Banca:Julio Cezar Franco de Oliveira / Mestre

Made available in DSpace on 2018-08-01T22:56:45Z (GMT). No. of bitstreams: 1 tese_6434_DISSERTAÇÃO GABRIELA PORFIRIO-PASSOS.pdf: 872492 bytes, checksum: 4693b33b1f6f17af259a9c2b1b3878cb (MD5) Previous issue date: 2013-02-26 / Com o objetivo de realizar o diagnóstico de leishmaniose tegumentar americana (LTA) em cães, foram utilizados métodos de cultura e isolamento, testes sorológicos de ELISA e Western Blot (WB) e pesquisa de DNA do parasito para dois grupos de animais, um grupo composto de animais sem lesões clínicas sugestivas de LTA, mas residentes em torno de casos clínicos humanos confirmados para LTA, e outro grupo de animais com lesões sugestivas de LTA que serviram como controle dos protocolos realizados. O estudo foi realizado no município de Iúna, ES, Brasil, região endêmica para enfermidade. No primeiro grupo, foram analisadas amostras de soro de 109 animais sem histórico ou lesões indicativas de LTA, estas foram submetidas às técnicas de ELISA e WB que resultaram em 20 animais sorologicamente positivos para as duas técnicas. O teste ELISA apresentou sensibilidade de 100,00% (IC95% - 0,83 a 1,00) e especificidade de 77,53% (IC95% - 0,67 a 0,86), em relação à técnica de WB. O teste WB apresentou maior acurácia e mostrou-se mais adequado para diagnóstico dos animais assintomáticos, enquanto a técnica de ELISA para a triagem. Para a pesquisa do DNA do parasito nos 20 animais assintomáticos e positivos pela técnica de WB utilizou-se sangue total e biópsia de tecido íntegro do pavilhão auricular pela técnica de reação em cadeia da polimerase (PCR). Os resultados para PCR da biópsia de tecido íntegro e PCR de tecido sanguíneo mostraram sensibilidade de 30,0% (IC95% - 0,12 a 0,54) e 20,0% (IC95% - 0,06 a 0,44), respectivamente. A especificidade, 99,0% (IC95% - 0,93 a 0,99) e 100% (IC95% - 0,96 a 1,00), para a PCR da biópsia de tecido íntegro e PCR de tecido sanguíneo quando comparadas ao WB. Os resultados mostram que tecido íntegro do pavilhão auricular e sangue de animais assintomáticos submetidos à técnica de PCR, apresentaram baixa sensibilidade e alta especificidade, assim, a PCR da biópsia de tecido íntegro é melhor indicador para animais assintomáticos que a PCR de tecido sanguíneo. Para o segundo grupo, foram identificados três animais com lesões sugestivas para LTA e sorologicamente positivo. A partir de uma amostra de biópsia de lesão sugestiva de LTA presente no pavilhão auricular, parte desta foi destinada a cultura e isolamento e outra parte para comparação de três protocolos de extração do DNA de tecido animal para diagnóstico da LTA canina. Os protocolos utilizados tiveram como base o Fenol-Clorofórmio, Acetato de Potássio e associação entre as duas metodologias. Em comparação com os padrões moleculares de concentração de DNA concluiu-se que o protocolo com Acetato de Potássio foi o mais indicado para o tipo de tecido empregado. Por fim, de posse dos dados apresentados para os animais sorologicamente positivos e assintomáticos estudados, foi possível concluir que estes não representaram potenciais reservatórios do parasito, o que evidencia ainda que para avaliação deste perfil da enfermidade seja indicada a associação de métodos de diagnóstico.

The dissertation is divided into two separate experiments that explore the effects of visual-spatial learning on PSD-95 dorsal hippocampal expression. Specifically, the aim of these studies was to explore the effect of learning an assay, the Hebb-Williams mazes, on the protein expression of PSD-95 in Fmr1 KO mice. PSD-95 is an important scaffolding protein hypothesized to be involved in learning and memory. In cellular models of Fragile X Syndrome it has been shown to be dysregulated but it has never been measured following behavioural learning. Establishment of a deficit using an ecologically valid behavioural assay could lead to the development of novel interventions. Study one employed a subset of the Hebb-Williams mazes of various levels of difficulty to evaluate PSD-95 protein expression in Fmrp intact and Fmr1 KO mice following learning. The results revealed significant increases in PSD-95 protein expression in control runners when compared to Fmr1 KO mice. There was a negative correlation between PSD-95 protein levels and mean total errors on the mazes meaning that as expression was increased, errors were decreased. The goals of study two were to reverse the molecular and behavioural deficits using pharmacological antagonist treatment shown to be effective in cellular models of Fragile X Syndrome. Fmr1 KO mice were treated with either saline or 20 mg/kg of a metabotropic glutamate receptor antagonist, 2-Methyl-6-(phenylethynyl) pyridine (MPEP). Relative to saline treated controls, drug treated Fmr1 KO mice made fewer errors on the same subset of Hebb-Williams mazes used in study one. Latency to complete these mazes did not differ between groups, indicating that MPEP treatment does not adversely affect motor functioning. Protein assessment revealed that PSD-95 was selectively rescued in MPEP treated mice and not saline controls. Similar to study one, a negative correlation between PSD-95 protein levels and mean total errors was observed. When taken together, these studies indicate that protein deficits are associated with a deficit of learning that can be reversed with a selective glutamate receptor antagonist. One of the strengths of the Hebb-Williams mazes is that performance is measurable without floor or ceiling effects, which plague other common behavioural assays. These data further suggest that pharmacological antagonist treatments may be promising in correcting the learning deficits in human Fragile X Syndrome patients.