Mitotane führt zur vermehrten Expression des CHOP-Proteins. Da es ebenfalls gelang weitere Expressionsveränderungen von am ER-Stress-beteiligten Proteinen nachzuweisen, kann von einer ER-Stress-Induktion durch Mitotane ausgegangen werden. Des Weiteren ließ sich zeigen, dass die gesteigerte CHOP-Expression nicht zelltypspezifisch ist, da sie sich ebenfalls in weiteren Zelllinien nachweisen ließ. Hierzu gehörten IMR32-, HeLaz91wt-, HepG2- und HEK293T-Zellen. Zudem kam es durch die Inkubation der NCI H295R-Zellen mit Mitotane zu einer Abnahme der Expression von SREBP 1 sowohl der Vorläuferstufe als auch der aktiven Form. Dies weist auf eine Herunterregulation des Lipidstoffwechsels durch Mitotane hin. Neben Mitotane gab es mit ATR 101 und AZD 3988 weitere Substanzen, die zu einer Zunahme der CHOP-Expression geführt haben. / Mitotane leads to increased expression of the CHOP protein. Since it was also possible to demonstrate further changes in the expression of proteins involved in ER stress, it can be assumed that mitotanes induce ER stress. Furthermore, it could be shown that the increased CHOP expression is not cell type specific, as it could also be detected in other cell lines such as IMR32, HeLaz91wt, HepG2 and HEK293T. Further, the incubation of NCI H295R cells with mitotane resulted in a decrease in the expression of SREBP 1, both the precursor and the active form. This indicates a down regulation of the lipid metabolism by Mitotane. In addition to mitotane, there were other substances with ATR 101 and AZD 3988 that led to an increase in CHOP expression.

Le champignon microscopique Aspergillus fumigatus provoque un nombre important de maladies graves. Parmi elles, l’aspergillose pulmonaire chronique (APC) et l’aspergillose broncho-pulmonaire allergique (ABPA) affectent 3 et 4,8 millions de personnes dans le monde, respectivement.L’APC est très souvent mortelle si elle n’est pas soignée. Elle se développe très souvent après une tuberculose. C’est donc une maladie des pays émergents, où il n’est souvent pas possible de la diagnostiquer à cause du coût trop important des techniques existantes.L’ABPA est une complication très grave de l’asthme et de la mucoviscidose, qui complique fortement ces maladies. Elle est très difficile à diagnostiquer.Notre travail a donc consisté à développer et évaluer deux tests, un test rapide permettant de poser le diagnostic d’APC sans avoir à utiliser de matériel de laboratoire à destination des pays émergents et un western blot qui permet la confirmation du diagnostic d’ABPA. / Aspergillus fumigatus is a microscopic fungus that can cause numerous diseases. Among them, chronic pulmonary aspergillosis (CPA) and allergic broncho-pulmonary aspergilloses (ABPA) affect 3 and 4.8 million people, respectively.CPA is often fatal if left untreated. It is often a complication of tuberculosis and therefore affect low and middle income countries. However, it is difficult to diagnose it in those countries, as the tests are too expansive.ABPA is a severe complication of asthma and cystic fibrosis, worsening those diseases. It’s very hard to diagnose it.Our work was to develop and evaluate two tests, a rapid test for the diagnosis of CPA that does not require laboratory equipment designed for low and middle income countries and a western blot for confirmation of ABPA diagnosis.

Acyl carrier proteins (ACPs) from Escherichia coli and Euglena were analyzed on Western blots using rabbit antibodies raised against E. coli ACP. Euglena ACP, unlike that from E. coli, behaves upon electrophoresis under denaturing conditions as its size would predict. Oligomeric forms of both ACPs were evident on Western blots, but the bacterial ACP had more tendency to aggregate. That the oligomeric forms were not due to impurities was shown by their regeneration from low-Mr protein, reaction with antibodies isolated from Iow-Mr protein, and by molecular weight determination of the ACP by low-angle laser light scattering.

Cancer remains one of the most common causes for death in the world today. Researchers are continuously trying to improve old, and develop new, methods in order to strife this global problem. Much research is being made trying to find new specific biomarkers that can be used to detect and diagnose cancer in an early stage. One candidate protein for possible future use as a biomarker is tripeptidyl-peptidase II (TPPII) which has previously been shown to be up-regulated in Burkitt´s lymphoma. This paper focuses on the expression of TPPII on an mRNA-level to see if there is any difference between expression in human leucocytes from patients with a leukemia diagnosis and a healthy volunteer, in order to evaluate if the expression of TPPII have any future use as a biomarker. Patient samples were analyzed using real time qPCR, to study the expression of mRNA, and Western blot, in order to correlate the mRNA findings with protein expression. Three different cell lines with different characteristics regarding expression and function of TPPII were also used to validate the methods used and for comparison with the patient samples analyzed. A difference in expression of mRNA were seen between the different patient samples, both individually and between larger groups of samples with the same diagnosis, indicating a large individual variation, thus making future use in a clinical setting difficult. However, seeing as only a few samples were analyzed in this study, more research must be done in order to draw any final conclusions.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-07-15T20:04:59Z No. of bitstreams: 1 Dissertação_ICS_Glauber Santos Ribeiro de Sena.pdf: 1217162 bytes, checksum: f7bea02af0d0becfe7772cc677cf106d (MD5) / Made available in DSpace on 2013-07-15T20:05:00Z (GMT). No. of bitstreams: 1 Dissertação_ICS_Glauber Santos Ribeiro de Sena.pdf: 1217162 bytes, checksum: f7bea02af0d0becfe7772cc677cf106d (MD5) / CAPES; FAPESB / Este trabalho teve como objetivo avaliar a presença dos LVPR em propriedades dos territórios de identidade baianos do Portal do Sertão, Sisal e Bacia do Jacuípe e caracterizar o perfil socioeconômico da caprino/ovinocultura nestas regiões. Em 134 propriedades foram coletados soros caprinos (n=1046) e ovinos (n = 704). Estes soros foram submetidos aos testes de IDGA, ELISA e Western Blot. Durante a coleta, aplicou-se um questionário para definir o perfil socioeconômico da caprino/ovinocultura dessas regiões. Os resultados desta pesquisa mostraram a ausência de animais positivos para CAEV através do IDGA, mas 10 animais positivos pelo ELISA e Western blot. Não foram encontrados ovinos positivos para Maedi-Visna pelo IDGA. A análise do questionário aplicado nas propriedades mostrou um perfil produtivo pouco tecnificado, com predomínio de pequenas propriedades com gestão familiar. Os rebanhos são pequenos, criados em sistema extensivo e formados principalmente por animais sem raça definida. Observou-se pouco investimento em práticas e instalações adequadas para o manejo alimentar, sanitário, reprodutivo e das crias que possibilitem o aumento da produtividade nas propriedades. A pequena produção tem como finalidade o corte (carne) e se destina a subsistência familiar e ao comércio local. Discute-se os alcances do desenvolvimento da caprino/ovinocultura na região. / Salvador

Vaccine Brucella abortus strain RB51, unlike the wild strain 2308 and another vaccine strain (strain 19) does not induce anti-O-chain antibodies. An efficacious vaccine strain that fails to produce an O-chain and thus a lack of an anti-O-chain humoral response greatly simplifies identification of vaccinated versus field strain infected animals. The three primary objectives of this research were the following: 1) to develop a serological assay to detect anti-RB51 antibodies in vaccinated elk (Cervus elaphus nelsoni), 2) to identify potential antigenic alterations in RB51 after vaccination of elk and BALB/c mice, and 3) to confirm the general stability of RB51. Elk were divided into four groups based upon gender and the route of inoculation (subcutaneous or ballistic) of RB51 bacteria. This study developed a highly reliable ELISA (using a monoclonal anti-bovine IgG 1 antibody and acetone killed whole RB51 bacteria) which can identify RB51-vaccinated elk. Also, isolates recovered from RB51-vaccinated elk were inoculated into female BALB/c mice whose spleens were then cultured. All elk and mice isolates were bacteriologically, biochemically, and serologically evaluated. This study showed that RB51 is a highly stable strain, which does not revert to smooth morphology or initiate synthesis of LPS-O-chain, maintains it biochemical characteristics, does not undergo detectable antigenic variations, and remains attenuated even after successive passages in elk and mice. Overall, this research indicates that RB51 is a vaccine candidate for the prevention of brucellosis in elk. Further studies are needed to determine the protective capabilities of RB51 in elk. / Master of Science

A proteinúria de origem renal é um indicador de lesão e atua na progressão da doença renal crônica em cães. Na rotina clínica, várias são as recomendações em relação à terapia de manutenção, que visam minimizar ou retardar a progressão da doença. Com o recente progresso na pesquisa sobre a terapia com as células tronco mesenquimais (CTM), tem-se discutido sobre a possibilidade de minimização dos mecanismos inflamatórios e imunológicos de autoperpetuação da lesão renal com a sua utilização, assim a análise sequencial das proteínas urinárias por métodos qualitativos, tais como a eletroforese em gel de poliacrilamida (SDS-PAGE) e a imunodetecção das proteínas por Western Blot pode trazer subsídios para esta avaliação. Portanto, como hipótese, suscita-se que a administração de CTM em cães com DRC possa trazer benefícios com o intuito de minimizar o desenvolvimento da proteinúria, contribuindo para o retardo na velocidade de progressão da doença renal. Trata-se de um estudo prospectivo, longitudinal e duplo-cego em que foram avaliados 22 cães com DRC, tratados com solução fisiológica (SF) ou CTM e avaliados a cada 30 a 45 dias em 12 momentos, divididos em grupos de acordo com o estágio da doença, grupo A (estágio 2, SF:n=6, CTM:n=3) e grupo B (estágio 3, SF:n=6, CTM:n=7). Observou-se que os cães do Grupo B apresentaram proteinúria mais intensa, maior porcentagem de proteínas de alto peso molecular, maior imunodetecção de albumina, proteínas ligadas ao retinol e a vitamina D e menor imunodetecção de proteína de Tamm-Horsfall ao longo do acompanhamento, quando comparado ao Grupo A. Os resultados obtidos nesse estudo não permitiram definir conclusões contundentes de que a terapia com a CTM tenha trazido alterações importantes que indicassem o seu benefício. Os dados sugerem que os cães nos estágios iniciais da DRC (estágio 2) poderiam ser os mais favorecidos com a referida terapia, entretanto mais estudos contemplando um número maior de animais, como o maior tempo de acompanhamento devem ser conduzidos para tal investigação. / Protein of renal origin is an indicator of injury and acts on the progression of chronic kidney disease in dogs. In the clinical routine, several recommendations regarding maintenance therapy are aimed at minimizing or delaying the progression of the disease. With the recent progress in the research on mesenchymal stem cell therapy (CTM), it has been discussed the possibility of minimizing the inflammatory and immunological mechanisms of self-perpetuation of the renal lesion with its use, thus the sequential analysis of the urinary proteins by qualitative methods such as polyacrylamide gel electrophoresis (SDS-PAGE) and the immunodetection of proteins by Western blot can prove the efficacy of the therapy. Therefore, as hypothesis, it is suggested that the administration of CTM in dogs with CKD can fetch benefits in order to minimize the development of proteinuria, contributing to the delay in the rate of progression of renal disease. This is a prospective, longitudinal, double-blind study in which 22 dogs with CKD were treated with physiological solution (SF) or CTM and evaluated every 30 to 45 days in 12 moments divided into groups according to (stage 2, SF: n = 6, CTM: n = 3) and group B (stage 3, SF: n = 6, CTM: n = 7). Group B dogs were found to have more intense proteinuria, a higher percentage of high molecular weight proteins, greater albumin immunodetection as well as retinol-binding protein and vitamin D-binding protein, and decreased Tamm-Horsfall protein immunodetection throughout follow-up, when compared to Group A. The results obtained in this study did not allow to draw conclusive conclusions that CTM therapy brought important changes that indicated its benefit. The data suggest that dogs in the early stages of CKD (stage 2) could be the most favored with such therapy, however more studies contemplating a larger number of animals, such as longer follow-up, should be conducted for such investigation.

A colibacilose é uma enfermidade importante na avicultura por causar prejuízos econômicos e danos à carcaça. Escherichia coli é um micro-organismo oportunista e a complexidade da infecção se deve a diversidade de sorotipos, variados fatores de virulência e baixa capacidade de estimular a imunidade cruzada. Assim, a compreensão dos mecanismos de virulência que ocorrem no hospedeiro, pode resultar no desenvolvimento de métodos diagnósticos e profiláticos. Neste estudo, 85 pintinhos foram divididos em quatro grupos, sendo três infectados com diferentes sorogrupos de Escherichia coli (O2, O78, O119) e um vacinado com Poulvac® E.coli (Pfizer®). As lesões sugestivas de colibacilose foram avaliadas e qualificadas aos 35 dias de idade, e em seguida realizada a colheita de sangue para a obtenção do soro. Foram obtidas proteínas in vitro dos sorogrupos de E. coli utilizados na infecção experimental e da vacina (mutante de Escherichia coli O78). Estas foram separadas por eletroforese unidimensional em gel de acrilamida utilizando dodecil sulfato de sódio (SDS) e foram submetidas a reação de western blot com a mistura de soros de cada grupo estudado e também com o pool de soros de aves livres de patógenos específicos (SPF), usadas como controle negativo. O perfil eletroforético revelou a presença de várias proteínas dos diferentes sorogrupos de E. coli quando cultivadas no meio casaminoácido e levedura. Houve diferenças entre os sorotipos, mas também proteínas comuns. Foi possível estabelecer uma relação entre virulência, quantidades de proteínas específicas e taxa de reisolamento. / The colibacillosis is a serious disease in poultry industry to cause economic losses and damage to carcass. Escherichia coli is an opportunistic microorganism and the complexity of infection is due to the diversity of serotypes, several virulence factors and low ability to stimulate cross-immunity. Thus understanding of virulence mechanisms that occur in the host, may result in development of diagnostic and prophylactic methods. In this study, 85 chicks were divided into four groups being infected with three different serogroups of E. coli (O2, O78, O119) and vaccinated with Poulvac E. coli ® (Pfizer ®). The lesions suggestive of colibacillosis were evaluated at 35 days of age, and then the blood sample are collected to obtain the serum. Proteins were obtained in vitro from serogroups of E. coli used in experimental infection and vaccine (mutant of Escherichia coli O78). These were separated by one-dimensional electrophoresis on acrylamide gels using sodium dodecyl sulfate (SDS) and then reacted by western blot with the pool of sera from each group and also with the pool of sera from specific pathogen free poultry used as negative control. The electrophoretic pattern showed the presence of many proteins of different serogroups of E. coli when grown in the broth casaminoacid and yeast. There were differences among serotypes, but also common proteins. It was possible to establish a relationship between virulence and reisolation rate.

A colibacilose é uma enfermidade importante na avicultura por causar prejuízos econômicos e danos à carcaça. Escherichia coli é um micro-organismo oportunista e a complexidade da infecção se deve a diversidade de sorotipos, variados fatores de virulência e baixa capacidade de estimular a imunidade cruzada. Assim, a compreensão dos mecanismos de virulência que ocorrem no hospedeiro, pode resultar no desenvolvimento de métodos diagnósticos e profiláticos. Neste estudo, 85 pintinhos foram divididos em quatro grupos, sendo três infectados com diferentes sorogrupos de Escherichia coli (O2, O78, O119) e um vacinado com Poulvac® E.coli (Pfizer®). As lesões sugestivas de colibacilose foram avaliadas e qualificadas aos 35 dias de idade, e em seguida realizada a colheita de sangue para a obtenção do soro. Foram obtidas proteínas in vitro dos sorogrupos de E. coli utilizados na infecção experimental e da vacina (mutante de Escherichia coli O78). Estas foram separadas por eletroforese unidimensional em gel de acrilamida utilizando dodecil sulfato de sódio (SDS) e foram submetidas a reação de western blot com a mistura de soros de cada grupo estudado e também com o pool de soros de aves livres de patógenos específicos (SPF), usadas como controle negativo. O perfil eletroforético revelou a presença de várias proteínas dos diferentes sorogrupos de E. coli quando cultivadas no meio casaminoácido e levedura. Houve diferenças entre os sorotipos, mas também proteínas comuns. Foi possível estabelecer uma relação entre virulência, quantidades de proteínas específicas e taxa de reisolamento. / The colibacillosis is a serious disease in poultry industry to cause economic losses and damage to carcass. Escherichia coli is an opportunistic microorganism and the complexity of infection is due to the diversity of serotypes, several virulence factors and low ability to stimulate cross-immunity. Thus understanding of virulence mechanisms that occur in the host, may result in development of diagnostic and prophylactic methods. In this study, 85 chicks were divided into four groups being infected with three different serogroups of E. coli (O2, O78, O119) and vaccinated with Poulvac E. coli ® (Pfizer ®). The lesions suggestive of colibacillosis were evaluated at 35 days of age, and then the blood sample are collected to obtain the serum. Proteins were obtained in vitro from serogroups of E. coli used in experimental infection and vaccine (mutant of Escherichia coli O78). These were separated by one-dimensional electrophoresis on acrylamide gels using sodium dodecyl sulfate (SDS) and then reacted by western blot with the pool of sera from each group and also with the pool of sera from specific pathogen free poultry used as negative control. The electrophoretic pattern showed the presence of many proteins of different serogroups of E. coli when grown in the broth casaminoacid and yeast. There were differences among serotypes, but also common proteins. It was possible to establish a relationship between virulence and reisolation rate.

A proteinúria de origem renal é um indicador de lesão e atua na progressão da doença renal crônica em cães. Na rotina clínica, várias são as recomendações em relação à terapia de manutenção, que visam minimizar ou retardar a progressão da doença. Com o recente progresso na pesquisa sobre a terapia com as células tronco mesenquimais (CTM), tem-se discutido sobre a possibilidade de minimização dos mecanismos inflamatórios e imunológicos de autoperpetuação da lesão renal com a sua utilização, assim a análise sequencial das proteínas urinárias por métodos qualitativos, tais como a eletroforese em gel de poliacrilamida (SDS-PAGE) e a imunodetecção das proteínas por Western Blot pode trazer subsídios para esta avaliação. Portanto, como hipótese, suscita-se que a administração de CTM em cães com DRC possa trazer benefícios com o intuito de minimizar o desenvolvimento da proteinúria, contribuindo para o retardo na velocidade de progressão da doença renal. Trata-se de um estudo prospectivo, longitudinal e duplo-cego em que foram avaliados 22 cães com DRC, tratados com solução fisiológica (SF) ou CTM e avaliados a cada 30 a 45 dias em 12 momentos, divididos em grupos de acordo com o estágio da doença, grupo A (estágio 2, SF:n=6, CTM:n=3) e grupo B (estágio 3, SF:n=6, CTM:n=7). Observou-se que os cães do Grupo B apresentaram proteinúria mais intensa, maior porcentagem de proteínas de alto peso molecular, maior imunodetecção de albumina, proteínas ligadas ao retinol e a vitamina D e menor imunodetecção de proteína de Tamm-Horsfall ao longo do acompanhamento, quando comparado ao Grupo A. Os resultados obtidos nesse estudo não permitiram definir conclusões contundentes de que a terapia com a CTM tenha trazido alterações importantes que indicassem o seu benefício. Os dados sugerem que os cães nos estágios iniciais da DRC (estágio 2) poderiam ser os mais favorecidos com a referida terapia, entretanto mais estudos contemplando um número maior de animais, como o maior tempo de acompanhamento devem ser conduzidos para tal investigação. / Protein of renal origin is an indicator of injury and acts on the progression of chronic kidney disease in dogs. In the clinical routine, several recommendations regarding maintenance therapy are aimed at minimizing or delaying the progression of the disease. With the recent progress in the research on mesenchymal stem cell therapy (CTM), it has been discussed the possibility of minimizing the inflammatory and immunological mechanisms of self-perpetuation of the renal lesion with its use, thus the sequential analysis of the urinary proteins by qualitative methods such as polyacrylamide gel electrophoresis (SDS-PAGE) and the immunodetection of proteins by Western blot can prove the efficacy of the therapy. Therefore, as hypothesis, it is suggested that the administration of CTM in dogs with CKD can fetch benefits in order to minimize the development of proteinuria, contributing to the delay in the rate of progression of renal disease. This is a prospective, longitudinal, double-blind study in which 22 dogs with CKD were treated with physiological solution (SF) or CTM and evaluated every 30 to 45 days in 12 moments divided into groups according to (stage 2, SF: n = 6, CTM: n = 3) and group B (stage 3, SF: n = 6, CTM: n = 7). Group B dogs were found to have more intense proteinuria, a higher percentage of high molecular weight proteins, greater albumin immunodetection as well as retinol-binding protein and vitamin D-binding protein, and decreased Tamm-Horsfall protein immunodetection throughout follow-up, when compared to Group A. The results obtained in this study did not allow to draw conclusive conclusions that CTM therapy brought important changes that indicated its benefit. The data suggest that dogs in the early stages of CKD (stage 2) could be the most favored with such therapy, however more studies contemplating a larger number of animals, such as longer follow-up, should be conducted for such investigation.