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.

Researchers have tried to identify the viral factor involved in making a transformed cell oncogenic in newborn hamsters. Although studies on the viral proteins from transformed cells failed to show a relationship to oncogenicity, this study intended to identify and map viral mRNAs from these transformed cell lines in order to test the same relationship. Northern blot hybridizations were used to study the AD5 E1 region specific mRNAs from ten transformed cell lines. These cell lines had been transformed with either virus, total restriction endonuclease digested viral DNA, or specific fragments of viral DNA from the left hand end of the genome. All of these lines were selected for their varying oncogenicity in newborn hamsters, and for the size of the transforming fragment. Ad5 DNA fragments inserted into recombinant plasmids were used as the probes for detection of mRNAs, providing tools for mapping transformed cell mRNAs to specific regions of the Ad5 genome. The results failed to show a relationship between E1 region mRNA production and oncogenicity, but did reveal unusual mRNA transcription patterns from most of the cell lines. All the transformed cell lines tested appeared to have only an E1B 22s mRNA as the major mRNA from that region. An E1A/E1B cotranscript was identified as well as viral/cell chimeric mRNAs. These chimeras were due to either run-off transcription or to preinitiation in cellular sequences. / Thesis / Master of Science (MS)

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.

Mitochondria are membrane bound organelles important for energy production in respiring cells through the process of oxidative phosphorylation. They have their own multi-copied mitochondrial DNA (mtDNA) genome separate from that in the nucleus that is needed for mitochondria to function properly and can exist in both wild type and mutant forms in the same cell. The integrity of the mtDNA is therefore of vital importance for the survival of the organism and as such understanding the mechanisms of mtDNA maintenance is relevant to human health and disease. This study employs a Southern blotting and non-radioactive probe method to examine various aspects of mtDNA maintenance. Restriction endonuclease mapping utilizing mtDNA-specific and nuclear DNA-specific digoxigenin (DIG)-labeled probes was performed to show that the synthesized probes are indeed specific for their target sequences. The DIG-labeled probes were used to quantitate mtDNA content from different DNA isolation methods. Whole-cell DNA extraction was found to yield higher levels of mtDNA compared to a commercially available spin-column kit. Next, Southern blots were used to analyze mtDNA copy number as well as mtDNA depletion in the hepatocarcinoma-derived cell line HepaRG following exposure to the nucleoside reverse transcriptase inhibitor 2’,3’-dideoxycytidine (ddC), a known mitochondrial toxicant. In comparison to proliferative HepaRG differentiated HepaRG contained about 2-fold more mtDNA. Relative to untreated control cells, proliferating HepaRG exposed to ddC had greater than a 96% reduction in mtDNA and had decreased cellular viability. Differentiated HepaRG cell viability was not affected after 13 days of ddC treatment; however, significant mtDNA depletion was observed. We estimate that differentiated HepaRG mtDNA depletion occurs quickly at about 20 molecules per hour.

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.

Creation of a reliable and easy to use serologic test would greatly improve ante mortem diagnosis of Mycobacterium avium subsp. avium and aid in the control of avian mycobacteriosis, particularly in captive birds. In order to determine whether serodiagnostics could be of value in testing ring-neck doves (Streptopelia risoria) for M. a. avium infection, Western blot analysis was used to assess the humoral response of ring-neck doves exposed to M. a. avium, and to evaluate whether an association could be made between humoral response and necropsy findings, histopathology, culture, and PCR testing. Western blot results were examined for reactivity patterns associating the humoral response with infection status, severity and type of lesions (diffuse vs multifocal granulomatous inflammation) and phenotype (white vs non-white). A sensitivity of 88.24% and a specificity of 100% were achieved utilizing Western blot analysis to detect M. a. avium infection in ring-neck doves, offering a negative predictive value of 93% and a positive predictive value of 100%. While Western blot analysis results did not reflect lesion severity, lesion type did partially correspond with the humoral response. The findings of the present study indicate that serologic testing can be used as a valuable ante mortem screening tool for identifying ring-neck doves infected with M. a. avium.

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

O Tomato chlorosis virus (ToCV) é uma espécie do gênero Crinivirus que causa danos, principalmente na cultura do tomateiro (Solanum lycopersicum). Foi primeiramente isolado e descrito em 1998, nos Estados Unidos, e em seguida foi reportado em doze países. No Brasil, foi constatado primeiramente no Estado de São Paulo, na região de Sumaré, em 2008, e posteriormente nos Estados da Bahia, Espírito Santo, Goiás, Minas Gerais e Rio de Janeiro. Há evidência da sua presença também nos Estados do Paraná e Santa Catarina. O ToCV pode infectar outras solanáceas além do tomateiro e, recentemente, foi observado infectando plantas de batata (Solanum tuberosum) no Brasil. Esse crinivirus é transmitido no Brasil principalmente pelo aleirodídeo (mosca branca) Bemisia tabaci MEAM1. Considerando o patossistema batateira/ToCV, não há estudos sobre a ocorrência, sintomatologia em diferentes variedades e danos provocados por esse crinivirus. Também não há antissoro policlonal para o isolado brasileiro do ToCV para uso na diagnose da doença em solanáceas. Esse trabalho teve por objetivos: purificar o ToCV e produzir antissoro policlonal; avaliar a reação de genótipos de batateira à infecção com o ToCV; avaliar o dano provocado por esse vírus em duas variedades de batateira. A purificação do vírus a partir de folhas de tomateiro e a produção de antissoro policlonal em coelho foram satisfatórias. No entanto, o antissoro não foi eficiente em ELISA, mas sim em dot-blot e somente na diluição de 1:20. Foi avaliada a reação de 21 genótipos de batateira à infecção com o ToCV, por meio da inoculação com B. tabaci MEAM1, com chance de escolha do vetor. Nenhum genótipo exibiu resistência à infecção; enquanto a variedade Camila foi assintomática e não apresentou alteração na fotossíntese. Plantas de batateira das variedades Ágata e Asterix sadias foram inoculadas com o ToCV, por meio da B. tabaci MEAM1 e ao final foram avaliadas a massa fresca da parte aérea, peso e número dos tubérculos colhidos. Em dois experimentos independentes, as reduções médias no peso fresco da parte aérea foram de 60,1% para Ágata e 46% para Asterix. Porém, as reduções nas produções dessas variedades, no primeiro experimento foram de 99,5% e 98,1%, respectivamente; enquanto no segundo os valores foram de 82,3% e 56,2%, respectivamente. / Tomato chlorosis virus (ToCV) is a species of the genus Crinivirus, which is causing considerable losses mainly on tomato crop (Solanum lycopersicum). It was first isolated and described on 1998 in the United States and subsequently reported in twelve countries. In Brazil, it was first reported in São Paulo State, in Sumaré region in 2008, and after that on the states of Bahia, Espírito Santo, Goiás, Minas Gerais and Rio de Janeiro. There is evidence of the presence of ToCV on the states of Paraná and Santa Catarina. ToCV can also infect other solanaceae and more recently, it was reported infecting potato plants (Solanum tuberosum) in Brazil. This crinivirus is transmitted by Bemisia tabaci MEAM1. Considering the patosystem potato/ToCV, there are no studies on the occurrence, symptomatology in different varieties, and damages caused by this crinivirus. In addition, there is no polyclonal antiserum for the Brazilian isolate of ToCV for use in diagnosis. The objectives of the present work were: to purify the virus and produce a polyclonal antiserum; to evaluate the reaction of potato genotypes to ToCV infection; to evaluate the yield loss caused by this crinivirus on two potato cultivars. The virus purification from tomato leaves and the production of polyclonal antiserum in rabbit were satisfactorily accomplished. However, the antiserum was not efficient on ELISA test, but in dot-blot, only when diluted 1:20. The reaction of 21 potato genotypes to infection with ToCV was evaluated by inoculation with B. tabaci MEAM1, with chance of choice for the vector. All genotypes were infected with ToCV and Camila was the only one asymptomatic. Plants of cultivars Ágata and Asterix were inoculated with ToCV, by means of viruliferous vector, and at the end were evaluated for the fresh mass of the aerial part, weight and number of harvested tubers. In two independent experiments, average reductions in aerial fresh weight were 60.1% for Ágata and 46% for Asterix. However, reductions in yield of these varieties in the first experiment were 99.5% and 98.1%, respectively; while in the second the values were 82.3% and 56.2%, respectively.