Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p.113-118).

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Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-26Bitstream added on 2014-06-13T20:50:00Z : No. of bitstreams: 1 vieira_gal_me_araiq.pdf: 2780789 bytes, checksum: 822f36edcc6ae68080d4ff7d0cca2cdf (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O protozoário hemoflagelado, Trypanosoma cruzi, transmitido principalmente pelo inseto triatomíneo, por infecção congênita ou por transfusão sanguínea, causa a tripanossomíase americana ou doença de Chagas, como é mais conhecida. Essa é uma séria doença parasitária que ocorre na América Latina, com considerável impacto social e econômico. Dois fármacos, nifurtimox e benzonidazol, são indicados no tratamento de pessoas infectadas, mas são pouco eficientes na fase aguda e praticamente ineficientes na fase crônica da doença. Devido a esses fatores, é de extrema importância que se encontre agentes quimioterápicos e/ou quimiopreventivos mais eficientes e eficazes. O presente trabalhou objetivou avaliar a influência no proteoma de T. cruzi de três substâncias extraídas de plantas da família Piperaceae, peperobtusina A e B de Peperomia obtusifolia e piplartina de Piper tuberculatum, através da eletroforese bidimensional (2D) – DIGE (Difference Gel Electrophoresis - GE Healthcare) associada à espectrometria de massas. O tratamento realizado com peperobtusina A de Peperomia obtusifolia em cepa Y de T. cruzi identificou proteínas de interesse, como proteína do bastão paraflagelar e triparedoxina peroxidase, importantes na composição flagelar e proteção natural do parasito ao estresse oxidativo, respectivamente. Os resultados obtidos com peperobtusina B de Peperomia obtusifolia na mesma cepa de T. cruzi mostraram proteínas que podem servir como interessantes alvos potenciais para futuro desenvolvimento de fármacos. São elas: calmodulina, tirosina aminotransferase e arginina quinase. Os resultados obtidos com o tratamento das cepas Y e Bol de T. cruzi pela piplartina de Piper tuberculatum demonstraram que apesar das diferenças de suscetibilidade dessas cepas em relação ao benzonidazol... / The protozoa hemoflagelate, Trypanosoma cruzi, transmitted mainly for the triatomíneo insect, congenital infection or sanguineous transfusion, cause American tripanosomiasis or Chagas disease, as more it is known. This is a serious parasitic illness that occurs in Latin America, with considerable social and economic impact. Two drugs, nifurtimox and benznidazol, are indicated in the treatment of infected people, but they are little efficient in the acute phase and practically inefficient in the chronic phase of the illness. Because of these factors, it is of extreme importance find efficient chemopreventive and/or chemotherapic agents. This work aims to evaluate the T. cruzi proteome influence of three substances extracted of plants belonging to Piperaceae family, being peperobtusine A and B of Peperomia obtusifolia and piplartine of Piper tuberculatum, through two dimensional electrophoresis (2D) - DIGE (Difference Gel Electrophoresis - GE Healthcare) and mass spectrometry. The treatment carried with peperobtusine A of P. obtusifolia in Y strain of T. cruzi identified interest proteins, as paraflagelar rod protein and triparedoxin peroxidase, important in the flagella composition and natural protection of oxidative stress, respectively. The results using peperobtusine B of the P. obtusifolia in Y strain of T. cruzi showed proteins that can be interesting potential targets for future development of drug. They are: calmodulin, tyrosine aminotransferase and arginine kinase. The results with the treatment of Y and Bol strains of T. cruzi with piplartine of P. tuberculatum demonstrated that although to the differences of susceptibility of these strains related to benznidazol, the piplartine was active for both strains as a similar way and modified the expression of enzymes involved in the protection of the parasitic to the... (Complete abstract click electronic access below)

Studies in animals and humans link both physical and psychological stress with an increased rate and severity of infections and onset of diseases. Stress is a very broad and complex topic. It can be defined as a condition occurring in response to adverse external influences capable of affecting physical health which leads to activation of a stress response in the body. There are two prominent stress responsive systems: the hypothalamic-pituitary-adrenal axis and the sympathetic adrenomedullary axis. These systems are responsible for the majority of the changes in the body, which occur in response to stress. Stress has been linked to many detrimental effects in cattle including immune suppression, increased disease susceptibility and decreased reproduction. These cause huge economic losses to the cattle industry every year. Weaning has been identified as one of the main stressors implicated in these negative effects. For this reason it is important to be able to identify animals stressed by weaning and do so using samples which are easily obtainable and useful for future diagnostic purposes. We hypothesize that weaning will cause sufficient stress in cattle to alter protein profiles in serum, which can be used to identify this type of stress. To do this we employed proteomic methodologies including two-dimensional gel electrophoresis and mass spectrometry to compare an abrupt weaned group of calves to a never weaned group and a previously weaned group (preconditioned). We have included a preconditioned group to examine the differences between this group and animals which have never been weaned. Preconditioned animals are typically used as a control group in weaning studies. A total of 83 distinct protein bands were identified after image analysis. Out of 83 protein bands, we found 9 spots which were significantly different in abundance among the treatment groups. Two out of 9 spots were significantly different between the abrupt weaned and the never weaned groups. Five protein bands were also found to be significantly different between the abrupt weaned group and the preconditioned group. Five protein bands were found to be significantly different between the never weaned group and the preconditioned group. Identification of these proteins, however, had limited success since the bovine protein database is not as extensive as that for humans or mice. Among the proteins identified were alpha-1-acid glycoprotein and collagen precursor. The differences in intensities found between the abrupt weaned group and the never weaned group may be useful as markers of calves going through weaning stress. We have also seen that animals who have undergone weaning and through the stress associated with that event are not exactly the same as animals which have never been weaned. This has implications to research where a preconditioned group is used as a control rather than a never weaned group. Despite the limitations of the methodology used for the current system, the overall results revealed specific changes in serum proteins which were associated with abrupt weaned animals. Future studies can be planned to determine the specificity of these protein changes and possibly identify the molecular basis of stress dependent disease susceptibility.

Studies in animals and humans link both physical and psychological stress with an increased rate and severity of infections and onset of diseases. Stress is a very broad and complex topic. It can be defined as a condition occurring in response to adverse external influences capable of affecting physical health which leads to activation of a stress response in the body. There are two prominent stress responsive systems: the hypothalamic-pituitary-adrenal axis and the sympathetic adrenomedullary axis. These systems are responsible for the majority of the changes in the body, which occur in response to stress. Stress has been linked to many detrimental effects in cattle including immune suppression, increased disease susceptibility and decreased reproduction. These cause huge economic losses to the cattle industry every year. Weaning has been identified as one of the main stressors implicated in these negative effects. For this reason it is important to be able to identify animals stressed by weaning and do so using samples which are easily obtainable and useful for future diagnostic purposes. We hypothesize that weaning will cause sufficient stress in cattle to alter protein profiles in serum, which can be used to identify this type of stress. To do this we employed proteomic methodologies including two-dimensional gel electrophoresis and mass spectrometry to compare an abrupt weaned group of calves to a never weaned group and a previously weaned group (preconditioned). We have included a preconditioned group to examine the differences between this group and animals which have never been weaned. Preconditioned animals are typically used as a control group in weaning studies. A total of 83 distinct protein bands were identified after image analysis. Out of 83 protein bands, we found 9 spots which were significantly different in abundance among the treatment groups. Two out of 9 spots were significantly different between the abrupt weaned and the never weaned groups. Five protein bands were also found to be significantly different between the abrupt weaned group and the preconditioned group. Five protein bands were found to be significantly different between the never weaned group and the preconditioned group. Identification of these proteins, however, had limited success since the bovine protein database is not as extensive as that for humans or mice. Among the proteins identified were alpha-1-acid glycoprotein and collagen precursor. The differences in intensities found between the abrupt weaned group and the never weaned group may be useful as markers of calves going through weaning stress. We have also seen that animals who have undergone weaning and through the stress associated with that event are not exactly the same as animals which have never been weaned. This has implications to research where a preconditioned group is used as a control rather than a never weaned group. Despite the limitations of the methodology used for the current system, the overall results revealed specific changes in serum proteins which were associated with abrupt weaned animals. Future studies can be planned to determine the specificity of these protein changes and possibly identify the molecular basis of stress dependent disease susceptibility.

Mammary gland samples from a large serial-slaughter Holstein heifer nutrition trial were used to determine the effects of stage of development and nutritional management on mitogenic activity of mammary extracts and mammary parenchymal composition. Stage of development and nutritional management of heifers had minimal effects on the mitogenic capacity of mammary extracts, and no significant effects on tissue composition. Two-dimensional proteome maps of heifer mammary extracts were constructed for heifers weighing 200 and 350 kg, respectively at slaughter. Proteins altered by stage of development and/or nutrition were quantified and identified; 820 total protein spots were analyzed. The expression of 131 protein spots differed by dietary treatment only. Stage of development altered the expression of 108 protein spots. Twenty-two protein spots were excised from gels for mass spectrometry analyses. Database searches for proteins with shared primary amino acid sequences were used to identify the proteins. Possible roles of these proteins in mammary development were described. In summary, heifers can be reared on high planes of nutrition without impairing mammary development, but mechanisms governing nutritional and temporal control of mammary development demand further investigation. / Master of Science

The unicellular parasite Giardia intestinalis is one of the organisms carrying mitochondrion-related organelle known as mitosome, which is adapted to the microaerobic lifestyle. The only known fuction of the mitosome is the synthesis of the iron-sulphur clusters. The research of the mitosomal proteome provides new information on the biogenesis and function of this unusual organelle. One of the means of the mitosome research is the analysis of the interactome of the known mitosomal proteins. The state-of- the-art method of the interactome approach is the use of the chemical crosslinking and the subsequent immunoaffinity isolation of the complexes, containing the protein of interest. In this thesis, the interactomes of GiTom40 and GiMOMP35 were characterized with the bioinformatic tools. The cellular localization of four of the chosen proteins was determined by the fluorescent microscopy. One of the proteins, the predicted dynein intermediate chain DIC6939, was phylogenetically classified as an axonemal dynein. The superresolution microsopy was utilized to observe the possible colocalization of DIC6939 with the mitosomes and blue native PAGE led to the visualization of its native complexes. In this work, the optimal conditions for DIC6939 interactome isolation were succesfully determined. The outcome...

Este trabalho teve como objetivo principal realizar um estudo proteômico diferencial aplicado à amostra Bradyrizobium elkanii (SEMIA 587). Isso se fez através da identificação de proteínas diferencialmente expressas nesta importante bactéria fixadora de nitrogênio quando cultivada em laboratório (in vitro) e quando inoculada em plantas de soja (in vivo). A caracterização das proteínas foi feita com a poderosa técnica de espectrometria de massas, e a análise proteômica com expressão diferencial foi comparada por meio de duas estratégias diferentes: MudPIT e Maldi-QToF. A avaliação das imagens dos géis bidimensionais permitiu verificar que a amostra de bactéria apresentou um número maior de proteínas expressas em relação ao bacterióide. Se comparada ao microrganismo na condição in vitro (bactéria), a espécie fixadora de nitrogênio (bacterióide) apresentou 19 proteínas induzidas e 230 reprimidas. O aumento de expressão observado para as proteínas do bacterióide foi pelo menos duas vezes maior do que o volume normalizado para 17 proteínas e, caiu pela metade (ou mais), para outras 26 proteinas em relação a bactéria. Através de ambas as técnicas (Maldi-QToF e ESI-QToF) empregadas na identificação das proteínas observou-se, comprovando a complementariedade existente entre as metodologias, que os padrões metabólicos tanto em bactéria como bacterióide foram mantidos. Ou seja, bactérias cultivadas em meio YML apresentaram um metabolismo voltado para a síntese celular, denotada pela produção de macromoléculas e metabolismo energético, ao passo que bacterióides, endosimbiontes isolados de nódulos de plantas de soja, apresentaram um metabolismo especialmente dedicado à fixação simbiótica do nitrogênio. / The main goal of this project was to perform a differential proteomic analysis of Bradyrizobium elkanii (SEMIA 587). It was made by the identification of differentially expressed proteins in this important nitrogen fixing bacteria grown under different conditions: in laboratory culture (in vitro) and symbiotically associated with soybean plants (in vivo). The characterization of proteins differentially expressed was done by the powerful technique of mass spectrometry, applying two different strategies: MudPIT and Maldi-QToF. It was possible, after computerized image analysis of 2D gels, to verify that bacteria had more expressed proteins than bacteroids. In vivo bacteria showed 19 proteins induced and 230 suppressed. The increase in expression observed for bacteroids macromolecules was at least two times greater than normalized volume for 17 proteins of the bacteria and two times smaller than 26 bacteria proteins. It was also observed, through Maldi-QToF and ESI-QToF techniques applied to protein identification, that bacteria and bacteroid main metabolic activities were maintained. This fact confirms that the techniques were complementary. The data showed that bacteria cultivated in YML medium had a metabolism direct to cellular synthesis, characterized by production of macromolecules and energetic metabolism. Endosimbiont bacteroids isolated from soyabean nodules showed a metabolism specially dedicated to nitrogen fixation. The results were in agreement with the ambient that each microorganism was originated. The fact that the identified proteins in bacteria were related to translation and aminoacids biosynthesis shows the need that heterotrophic microorganisms have to synthesize its own proteins and enzymes useful for their metabolism. On other hand, since the bacteroids are symbiotically associated with soybean reducing atmospheric nitrogen in an extremely energy-intense process, it is expected that its proteins should be mainly related to biological nitrogen fixation and energetic metabolism.

The immune system has the ability to discriminate self from non-self proteins and also make appropriate immune responses to pathogens. A fundamental problem is to understand the genomic differences and similarities among the sets of self peptides and non-self peptides. The sequencing of human, mouse and numerous pathogen genomes and cataloging of their respective proteomes allows host self and non-self peptides to be identified. T-cells make this determination at the peptide level based on peptides displayed by MHC molecules.<p>In this project, peptides of specific lengths (k-mers) are generated from each protein in the proteomes of various model organisms. The set of unique k-mers for each species is stored in a library and defines its "immunological self". Using the libraries, organisms can be compared to determine the levels of peptide overlap. The observed levels of overlap can also be compared with levels which can be expected "at random" and statistical conclusions drawn.<p>A problem with this procedure is that sequence information in public protein databases (Swiss-PROT, UniProt, PIR) often contains ambiguities. Three strategies for dealing with such ambiguities have been explored in earlier work and the strategy of removing ambiguous k-mers is used here.<p>Peptide fragments (k-mers) which elicit immune responses are often localized within the sequences of proteins from pathogens. These regions are known as "immunodominants" (i.e., hot spots) and are important in immunological work. After investigating the peptide universes and their overlaps, the question of whether known regions of immunological significance (e.g., epitope) come from regions of low host-similarity is explored. The known regions of epitopes are compared with the regions of low host-similarity (i.e., non-overlaps) between HIV-1 and human proteomes at the 7-mer level. Results show that the correlation between these two regions is not statistically significant. In addition, pairs involving human and human viruses are explored. For these pairs, one graph for each k-mer level is generated showing the actual numbers of matches between organisms versus the expected numbers. From graphs for 5-mer and 6-mer level, we can see that the number of overlapping occurrences increases as the size of the viral proteome increases.<p>A detailed investigation of the overlaps/non-overlaps between viral proteome and human proteome reveals that the distribution of the locations of these overlaps/non-overlaps may have "structure" (e.g. locality clustering). Thus, another question that is explored is whether the locality clustering is statistically significant. A chi-square analysis is used to analyze the locality clustering. Results show that the locality clusterings for HIV-1, HIV-2 and Influenza A virus at the 5-mer, 6-mer and 7-mer levels are statistically significant. Also, for self-similarity of human protein Desmoglein 3 to the remaining human proteome, it shows that the locality clustering is not statistically significant at the 5-mer level while it is at the 6-mer and 7-mer levels.