Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 129-143).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 117-127).

Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic but some activity is also present in the nuclear fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells (MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion chromatography on Sephacryl S-300 and glycerol density gradient sedimentation. The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc. Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12 different polypeptide components compared to the 28 different polypeptide components of MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not have any obvious sequence homology with the subunits of proteasomes from other cells. Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct. The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes. MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes. Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell types are activated by endogenous high molecular mass complexes such as the bovine 19S complex called PA700. These complexes form end-on associations with the 20S proteasome. However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited unique end-on associations between individual units forming long (up to 200 nm) ribbon-like chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism for controlling their proteasome activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [87]-96).

Berlin : Freie Univ., Diss., 2005 / Dateiformat: zip, Dateien im PDF-Format. - Erscheinungsjahr an der Haupttitelstelle: 2005

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 132-145).

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 3 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-09-03T16:36:35Z No. of bitstreams: 1 monete_r_gomes_ioc_bcs_0002_2010.pdf: 4408396 bytes, checksum: dc300c2a3e79c9521a3d4a99c18e5e9d (MD5) / Made available in DSpace on 2012-09-03T16:36:35Z (GMT). No. of bitstreams: 1 monete_r_gomes_ioc_bcs_0002_2010.pdf: 4408396 bytes, checksum: dc300c2a3e79c9521a3d4a99c18e5e9d (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Leishmania major, Trypanosoma brucei e Trypanosoma cruzi (Tritryps) são protozoários unicelulares que causam a leishmaniose, a doença do sono e a doença de Chagas, respectivamente. Essas doenças causam ônus econômicos principalmente em regiões subtropicais e tropicais. Atualmente, não existem vacinas comercialmente disponíveis e não há tratamento eficaz para tais doenças. Isso se deve ao fato dos fármacos disponíveis apresentarem muitos efeitos colaterais e estarem propensas ao desenvolvimento de resistência. A maioria desses fármacos foi descoberta através da seleção de um grande número de compostos contra parasitas íntegros. Porém, nos últimos anos, uma nova abordagem vem ganhando espaço sob o termo de “desenho racional de fármacos”. Este termo representa a busca por compostos contra alvos moleculares específicos, visando diferenças bioquímicas e fisiológicas entre o parasita e o hospedeiro. A era pós-genômica gerou uma grande quantidade de informações que permitem a identificação ótima de novos alvos. Neste contexto, a partir de dados públicos dos genomas de Tritryps, reconstruímos as vias de processamento da informação genética (com ênfase nas vias de replicação e reparo, transcrição e tradução) nesses organismos, para adquirir uma melhor representação das enzimas envolvidas nestes processos. Estas análises permitiram estudos comparativos para identificar candidatos a novos alvos terapêuticos. Em nossa metodologia utilizamos a ferramenta AnEnPi (http://bioinfo.pdtis.fiocruz.br/AnEnPi/) para buscar nas seqüências genômicas por enzimas análogas. Utilizando os dados provindos do KEGG, primeiro houve uma etapa de clusterização das estruturas primárias de todas as enzimas desse banco de dados anotadas com o mesmo EC. Para isso utilizou-se uma pontuação (score) de similaridade no Blastp de 120, como parâmetros de corte. Encontramos 830 grupos de ECs com mais de um cluster e 1430 com um único cluster. Após isso, foi realizado um passo de reanotação. Para isto, foi rodado um novo Blastp, assumindo como ponto de corte um e-value de 10e-20, entre todas as proteínas preditas nos genomas de cada Tritryp contra todos os clusters. Desses dados geramos mapas das vias de interesse para esses organismos e os comparamos aos mapas que o KEGG disponibiliza como padrão. Identificamos alguns casos de analogia nestas vias entre seres humanos e Tritryps que podem vir a ser utilizados como novos alvos terapêuticos para o desenvolvimento de fármacos contra esses parasitas. Foi feita a modelagem por homologia de um análogo (6.1.1.-, de T. brucei), utilizando a ferramenta MHOLline. Além disso, buscamos no banco de alvos terapêuticos para doenças negligenciadas, TDRTARGETS (http://tdrtargets.org/), pelos ECs identificados como possíveis novos alvos, e não encontramos nenhuma ocorrência. Tal fato pode indicar que com a metodologia aplicada conseguimos identificar novos candidatos a alvos terapêuticos contra estes parasitas. Em análises futuras, vamos testar e-values mais restritivos na etapa de reanotação, para assim, testar o potencial de reanotação da ferramenta. / Leishmania major, Trypanos oma brucei e Trypanosoma cruzi (Tritryps) are unicellular protozoa that cause leishmaniasis, sleeping sickness and Chagas disease, respectively. These diseases cause economic burden mainly in subtropical and tropical regions. Currently, there are no commer cially available vaccines and no effective treatment for such diseases. This is because the available drugs present many side effects and are willing to develop resistance. Most of these drugs were discovered through the screening of large numbers of compo unds against whole parasites. However, a new approach has been gaining ground under the term "rational drug design", recently. This term represents the search for compounds against specific molecular targets, aiming physiological and biochemical difference s between parasites and hosts. The post - genomic era generated a lot of information that allow optimal identification of new targets. In this context, from public data of the Tritryps’ genomes, we reconstructed the genetic information processing pathway (wi th emphasis on replication and repair, transcription and translation) of these organisms, to obtain a better representation of enzymes involved in these processes. These analyses allowed comparative studies to identify candidates for new therapeutic target s. In our methodology we used the AnEnPi tool ( http://bioinfo.pdtis.fiocruz.br/AnEnPi/ ) to search the genomes for analogous enzymes. Using the data coming from KEGG, there was first a clustering step of the primary structures of all enzymes annotated with the same EC in this database. For this we used a Blastp similarity score of 120 as threshold. We found 830 groups of ECs with more than one cluster and 1430 with only one cluster. After that, we performed a reannotation step. For this task a new Blastp was done, assuming an e - value cutoff of 10e - 20 , among all pr edicted proteins, from each Tritryp genome, against all clusters. From these data we generated maps, for Tritryps, of the pathways of interest and compared them to the KEGG standard maps. We identified some cases of analogy in these pathways between humans and Tritryps that may be used as new therapeutic targets for developing drugs against these parasites. The homology modeling was done for an analog (6.1.1. - , T. brucei ), using the tool MHOLline. Furthermore we also searched in the therapeutic targets data base for neglected disease, TDRTARGES (http://tdrtargets.org/), for the ECs identified as possible new targets, and no occurrences were found. This may indicate that with the methodology applied we managed to identify new candidates for therapeutic targets against these parasites. In further analysis, we will test more restrictive e - values on the reannotation step to test the potential of reannotation of the tool.