Dissertations / Theses on the topic 'Type II topoisomerase; Enzyme'

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007.
Title from title page screen (viewed on July 29, 2008). Research advisor: Mary-Ann Bjornsti, Ph.D. Document formatted into pages (xiii, 175 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 161-175).

Doenças tropicais negligenciadas compartilham características que permitem que elas persistam nas condições de pobreza, onde se aglomeram e se sobrepõe frequentemente. Mais de um bilhão de pessoas sofrem de uma ou mais doenças tropicais negligenciadas. Entre estas está a doença de Chagas, resultante da infecção pelo protozoário parasito hemoflagelado Trypanosoma cruzi, tendo insetos triatomíneos como vetores. Topoisomerases são enzimas envolvidas na regulação do superespiralamento do DNA resolvendo problemas topológicos associados com replicação, transcrição, recombinação e reparo. As topoisomerases do tipo II são essenciais para tripanossomatídeos por atuarem na replicação e organização do DNA contido em uma região especializada da mitocôndria denominada cinetoplasto. O estudo da Topoisomerase II Mitocondrial (TcTopoIImit) foi realizado através de análises sequenciais e estruturais de outras topoisomerases para a determinação de construções para a clonagem. As construções para o domínio N-terminal foram clonadas no vetor pTZ57R/T e subclonadas em vetores do sistema pET para expressão proteica em E. coli. Experimentos de expressão foram realizados em diferentes cepas, vetores, soluções tampão e outros parâmetros variáveis visando a obtenção dos produtos recombinantes na forma solúvel, porém, tais resultados não foram obtidos. A flavoproteína Old Yellow Enzyme de Trypanosoma cruzi (TcOYE) é uma oxidoredutase que usa NAD(P)H como cofator. Esta enzima é clinicamente relevante devido ao seu papel no mecanismo de ação de alguns agentes tripanocidas, utilizados no tratamento da doença de Chagas, por produzirem espécies reativas de oxigênio. Neste trabalho, a enzima recombinante TcOYE foi produzida, purificada, cristalizada pelo método de difusão de vapor e sua estrutura cristalográfica, resolvida por substituição molecular e refinada. A TcOYE foi cristalizada nas formas cristalinas P212121 e P21 e os dados de difração foram coletados para o máximo de resolução de 1,27 e 2,00 Å, respectivamente. As coordenadas atômicas e fatores de estrutura das estruturas da TcOYE nas formas cristalinas P212121 e P21 foram depositados no Banco de Dados de Proteínas com os códigos de acesso 4E2B e 4E2D, respectivamente. A TcOYE apresenta um enovelamento canônico em um barril (α/β)8 com o grupo prostético localizado na cavidade maior do sítio ativo. Após a obtenção dos modelos cristalográficos análises sequenciais e estruturais foram realizadas para a TcOYE e outras OYEs. A região 141-156 do subdomínio capping em TcOYE, assim como em outras OYEs, é intrinsecamente desestruturada exibindo altos valores de fatores B. A região mais divergente entre as OYEs é o loop estendido (289-297), que pode variar em comprimento e composição mudando o volume e a acessibilidade ao sítio ativo.
Neglected tropical diseases share features that allow them to persist in conditions of poverty, which clump together and frequently overlap. More than 1 billion people suffer from one or more neglected tropical diseases. Among them is the Chagas disease is an important parasitic disease resulting from infection by the flagellate protozoan parasite Trypanosoma cruzi, with triatomine insects as vectors. Topoisomerases are ubiquitous enzymes involved in the regulation of DNA supercoiling and overcoming topological barriers during replication, transcription, recombination and repair. The type II topoisomerases are essential for trypanosomatids since, in addition to their role in nuclear DNA metabolism, these enzymes might also play an important role in the replication and organization of the DNA contained in the specialized region of the mitochondrion known as the kinetoplast. The study of Mitochondrial Topoisomerase II from T. cruzi (TcTopoIImit) was performed by sequence and structural analyses into topos members for determination of domains constructions for cloning. Constructions for the N-domain were then cloned in pTZ57R/T vector and subcloned into pET system vectors for protein expression in E. coli. The protein expression experiments were performed by different strain cells, vectors, buffers solutions and others adjustable parameters for improve the solubility but recombinant products in soluble form were not obtained. The flavoprotein Old Yellow Enzyme from Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced, purified, crystallized by the vapour diffusion method and its crystallography structure was solved for molecular replacement and refined. TcOYE was crystallized in two crystalline forms, P212121 and P21 and diffraction data were collected to a maximum resolution of 1.27 and 2.00 Å, respectively. The atomic coordinates and structure factors of the TcOYE structure in P212121 and P21 crystalline forms have been deposited in the Protein Data Bank with the accession codes 4E2B and 4E2D, respectively. TcOYE displays a canonical (α/β)8-barrel fold with a FMN prosthetic group located at the large active-site cavity. Afterwards, sequential and structural analyses were carried out for TcOYE and others OYEs. The region 141-156 of the capping subdomain in TcOYE as well as in others OYEs is intrinsically flexible exhibiting high B-factor values. The most divergent region among these OYEs is the extended loop (289-297), which can vary in length and composition changing the volume and accessibility to the active site.

Thesis (Ed. D.)--Illinois State University, 1990.
Title from title page screen, viewed November 28, 2005. Dissertation Committee: Herman E. Brockman (chair), Alan J. Katz, Lynne A. Lucher, Radheshyam K. Jayaswal, David F. Weber, Anthony E. Liberta. Includes bibliographical references (leaves 114-131) and abstract. Also available in print.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior
Os bufodienolÃdeos sÃo esterÃides cardioativos de 24 carbonos, isolados originalmente de um extrato de pele de sapos da famÃlia Bufonidae utilizado na medicina chinesa. Os bufodienolÃdeos possuem grande variedade de atividades biolÃgicas, incluindo atividades antineoplÃsicas. Em relaÃÃo à atividade antitumoral, os bufodienolÃdeos tem demonstrado inibir o crescimento de vÃrias linhagens de cÃlulas cancerÃgenas humanas por induzir apoptose e parada do ciclo celular. O presente estudo avaliou o potencial citotÃxico e genetÃxico de seis bufodienolÃdeos em seis linhagens tumorais humanos, trÃs linhagens murinas normais e cÃlulas mononucleadas do sangue perifÃrico (CMSP) humano. Todos os seis bufodienolÃdeos foram citotÃxicos para todas as linhagens tumorais e CMSP com valores de IC50 variando entre 0,002 e 3,17 ÂM. Os bufodienolÃdeos testados nÃo apresentaram citotoxicidade para linhagens murinas normais. Desta forma, o composto hellebrigenina foi escolhido para se determinar o mecanismo de aÃÃo envolvido. Uma sequÃncia de experimentos in vitro foram realizados utilizando-se a linhagem leucÃmica HL-60. As cÃlulas foram tratadas em diferentes concentraÃÃes da amostra hellebrigenina (0,03, 0,06 e 0,12 ÂM) por 24 horas. A viabilidade das cÃlulas (nÃmero de cÃlulas viÃveis e integridade de membrana) HL-60 avaliada por citometria de fluxo, mostrou que o nÃmero de cÃlulas reduziu a partir da menor concentraÃÃo (0,03 ÂM) testada e a porcentagem de cÃlulas com membrana integra reduziu a partir da concentraÃÃo 0,06 ÂM. A anÃlise morfolÃgica por citometria de fluxo revelou aumento de cÃlulas com padrÃo apoptÃtico a partir da concentraÃÃo de 0,06 ÂM. Jà a anÃlise do conteÃdo nuclear, nos mostrou aumento de fragmentaÃÃo de DNA sub-G1 indicativo de apoptose e acÃmulo de cÃlulas na fase G2/M a partir das concentraÃÃes de 0,03 e 0,06 ÂM, respectivamente. Outros testes por citometria de fluxo revelaram que houve externalizaÃÃo da fosfatidilserina, despolarizaÃÃo mitocondrial, ativaÃÃo da caspase iniciadora 8 e consequente ativaÃÃo das caspases efetoras 3 e 7. Estes dados indicam um mecanismo citotÃxico por induÃÃo de mais de uma via apoptÃtica. Hellebrigenina nÃo foi capaz de causar danos ao DNA de HL-60 e de CMSP e nem o surgimento de aberraÃÃes cromossÃmicas em CMSP. Por meio dos estudos de docking molecular foi possÃvel predizer a ligaÃÃo entre hellebrigenina e topoisomeraseIIα humana, resultado compatÃvel com a possÃvel inibiÃÃo dessa enzima. De forma geral, os resultados apontam o potencial citotÃxico do bufodienolÃdeo hellebrigenina
Bufodienolides are cardioactive steroids of 24 carbons, originally isolated from a frogâs skin extract of the family Bufonidae used in Chinese medicine. Bufodienolides shows many biological activities, including anticancer activities. Related to antitumor activity, the bufodienolÃdeos has been shown to inhibit the growth of several human cancer cell lines by inducing apoptosis and cell cycle arrest. This study evaluated the potential cytotoxicity and genotoxicity of six bufodienolides, in six human tumor cell lines, three normal murine lineages and PBMC (peripheral blood mononuclear cells). All six bufodienolides were cytotoxic to all cell lines and tumor PBMC with IC50 values ranging from 0.002 to 3.17 ÂM. Bufodienolides showed no cytotoxicity for normal murine strains. Thus, the compound hellebrigenin was chosen to determine the action mechanism involved, a sequence of in vitro experiments were performed using HL-60 leukemia cell line. Cells were treated at different concentrations of hellebrigenin (0.03, 0.06 and 0.12 ÂM) for 24 hours. Cell viability (viable cell number and membrane integrity) HL-60 assessed by flow cytometry showed that the number of cells decreased from the lower concentration (0.03 ÂM) tested and the percentage of cells with reduced membrane integrity from 0.06 ÂM concentration. Morphological analysis by flow cytometry revealed increased apoptotic cells starting at concentrations of 0.06 ÂM. The analysis of nuclear content, showed an increase in DNA fragmentation indicative of sub-G1 apoptosis and accumulation of cells in G2 / M phase from the concentrations of 0.03 and 0.06 ÂM, respectively. Other tests by flow cytometry revealed that there was an externalization of phosphatidylserine, mitochondrial depolarization, activation of caspase 8 and initiating subsequent activation of effector caspases 3 and 7. These data indicate a cytotoxic mechanism induced by over an apoptotic pathway. Hellebrigenin was not able to cause DNA damage in HL-60 and PBMC nor the emergence of chromosomal aberrations in PBMC. Through the studies of molecular docking was possible to predict the connection between hellebrigenina and human topoisomeraseIIα, showing a result that is compatible with a possible inhibition of this enzyme. Overall, the results indicate the potential cytotoxicity of hellebrigenin

uinolones are a family of drugs used to treat bacterial infections by targeting bacterial type II topoisomerases (TOP2s), DNA gyrase and topoisomerase IV. Recent studies have shown that quinolones can cause genotoxicity in mammalian cells. Genotoxicity occurs when an agent causes damage to the genetic apparatus of a cell. Due to the similarities between the mammalian and bacterial TOP2 enzymes it is thought that the quinolones are targeting mammalian TOP2 to produce their genotoxic response. The aim of this study was to investigate if quinolone genotoxicity involves mammalian TOP2. Using the micronucleus assay, the genotoxicity of two quinolones ciprofloxacin and gemifloxacin was tested in three Nalm-6 cell lines containing varying amounts of TOP2. With ciprofloxacin only the Nalm-6TOP2A+/- cells showed genotoxicity, whereas for gemifloxacin only the Nalm-6 WT cells showed genotoxicity, suggesting that for gemifloxacin the removal of TOP2A or TOP2B lowers the genotoxicity of the quinolone. A selection of quinolones were tested using the Trapped in AgaRose DNA ImmunoStaining (TARDIS) assay to determine whether they stabilise the TOP2-DNA complexes in mammalian cells as known TOP2 poisons do. The analysis showed that after three hours incubation the level of complexes increased, indicating that the quinolones are able to stabilise TOP2-DNA complexes. Taken together the micronucleus and TARDIS assay data show that the quinolones are targeting mammalian TOP2 at high concentrations.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Objetivos: Estudar a expressao imuno-histoquimica de topoisomerase IIƒ¿ e de caspase-3 ativada, marcadores de proliferacao e de apoptose, respectivamente, a deteccao de DNA HPV e a evolucao da lesao cervical em mulheres portadoras de lesao intra-epitelial escamosa de baixo grau (LBG). Metodos: Foram avaliadas 40 mulheres portadoras de LBG e 32 sem neoplasia cervical, diagnosticadas por exame cito-colpo-histopatologico, quanto a imunoexpressao de topoisomerase IIƒ¿ e de caspase-3 ativada e quanto a deteccao de DNA HPV por PCR consensual (GP5+/GP6+) em material de esfregaco cervico-vaginal. Os achados foram relacionados as variaveis clinicas das pacientes e a evolucao clinica das lesoes cervicais em 12 meses. As pacientes assinaram termo de consentimento livre e esclarecido. Resultados: A media percentual de celulas imunomarcadas por topoisomerase foi de 11,71% e 4,13%, no grupo com LBG e controle, respectivamente, com diferenca estatisticamente significante. Observou-se que houve expressao de caspase-3 em 17 (42,5%) e em 5 (15,63%) pacientes com e sem LBG, respectivamente, com diferenca estatisticamente significante. Foi detectado HPV DNA em 65% das pacientes com LBG e em 59,4% das pacientes sem lesao cervical, sem relacao com a expressao de topoisomerase IIƒ¿ ou caspase-3. Na presenca de DNA-HPV, a expressao de topoisomerase IIƒ¿ no grupo com LBG foi significativamente maior do que em fragmentos sem lesao. Nao foi observada diferenca quanto a evolucao da lesao cervical em 12 meses de acordo com a imunoexpressao de topoisomerase IIƒ¿. Com relacao a caspase-3 ativada, a maioria das pacientes com imuno-histoquimica negativa teve regressao da lesao cervical. Conclusoes: A imunoexpressao de topoisomerase IIƒ¿ e de caspase-3 ativada podem ser considerados marcadores de proliferacao e de apoptose em lesao cervical de baixo grau, sem relacao com a presenca de DNA-HPV.
Purpose: To evaluate the correlation between the expression of topoisomerase II alpha, active caspase-3 and infection with human papillomavirus in low-grade cervical intraepithelial lesion and in the normal cervix, and whether they might influence susceptibility to, or evolution of, cervical lesion. Patients and methods: Forty cervical biopsies patients with low-grade cervical intraepithelial lesion and thirty-two with normal cervix were stained by immunohistochemistry for topoisomerase IIá and active caspase-3 and were investigated for the presence of HPV on exfoliated cells by general primer GP5+/6+ PCR amplification of DNA. These findings were correlated with clinicopathological features of the patients including their clinical outcome after twelve months. Subjects provided written informed consent. Results: Low-grade CIN patients as a group had a significantly higher expression of topoisomerase II alpha compared to controls, without correlation to disease outcome at 12 months. Caspase-3 was expressed in 42.5% of CIN patients and in 15.63% without disease, and most of women without caspase-3 receded cervical lesion. HPV DNA testing was positive in 65% of the patients with cervical lesion, and in 59.4% of the control group and was not associated to the expression of topoisomerase IIá or active caspase-3. In the presence of a positive HPV DNA testing, women with cervical lesion had a significantly higher expression of topoisomerase II alpha compared to controls. Conclusion: Topoisomerase II alpha and active caspase-3 might be useful diagnostic and prognostic markers in low-grade cervical lesions, delaying a better follow-up.
CNPq: 134106/2005-9
TEDE
BV UNIFESP: Teses e dissertações

Interactions between the retinoic acid receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In acute promyelocytic leukemia (APL), RARalpha is fused with the promyelocytic leukemia (PML) gene, resulting in the expression of the fusion protein PML/RARalpha. Here, I report that topoisomerase II beta (topoIIbeta) associates with and negatively modulates PML/RARalpha and RARalpha transcriptional activity, and increased levels and association of topoIIbeta cause resistance to retinoic acid (RA) in APL cell lines. Knock down of topoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA-gene expression. Overexpression of topoIIbeta, in clones from an RA-sensitive cell line, conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicate that topoIIbeta is bound to an RA-response element, and inhibition of topoIIbeta causes hyper-acetylation of histone 3 at lysine 9 and activation of transcription. These results identify a novel mechanism of resistance in APL and provide further insights to the role of topoIIbeta in gene regulation and differentiation.
Studies to determine the mechanism by which topoIIbeta protein is regulated found that levels of protein kinase C delta (PKCdelta) correlated with topoIIbeta protein expression. Moreover, activation of PKCdelta, by RA or PMA, led to an increase of topoIIbeta protein levels. Most notably, in NB4-MR2 cells, we observed increased phosphorylation levels of threonine 505 on PKCdelta, a marker of activation. Inhibition of PKCdelta was able to overcome the topoIIbeta repressive effects on RA-target genes. In addition, the combination of RA and PKCdelta inhibition led to increased expression of the granulocytic marker, CD11c, in NB4 and NB4-MR2 cells. These results suggest that PKCdelta regulates topoIIbeta expression, and a constitutively active PKCdelta in the NB4-MR2 cell line leads to overexpression of topoIIbeta.
In conclusion, these studies demonstrate that topoIIbeta associates with RARalpha, binds to RAREs and plays a critical role in RA dependent transcriptional regulation and granulocytic differentiation. In addition, I show that topoIIbeta overexpression leads to RA resistance and provide evidence that topoIIbeta protein levels are regulated via a mechanism involving the PKCdelta pathway. This work has contributed to an enhanced understanding of the role of topoIIbeta in gene regulation and brings novel perspectives in the treatment of RA-resistance in APL.

De nos jours, un des objectifs importants de la recherche bioinorganique moderne est le développement d'enzymes artificielles. L'étude séquentielle des acides aminés présents dans le centre actif des métalloenzymes peut présenter une voie possible de la stratégie de modélisation enzymatique. Cependant, les peptides linéaires ont leurs limites lors de la reconstitution des centres actifs des métalloenzymes : ils ne possèdent pas la structure tridimensionnelle bien définie, par conséquent leur structure est vulnérable vis-à-vis de la coordination ou de l’hydrolyse des azotes amidiques. La capacité de coordination des métaux par des peptides linéaires peut être améliorée, par exemple, en les attachant à une plateforme tripodale. Les composés tripodaux peuvent assurer une organisation structurale rigide ou moins flexible pour des chaînes latérales des acides aminés, créant ainsi des sites de coordination pré-organisés pour les métaux. Dans cette thèse, la synthèse et la caractérisation des ligands peptidiques tripodaux contenant de l'histidine et la formation des complexes en présence de cuivre(II) et de zinc(II) sont présentées. Les propriétés acido-basiques ont été étudiées par potentiométrie et différentes techniques spectroscopiques ont été utilisées pour la caractérisation structurale (UV-Vis, CD, ESR, RMN et MS). Outre que la caractérisation thermodynamique et structurale, des propriétés catalytiques des complexes en réaction enzymatiques (oxydation du catéchol, dismutation du superoxyde) ont également été étudiées. Nos résultats ont démontré que les ligands peptidiques tripodaux sont capables d'améliorer la stabilité des complexes métalliques et qu'ils peuvent fournir des structures adéquates pour mimer efficacement les fonctions catalytiques des enzymes. Grâce aux études approfondies et systématiques des propriétés acido-basiques et spectroscopiques, nous avons mis en évidence les forces motrices de la coordination des métaux et établi l'impact de la structure tripodale sur la stabilité, la structure et les propriétés catalytiques des complexes formés. Nos résultats confirment l'effet bénéfique des plateformes tripodales durant la complexation des métaux, et soulignent les possibilités qui s’offrent aux peptides tripodaux dans le domaine de la biomimétisme
One of the most important directions of modern bioinorganic research is the development of artificial enzymes. One pathway of enzyme modeling strategy is the study of amino acid sequences present in the active centers of metalloenzymes. Linear peptides, however, have their limitations in reconstituting the active centers of metalloenzymes, since they do not possess the well-defined three dimensional structure, therefore their structure is vulnerable towards amide nitrogen coordination/hydrolysis. Improvement of metal binding capabilities of linear peptides can be obtained by e.g. their functionalization with tripodal ligands. Tripodal compounds may provide a rigid, less flexible platform for the coordinating amino acid side chains, creating pre-organized metal binding sites. In my thesis, I present synthesis and characterization of histidine containing tripodal peptide ligands and their complex formation in presence of copper(II) and zinc(II). Solution equilibrium was studied with pH potentiometric measurements, and several spectroscopic methods were used for structural characterization (UV-Vis, CD, ESR, NMR and MS methods). Beside thermodynamic and structural characterization, enzyme mimicking catalytical properties of the complexes have also been investigated (catechol oxidation, superoxide dismutation). Our results demonstrated that tripodal peptide ligands are capable of enhancing the stability of metal-peptide complexes, and they may provide convenient structures to efficiently mimic the catalytic functions of enzymes. With thorough and systematical solution equilibrium and spectroscopic studies, we uncovered the driving forces of metal coordination, and established the impact of the tripodal structure in stability, structure and catalytic properties of the forming complexes. Our findings confirm the beneficial effect of tripodal scaffolds in peptide-type ligand-metal complexes, and emphasize the possibilities lying within tripodal peptides in the field of enzyme mimicking

The present studies tested the hypothesis that 17beta-estradiol (E2) downregulates ACE and AT1R in several tissues important to cardiovascular regulation, including the brain and heart, and that this downregulation attenuates the progression of LV dysfunction following myocardial infarction (MI). In Experiment 1, female Wistar rats were randomized into one of four groups: (1) sham-ovariectomized (OVX); (2) OVX+vehicle (veh); (3) OVX+E2 replacement at physiological levels and (4) OVX+high E2. Five weeks following OVX, ACE and ATIR were increased 15-90% in the heart, several cardiovascular nuclei of the brain, kidney, abdominal aorta, adrenal and lung. These increases were prevented in all cases by E2 replacement at physiological levels and in most cases reversed to decreases by high E2. In Experiment 2, age-matched female Wistar rats underwent 1 of 3 treatments: no surgery (ovary-intact), OVX+veh treatment for two weeks, or OVX+high E2 treatment for two weeks. (Abstract shortened by UMI.)

Introdução: As mucopolisaccaridoses tipo I (MPS I), tipo II (MPS II) e tipo VI (MPS VI) são doenças lisossômicas (DL) para as quais está disponível a terapia de reposição enzimática (TRE) com laronidase, idursulfase e galsufase, respectivamente. Objetivo Primário: Analisar a frequência anual de intervenções médicas (número de consultas, internações, cirurgias, exames solicitados, medicamentos prescritos, equipamentos de uso crônico e outras formas de terapia) em uma amostra de pacientes brasileiros com MPS I, II e VI e, desta forma, contribuir para o conhecimento dos aspectos farmacoeconômicos relacionados a essas doenças. Metodologia: Estudo exploratório, retrospectivo, de base hospitalar, baseado em revisão de prontuário, com amostragem por conveniência, e que foi realizado em duas etapas (etapas 1 e 2). Um instrumento específico para a coleta de dados de ambas as etapas foi construído pela equipe do estudo, que é multidisciplinar. Os desfechos de interesse foram as frequências anuais de intervenções médicas (consultas, exames, cirurgias, internações, medicamentos utilizados, outras formas de terapia). A etapa 1 consistiu em estudo pré-experimental, realizado no Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre (SGM-HCPA), e que comparou as variáveis de interesse, para o mesmo grupo de pacientes, entre o período pré e pós-TRE. Os critérios de inclusão dessa etapa foram: ter diagnóstico confirmado de MPS I; estar em acompanhamento regular no SGM-HCPA desde o diagnóstico; estar em TRE por pelo menos um ano; e não ter participado de ensaio clínico envolvendo TRE ou ter realizado transplante de células-tronco hematopoiéticas. A etapa 2 foi transversal, multicêntrica (centros incluídos: SGMHCPA, Departamento de Genética Médica da Universidade Estadual de Campinas - UNICAMP, Pontifícia Universidade Católica de Campinas – PUC-Campinas, e Departamento de Pediatria da Universidade Estadual do Rio de Janeiro - UERJ), e comparou as variáveis de interesse entre grupos diferentes de pacientes (aqueles recebendo TRE e aqueles não recebendo TRE). Para essa etapa, foram considerados somente os dados relativos a 2010, sendo os seguintes os critérios de inclusão dos pacientes: ter diagnóstico confirmado de MPS I, II e VI; não estar participando de nenhum ensaio clínico envolvendo TRE ou ter realizado transplante de células-tronco hematopoiéticas; estar em TRE por pelo menos 12 meses antes do início da coleta, ou em acompanhamento por pelo 12 meses antes do início da coleta. Resultados: Etapa 1 - Nove pacientes (graves=3; atenuados=6) com MPS I foram incluídos no estudo, com mediana de idade de diagnóstico de 4,4 anos. Somente o número de cirurgias/ano/paciente foi dependente do tempo de doença (p=0,0004) e da gravidade do fenótipo (p=0,014). Com relação às comparações pré e pós-TRE, as variáveis que apresentaram diferença significativa (média do número/ano/paciente) foram: exames (pré-TRE=10,2+2,7; pós-TRE=22,5+2,1; p=0,005) e internações (pré-TRE=0,05+0,04; pós-TRE=0,30+0,11; p=0,013). Para as demais variáveis, não foi encontrada associação. Etapa 2 - Trinta e quatro pacientes com MPS I (n=12), II (n=17) e VI (n=5) foram incluídos no estudo. Desses, sete não utilizavam TRE (grupo “sem TRE") e 27 faziam uso de tratamento específico (grupo “com TRE"). Não foi encontrada correlação significativa entre tempo de doença e as variáveis estudadas. Considerando a amostra total, foi encontrada diferença entre o grupo “sem TRE” e o grupo “com TRE” em relação à mediana de internações hospitalares e de cirurgias realizadas [1(0-2) vs. 0 (0-1), p=0,015; e 0 (0-2) vs. 0 (0-0), p=0,040, respectivamente]. Para as crianças/adolescentes (<18 anos), não foi encontrada diferença estatística entre os grupos. Os pacientes com comprometimento cognitivo utilizavam mais medicamentos que os demais (p=0,024). Encontrou-se correlação negativa entre as variáveis duração da TRE e número anual de internações (r= -0,504; p=0,007). Discussão/ Conclusões: Este é um dos primeiros estudos a avaliar aspectos relacionados à farmaconomia da TRE para as MPS. De acordo com os resultados obtidos na etapa 2, verifica-se que, desconsiderando-se o custo associado às infusões, o custo do tratamento de pacientes com MPS parece ser menor para aqueles pacientes que utilizam a TRE do que para os pacientes que fazem somente tratamento sintomático. Entretanto, de acordo com a etapa 1 do estudo, a TRE parece não impedir a evolução da doença, pelo menos em relação à MPS I, e, assim, a cada ano de vida do paciente ocorreria um incremento do custo associado ao tratamento. Estudos adicionais, com maior tamanho amostral, deverão ser realizados para confirmar nossos achados.
Introduction: The mucopolysaccharidoses type I (MPS I), II (MPS II) and VI (MPS VI) are lysosomal disorders (LSD) for which enzyme replacement therapy (ERT) with laronidase, idursulfase and galsulfase, respectively, are available. Principal objective: To analyze the annual frequency of medical interventions (number of medical appointments, hospital admissions, surgical procedures, exams performed, medications prescribed, ancillary therapies and the use of medical devices) in a sample of Brazilian patients with MPS I, II and VI, and thus, contribute to the understanding of some pharmacoeconomic aspects related to these diseases. Methodology: Retrospective, exploratory, hospital-based study, based on medical records review, with convenience sampling, which was conducted in two steps (steps 1 and 2). A specific data collection instrument for both steps was designed by the study team, which is multidisciplinary. The chosen outcomes were: annual frequencies of medical interventions (medical appointments, exams, surgical procedures, hospital admissions, medications used and ancillary therapies). Step 1 was a pre-experimental study conducted at the Medical Genetics Service of Hospital de Clínicas de Porto Alegre (SGM-HCPA), and compared the variables of interest between the pre and post-ERT periods for the same group of patients. The patient inclusion criteria were: a biochemical diagnosis of MPS I and regular follow-up at SGM-HCPA since diagnosis; ERT for at least 1 year; no enrollment in any clinical trials involving ERT, and no history of hematopoietic stem cell transplantation. Step 2 was a cross-sectional and multicentric estudy (Centers included: SGM-HCPA), the Department of Medical Genetics of Universidade Estadual de Campinas - UNICAMP, Pontifícia Universidade Católica de Campinas - PUC-Campinas, and the Department of Pediatrics at Universidade Estadual do Rio de Janeiro – UERJ, which compared the variables of interest between different groups of patients (those receiving and those not receiving ERT). For this step only data from 2010 were considered. The inclusion patient criteria were: a biochemical diagnosis of MPS I, II or VI; no enrollment in any clinical trials involving ERT, and no history of hematopoietic stem cell transplantation, to be on ERT for at least 12 months before the start of data collection or to undergo regular follow-up for at least 12 months before the start of data collection. Results: Step 1 – Nine MPS I patients (severe=3; attenuated phenotype=6) were included in the study with median age at diagnosis was 4.4 years. Only the number/year/patient of surgeries was found to be dependent on length of disease (p=0.0004) and on severity of phenotype (p=0.014). Regarding pre- and post-ERT comparisons, the variables for which a significant difference was detected (mean number/year/patient) were exams (pre-ERT, 10.2±2.7; post-ERT, 22.5±2.1; p=0.005) and hospital admissions (pre-ERT, 0.05±0.04; post-ERT, 0.30±0.11; p=0.013). For the other variables, no association was found. Step 2: Thirty-four patients with MPS I, II and VI were included (I=12, II=17, VI=5). From them, 27 on ERT (“ERT group”) and 7 receiving supportive care only (“non-ERT group”). There were no significant correlation between length of disease and any of the variables of interest. There were significant between-group differences in the median number of hospital admissions and surgical procedures, both of which were higher in the non-ERT group [1(0-2) vs. 0 (0-1), p=0,015; e 0 (0-2) vs. 0 (0-0), p=0,040, respectively]. There were no significant between-group differences when only children and adolescents (<18 years) were taken into account. Patients with cognitive involvement used more medications than the others (p=0.024). A correlation was detected between time on ERT and the hospital admissions variable (r= -0.504; p=0.007). Discussion/conclusions: This was one of the first studies to evaluate aspects related to pharmacoeconomics of ERT for MPS. According to the results of step 2, and not acknowledging the costs associated with recombinant enzyme infusions, patients with MPS who undergo ERT generate less cost to SUS than patients on symptomatic treatment. On the other hand, according to the results of step 1, ERT seems not to stop the disease progress, at least in respect to MPS I, and thus, for each year of a patient life occurred an increase in cost associated with treatment. Additional studies with larger sample size are needed to confirm our findings.

Diet-induced obesity promotes type 2 diabetes (T2D). Drugs that inhibit the renin-angiotensin system (RAS) have been demonstrated in clinical trials to decrease the onset of T2D. Previously, we demonstrated that mice made obese from chronic consumption of a high-fat (HF) diet have marked elevations in systemic concentrations of angiotensin II (AngII). Pancreatic islets have been reported to possess components of the renin-angiotensin system (RAS), including angiotensin type 1a receptors (AT1aR), the primary receptor for AngII, and angiotensin converting-enzyme 2 (ACE2), which negatively regulates the RAS by catabolizing AngII to angiotensin-(1-7) (Ang-(1-7)). These two opposing proteins have been implicated in the regulation of β-cell function. We hypothesized that the RAS contributes to the decline of β-cell function during the development of T2D with obesity. To test this hypothesis we first examined the effects of whole-body deficiency of ACE2 in mice on β-cell function in vivo and in vitro during the development of T2D. Whole-body deficiency of ACE2 resulted in impaired β-cell adaptation to insulin resistance with HF-feeding and a reduction of in vivo glucose-stimulated insulin secretion (GSIS) associated with reduced β- cell mass and proliferation. These results demonstrate that ACE2 plays a role in the adaptive response to hyperinsulinemia with obesity. In islets from HF-fed mice, AngII inhibited GSIS. In mice with pancreatic-specific deletion of AT1aR, AngII-induced inhibition of GSIS in vitro from islets of HF-fed mice was abolished. However, there was no effect of pancreatic AT1aR-deficiency on glucose homeostasis in vivo in HF-fed mice exhibiting pronounced hyperinsulinemia. Notably, pancreatic weight, insulin content and basal and glucose-stimulated insulin secretion from islets were decreased in mice with pancreatic AT1aR deficiency. These results suggest that AT1aR may contribute to pancreatic cell development, and also contribute to AngII-induced reductions in GSIS from islets of HF-fed mice. Overall, these studies suggest a role for the RAS in the regulation of β-cell function in T2D.

Oxidative stress is associated with development and progression of cardiovascular disease. Angiotensin II produces oxidative stress and endothelial dysfunction, and its actions may be attenuated by the activity of angiotensin converting enzyme type 2 (ACE2) which converts angiotensin II to the vasoprotective peptide angiotensin (1-7). Similarly, increased oxidative stress is associated with aortic valve stenosis in humans and mice. In my thesis studies, I explore mechanisms of modulation and generation of oxidative stress in cerebral arteries and heart valves. First, I tested the hypothesis that ACE2 deficiency increases oxidative stress and vasomotor dysfunction in cerebral arteries, and examined the role of ACE2 in vascular aging. Vasomotor function was assessed in the basilar artery ex vivo of adult and old ACE2 deficient (ACE2-/y) and wild type (WT or ACE2+/y) mice. ACE2 was present, but at relatively low levels in cerebral arteries. Systolic blood pressure was similar in adult and old ACE2-/y and ACE2+/y mice. Maximal dilatation to acetylcholine was impaired in the basilar artery from adult ACE2-/y mice compared to adult ACE2+/y. In old mice, maximal vasodilatation to acetylcholine was impaired in ACE2+/y mice and severely impaired in ACE2-/y mice. The antioxidant tempol improved responses to acetylcholine in adult and old ACE2-/y and ACE2+/y mice. Nitrotyrosine staining in the basilar artery was increased in adult ACE2-/y mice and in old ACE2-/y and ACE2+/y mice relative to adult ACE2+/y, which indicates that oxidative stress was higher in cerebral arteries from ACE2 deficient mice and old mice. Expression of NADPH oxidase subunits Nox2 and p47phox, and of pro-inflammatory molecules Rcan1 and TNF alpha; was increased in cerebral arteries from old ACE2-/y and ACE2+/y mice. Additionally, I tested the hypothesis that serotonin induces oxidative stress in human heart valves, and examined mechanisms by which serotonin may increase reactive oxygen species (ROS). Superoxide (O2.-) was measured in heart valves from explanted human hearts that were not used for transplantation. Superoxide levels (lucigenin-enhanced chemiluminescence) were increased in homogenates of cardiac valves and pulmonary artery after incubation with serotonin. A non-specific inhibitor of flavin-oxidases (DPI), or inhibitors of monoamine oxidase-MAO (tranylcypromine and clorgyline), prevented serotonin-induced increase in O2.-. Dopamine, another MAO substrate which is increased in patients with carcinoid syndrome, also increased superoxide levels in heart valves, and this effect was attenuated by clorgyline. Apocynin did not prevent increases in O2.- during serotonin treatment. Addition of serotonin to recombinant human MAO-A generated superoxide, and this effect was prevented by an MAO inhibitor. In conclusion, I have demonstrated that ACE2 deficiency impairs vasomotor function in cerebral arteries from adult mice and augments endothelial dysfunction during aging. Oxidative stress plays a critical role in cerebrovascular dysfunction induced by ACE2 deficiency and aging. I have also identified a novel mechanism whereby MAO-A can contribute to increased oxidative stress in human heart valves and pulmonary artery exposed to serotonin and dopamine.

INTRODUÇÃO: A endometriose, uma doença benigna, tem características invasivas com potencial proliferativo. O desenvolvimento das lesões pode ocorrer em decorrência de crescimento celular glandular e/ou estromal ou de alterações na cinética celular. Cinética celular refere-se ao equilíbrio entre a morte celular, ou apoptose, e a proliferação celular, que pode ser avaliada pela expressão de fatores de crescimento como, por exemplo, a topoisomerase 2-alfa (TOP2A). Também influenciam a cinética celular oncoproteínas como p53 e c-erB2, conhecidas por interferir na apoptose, podendo resultar em oncogênese. OBJETIVOS: O objetivo principal deste estudo foi comparar a cinética celular da endometriose infiltrativa de retosigmoide com a do endométrio eutópico de pacientes sem endometriose. Para tanto, foi avaliada a expressão de apoptose e de TOP2A bem como das oncoproteínas p53 e c-erB2. MÉTODOS: Foram obtidas amostras de lesões de endometriose envolvendo o reto-sigmoide de 60 mulheres com a doença e amostras de endométrio eutópico de 20 mulheres sem endometriose. A expressão de TOP2A e das proteínas p53 e c-erB2 foram quantificadas por técnica imuno-histoquímica. Método TUNEL foi utilizado para analisar os padrões de apoptose, que resultaram em índice de apoptose (IA). Índice de proliferação celular (IP) foi determinado a partir do nível de expressão de TOP2A. Índice de renovação celular (IRC) foi calculado pela razão entre IP e IA. As análises imuno-histoquímicas foram realizadas tanto no tecido endometrial como um todo, quanto nos componentes estromal e glandular separadamente. Coeficiente de Correlação de Spearman foi aplicado para identificar eventuais correlações entre variáveis clínicas, morfológicas (tamanho, quantidade e nível de invasão das lesões) e experimentais. RESULTADOS: Na análise da amostra do tecido como um todo, não foram evidenciadas diferenças entre os grupos experimental e controle em relação ao IA (p = 0,389). Por outro lado, o IP foi significativamente maior nas amostras-controle (p < 0,001). Na avaliação em que se sepaxii raram as células estromais dos componentes glandulares, tanto o IP quanto o IRC foram significativamente maiores no grupo-controle em comparação com o grupo experimental (IP estromal: p = 0,006; IP glandular: p = 0,001; IRC estromal: p =0,032; IRC glandular: p = 0,007). Nas pacientes com endometriose, foi encontrada correlação entre IP e IRC glandular e o número de lesões (p = 0,003). Também foi observada correlação entre o IRC glandular e o tamanho das lesões (p = 0,006). Não houve diferença entre os grupos no que se refere à expressão de p53 e cerB2. CONCLUSÕES: A cinética celular se mostrou alterada em pacientes com endometriose do reto-sigmoide, conforme demonstrado pela redução nos níveis e na frequência de TOP2A, e pelos IP e IRC mais baixos; entretanto, apoptose e as expressões de p53 e c-erB2 se mostraram inalteradas
BACKGROUND: Endometriosis, a benign disease, has invasive features with its proliferative potential. Development of lesions may occur due to stromal and/or glandular cell growth and to alterations in cellular kinetics. Cellular kinetics involves a balance between the regulation of cell death, or apoptosis, and cell growth, that can be evaluated by the expression of growth factors, such as topoisomerase 2- alpha (TOP2A). Oncoproteins, such as p53 and c-erB2, known to affect apoptosis resulting in oncogenesis, also influence cellular kinetics. OBJECTIVES: The main objective of this study was to compare the cellular kinetics in deep endometriosis involving the recto-sigmoid to eutopic endometrium from patients without endometriosis. Apoptosis and TOP2A expression were primarily evaluated, as well as p53 and c-erB2 expression. METHODS: Study samples were obtained from endometriosis lesions involving the recto-sigmoid in 60 women, and control samples were obtained from eutopic endometrium from 20 women without endometriosis. The expression of TOP-2A, p53 and c-erB2 proteins were quantified using immuno-histochemistry. TUNEL method was used in the analysis of apoptosis patterns, and the apoptosis index (AI) was derived. The proliferation index (PI) was derived from the level of expression of TOP-2A. Cellular renew index (CRI) was calculated from the ratio of the PI and AI. Immunohistochemical analyses were performed in two ways: on the tissue collectively, and on the stromal and glandular components separately. Spearmans correlation coefficient was used to identify the correlation between clinical, morphological (size, number and level of invasion of lesions) and the study variables. RESULTS: When looked at collectively, there was no difference in the AI between study and control groups (p = 0.389). PI, however, was noted to be significantly higher in the control samples (p < 0.001). When evaluating the stromal cells separately from the glandular components, the PI and CRI were both significantly xiv higher in the control group compared to the study group (Study stromal PI vs control stromal PI; p = 0.006; Study glandular PI vs study glandular PI; p = 0.001; Study stromal CRI vs control stromal CRI; p = 0.032; study glandular CRI vs control glandular CRI; p = 0.007). In patients with endometriosis, a correlation was found between glandular PI, CRI and number of lesions (p = 0.003). The same result was observed in the analysis of stromal CRI and lesion size (p = 0.006). There was no difference in expression of p53 and c-erB2 between groups. CONCLUSIONS: Cellular kinetics is altered in endometriosis of the recto-sigmoid, as shown by the decrease in the levels and frequency of TOP2A expression, and lower PI and CRI; however, apoptosis and p53 and c-erB2 expression were unaffected

Palladium-catalysed carbonylative multicomponent reactions have proven useful for the synthesis of structurally diverse compounds. Carbon monoxide serves as an atom-efficient, one-carbon building block, which allows for further structural elaboration of the carbonyl compound. By varying the components of the carbonylative multicomponent reaction, considerable product diversity can readily be attained. However, due to the reluctance to use toxic CO gas, considerable efforts have been directed at exploring non-gaseous approaches. The work described in this thesis has mainly focused on the development of palladium(0)-catalysed, carbonylative multicomponent synthetic methodology, using the non-gaseous CO source molybdenum hexacarbonyl, in the synthesis of heterocycles and other biologically relevant functional groups. The first part of this work describes the development of a non-gaseous carbonylative Sonogashira cross-coupling of bifunctional ortho-iodoanilines and terminal alkynes. Where 4-quinolones were synthesised via a carbonylation/cyclisation sequence. Using a similar synthetic strategy, three different N-cyanobenzamide intermediates were prepared by palladium-catalysed carbonylative couplings of various aryl halides and bromides and cyanamide. The formed intermediates provided a basis for further chemical transformations. First, ortho-iodoanilines were carbonylatively coupled with cyanamide and subsequently cyclised to yield heterocyclic 2-aminoquinazolinones. Next, building on those findings, the same synthetic strategy was applied to ortho-halophenols to provide a highly convenient domino carbonylation/cyclisation method for the preparation of benzoxazinones. The developed method was used to evaluate the efficiency of various non-gaseous CO sources. Third, the palladium-catalysed carbonylative synthesis of N-cyanobenzamides, was used to produce biologically relevant N-acylguanidines with considerable product diversity. Finally, one of the developed carbonylative methodologies was used in the preparation of potential NDH-2 inhibitors based on a quinolinyl pyrimidine scaffold. The prepared compounds were biologically evaluated in terms of inhibition of oxidoreductase NDH-2 and antibacterial activity on Gram-negative bacteria, S. aureus and Mtb. The biological evaluation revealed that some of the quinolinyl pyrimidines exerted inhibitory activity on the NDH-2 enzyme and possessed antibacterial properties. The work described in this thesis has been devoted to the development of non-gaseous one-pot, multicomponent carbonylation/cyclisation and carbonylation/amination reactions. The described methods offer highly attractive synthetic strategies that can be of great value to synthetic and medicinal chemists.

碩士
國立中興大學
生物化學研究所
91
Type II DNA topoisomerases are essential cellular enzymes which alter the topological state of DNA. They are involved in all aspects of DNA metabolism including DNA replication, transcription, recombination, and chromosome segregation. All type II topoisomerases use an ATP-coupled DNA transport reaction in which the enzyme cleaves and generates a "gate" on one DNA duplex (termed G-segment), transports a second duplex (termed T-segment) through the break, and subsequently religates the cleaved DNA. Many antitumor and antibacterial drugs inhibit type II DNA topoisomerase by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for the cytotoxicity of these drugs. Although type II enzymes interact extensively with DNA, the protein-DNA interaction is yet to be structurally characterized. This experiment is aimed to at determing the following crystal structures: 1) the crystal structure of the DNA cleavage/ rejoin domain of bacteriophage T2 topoisomerase (gene 52 protein), 2) the structures of the protein-DNA complex(es), 3) structure(s) of protein-DNA-drug ternary complex(es). Previous studies show that the gene 52 protein has a disorder region in the N- terminus that may interfere with crystallization, therefore I use a truncation mutant of gene 52 protein, (termed Tr-11, in which the 11 N-terminal residues were removed) for structural analysis. Large quantity of Tr-11 protein can be obtained following ammonium sulfate precipitation, hydroxyapatite chromatography, and gel filtration chromatography, and crystals of Tr-11 protein were found in the condition consisted of 15% PEG 4K、0.1 M sodium citrate pH 5.6、0.2 M ammonium acetate. However, only low resolution diffraction was observed from these crystals. Finally I used a internal deletion mutant of gene 52 protein (termed ΔT2-52-5g, in which residues 342 to 418 were replaced by a glycine-linker) for crystallization and crystals were found in 18% PEG 8K、0.1M sodium cacodylate pH 6.5、0.2M calcium acetate .In addition, crystallization of ΔT2-52-5g/DNA complex has also been initated.

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs, and are responsible for conferring trimethoprim resistance to the host bacterial strain. An overproduced derivative of R388 DHFR, RBG200 DHFR, was purified to apparent homogeneity. The pH versus activity profile of RBG200 DHFR was found to be similar to that previously reported for R388 DHFR, but different from that of chromosomal DHFRs. Gel filtration and equilibrium ultracentrifugation experiments suggested that at pH 6, where the enzyme is most active, RBG200 DHFR is in equilibrium between tetramer and other protein species. Gel filtration studies suggested that the loss of enzyme activity between pH 6 and pH 5 is not due to tetramer dissociation. Gel filtration experiments with R67 DHFR produced similar results. RBG200 DHFR was found to catalyze the transfer of the pro-R hydrogen of NADPH to dihydrofolate, making it a type-A dehydrogenase, along with the chromosomal DHFRs. Addition of NADP$\sp+$ to RBG200 DHFR results in the formation of an initial binary complex which slowly interconverts to a second binary complex, with an apparent first order rate constant of 1.0 $\times$ 10$\sp{-4}$ s$\sp{-1}$, at 25$\sp\circ$C. The binding of NADP$\sp+$ to RBG200 DHFR in the second binary complex was found to be weak, K$\sb{\rm D}$ = 1.9 $\pm$ 0.4 mM. The stoichiometry for coenzyme binding was found to be approximately one coenzyme per tetramer of RBG200 DHFR. Transferred NOEs were used to estimate internuclear distances, and it was found that NADP$\sp+$ binds to RBG200 DHFR in the initial and final RBG200 DHFR$\cdot$NADP$\sp+$ binary complexes, as well as in the ternary complex with folate, with a syn conformation about the nicotinamide-ribose glycosidic bond and an anti conformation about the adenine-ribose glycosidic bond. From the stereochemistry of hydride transfer and the conformation of the enzyme-bound cofactor, a model was proposed for the orientation of coenzyme and substrate as the active site of RBG200 DHFR, which differs from that observed at the active site of chromosomal DHFRs, and may be responsible for the resistance of type II DHFRs to several antifolates.

碩士
國立臺灣大學
生物化學暨分子生物學研究所
97
The intertwined structure of duplex DNA is known to cause topological problems during DNA transactions, in which processive strand unwinding generates the under- and over-winding on the flanking duplex regions. If not removed, the accumulation of superhelical strains will halt DNA duplication and gene expression. In addition, newly replicated daughter chromosomes are interlinked and must be segregated prior to cell division. In bacteria, two closely related yet functionally distinct type IIA topoisomerases (topos), DNA gyrase and topo IV, are responsible for resolving these topological problems. With the unique (-) supercoiling activity, gyrase removes the (+) supercoils in front of the replication forks and maintains bacterial genomes in a slightly underwound state, promoting the initiation of transcription and replication. In contrast, topo IV is specialized for decatenation of tangled daughter chromosomes. For most bacteria, these two enzymes are both essential and work in concert to maintain the superhelical homeostasis. However, a few eubacteria possess only one type II topo. It remains controversial whether this lone type II topo functions mainly as a gyrase or a topo IV, or if it possesses both the (-) supercoiling and decatenation activities, to sustain cell viability. Here we characterized the lone type II topo encoded by the genome of Aquifex aeolicus, a hyperthermophilic eubacterium. This enzyme is currently annotated as a gyrase, composed of the GyrB and GyrA subunits. However, sequence analysis reveals that the C-terminal domain of A. aeolicus GyrA (aeGyrA-CTD), a domain known to confer the (-) supercoiling activity of gyrase, is very different from other GyrA-CTD. Specifically, the aeGyrA-CTD is significantly shorter in length and lacks a characteristic GyrA box (QRRGGKG) found in all gyrases. These differences may contribute to its unique activity as the sole type II topo. The A. aeolicus topo II was purified to homogeneity, whose optimal activity was observed at around 85℃, consistent with the extremely high growing temperature of this bacterium. At 85℃ A. aeolicus topo II was found to introduce (-) supercoils into relaxed DNA molecules, indicating that it may function as a gyrase. Surprisingly, while other gyrases require ATP to drive this reaction, A. aeolicus topo II is not strictly ATP-dependent. Furthermore, when the (-) supercoiled DNA was incubated with A. aeolicus topo II, it becomes more relaxed even in the presence of ATP. These properties are distinct from those of E. coli gyrase, whose (-) supercoiling activity requires ATP and it does not exhibit relaxation activity in the presence of ATP. In addition, A. aeolicus topo II efficiently resolves interlinked kDNA network, suggesting that it may act as a topo IV-like decatenase in vivo. Therefore, A. aeolicus topo II is unique in that, depending on substrates, either (-) supercoiling or relaxation activity can be observed, with both activities being accelerated by ATP hydrolysis. Moreover, removal of aeGyrA-CTD merely reduces the reaction rate but the catalytic pattern of A. aeolicus topo II on different substrates was not affected, indicating that this domain does not play a dominant role in mediating the holoenzyme activity. The unique ATP-independent supercoiling activity at high temperature can be explained by relaxation of (+) supercoils. Due to the emergence of compensatory (+) supercoils on relaxed closed circular DNA associated with DNA unwinding at high temperature, A. aeolicus topo II releases the free energy stored as (+) supercoils to achieve DNA relaxation. Therefore, it appears that A. aeolicus topo II possesses merely relaxation and no (-) supercoiling activity, and the relaxation process is further facilitated by ATP hydrolysis, where the nucleotide binding-induced structural changes leads to more efficient capture of a second DNA segment for strand passage. The biased activity toward DNA relaxation of A. aeolicus topo II seems reasonable with the concomitant presence of the hyperthermophilic-specific reverse gyrase in vivo. Reverse gyrase actively generates (+) supercoils to overcome DNA denaturation, wheras A. aeolicus topo II functions as a decatenase to support chromosome segregation. In addition, this enzyme also relaxes excessive superhelical strains to maintain DNA superhelical homeostasis. By harboring efficient relaxation and decatenation activities, A. aeolicus topo II is functionally more related to bacterial topo IV. Being one of the earliest diverging eubacteria, our result implicates that the relaxation/decatenation activity evolved earlier in the prototype of topo II, and (-) supercoiling activity may not be required for the growth of ancient bacteria. Further studies on other type II topos from a wide range of bacteria will provide more information regarding how the topoisomerase activity can be fine-tuned to meet specific physiological requirements.

博士
國立交通大學
生物科技系所
96
Dengue viruses (DVs) are mosquito-borne infectious pathogens. They have become an expanding public health problem in the tropics and subtropics. The dengue envelope (E) protein is one of the viral structure proteins responsible mainly for the virus attachment and entry onto host cells. It is also the major immunogen for virus neutralization. In chapter I, I have constructed a recombinant plasmid expressing a truncated E protein of DV-2 virus PL046 strain. The C-terminal hydrophobic domain of the E protein was removed and replaced with the sequence of S peptide to facilitate expression and purification. When expressed in Escherichia coli, the recombinant E proteins were found to be in the form of aggregated state. Through denaturation and dialysis processes, the receptor-interacting function of the purified recombinant E proteins was maintained, which was demonstrated by its ability to inhibit the DV-2 plaque-forming efficiency on mammalian BHK-21 host cells. In chapter II, to identify the human cellular proteins interacting with the envelope protein of dengue virus serotype 2 inside host cells, I have performed a screening with the yeast two-hybrid-based “Functional Yeast Array”. Interestingly, the Small Ubiquitin-like Modifier-1 Conjugating Enzyme 9 protein, modulating cellular processes such as those regulating signal transduction and cell growth, was one of the candidates interacting with the dengue virus envelope protein. With co-precipitation assay, it is demonstrated that the dengue envelope protein indeed could interact directly with the Ubc9 protein. Site-directed mutagenesis has demonstrated that Ubc9 might interact with the E protein via amino acid residues K51 and K241. Furthermore, immunofluorescence microscopy has shown that the DV2E-EGFP proteins tended to progress toward the nucleus membrane and co-localized with Flag-Ubc9 proteins around the nucleus membrane in the cytoplasm side, and DV2E-EGFP also shifted the distribution of Flag-Ubc9 from evenly in the nucleus toward concentrating around the nuclear membrane in the nucleic side. In addition, over-expression of Ubc9 could reduce the plaque formation of the dengue virus in mammalian cells. This is the first report that DV envelope proteins can interact with the protein of sumoylation system and Ubc9 may involve in the host defense system to prevent virus infection.

碩士
國立陽明大學
衛生福利研究所
95
Context--Diabetic chronic kidney disease (CKD) is the largest cause of end-stage renal disease (ESRD). Angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) have been shown to prevent ESRD or mortality among type 2 diabetic CKD patients. The optimal ACEIs/ARBs use and the association between ACEIs/ARBs use and ESRD or mortality in Taiwan is unknown. Objectives--To describe the use of ACEIs/ARBs and evaluate the association between ACEIs/ARBs use and ESRD or mortality in type 2 diabetic CKD patients. Methods--Using National Health Insurance (NHI) databases in Taiwan, excluding those with chronic renal failure, 13,673 new cases of type 2 diabetic CKD patients were identified in 2001. Subgroups using ACEIs, ARBs, or combination were identified by the same medication and at least 2 dispensing during 4 months. Subjects were prospectively followed until death, ESRD, or the end of 2003. Analyses of predictors for ACEIs/ARBs prescription were performed by generalized estimating equation (GEE) approaches for cluster effect. Cox regression models were used to assess differences in ESRD or mortality between subgroups. Results--ACEIs/ARBs were prescribed only 56.02％ of this ideal cohort. Concurrent comorbidities were strongly associated with ACEIs/ARBs prescription, with OR and 95% CI for congestive heart failure of 1.49 (1.30-1.72). We observed significantly fewer ESRD cases in the ARBs group compared with the ACEIs group, with HR and 95% CI of 0.51(0.25-0.98). ARBs prescription was associated with low risk of death (HR 0.69, 95% CI 0.56-0.86).Cardiovascular-related mortality was also reduced (HR 0.42, 95% CI 0.24-0.75). Conclusions--National rates of ACEIs/ARBs use are disturbing low. The use of ARBs was associated with a significant reduction in ESRD or mortality in a broad spectrum of type 2 diabetic CKD patients. Educational efforts to patients and physicians should be highlighted to further increase rates of ACEIs/ARBs use. ARBs should be indicated in virtually type 2 diabetic CKD patients in Taiwan.