Rozprawy doktorskie na temat „RecR”

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Brasil, desde 1996, levou ao aparecimento de populações de mosquitos resistentes a esse composto. Apesar disso, o produto continua sendo usado pelo governo, exceto nos locais de detecção da resistência, onde foi substituído por larvicidas biológicos. O conhecimento sobre a forma de desenvolvimento e reversão da resistência em campo, bem como os mecanismos que modulam sua manifestação, pouco avançou nos últimos anos, apesar destas informações serem necessárias para a elaboração de esquemas seguros de manejo da resistência. Este trabalho se propôs a avaliar, utilizando uma linhagem de A. aegypti resistente ao temephos, os mecanismos responsáveis, ao menos em parte, por esta resistência, a possibilidade de respostas cruzadas com outros inseticidas e a reversão à susceptibilidade a este composto, em diferentes situações que simulam a realidade em campo. Assim, diferentes gerações da linhagem de A. aegypti, Recife-Resistente, RecR (14ª e 17ª gerações) mantidas sob forte pressão de seleção ao temephos, foram utilizadas. Como controle, utilizou-se uma linhagem padrão de susceptibilidade, a Rockefeller. Ensaios in vivo com concentrações múltiplas do temephos foram realizados para calcular a CL50 e CL90 e definir a razão de resistência (RR) nas diferentes gerações da RecR. A susceptibilidade da RecR a outros inseticidas, como o regulador de crescimento pyriproxyfen e os adulticidas malathion (organofosforado), deltametrina e cipermetrina (piretróides) foi verificada através de bioensaios dose-resposta (DR) e dosediagnóstica (DD). Para estudos preliminares dos mecanismos que conferem resistência, a atividade de enzimas associadas à detoxificação de inseticidas, como a glutationa S-transferase (GST s), esterases (EST s) α e β e oxidases de função mista (MFO s), também foi analisada na RecR. Para o estudo da reversão da resistência foram estabelecidas três sublinhagens. Duas delas foram provenientes da 14ª geração da RecR (RecRF14), sendo que uma foi mantida sem exposição ao temephos (RecRev1) e a outra sem exposição e com introdução de 30% de indivíduos com baixa resistência (RecRev2). A terceira sublinhagem, proveniente da 17ª geração da RecR (RecRF17), além de não ter sido exposta contou com a introdução de 50% de indivíduos susceptíveis-Rockefeller (RecRev3), a cada nova geração. Os resultados demonstraram que a RecR, apesar de altamente resistente ao temephos, apresentou resposta alterada ao pyriproxyfen e à cipermetrina e susceptibilidade à deltametrina e ao malathion, o que revela a inexistência de resistência cruzada aos dois últimos compostos. Todas as enzimas, em especial as GST s, mostraram atividade alterada nas fases adulta e larvária da RecRF17, exceto as MFO s, portanto é possível sugerir o envolvimento do mecanismo metabólico na resistência ao temephos. Quanto à reversão da resistência, observou-se que cessada a pressão de exposição ao temephos, após nove gerações consecutivas, houve uma redução na RR90 de 14 vezes (8,7) e 42 vezes (3,0) para RecRev1 e RecRev2, respectivamente. A RecRev3 recuperou a susceptibilidade ao composto na F3. Estes resultados demonstraram uma queda drástica na RR nas três condições avaliadas, mas revelam que a resistência ao composto não regride rapidamente diante da simples interrupção de seu uso, como observado na RecRev1, que permaneceu com nível intermediário de resistência (RR= 8,7). Por outro lado, os esquemas que tentaram simular condições de campo relativas à migração de indivíduos susceptíveis ou com baixa resistência mostraram-se mais eficientes na recuperação da susceptibilidade, revelando o caráter instável desta resistência. É possível sugerir, por fim, que a resistência ao composto é reversível e que métodos baseados na liberação de machos susceptíveis possam representar mais uma forma de manejar a resistência ao temephos em campo

Uppsatsen avhandlar en undersökning om evenemangsorganisationers ledarskapsstrategier vid rekrytering och ledning av volontärer. Då tidigare forskning inom området tenderar att utgå från volontärernas perspektiv fanns det ett intresse att utföra en studie utifrån ett organisatoriskt perspektiv. Detta möjliggjordes genom semistrukturerade intervjuer med evenemangsorganisationer. Syftet med uppsatsen var att undersöka olika evenemangsorganisationers strategier för att rekrytera och leda volontärer. För att uppnå studiens syfte utformades följande frågeställningar: Vilka strategier använder evenemangsorganisationer för att rekrytera volontärer? Vilka strategier använder evenemangsorganisationer för att leda volontärer? Vilka likheter och skillnader finns mellan de olika evenemangsorganisationernas strategier vid rekrytering och ledning av volontärer? För att besvara våra frågeställningar utgick vi från fyra teoretiska utgångspunkter. Första utgångspunkten som presenteras är modellen Volunteer functions inventory, ett index som används för att förklara hur anpassade ledarskapsstrategier i relation till volontärers motivationsfaktorer kan bidra till tillfredsställda volontärer. Därefter redogörs teorin om De fem praktikernas ledarskap, som förklarar hur ledare ska gå tillväga för att uppnå ett idealistiskt ledarskap. Sedan presenteras en teori om ett idealläge vid rekrytering av volontärer, Volunteer recruitment. Avslutningsvis beskrivs Transformativt ledarskap, en ledarskapsstil med syfte att involvera volontärer i beslutsprocesser. Studiens resultat påvisade att valda evenemangsorganisationer till viss mån utformar strategier för hur volontärer ska rekryteras och ledas, där strategierna både skiljer sig och liknar varandra. Det går att identifiera att motivationsfaktorer, kravställande, mål och visioner, involvering och kommunikation är de strategier som evenemangsorganisationer belyser är av betydelse vid rekrytering och ledning av volontärer. Resultatet kommer att ligga till underlag för hur organisationer genom sitt ledarskap kan rekrytera och leda volontärer vid evenemang, med syfte att skapa tillfredsställda volontärer. Studien ska även bidra till förståelse för de strategier organisationer inom evenemangsbranschen kan tillämpa för att rekrytera nya volontärer samt för att behålla befintliga volontärer.
This study examines the leadership strategies of event organizations in their work with volunteers. Since earlier studies tend to be based solely on volunteers' perspectives, we found it interesting to conduct a study from an organizational perspective, which was made possible by semi-structured interviews with event organizations. The purpose of this thesis is to investigate the leadership strategies that different event organizations applies when recruiting and leading volunteers. Three research questions were asked to answer our purpose: Which strategies do event organizations use when recruiting volunteers? Which strategies do event organizations use when leading volunteers? Which similarities and differences are there between the strategies that the event organizations use when recruiting and leading volunteers? This thesis is using four theoretical frameworks that help us to answer our research questions. Firstly, the framework Volunteer functions inventory, used an index to investigate how strategies are related to volunteer motivation factors. Secondly, the theory of The five practices of exemplary leadership, explaining how leaders will achieve idealistic leadership is presented. Thirdly, a theory for ideal strategy when recruiting volunteers, volunteer recruitment is explained. Lastly, Transformational leadership that describes a leadership style with the aim of involving volunteers in decision-making processes is presented. The study results indicate that event organizations formulates strategies for deciding how volunteers should be recruited and managed and that there are many similarities and differences found between these strategies. It is possible to identify that motivational factors, requirements, goals and visions, involvement and communication are the main factors that event organizations highlight as important to attract and satisfy volunteers. The result should be the basis for how organizations can recruit and lead volunteers through events via their leadership to create the result that event organizations strives for. The study will also help to understand if organizations in the event industry use strategies for how to recruit new volunteers and retain existing volunteers. The language of this thesis is Swedish.

La réplication fidèle de l’ADN au cours du cycle cellulaire est essentielle au maintien de l’intégrité du génome à travers les générations. Toutefois, de nombreux éléments peuvent perturber et compromettre la réplication et donc cette intégrité. La mitomycine C (MMC) est une molécule génotoxique utilisée en chimiothérapie. Elle forme des liaisons covalentes entre les deux brins d’ADN, ce qui est un obstacle à la bonne réplication de l’ADN. La rencontre de la fourche de réplication avec une liaison covalente entre les deux brins d’ADN va aboutir à une cassure double brin. Escherichia coli (E.coli) est un modèle d’étude très étendu car facile d’utilisation, permettant d’aborder des notions complexes. E coli possède divers mécanismes pour réparer ces lésions dont le régulon SOS. Le régulon SOS est un ensemble de gènes sous contrôle d’un promoteur réprimé par la protéine LexA. En réponse à des dommages à l’ADN, LexA est dégradé et les gènes du régulon sont activés.En utilisant une technique de biologie moléculaire qui permet de quantifier l’interaction entre deux chromatides sœurs restées cohésives derrière la fourche de réplication (étape appelée cohésion des chromatides sœurs), nous avons montré qu’en réponse à des cassures double brin générées par la MMC, la cohésion entre les chromatides sœurs nouvellement répliquées est maintenue. Ce phénomène est dépendant de RecN, une protéine induite de façon précoce dans le régulon SOS. RecN est une protéine de type SMC (structural maintenance of chromosomes), un groupe de protéines impliqué dans la dynamique et la structure du chromosome. En parallèle, des techniques de microscopie confocale et de marquage du chromosome par des protéines fluorescentes ont permis de montrer que la protéine RecN est impliquée dans une condensation globale du nucléoide suite à un traitement par la MMC. Cette condensation du nucléoide s’accompagne d’un rapprochement des chromatides sœurs ségrégées. Ces deux phénomènes, médiés par RecN pourraient permettre une stabilisation globale des nucléoides et favoriser l’appariement des chromatides sœurs pour permettre la recombinaison homologue.De façon intéressante, l’inhibition de Topoisomérases de type II (Topoisomerase IV et Gyrase) permettent de restaurer le phénotype d’un mutant recN en viabilité et en cohésion des chromatides sœurs. Les Topoisomérases sont des protéines qui prennent en charge les liens topologiques générés par la réplication et la transcription). Les liens topologiques non éliminés par les Topoisomerases permettraient de garder les chromatides sœurs cohésives et favoriser la réparation, même en l’absence de RecN.De plus, une expérience de RNA seq (séquençage de tout le transcriptome de la bactérie) a révélé que dans un mutant recN, le régulon SOS est moins induit que dans les cellules sauvages. Ceci va de pair avec une déstructuration des foci de réparation RecA. Il est possible que le rapprochement des chromatides sœurs médié par RecN permettrait de stabiliser le filament RecA et donc l’induction du SOS.L’ensemble de ces résultats suggère que RecN, une protéine de type SMC, permet de maintenir la cohésion entre les chromatides sœurs nouvellement répliquées, favorisant la réparation de cassures double brins par recombinaison homologue
Maintaining genome integrity through replication is an essential process for the cell cycle. However, many factors can compromise this replication and thus the genome integrity. Mitomycin C is a genotoxic agent that creates a covalent link between the two DNA strands. When the replication fork encounters the DNA crosslink, it breaks and creates a DNA double strand break (DSB). Escherichia coli (E.coli) is a widely used model for studying complex DNA mechanisms. When facing a DNA DSB, E. coli activates the SOS response pathway. The SOS response comprises over 50 genes that are under the control of a LexA-repressed promoter. Upon a DSB induction, RecA, a central protein of the SOS response will trigger the degradation of LexA and all the SOS genes will be expressed.We have developed a novel molecular biology tool that reveals contacts between sister chromatids that are cohesive. It has been shown in the lab (Lesterlin et al. 2012) that during a regular cell cycle, the two newly replicated sister chromatids stay in close contact for 10 to 20 min before segregating to separate cell halves thanks to the action of Topoisomerase IV. This step is called sister chromatid cohesion. We have used this molecular biology tool to study sister chromatid cohesion upon a genotoxic stress induced by mitomycin C (MMC). We have shown that sister chromatid cohesion is maintained and prolonged when the cell is facing a DSB. Moreover, this sister chromatid cohesion is dependent on RecN, an SOS induced structural maintenance of chromosome-like (SMC-like) protein. In the absence of RecN, the proximity between both sister chromatids is lost and this has a deleterious effect on cell viability. By tagging the chromosome with fluorescent proteins, we have revealed that RecN can also mediated a progressive regression of two previously segregated sister chromatids and this is coordinated with a whole nucleoid compaction. Further studies showed that this genome compaction is orderly and is not the result of a random compaction in response to DNA damage.Interestingly, inhibiting TopoIV in a recN mutant fully restores viability and sister chromatid cohesion suggesting that RecN’s action is mainly structural. Preserving cohesion through precatenanes is sufficient to favor repair and cell viability even in the absence of RecN.An RNA-seq experiment in a WT strain and a recN mutant revealed that the whole SOS response is downregulated in a recN mutant. This suggests that RecN may have an effect on the induction of the SOS response and thus RecA filament formation. This is in good agreement with the change in RecA-mcherry foci formation we observed. In the WT strain, the RecA-mcherry foci are defined as described in previous work. However, in the recN, the RecA-mcherry foci seemed to form bundle like structures. These RecA bundles were previsously described by Lesterlin et al. in the particular case of a DSB occurring on a chromatid that has already been segregated from its homolog. This could mean that in the absence of recN, the sister chromatids segregate and RecA forms bundle like structures in order to perform a search for the intact homologous sister chromatid.Altogether, these results reveal that RecN is an essential protein for sister chromatid cohesion upon a genotoxic stress. RecN favors sister chromatid cohesion by preventing their segregation. Through a whole nucleoid rearrangement, RecN mediates sister chromatid regression, favoring DNA repair and cell viability

Le génome de Deinococcus deserti, une bactérie très radiotolérante, a été analysé et comparé à ceux de D. Radiodurans et D. Geothermalis. Environ 230 protéines sont spécifiquement conservées chez ces 3 espèces, dont IrrE, un régulateur essentiel pour la radiotolérance. D. Deserti possède plusieurs gènes supplémentaires liés à la réparation de l’ADN, dont imuY et dnaE2 (ADN polymérases translesionnelles). En plus, D. Deserti a 3 recA qui codent pour 2 protéines RecA différentes (RecAC et RecAP). Pour étudier ces gènes, des outils génétiques ont été mis au point. Différents résultats suggèrent qu’IrrE, nécessaire pour l’induction de plusieurs gènes après irradiation, a une activité peptidase. Les 2 RecA sont fonctionnelles pour la réparation de l’ADN. D. Deserti est mutable par UV, ce qui nécessite ImuY, DnaE2 et RecAC, mais pas RecAP
The genome of Deinococcus deserti, a highly radiation-tolerant bacterium, was analyzed and compared to those of D. Radiodurans and D. Geothermalis. About 230 proteins are specifically conserved in these 3 species, including IrrE, a regulator protein essential for radiotolerance. D. Deserti has several supplementary DNA repair genes, like imuY and dnaE2 (translesion DNA polymerases). Moreover, D. Deserti has 3 recA that code for 2 different RecA proteins (RecAC et RecAP). To study these genes, genetic tools were developed for D. Deserti. Different results suggest that IrrE, required for the induction of several genes after irradiation, has peptidase activity. The 2 RecA proteins are functional for DNA repair. D. Deserti is mutable by UV, which requires ImuY, DnaE2 and RecAC, but not RecAP

Orientador: Hernandes Faustino de Carvalho
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A próstata tem merecido crescente atenção devido à maior incidência de câncer prostático e outras afecções do órgão, que resultam do aumento na longevidade dos indivíduos do sexo masculino em todo o mundo. Além disto, o desenvolvimento e crescimento prostático normal apresenta regulação androgênica e está sujeito a uma série de disruptores endócrinos que afetam o seu crescimento e função, assim como predispõem ao desenvolvimento tumoral. Nosso interesse reside principalmente na remodelação prostática seguida à castração e nas interações epitélio estroma que ocorrem neste órgão. Neste trabalho, investigamos a expressão do inibidor de metaloproteinases (MMPs) RECK, em nível de RNAm, procurando correlacioná-Io com o desenvolvimento pós-natal e com a regressão prostática seguida à castração. Para isto, foram utilizadas técnicas de RT-PCR semiquantitativo, Real time RT-PCR e de hibridação in situ,pareados sempre que possível com a expressão do RNAm e com a atividade de algumas MMPs. Os resultados demonstram que o gene RECK é expresso na próstata ventral de ratos, que existe uma significativa redução na sua expressão ao longo do desenvolvimento pós-natal, que há mecanismos diferenciados controlando a expressão dos pares RECKlMMP-2 e MMP-7/MMP-14. Foi observado também um crescente aCÚInulo da forma ativa da MMP-9, conforme o animal se aproxima da idade adulta. Utilizando RT-PCR semiquantitativo, pudemos determinar que o conteúdo relativo do RNAm para o RECK após a castração não muda, embora haja uma inversão no balanço entre a expressão epitelial (células epiteliais) e estromal (células musculares lisas e fibroblastos), nesta situação. No conjunto, os resultados sugerem que o RECK é expresso por diferentes tipos celulares da próstata ventral de ratos, com mecanismos de regulação complexos provavelmente oriundos da existência de diferentes compartimentos no órgão, ao contrário do que se observa para células isoladas
Abstract: The prostate has deserved increasingly attention due to the growing incidence of prostatic cancer and other prostatic diseases, which can be related to the longevity increase of men around the world. Besides, the normal prostatic development is under androgen regulation and as so is subject to a series of endocrine disruptors which affect its growth and function and predisposes to prostate cancer. Our interest resides on the prostatic remodelling following castration and on the epithelial-stromal relationships known to occur in the organ. In this work, we have investigated the expression of the matrix metalloproteinase inhibitor RECK, at the rnRNA leveI, trying to correlate its expression with the post natal prostatic development and regression after castration, using semiquantitative RT-PCR, Real time RT-PCR and in situ hybridization, paralleled with the determination of some MMPs expression and activity. Tbe results demonstrate that RECK is expressed in the rat ventral prostate, that there is a significative reduction in its expression during the post natal development, which is paralleled by the expression of some MMPs and that the mechanisms controling the pairs RECKJMMP-2 and MMP-7/MMP-14 are different. It was also observed an increased proportion of the active form of MMP-9, as the animal approaches adulthood. Using semiquantitative RT-PCR, we could determine that the relative content ofRECK rnRNA remains unchanged by castration, spite detecting an inversion in the balance between the epithelial (epithelial cells) and stromal (smooth muscle cells and fibroblasts) in this situation. Taken together, the results indicate that RECK is expressed by different cell types of the rat ventral prostate, with regulatory mechanisms appearing more complex, likely resulting ftom the existence of different compartments in the organ opposing what was seen for isolated cells
Mestrado
Biologia Celular
Mestre em Biologia Celular e Estrutural

Recycling is an important part of the automotive industry and this thesis was made to examine the environmental impact from the production of a new produced rear subframe compared to a remanufactured subframe. A life cycle assessment has been done to investigate the inputs and outputs of the processes surrounding the new production and remanufacturing. The emissions from the processes have been categorized into four environmental categories. Based on the categories a comparison have been made to evaluate the environmental impact and conclude the differences between the two processes.

We consider an economy under a fixed exchange rate system, but with bounds (a minimum level or a band) on the real exchange rate. The international price of the tradable good is characterized by the continuous arrival of shocks that change its level. In a model with microfoundations, we investigate the effects of targeting the real exchange rate through nominal exchange rate changes that preclude the real exchange from trespassing the imposed bounds. A stochastic general model with two goods and fixed non-tradable goods price level is developed. We analyze the cases in which a lower bound or a band on the real exchange rate is introduced. The general conclusion is that when bounds are established, then welfare effects can be expected, which are generated at the expense of the levels of consumption that go in the opposite direction than what policy intended. This short-run effect is present even in the case the targeting policy is never exercised. This result is similar to the one we find in the target zones literature, in the sense that just the existence of this tolerance band changes the behavior of the economy. An interesting result is that, in the case in which home goods prices are fixed, the imposition of the band on the real exchange rate does not change its behavior within the band. However, this result is not true of other real variables in the economy. In other words, although the targeted variable within the band behaves identically to the case in which there are no bounds, the rest of the real variables in the economy behave differently, even if the targeted variable remains within the band and the escape clause is not triggered.

Gliomas são tumores altamente invasivos, resistentes aos tratamentos disponíveis atualmente e com alta taxa de mortalidade. A superexpressão de RECK na linhagem de glioma humano T98G comprometeu a capacidade das células de migrar e invadir in vitro, com rearranjo do citoesqueleto e alteração na distribuição espacial de FAK fosforilado. Entretanto, o possível mecanismo envolvido na inibição da migração mediada por RECK não foi desvendado. Para estudarmos os mecanismos envolvidos nesta alteração da capacidade migratória, as células T98G foram transfectadas com o vetor plasmidial pCXN2-hRECK (RECK+). A via das integrinas, a atividade de alguns membros da família das RhoGTPases e elementos do citoesqueleto foram avaliados através de imunoblotting, imunomarcação e ensaios de pull-down para as células RECK+ em comparação com células T98G não-transfectadas (WT), células T98G transfectadas com vetor pCXN2 na ausência do gene RECK (vetor) e fibroblastos primários humanos (FF287). Nossos resultados mostram um aumento na expressão de integrina β1 e uma diminuição da fosforilação de FAK no sítio de auto-fosforilação Tyr397 que, juntamente com o aumento das fibras de estresse e a diminuição dos lamelipódios, sugerem um fenótipo menos migratório da célula. Porém, quando avaliada a atividade de Rac1, esta se mostrou aumentada, embora uma das vias de ativação de Rac1 seja através da fosforilação de FAK levando à formação dos lamelipódios. A hipótese é que RECK inibe a quebra das adesões focais que participam do processo de migração, dificultando a mobilidade celular. Como as células continuam recebendo o estímulo para migrar, estas ativam Rac1 através de uma via independente de FAK. Além disso, a imunomarcação de paxilina mostrou um aumento no tamanho das adesões focais nas células RECK+, indicando que RECK pode influenciar nas estruturas responsáveis pelo contato célula-matriz.
Gliomas are highly invasive, treatment-resistant and lethal tumors. Overexpression of RECK in human glioma cell line T98G decreased cell migration and invasion in vitro, lead to cytoskeleton rearrangement and caused changes in phospho-FAK distribution. However, the pathway involved in RECK-mediated inhibition of cell migration has not been elucidated yet. To study the mechanisms by which RECK affects cell motility, T98G cells were transfected with pCXN2-hRECK vector (RECK+). Some proteins involved in the integrin pathway, activity of some proteins of RhoGTPase family and cytoskeleton proteins were analyzed through immunoblotting, immunostaining and pull-down assay in RECK+ cells and compared with non-transfected T98G cells, T98G transfected with pCXN2 without RECK gene and human primary fibroblasts (FF287). Our results showed an increase in integrin β1 expression and a decrease in FAK phosphorylation in the Tyr397 site, which together with the increase of stress fibers and decrease of lamellipodia, suggest a less migratory phenotype. Despite this, Rac1 activity was increased even though one of Rac activation pathways is through phospho-FAK, leading to lamellipodium formation. Our hypotheses is that RECK affects focal adhesion turnover, diminishing cell motility. As cells are still receiving a positive signal to migrate, they activate Rac1 through a FAK-independent pathway. Besides that, paxillin immunostaining showed that focal adhesions are larger in RECK+ cells, indicating that RECK can influence structures related with cell-matrix contact.

Os tumores da cérvice-uterina, que representam uma das principais doenças ginecológicas em mulheres na idade reprodutiva em todo o mundo, estão etiologicamente associados com a infecção pelo papilomavírus humano (HPV). A progressão de uma lesão intraepitelial escamosa de baixo-grau (LSIL) a um carcinoma invasivo de cérvix uterina está acompanhada da degradação da matriz extracelular (MEC) devido à ação progressiva das metaloproteinases de matriz (MMP-2, MMP-9 e MMP-14) no processo de invasão e metástase. Entretanto, o balanço entre as MMPs e seus reguladores como RECK e TIMPs é necessário para controlar esta invasão. O objetivo deste projeto consiste em avaliar a atividade e a expressão das metaloproteinases 2, 9, e 14, e caracterizar a expressão do gene supressor de metástase RECK e do inibidor tecidual de metaloproteinases (TIMP-2), em modelo de queratinócitos humanos infectados com retrovírus recombinantes que expressam os oncogenes E6 e/ou E7 de HPV 16, em culturas cultivadas em monocamada e organotípicas. Para isso, utilizamos ensaios de real-time PCR, zimografia, western blot, imunocitoquímica, ensaio de ELISA e imunohistoquímica. Em culturas em monocamada observamos que as células que expressam as oncoproteínas E6E7 de HPV16 apresentaram menores níveis protéicos de RECK e TIMP-2 em relação ao controle pXLSN. Quando analisamos as culturas organotípicas, também observamos esta diminuição dos níveis de RNAm e protéicos de RECK em rafts que expressam E6E7, acompanhado pelo aumento da atividade de MMP-9, em relação ao controle. Também observamos que o tratamento das culturas com a citocina TNF aumenta a expressão gênica, protéica e atividade de MMP-9 em todas as linhagens analisadas. Além disso, os oncogenes E6 e/ou E7 não afetam a expressão e/ou atividade de MMP-2, MT1-MMP. Nossos dados demonstraram que a expressão das oncoproteínas E6E7 de HPV16 estão relacionadas com o desequilíbrio entre MMPS e seus inibidores, sugerindo que em uma fase pré-invasiva do carcinoma cervical, não somente as MMPs, mas, principalmente seus inibidores são críticos para início da progressão tumoral.
Cervical cancer is etiologically associated with to high-risk human papillomavirus (HPV) infection. It has been observed that matrix metalloproteinases (MMPs) -2, -9, and MT1-MMP are required for basement membrane degradation during cervical carcinoma progression. Moreover, a counterbalancing among MMPs and their regulators, such as TIMPs and RECK, is necessary to modulate invasion. In order to study the effect of HPV oncogenes on MMPs expression, primary human keratinocytes (PHKs) were infected with recombinant retroviruses expressing wild-type HPV16 E6 and/or E7 oncogenes and were used to seed monolayers and organotypic cultures. Quantitative real-time PCR (Q-PCR), western blot, zimography, immunocitochemistry, ELISA assay and immunohistochemistry were used to determine the expression level and activity of MMP-2, MMP-9, MT1-MMP and their inhibitors RECK and TIMP-2. We observed that cultures expressing E6E7 presented lower RECK and TIMP-2 protein levels than control keratinocytes. In addition, rafts cultures presented the same lower RECK levels additionally presenting higher MMP-9 activity than control. Furthermore, we observed that expression of E6 and/or E7 proteins do not affect MMP-2 and MT1-MMP protein levels and/or activity. We also observed that TNF treatment enhance the MMP-9 gene and protein expression and activity in all studied cell lines. Taken together, our results demonstrate that HPV16E6E7 expression is related with the unbalance between MMPs and their inhibitors, suggesting that in the initial steps of HPV-related cervical disease, not only MMPs but also RECK and TIMP-2 are critical for tumor progression.

La protéine RecA intervient dans la réparation des lésions de l’ADN (i) en clivant le répresseur LexA et en déréprimant les gènes SOS qui sont sous le contrôle de LexA, (ii) en clivant la protéine UmuD dont seule la forme clivée est active dans la réparation fautive, (iii) en participant directement à la recombinaison post-replicative et à la réparation fautive. Nous avons étudié l'importance des différentes activités de la protéine RecA dans la régulation de la réparation SOS grâce à l’emploi de mutants ponctuels. Dans les résultats rapportés dans le chapitre l, l'activité coprotéase de différents mutants recA a été estimée par leur capacité à induire des mutants du phage λ. Ces phages codent pour des répresseurs présentant des sensibilités différentes au clivage protéolytique. Nos résultats montrent que l'activité coprotéase dépend in vivo (i) du nombre de lésions et de l'aptitude de la cellule à éliminer ces lésions (ii) de la quantité de molécules RecA (iii) de la capacité de la protéine RecA à interagir avec les cofacteurs (NTP et ADN simple brin) pour former un complexe actif, (iv) de l'affinité du répresseur pour la protéine RecA. Le chapitre suivant concerne l'interaction de la protéine RecA avec les différentes protéines dont elle stimule le clivage: LexA, UmuD, λcI et Φ80cl. De nouveaux mutants recA ont été isolés. Leur étude montre que le changement d'un acide aminé affecte spécifiquement le clivage de certains répresseurs. En effet, le mutant recA1730 ne clive pas la protéine LexA, le mutant recA1734 ne clive pas UmuD et Φ80cl et le mutant recA430 clive partiellement LexA et pas du tout UmuD et λcI. Bien que les changements dans ces trois protéines ne concernent qu'un acide aminé, ils altèrent également l’activité de recombinaison de la protéine RecA. Deux classes de mutants altérés dans la recombinaison peuvent être distinguées : les mutants dont l'activité de recombinaison est partiellement restaurée par la surproduction de protéine RecA dans des bactéries LexA (Def) et ceux qui sont insensibles à cet effet. Le mutant recA1735 qui est décrit dans le 3ème chapitre présente un phénotype nouveau : la protéine RecA1735 amplifiée dans une bactérie LexA (Def) est létale. La protéine RacA1735 présente une activité coprotéase normale et une activité de recombinaison réduite. Un excès des protéines UmuCD impliquées dans la réparation fautive inhibe l'effet létal de l’amplification de la protéine RecA1735. Nous montrons que l'accumulation de protéine UmuCD réduit l'activité recombinatrice de la protéine RecA+. L'effet létal de la protéine RecA1735 pourrait être dû à des « accidents » au cours de la recombinaison qui causeraient des dommages irréversibles de l'ADN. Le dernier chapitre porte sur la comparaison des gènes PsiB portés par les plasmides F et R6-5. La présence d'un plasmide R6-5 inhibe l'activité coprotéase de la protéine RecA alors qu'aucune inhibition n'est détectée en présence du plasmide F. Bien que les protéines Psi B produites par les deux plasmides soient très semblables (seulement 4 acides diffèrent sur 152) seul le plasmide R6-5 Inhibe l'induction du système SOS. Nous montrons que cette activité anti-SOS est due à la production de protéine PsiB. J'ai résolu le paradoxe des différences d'inhibition par les plasmides F et R6-5 en montrant que les séquences de régulation du gène PsiB diffèrent dans une région de 71 nucléotides comprenant les sites -10 et -35 de reconnaissance de la RNA polymérase. L'activité des deux promoteurs a été mesurée grâce à leur fusion à un fragment du gène lacZ. Le promoteur du gène PsiB du plasmide R6-5 est trois fois plus actif que celui du plasmide F ce qui pourrait expliquer les différences d'inhibition observées.

碩士
國立陽明大學
遺傳學研究所
84
recF，recO和recR基因是原先被發現參與RecF途徑之基因，目前有許多證據顯示此三個基因產物作用於DNA重組的同一步驟。現今有關RecF，RecO和RecR蛋白的在DNA重組途徑中所扮演的角色仍不清楚。 在本論文中，我們針對已純化的RecF，RecO和RecR蛋白的生化功能作進一步之探討，以期瞭解這些蛋白在DNA重組和修復上扮演的角色。主要的工作包括：(1)探討RecF，RecO和RecR與DNA結合之能力；(2)探討RecF，RecO和RecR的helix-unwinding活性。 首先，在RecF，RecO和RecR與DNA結合能力方面，我們證實單獨的RecF或RecO可以與ssDNA結合，而RecR不能。但在有RecO存在下，則RecR可與ssDNA結合。當RecF，RecO和RecR同時與ssDNA作用時，發現這三個蛋白皆可與ssDNA結合。 而在DNA unwinding活性方面，我們發現RecO有明確的活性，RecO的helix-unwinding活性需要有ATP及鎂離子，ATP之需求可以dATP取代，但無法以ATPγS或其它NTP替代，鎂離子之需求可以錳離子取代，但無法以鈣離子或鋅離子取代，RecOunwind helix的能力與RecO蛋白質之濃度成正比，當RecO之量可飽和結合至ssDNA時，其unwilnd DNA之活性最高，最後此活性會受到高濃度之鎂離子或鹽的抑制。根據我們發現RecO具有helix-unwinding活性，認為RecFOR在DNA重組作用中扮演新的角色。在本文中有將有詳細討論。 The recF, recO and recR genes were originally identified as those affecting the RecF pathways of recombination in Escherichia coli. Several lines of genetic evidence suggest that the recF, recO and recR gene products function at the same step of recombination, possibly at an early presynaptic step. The exact role of RecFOR in DNA recombination are not known. In this work, the interactions of RecF, RecO and RecR with ssDNA and the helix-unwinding activities of RecF, RecO and RecR were examined. We observed that single RecF or RecO can bind to ssDNA while single RecR cannot. In the presence of RecO, but not RecF, the RecR was found to associate with ssDNA. When the RecF, RecO and RecR were reacted with ssDNA, all three proteins were found to associate with DNA. With regards to helix-unwinding activity, we observed that RecO possesses such an activity. The helix-unwinding activity of RecO requires the presence of MgCl2 and ATP in the reaction mixture. The requirement of ATP can be substituted by dATP, but cannot be substituted by ATPγS or other NTPs. The requirement of magnesium can be substituted by manganese, but not by calcium or zinc. The unwinding activity is proportional to the concentration of RecO protein and is sensitive to high concentration of MgCl2 or NaCl. Maximal unwinding was observed when the RecO protein to DNA nucleotides was greater than 0.05. Our finding that RecO possesses helix-unwinding activity suggests a new role of ReeFOR in DNA recombination. A model for the possible involvement of RecFOR in DNA recombination is presented.

碩士
國立陽明大學
遺傳學研究所
82
recR基因是大腸桿菌細胞中與DNA重組有關的基因之一，其基因產物則為參與RecF重組途徑之作用成員之一。recR基因之發現雖然早在1989年，其選殖與定序亦於稍後陸續完成，但是關於Recf蛋白生化功能之研究與探討卻至今仍無明顯之進展。 本篇論文所探討的主題是recR基因之表現，目的是希望能大量表現RecR蛋白以利其純化及將來生化功能之探討。另外，本文也分析RecR蛋白質大量表現的情況下對於細胞生理功能之影響。為了達成上述目的，在我們的研究中選用兩種具有不同啟動子的表現載體：分別是具有Ptac啟動子的pKK388-1以及具有T7ψ10啟動子的PET-3d。並利用分子選殖技術將recR基因序列之結構部份接於兩種表現載體之上。 經由IPTG誘導後以SDS-PAGE分析細胞中RecR蛋白之表現，我們發現具有T7ψ10啟動子之表現載體可大量表現RecR，而其有Ptac啟動子的載體則僅微量表現RecR。另在誘導RecR蛋白表現的情況下，我們發現細胞之DNA修復作用及細胞存活性之變化與基礎表現狀態下比較並無明顯之差異。此結果顯示RecR之大量表現對於細胞之正常生理功能並無明顯之影響。 為進一步了解RecR蛋白之生化功能，我們從經IPTG誘導之細胞萃取液純化RecR。在經由Pheny1 sepharose CL-4B column以及PBE94 column加以純化之後，我們已可得到純度約80%的RecR蛋白質，此純化之RecR蛋白在體外DNA重組作用扮演之角色將於日後探討。 The recR gene was identified in 1989 as one of the RecF path-way recombination genes in Escherichia coli. Although the recR gene has been cloned and its product identified, the biochemical functions of RecR protein and its role in homologous recombination remain unknown. In this Study, the Structural portion of the recR gene was Synthesized by polymerase chain reaction (PCR) and cloned onto expression vectors which contain a Nco I fusion cloning site. Follow- ing IPTG induction, the expression of RecR was analyzed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the recombinant plasmids carrying a T7 promoter (pVW62 and pVW63) overexpressed RecR in large quantity while the recombinant plasmid carrying a Ptac (pVW61) expressed RecR poorly. The effects of RecR overexpression on cell viability and U.V. Sensitivity were alSo examined. It was observed that the overexpression of RecR did not lead to cell death, nor did it affect the U.V. Sensitivity of uvrA recBC sbcBC recR cells. Finally, aS a prelude to understand the biochemical functions of RecR protein, we have purified RecR protein from cells which overexpressed RecR. After chromatography with Phenyl SepharoSe CL -4B and PBE 94, RecR with about 80% purity was obtained. Further works on the biochemical functions of RecR will be pursued.

The work reported in this thesis includes structural and functional studies on thymidylate kinase and RecFOR pathway proteins from Thermus thermophilus HB8. In the first part, a study was performed on the thymidylate kinase from the thymidine tri-phosphate biosynthesis pathway. Thymidylate kinase is an important enzyme in DNA synthesis. It catalyzes the conversion of thymidine monophosphate (TMP) to thymidine diphosphate (TDP), with ATP as the preferred phosphoryl donor, in the presence of Mg2+. In this study, the dynamics of the active site and the communication paths between the substrates, ATP and TMP, are reported for thymidylate kinase from Thermus thermophilus. Conformational changes upon ligand binding and the path for communication between the substrates and the protein are important in understanding the catalytic mechanism of the enzyme. High-resolution X-ray crystal structures of thymidylate kinase in apo and ligand-bound states were solved. Structural analyses provide an insight into the mode of substrate binding at the active site. The residues involved in communication between the substrates were identified through network analysis using molecular dynamics simulations. The analyses of mutants suggest that the proper positioning of TMP is important for catalysis and provide an insight into the phosphoryl-transfer mechanism. The substrate binding, catalysis or the product release could be the rate-limiting step in the enzyme catalysis. Most of the studies on thymidylate kinase have either focused on understanding the mode of substrate binding or the mechanism of catalysis, but the product release event remains largely unexplored. This work reports four high-resolution crystal structures of thymidylate kinase from T. thermophilus in apo and product bound states. Random accelerated molecular dynamics (RAMD) simulations were performed to study the product release from the product bound high-resolution crystal structures of thymidylate kinase from T. thermophilus and human. The water molecules present around the Mg2+ ion contribute to the sequential release of the products. The presence of ADP-Mg2+ complex has a minor effect on the release of TDP. Thus, the release of the products from the active site could be random in order. The second part deals with the homologous recombination pathway, the RecFOR pathway. RecF, RecO and RecR proteins mediate the binding of RecA protein on the single strand binding (SSB) protein coated 3’ overhang of DNA. However, their interaction with each other and the DNA molecule is not clear. RecF exists as a monomer in solution but exhibits ATP dependent dimerization and DNA dependent ATP hydrolysis. The interaction assembly of RecF with RecR and DNA is not clear. RecR exists as a dimer in solution, although the crystallographic assembly suggests a tetramer. Thus, the dimeric assembly and the tetrameric assembly of RecF and RecR were stabilized by cysteine mutations at the interface residues and their interactions have been studied

E. coli RecA is a multifunctional protein known to be associated with the cell membrane, forming foci often located at the cell poles, which gets redistributed along the length of the cell during SOS response. Several lines of evidence suggest that RecA foci is occluded from the nucleoid during SOS response and positioned at the polar-proximal end of the cell, implicating RecA-membrane interactions. Compared to the E. coli RecA paradigm, very little is known about the in vivo properties of mycobacterial RecA proteins. Furthermore, the distribution of RecA in the cell or its interaction with the constituents of the mycobacterial cell membrane and its role in cell physiology remains to be investigated. The mycobacterial cell wall is a rigid structure that contains distinctive lipids and glycolipids, including mycolic acids, phosphatidyl inositol mannosides, phthiocerol dimycocerosates and lipoglycans. In this study, we demonstrate that mycobacterial RecA proteins specifically interact with phosphatidylinositol (PI) and cardiolipin (CL) among other acidic lipids. Interestingly, these interactions had no discernible effect on the ability of RecA proteins to bind single-strand DNA or ATP. However, PI and CL abrogated the DNA-dependent ATP activity of mycobacterial as well as E. coli RecA proteins. Further, RecA-phospholipid interaction had no significant effect on their capacity to promote strand exchange between linear double-stranded and single-stranded DNA. Furthermore, GFP-tagged RecA foci were localized at the polar ends, which are redistributed in the cell upon DNA damage and then exclusively associated with the nucleoid. The interaction of RecA protein with CL and PG, which represent two of the three major phospholipids of the M. smegmatis cell membrane, may be physiologically relevant because it provides a possible mechanism by which the membrane components may regulate RecA levels and function during the SOS response and recombinational DNA repair. Altogether, these and other findings indicate that the interaction of RecA proteins with CL and PI, the major acidic constituents of the mycobacterial membrane, may be physiologically relevant, as they provide a possible mechanism for storage of RecA in the cell and regulate recombinational DNA repair during the SOS response. This work sets the stage for future studies on the broader role of the mycobacterial cell membrane and provides a framework for further investigations into the role of cell membrane components in RecA function.

Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA. X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures. Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue, in a way similar to that caused by nucleotide-binding. The ordering of the DNA-binding loops L1 and L2, which present an ensemble of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide-binding presumably on account of the movement of the switch residue which forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications to intermolecular communications within the RecA filament. The structures resulting from different orientations of the C-domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA molecule at different phases of activity and provide insights into the mechanism of action of RecA. Crystal structures of mutants of MsRecA involving changes of Gln 196 from glutamine to alanine, asparagine and glutamic acid, wild type MsRecA and several of their nucleotide complexes were subsequently determined using mostly low temperature and partly room temperature X-ray data. At both the temperatures, nucleotide binding results in a movement of Gln 196 towards the bound nucleotide in the wild type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 25 crystal structures reported in this thesis, along with the 5 MsRecA structures reported earlier, provide further elaboration of the relation among the pitch of the `inactive´ RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low temperature structures define one extreme of the range of positions the C-domain can occupy. The movement of the C-domain is correlated to those of the LexA binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the `active´ filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. This work contributes to the description of the early stages of this trajectory and provides insights into structures relevant to the later stages. The interesting results observed in the case of MsRecA prompted similar studies on the RecA from Mycobacterium tuberculosis (MtRecA). In this study, the crystals were grown at slightly different conditions and examined at different relative humidities and temperatures. Surprisingly, in spite of the 92% sequence identity between the two proteins, the structures indicated MtRecA to be substantially less plastic than MsRecA. The crystal structures do not provide an obvious explanation for this difference. Further studies are warranted to explain the molecular basis of the difference. RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of four crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures determined as part of the doctoral programme and those reported earlier bring to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in HJ binding. This role along with its role in oligomerization could have important biological implications. In addition to the work on RecA and RuvA, which forms the body of the thesis, the author was also involved in a structural bioinformatics study in which several carbohydrate binding proteins were probed to identify common minimum principles required for binding mannose, glucose and galactose. The study, presented in an Appendix, identified interactions that were specific to particular sugars, leading to individual fingerprints. These fingerprints were then used for exploring lead compounds, using a fragment based approach. This investigation helped the author to familiarize himself with the analysis of protein structures and ligand design based on them.

Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA. X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures. Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue, in a way similar to that caused by nucleotide-binding. The ordering of the DNA-binding loops L1 and L2, which present an ensemble of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide-binding presumably on account of the movement of the switch residue which forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications to intermolecular communications within the RecA filament. The structures resulting from different orientations of the C-domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA molecule at different phases of activity and provide insights into the mechanism of action of RecA. Crystal structures of mutants of MsRecA involving changes of Gln 196 from glutamine to alanine, asparagine and glutamic acid, wild type MsRecA and several of their nucleotide complexes were subsequently determined using mostly low temperature and partly room temperature X-ray data. At both the temperatures, nucleotide binding results in a movement of Gln 196 towards the bound nucleotide in the wild type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 25 crystal structures reported in this thesis, along with the 5 MsRecA structures reported earlier, provide further elaboration of the relation among the pitch of the `inactive´ RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low temperature structures define one extreme of the range of positions the C-domain can occupy. The movement of the C-domain is correlated to those of the LexA binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the `active´ filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. This work contributes to the description of the early stages of this trajectory and provides insights into structures relevant to the later stages. The interesting results observed in the case of MsRecA prompted similar studies on the RecA from Mycobacterium tuberculosis (MtRecA). In this study, the crystals were grown at slightly different conditions and examined at different relative humidities and temperatures. Surprisingly, in spite of the 92% sequence identity between the two proteins, the structures indicated MtRecA to be substantially less plastic than MsRecA. The crystal structures do not provide an obvious explanation for this difference. Further studies are warranted to explain the molecular basis of the difference. RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of four crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures determined as part of the doctoral programme and those reported earlier bring to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in HJ binding. This role along with its role in oligomerization could have important biological implications. In addition to the work on RecA and RuvA, which forms the body of the thesis, the author was also involved in a structural bioinformatics study in which several carbohydrate binding proteins were probed to identify common minimum principles required for binding mannose, glucose and galactose. The study, presented in an Appendix, identified interactions that were specific to particular sugars, leading to individual fingerprints. These fingerprints were then used for exploring lead compounds, using a fragment based approach. This investigation helped the author to familiarize himself with the analysis of protein structures and ligand design based on them.

碩士
亞洲大學
休閒與遊憩管理學系碩士班
95
Dog’s utility is stressing on working functionally in elder human society. But with society changing ,people living style changing, and the quality of spirit lives raising, dog became merchandise ,the most loyal friend、companion、comforter, and a subject of playing. Raising of pet’s life value and characteristic, dog is not just a tool when people have time to perform hobby, its vitality and good well performance boost its value and importance, making people regard pets as accompany and personification, even making owner become pet’s parents. At the same time, this kind of relationship between pets and owner is closer, it is worthy to discuss and research. However, this research is based on quantification and deeply interview is auxiliary. Taichung city is the major base, there are 407 samples, deeply discussing pet’s role and importance in leisure time in order to understand the owner’s rearing motivation、rearing behavior, and rearing barrier that counteracts owner’s leisure time and influence. Based on theory of planned behavior, we discuss the causes that make owners to create rearing behavior. In addition, we also depend on different rearing motivation types and then distinguish those into many districts. In the end, this research we depend on SPSS 10.0 analysis result to make conclusion and proposition; it is hoped that the research data can be provided and consulted to related departments and industries when they are making policies or marketing programs.

碩士
國立臺灣大學
土木工程學研究所
96
The objective of this study is to construct the rear-end collision avoidance and warning system of advance safety vehicle (ASV) by rear-end parking camera. Based on image processing technology to get and analyze the driving environmental data, we developed the rear-end collision warning logic and system. Since the automobile electronics industry developed faster, the parking assist system, like the ultrasonic sensors and rear-end camera, almost become the basic equipment in vehicles. However, the parking assist equipments are only turned on when in parking mode. Therefore, to provide the following vehicle information for subjective one, this study tends to apply the rear-end camera in vehicle when moving forward. It not only reduces the equipment cost of the vehicle but also increases the safety for drivers. The main idea of warning system is to prevent accidents which caused by inattentive of drivers. This study used the image processing to detect the longitudinal data of the following vehicle, including the relative distance, velocity, and acceleration. We developed the dynamic thresholds by the relative data and the drivers’ perceptive reaction time to issue the warning signal for drivers. The α-β-γ filter was applied in this section to get the smoother relative data. This study built up a rear-end collision avoidance and warning system by rear-end camera. We constructed the warning program by Borland C++ Builder. The warning hardware of the system was used the industry personal computer and on-board video equipment. The experiment result was successful for the off-line video test. In other words, this study proves that the developed rear-end warning system could appropriately issue the warning signal for drivers.