Dissertation (Ph.D.)--University of Toledo, 2009. / Typescript. "Submitted as partial fulfillment of the requirements for the Doctor of Philosophy degree in Biology." Bibliography: leaves 54-56, 91-95, 118-119, 133-139.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: Charles W. Caldwell. "May 2008" Includes bibliographical references

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed Sept. 23, 2009). Includes bibliographical references (p. 66-77).

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 101-124) Also available in print.

Thesis (M.S.)--Northern Michigan University, 2008. / "14-57395." Bibliography: leaves 41-42.

Thesis (M. Phil.)--University of Hong Kong, 200. / Also available online.

The purpose of this study was to explore the relationship between identification with feminist beliefs and sexual assertiveness, by examining three potential mediators of that relationship: self-objectification, empowered entitlement, and self-silencing. Cross-sectional survey data were collected via online survey from 188 women. Results from correlational analyses indicated that active commitment to feminist beliefs was significantly related to lower levels of self-objectification and self-silencing and higher levels of empowered entitlement, but was not related to sexual assertiveness. Identification with nonfeminist beliefs (passive acceptance of sexism) was significantly related to higher levels of self-objectification and self-silencing and decreased empowered entitlement, as well as lower levels of sexual assertiveness. Baron and Kenny's (1986) regression approach was used to explore potential mediators of the relationship between identification with nonfeminist beliefs and sexual assertiveness. In individual regression analyses, self-silencing fully mediated the relationship between identification with nonfeminist beliefs and sexual assertiveness. Regression analyses examining empowered entitlement as a mediator approached significance and analyses examining self-objectification as mediator were non-significant. An integrative analysis utilizing Preacher and Hayes' (2008) method for evaluating indirect effects in multiple mediator models was used to further explore the impact of all three mediator variables and two covariates (age and education level) on the relationship between nonfeminist beliefs and sexual assertiveness. The overall model accounted for a significant portion of the variance in sexual assertiveness and the total indirect effect of nonfeminist beliefs on sexual assertiveness through the set of mediators was significant, whereas the direct effect of nonfeminist beliefs on sexual assertiveness was not significant, indicating that, after controlling for covariates, the set of three mediators together (self-objectification, empowered entitlement, and self-silencing) fully mediated the relationship between nonfeminist beliefs and sexual assertiveness. However, self-silencing appeared to contribute the only unique significant mediation in the model, accounting for approximately 84% of the total indirect effect. Unique indirect effects for self-objectification and empowered entitlement were not significant. Implications for understanding the relationship between identification with nonfeminist beliefs and sexual assertiveness and directions for future research are discussed.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A expressÃo heterÃloga de genes que codificam proteÃnas relacionadas à patogÃnse (PR-proteÃnas) representa uma alternativa promissora para o desenvolvimento de plantas resistentes ao ataque de fungos. Dentre as PR-proteÃnas com grande potencial biotecnolÃgico, estÃo as quitinases, hidrolases que catalisam a degradaÃÃo da quitina, que à um polÃmero constituinte da parede celular de muitas espÃcies de fungos fitopatogÃnicos. AlÃm da funÃÃo relacionada aos mecanismos de defesa, acredita-se que essas enzimas podem desempenhar outros papÃis, como a regulaÃÃo de processos de crescimento e desenvolvimento em plantas. O objetivo deste trabalho foi estudar o papel de uma quitinase de classe I de feijÃo-caupi (VuChiI) na defesa e no desenvolvimento vegetal e para isso duas abordagens foram conduzidas. A primeira consistiu no silenciamento do gene que codifica esta proteÃna em plantas de feijÃo-caupi. Nos experimentos de silenciamento gÃnico foram utilizados meristemas apicais de plÃntulas de feijÃo-caupi, que foram entÃo bombardeados com micropartÃculas portando um vetor de silenciamento (pKANNIBAL), onde foi clonada uma construÃÃo em grampo (intron harpin) do gene de VuChiI. As plantas regeneradas em meio de cultura de Murashige e Skoog (MS) contendo o herbicida imazapyr, como agente de seleÃÃo, foram analisadas por PCR, o que resultou na confirmaÃÃo da transformaÃÃo genÃtica de cinco plantas, a partir de 1.092 meristemas apicais bombardeados. O RNA foi extraÃdo das plantas transformadas e utilizado em anÃlises de Northern blot que possibilitou a detecÃÃo de siRNAs no RNA total extraÃdo das plantas putativamente transgÃnicas. Apenas uma das plantas transformadas produziu uma vagem contendo seis sementes, das quais apenas uma germinou. As plantas transformadas apresentaram ciclo de vida de mais de 365 dias enquanto que o esperado à de 65-70 dias. Com base nos resultados sugere-se que o silenciamento da quitinase de classe I de feijÃo-caupi pode ter promovido alteraÃÃo no desenvolvimento da planta. A segunda estratÃgia de transformaÃÃo genÃtica consistiu no co-cultivo de discos foliares de tabaco com suspensÃo de Agrobacterium tumefaciens LB4404, com o intuito de expressar a proteÃna recombinante no espaÃo extracelular das plantas regeneradas. Essa estratÃgia tem como objetivo fazer com que a proteÃna entre em contato com o fungo antes mesmo que este agente patogÃnico penetre na cÃlula vegetal. Para tanto, o inserto referente à quitinase de feijÃo-caupi foi clonado no vetor binÃrio pCAMBIA1305.2 sob controle do promotor CaMV35S e fundido ao peptÃdeo sinal GRP (glycine-rich protein de Oryza sativa), que permite a secreÃÃo da proteÃna recombinante via apoplasto. Foram produzidas trÃs linhagens de plantas que tiveram a transformaÃÃo genÃtica confirmada por PCR. As sementes obtidas das plantas transformadas, geraÃÃo R1, foram semeadas em meio seletivo para a anÃlise da transmissÃo do transgene. O teste de X2 indicou que o transgene foi transmitido para a progÃnie em proporÃÃes Mendelianas. Os modelos experimentais desenvolvidos neste trabalho poderÃo contribuir para a elucidaÃÃo dos papÃis biolÃgicos da quitinase de classe I de feijÃo-caupi. / Heterologous expression of genes encoding pathogenesis related proteins (PR-proteins) represents a promising alternative for the development of plants resistant to fungi. Among the PR-proteins with great biotechnological potentials are the chitinases, a class of hydrolases which catalyze the degradation of chitin, a polymer constituent of cell wall of several species of pathogenic fungi. Besides their function related to defense mechanisms, it is believed that these enzymes may play other roles, such as regulation of growth and development processes in plants. Using two approaches, this study sought to investigate the role of a class I chitinase of cowpea (VuChiI) in defense and plant development. The first approach consisted of silencing of the gene encoding for VuChiI in cowpea. Apical meristems excised from cowpea seeds were bombarded with microparticles containing a silencing vector (pKANNIBAL), which was cloned with a construct harboring an intron harpin of the gene VuChiI. Plants regenerated in a Murashige and Skoog (MS) media containing imazapyr as a selection agent, were analyzed by PCR, resulting in confirmation of genetic transformation of five plants from the 1092 apical meristems bombarded. Northern blot analysis of total RNA extracted from the putative transgenic plants allowed for the detection of siRNAs. Upon regeneration and germination, only one of the five transformed plants produced a pod containing six seeds. Of these, only one germinated when further planted. The transformed plants exhibited a life cycle of over 365 days, as against the normal cycle of 65-70 days. Based on these results we posit that silencing of class I cowpea chitinase may have led to changes in plant development. The second strategy consisted of co-cultivation of tobacco leaf discs with Agrobacterium tumefaciens strain LB4404 suspension to express recombinant protein in the extracellular space of the regenerated plants in order to expose the protein fungus even before its penetration into the plant cell. To achieve this, a sequence of cowpea chitinase insert was cloned into the binary vector pCAMBIA1305.2 under the control of CaMV35S promoter and signal peptide fused to glycine-rich protein of Oryza sativa (GRP), which allowed the secretion of recombinant protein via apoplast. This led to the generation of three strains as confirmed by PCR. Seeds obtained from transformed R1 generation of the plants were germinated and analyzed for transmission of the transgene using X2 test, which confirmed its transmission to the progeny in a Mendelian fashion. The experimental models developed in this work may serve to further assess the biological roles of class I chitinase from cowpea.

Orientadores: Michel Georges Albert Vincentz, Fabio Tebaldi Silveira Nogueira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T11:23:48Z (GMT). No. of bitstreams: 1 Zanca_AlmirSamuel_M.pdf: 11034884 bytes, checksum: 0545b58df6802e07009aef761dda3003 (MD5) Previous issue date: 2009 / Resumo: RNAs não-codificantes de 20-27 nucleotídeos (nt) regulam transcricionalmente ou pós-transcricionalmente a expressão de genes endógenos, modelando o transcriptoma e a produção de proteínas. Dentre estes, microRNAs (miRNAs) desempenliam papel chave no desenvolvimento vegetal, observação comprovada pela avaliação fenotípica e molecular de plantas transgênicas e de mutantes defectivos na produção de tais RNAs. MiRNAs são produzidos a partir de precursores longos (pri-miRNAs), os quais são posteriormente processados por enzimas específicas, gerando o miRNA maduro (20-22 nt). O miRNA maduro, por sua vez, guia a clivagem do mRNA de genes-alvo e bloqueia a tradução de proteínas, afetando diversos aspectos do desenvolvimento. O sequênciamento de populações de RNAs regulatórios possibilitou a identificação de miRNAs conservados e específicos em diferentes espécies vegetais, embora estudos em plantas de importância econômica sejam ainda incipientes. Atualmente, existem diversos bancos públicos de sequências ESTs disponíveis. Esses bancos possuem um grande número de sequências não-codíficantes, dentre as quais podem estar presentes pri-miRNAs, os quais são também são moléculas poliadeniladas similares a mRNAs codifícantes. O banco público de ESTs de cana-de-açúcar TIGR Gene Index foi usado como base para uma busca de miRNAs. O processo criado possibilitou a identificação de 20 precursores de miRNAs, agrupados em 15 famílias distintas. No presente trabalho desenvolveu-se também ferramenta para predição de potenciais alvos para os miRNAs encontrados. As famílias de miRNAs de cana-de-áçucar e a ferramenta de predição de genes-alvo estão integrados em banco de dados que estará disponível brevemente. Análise de expressão gênica demonstrou que precursors de miRNAs de cana-de-açúcar acumulam em níveis variáveis em distintos tecidos/órgãos. Além disso, tanto o acúmulo do miRNA maduro quanto a degradação do mRNA-alvo foram avaliados para alguns casos estudados. A caracterização de um miRNA específico de monocotiledôneas (miR528) e a confirmação de seu alvo, um gene comum em angiospermas, predito pela primeira vez neste trabalho, gera um interessante questionamento sobre a regulação desse gene via miRNA apenas em monocotiledôneas / Abstract: No-coding RNAs of 20-27 nucleotides (nt) transcriptional or posttranscriptionally regulate endogenous gene expression, affecting the cellular output of transcripts and proteins. Among these RNAs, microRNAs (miRNAs) play an important role in plant development as confirmed by phenotypic and molecular evaluation of transgenic plants and knockout mutants defective in miRNA biogenesis and function. miRNAs are produced from long precursors (pri-miRNAs), which are processed by specific enzymes into the mature miRNA (20-22 nt). The mature miRNA guides the cleavage of target genes as well as impairs protein translation, affecting several development processes. Deep sequencing of small RNAs identified conserved and species-specific miRNAs. Nevertheless, studies on crops are still in their infancy. Public ESTs databases are an important source of no-coding sequences, in which we can find miRNAs precursors, which are polyadenilated RNAs as messenger RNAs. In this work, the public sugarcane EST database TIGR Gene Index was used to search conserved miRNAs. The pipeline developed in this work made possible the identification of 20 miRNAs precursors, grouped into 15 families. It was also developed a search tool for potential miRNAs targets. Sugarcane miRNAs precursors displayed tissue/organ differential expression profiles. Moreover, a new identified miRNA target was confirmed experimentally. This new target is regulated by a monocot specific miRNA, miR528. Interestingly, this miRNA target is conserved in eudicots and monocots, even though its regulation by miRNA is not. This finding raises the question of why this gene has evolved in having a miRNA-mediated posttranscriptional regulation only in monocots / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

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