IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning.

Something real: A social marketing approach to smoking prevention among Montevideo youth

Uruguay has some of the strictest tobacco-control laws in Latin America. Despite this, youth smoking rates in Uruguay are amongst the highest in South America. Thus, it is important to identify strategies to prevent youth smoking in Uruguay. The current qualitative research study sought to identify intrapersonal and socioenvironmental factors that are associated with smoking among middle school youth in Uruguay. It also sought to develop potential prevention strategies and media messages that would resonate with youth for a social media campaign. The study was grounded in social cognitive theory and the theory of reasoned action/planned behavior, among other behavioral science theories; anthropological perspectives were also considered. To achieve these goals, 29 group and individual structured interviews were conducted in two private middle schools catering to lower and higher SES youth in Montevideo, Uruguay during the summer of 2012. One hundred and three study participants, including students, parents, and teachers, were interviewed. The structured interviews were recorded, transcribed, translated, back translated, coded and analyzed. The study findings show that positive attitudes towards smoking (i.e. to be seen, to increase status, to ensure women's equality, to looking old, and to service as a rite of passage), delinquent behavior (i.e. transgression/deviant behavior), social norms that support smoking (i.e. peer pressure and modeling, group membership/sense of belonging, parental modeling, and family support), easy access and availability to tobacco (i.e. retails stores) were factors associated with youth smoking. Potential protective factors may include parental support, negative attitudes towards smoking, sports/music, and smoke-free environments. Because study participants are accustomed to government-sponsored strong countermarketing graphic imaging, study participants selected even stronger images and messages as the preferred way to receive tobacco prevention messages. Something Real ("Algo Real") was a theme that resonated with the participants and chosen as the name for the proposed campaign. This campaign was designed as a multiple component intervention that included mass, school base, and family based strategies to prevent tobacco use. Some intervention materials specific to these intervention components were developed to target relevant intrapersonal and socioenvironmental factors identified above. These materials will be tested in future pilot studies and larger scale evaluation with this population, outside the scope of this dissertation. ^