UNDERSTANDING NANOG'S ROLE IN CANCER BIOLOGY

Understanding Nanog’s Role in Cancer Biology Mark Daniel Badeaux Supervisory Professor Dean Tang, PhD The cancer stem cell model holds that tumor heterogeneity and population-level immortality are driven by a subset of cells within the tumor, termed cancer stem cells. Like embryonic or somatic stem cells, cancer stem cells are believed to possess self-renewal capacity and the ability to give rise to a multitude of varieties of daughter cell. Because of cancer’s implied connections to authentic stem cells, we screened a variety of prostate cancer cell lines and primary tumors in order to determine if any notable ‘stemness’ genes were expressed in malignant growths. We found a promising lead in Nanog, a central figure in maintaining embryonic stem cell pluripotency, and through a variety of experiments in which we diminished Nanog expression, found that it may play a significant role in prostate cancer development. We then created a transgenic mouse model in which we targeted Nanog expression to keratin 14-expressing in order to assess its potential contribution to tumorigenesis. We found a variety of developmental abnormalities and altered differentiation patterns in our model , but much to our chagrin we observed neither spontaneous tumor formation nor premalignant changes in these mice, but instead surprisingly found that high levels of Nanog expression inhibited tumor formation in a two-stage skin carcinogenesis model. We also noted a depletion of skin stem cell populations, which underlies the wound-healing defect our mice harbor as well. Gene expression analysis shows a reduction in c-Jun and Bmp5, two genes whose loss inhibits skin tumor development and reduces stem cell counts respectively. As we further explored Nanog’s activity in prostate cancer, it became apparent that the protein oftentimes was not expressed. Emboldened by the competing endogenous RNA (ceRNA) hypothesis, we identified the Nanog 3’UTR as a regulator of the tumor suppressive microRNA 128a (miR-128a), which includes known oncogenes such as Bmi1 among its authentic targets. Future work will necessarily involve discerning instances in which Nanog mRNA is the biologically relevant molecule, as well as identifying additional mRNA species which may serve solely as a molecular sink for miR-128a.

Temporal and spatial regulation of cell division by the nucleoid in Escherichia coli

Selection of division sites and coordination of cytokinesis with other cell cycle events are critical for every organism to proliferate. In E. coli, the nucleoid is proposed to exclude division from the site of the chromosome (nucleoid occlusion model). We studied the effect of the nucleoid on timing and placement of cell division. An early cell division protein, FtsZ, was used to follow development of the division septum. FtsZ forms a ring structure (Z ring) at potential division sites. The dynamics of Z ring was visualized in live cells by fusing FtsZ with a green fluorescent protein (GFP). Emanating FtsZ-GFP polymers from the constricted septum or aggregates in daughter cells were also observed, probably representing the FtsZ depolymerization and immature FtsZ nucleation processes. We next examined the nucleoid occlusion model. Mutants carrying abnormally positioned chromosomes were employed. In chromosomal partition mutants, replicated chromosomes cannot segregate. The Z ring was excluded from midcell to the edge of the nucleoid. This negative effect of nucleoids was further confirmed in replication deficient dnaA mutants, in which only a single chromosome is present in the cell center. These results suggest that the nucleoid, replicating or not, inhibits division in the area where the chromosome occupies. In addition, increasing the level of FtsZ does not overcome nucleoid inhibition. Interestingly in anucleate cells produced by both mutants, the Z ring was localized in the central part of the cell, which indicates that the nucleoid is not required for FtsZ assembly. Relaxation of chromosomes by reducing the gyrase activity or disruption of protein translation/translocation did not abolish the division inhibition capacity of the nucleoid. However, preventing transcription did compromise the nucleoid occlusion effect, leading to formation of multiple FtsZ rings above the nucleoid. In summary, we demonstrate that nucleoids negatively regulate the timing and position of division by inhibiting FtsZ assembly at unselected sites. Relief of this inhibition at midcell is coincident with the completion of DNA replication. On the other hand, FtsZ assembly does not require the nucleoid. ^

Family stress, coping, communication, and adaptation in unintended adolescent pregnancy

Objective. This study examines post-crisis family stress, coping, communication, and adaptation using the Double ABC-X Model of Family Adaptation in families with a pregnant or postpartum adolescent living at home. ^ Methods. Ninety-eight pregnant and parenting adolescents between ages 14 and 18 years (Group 1 at 20 or more weeks gestation; Group 2 at delivery and 8 weeks postpartum) and their parent(s) completed instruments congruent with the model to measure family stress, coping, communication, and adaptation. Descriptive family data was obtained. Mother-daughter data was analyzed for differences between subjects and within subjects using paired t-tests. Correlational analysis was used to examine relationships among variables. ^ Results. More than 90% of families were Hispanic. There were no significant differences between mother and daughter mean scores for family stress or communication. Adolescent coping was not significantly correlated to family coping at any interval. Adolescent family adaptation scores were significantly lower than mothers' scores at delivery and 8 weeks postpartum. Mean individual ratings of family variables did not differ significantly between delivery and 8 weeks postpartum. Simultaneous multiple regression analysis showed that stress, coping, and communication significantly influenced adaptation for mothers and daughters at all three intervals. The relative contributions of the three independent variables exhibited different patterns for mothers and daughters. Parent-adolescent communication accounted for most of the variability in adaptation for daughters at all three intervals. Daughters' family stress ratings were significant for adaptability (p = .01) during the pregnancy and for cohesion (p = .03) at delivery. Adolescent coping (p = .03) was significant for cohesion at 8 weeks postpartum. Family stress was a significant influence at all three intervals for mothers' ratings of family adaptation. Parent-adolescent communication was significant for mother's perception of both family cohesion (p < .001) and adaptability (p < .001) at delivery and 8 weeks, but not during pregnancy. ^ Conclusions. Mothers' and daughters' ratings of family processes were similar regarding family stress and communication, but were significantly different for family adaptation. Adolescent coping may not reflect family coping. Family communication is a powerful component in family functioning and may be an important focus for interventions with adolescents and parents. ^

Identification and analysis of novel mitotic inhibitors of the Caenorhabditis elegans Aurora B kinase

Proper execution of mitosis requires the accurate segregation of replicated DNA into each daughter cell. The highly conserved mitotic kinase AIR-2/Aurora B is a dynamic protein that interacts with subsets of cofactors and substrates to coordinate chromosome segregation and cytokinesis in Caenorhabdiris elegans. To identify components of the AIR-2 regulatory pathway, a genome-wide RNAi-based screen for suppressors of air-2 temperature-sensitive mutant lethality was conducted. Here, I present evidence that two classes of suppressors identified in this screen are bona fide regulators of the AIR-2 kinase. The strongest suppressor cdc-48.3, encodes an Afg2/Spaf-related Cdc48-like AAA+ ATPase that regulates AIR-2 kinase activity and stability during C. elegans embryogenesis. Loss of CDC-48.3 suppresses the lethality of air-2 mutant embryos, marked by the restoration of the dynamic behavior of AIR-2 and rescue of chromosome segregation and cytokinesis defects. Loss of CDC-48.3 leads to mitotic delays and abnormal accumulation of AIR-2 during late telophase/mitotic exit. In addition, AIR-2 kinase activity is significantly upregulated from metaphase through mitotic exit in CDC-48.3 depleted embryos. Inhibition of the AIR-2 kinase is dependent on (1) a direct physical interaction between CDC-48.3 and AIR-2, and (2) CDC-48.3 ATPase activity. Importantly, the increase in AIR-2 kinase activity does not correlate with the stabilization of AIR-2 in late mitosis. Hence, CDC-48.3 is a bi-functional inhibitor of AIR-2 that is likely to act via distinct mechanisms. The second class of suppressors consists of psy-2/smk-1 and pph-4.1, which encode two components of the conserved PP4 phosphatase complex that is essential for spindle assembly, chromosome segregation, and overall mitotic progression. AIR-2 and its substrates are likely to be targets of this complex since mitotic AIR-2 kinase activity is significantly increased during mitosis when either PSY-2/SMK-1 or PPH-4.l is depleted. Altogether, this study demonstrates that during the C. elegans embryonic cell cycle, regulators including the CDC-48.3 ATPase and PP4 phosphatase complex interact with and control the kinase activity, targeting behavior and protein stability of the Aurora B kinase to ensure accurate and timely progression of mitosis. ^

Parental acculturation and adolescent HPV uptake among Hispanics: Results from the California Health Interview Survey

Background. The Centers for Disease Control and Prevention (CDC), the American Cancer Society (ACS), and the American College of Obstetricians and Gynecologists (ACOG) all recommend the HPV vaccine for girls 11-12. The vaccine has the potential to reduce cervical cancer disparities if it is used by populations that do not participate in screening. Evidence suggests that incidence and mortality are higher among Hispanic women compared to non-Hispanic white women because they do not participate in screening. Past literature has found that acculturation has a mixed effect on cervical cancer screening and immunization. Little is known about whether parental acculturation is associated with adolescent HPV vaccine uptake among Hispanics and the mechanisms through which acculturation may affect vaccine uptake.^ Aims. To examine the association between parental acculturation and adolescent HPV uptake among Hispanics in California and test the structural hypothesis of acculturation by determining if socioeconomic status (SES) and health care access mediate the association between acculturation and HPV vaccine uptake.^ Methods. Cross-sectional data from the 2007 California Health Interview Survey (CHIS) were used for bivariate and multivariate logistic regression analyses. The sample used for analysis included 1,090 Hispanic parents, with a daughter age 11-17, who answered questions about the HPV vaccine. Outcome variable of interest was HPV vaccine uptake (≥1dose). Independent variables of interest were language spoken at home (a proxy variable for acculturation), household income (percent of federal poverty level), education level, and health care access (combined measure of health insurance coverage and usual source of care).^ Results. Parents who spoke only English or English and Spanish in the home were more likely to get the HPV vaccine for their daughter than parents who only spoke Spanish (Odds Ratio [OR]: 0.55, 95% Confidence Interval [CI]: 0.31-0.98). When SES and health care access variables were added to the logistic regression model, the association between language acculturation and HPV vaccine uptake became non-significant (OR: 0.68, 95% CI: 0.35-1.29). Both income and health care access were associated with uptake. Parents with lower income or who did not have insurance and a usual source of care were less likely to have a vaccinated daughter.^ Discussion. Socioeconomic status and health care access have a more proximal effect on HPV vaccine uptake than parental language acculturation among Hispanics in California.^ Conclusion. This study found support for the structural hypothesis of acculturation and suggest that interventions focus on informing low SES parents who lack access to health care about programs that provide free HPV vaccines.^

Process evaluation of intervention program for prevention of early sexual activity and alcohol use among Hispanic teenagers

Hispanic teens are a high-risk population for initiation of early sexual activity and alcohol use which in turn has numerous social and health consequences. One strategy to address prevention of these behaviors is to implement a capacity building intervention that promotes parent child communication, encompasses their cultural values and community participation. This study describes the process evaluation of a pilot intervention program amongst Hispanic teens and their families living along the Texas-Mexico border. “Girls Lets Talk” is a small group intervention with 10-14 year old teens and their female adult family members that involves education regarding effects of alcohol use and sexual activity as well as activities for monitoring and refusal skills to prevent risky behaviors. Two waves of the program each consisting of at least seven mother daughter dyads were conducted. During the designing process, community advisory board meetings and focus groups were held to review course materials and ensure they were appropriate to the Mexican American culture. Parent and adolescent surveys were administered at the beginning and end of the intervention to assess for psychosocial outcome variables. All sessions received high mean satisfactory scores (mean of 4.00 or better on a five point scale) for both adult and adolescent participants. Qualitative feedback was obtained via debriefing sessions to evaluate experience as well as alter recruitment strategies. A Wilcoxon Sign Rank analysis of the pre and post intervention surveys was done that showed significant changes in some outcome variables such as intentions and confidence for monitoring behaviors for adults and beliefs regarding sexual activity. “Girls Lets Talk” is a promising example of how a process evaluation plan can help develop a theory based health promotion program using the community based participatory research approach. The intervention may also be effective in altering intentions and enhancing self-efficacy among parents and teens in order to decrease risky behaviors such as early sexual activity and alcohol use.^

Characterization of a Novel Role for the Tousled- like Kinase in Kinetochore Assembly and Function in Caenorhabditis elegans

Chromosome segregation is a critical step during cell division to avoid aneuploidy and promote proper organismal development. Correct sister chromatid positioning and separation during mitosis helps to achieve faithful transmission of genetic material to daughter cells. This prevents improper chromosome partitioning that can potentially result in extrachromosomal fragments, increasing the tumorigenic potential of the cells. The kinetochore is a protenaicious structure responsible for the initiation and orchestration of chromosome movement during mitosis. This highly conserved structure among eukaryotes is required for chromosome attachment to the mitotic spindle and failure to assemble the kinetochore results in aberrant chromosome segregation. Thus elucidating the mechanism of kinetochore assembly is important to have a better understanding of the regulation that controls chromosome segregation. Our previous work identified the C. elegans Tousled-like kinase (TLK-1) as a mitotic kinase and depletion of TLK-1 results in embryonic lethality, characterized by nuclei displaying poor mitotic chromosome alignment, lagging chromosome, and chromosome bridges during anaphase. Additionally, previous studies from our group revealed that TLK-1 is phosphorylated independently by Aurora B at serine 634, and by CHK-1 at threonine T610. The research presented herein reveals that both phosphorylated forms of TLK-1 associate with the kinetochore during mitosis. Moreover, by systematic depletion of kinetochore proteins, I uncovered that pTLK-1 is bona fide kinetochore component that is located at the outer kinetochore layer, influencing the microtubule-binding interface. I also demonstrated that TLK-1 is necessary for the kinetochore localization of the microtubule interacting proteins CLS-2 and LIS-1 and I show that embryos depleted of TLK-1 presented an aberrant twisted kinetochore pattern. Furthermore, I established that the inner kinetochore protein KNL-2 is an in vitro substrate of TLK-1 indicating a possible role of TLK-1 in regulating centromeric assembly. Collectively, these results suggest a novel role for the Tousled-like kinase in regulation of kinetochore assembly and microtubule dynamics and demonstrate the necessity of TLK-1 for proper chromosome segregation in C. elegans.

Exploring parental factors and their influence on early adolescent sexual health

Black and Hispanic youth experience the largest burden of sexually transmitted infections, teen pregnancy, and childbirth (Hamilton, Martin, & Ventura, 2011). Minority youth are disporportionately more likely to sexually debut at every age and debut before the age of 13 compared to whites (Centers for Disease Control and Prevention, 2011). However, there is little known about pre-coital sexual activity or protective parental factors in early adolscent minority youth. Parental factors such as parent-child communication and parental monitoring influence adolescent sexual behaviors and pre-coital sexual behaviors in early adolescence. Three distinct methods were used in this dissertation. Study one used qualitative methods, semi-structured, in-depth, individual interviews, to explore parent-child communication in African American mother-early adolescent son dyads. Study two used quantitative methods, secondary data analysis of a cross sectional study, to conduct a moderation analysis. For study three, I conducted a systematic review of parent-based adolescent sexual health interventions. Study one found that mothers feel comfortable talking about sex with adolescents, provide a two-prong sexual health message, and want their sons to tell their when they are thinking of having sex. Study found that parental monitoring moderates the relation between parent-child communication and pre-coital sexual behaviors. Study three found that interventions use a variety of theory, methods, and strategies and that no parent-based programs target faith-based organizations, mother-son or father-daughter dyads, or parents of LGBTQ youth. Adolescent sexual health interventions should consider addressing youth-to-parent disclosure of sexual activity or intentions to debut, addressing both parent-child sexual health communication and parental monitoring, and using a theoretical framework.^