Theses and Dissertations
This collection includes both ASU Theses and Dissertations, submitted by graduate students, and the Barrett, Honors College theses submitted by undergraduate students.
Reducing the amount of error and introduced data variability increases the accuracy of Western blot results. In this study, different methods of normalization for loading differences and data alignment were explored with respect to their impact on Western blot results. GAPDH was compared to the LI-COR Revert total protein stain as a loading control. The impact of normalizing data to a control condition, which is commonly done to align Western blot data distributed over several immunoblots, was also investigated. Specifically, this study addressed whether normalization to a small subset of distinct controls on each immunoblot increases pooled data variability compared to a larger set of controls. Protein expression data for NOX-2 and SOD-2 from a study investigating the protective role of the bradykinin type 1 receptor in angiotensin-II induced left ventricle remodeling were used to address these questions but are also discussed in the context of the original study. The comparison of GAPDH and Revert total protein stain as a loading control was done by assessing their correlation and comparing how they affected protein expression results. Additionally, the impact of treatment on GAPDH was investigated. To assess how normalization to different combinations of controls influences data variability, protein data were normalized to the average of 5 controls, the average of 2 controls, or an average vehicle and the results by treatment were compared. The results of this study demonstrated that GAPDH expression is not affected by angiotensin-II or bradykinin type 1 receptor antagonist R-954 and is a less sensitive loading control compared to Revert total protein stain. Normalization to the average of 5 controls tended to reduce pooled data variability compared to 2 controls. Lastly, the results of this study provided preliminary evidence that R-954 does not alter the expression of NOX-2 or SOD-2 to an expression profile that would be expected to explain the protection it confers against Ang-II induced left ventricle remodeling.
Exploration of a mouse model (C57BL/6J) capable of demonstrating behavioral changes after adolescent social isolation that are consistent with prior findings may prove beneficial in later research. This study examined 2 proposed long-term effects of isolated housing (one mouse/cage), when compared to group housing (two mice/cage) during adolescence. Mice were placed in their respective housing conditions after weaning (PND 21) and remained in those conditions until PND 60. The same cohorts were used in both phases of the experiment. Phase 1 sought to confirm previous findings that showed increases in ethanol intake after adolescent social isolation using a 2-bottle preference Drinking-in-the-Dark (DID) design over a 4-day period (PND 64-PND 67.). Phase 2 sought to elucidate the effects present after adolescent social isolation, as measured using response inhibition capabilities demonstrated during fixed-minimum interval (FMI) trials (PND 81-PND 111). Findings in phase 1 of the experiment were non-significant, save a strong tendency for female mice in both housing conditions to drink more as a proportion of their bodyweight (g/kg). However, a trend of lower bodyweight in single housed mice did exist, which does suggest that detrimental stress was applied via the used of adolescent isolation in that housing condition. Findings in phase 2 showed little effect of adolescent social isolation on mean inter-response time (IRT) at any criterion used (FMI-0, FMI-4, FMI-6). Evaluation of mean interquartile range (IQR) of IRTs showed a significantly greater amount of variation in IRT responses within single housed mice at the highest criterion (FMI-6), and a trend in the same direction when FMI-4 and FMI-6 were tested concurrently. Taken as a whole, the findings of this experiment suggest that the effect of adolescent social isolation on ethanol intake is far less robust than the effect of sex and may be difficult to replicate in a low-power study. Additionally, adolescent social isolation may interfere with the ability of mice to show consistent accuracy during FMI tasks or a delay in recognition of FMI criterion change.
Midlife is a unique period of development during which individuals are simultaneously engaging in multiple roles. Despite this, there is a surprisingly small amount of research on this period of the life course. In order to examine sources of adversity during this period, we analyzed interviews with individuals in midlife about their greatest challenge. The most common themes for types of adversity included relationships, health, and work, reflecting the unique combination of roles in midlife.
Using in vitro experimental models of both SAOS-2 (non-metastatic) and 143-b (metastatic) osteosarcoma cell lines and Western blot analysis, we have demonstrated that basal levels of molecular chaperone BiP (Binding immunoglobulin protein, or GRP-78) and peIF2α (phospho-eukaryotic initiation factor 2 alpha), both markers of the UPR, were higher in SAOS-2 than 143-b cells. We also show that both these markers were further up-regulated upon exposure to hypoxia, as evidenced by the increase in banding intensity in both SAOS-2 and 143-b cells. Furthermore, analysis of another UPR marker, ATF6 (activating transcription factor 6) showed that basal levels of active nuclear ATF6 were slightly higher in SAOS-2 cells than in 143-b cells. However, unlike the other UPR markers these levels were significantly reduced upon exposure to hypoxia (0.1% O2). In addition to hypoxia, treatment with Cisplatin also had similar effects on the expression of aforementioned UPR markers: BiP and peIF2α. We found that the 143-b OS cells were more sensitive to the Cisplatin treatment than the SAOS-2 OS cells, and thus more prone to cell-mediated death.
Our findings shed light on the unknown mechanisms underlying chemotherapeutic drug resistance in osteosarcoma patients. Our research may lead to novel therapies that seek out and destroy the chemoresistant OS cells within the hypoxia core of tumors, thereby preventing survival and metastasis, and ultimately improving the chances of survival amongst OS patients.