Inhibition of ErbB2 and Thymidylate Synthase by a Multi-Targeted Small-Interfering RNA in Human Breast Cancer Cell Lines

The therapeutic potential of a novel multi-targeted small-interfering RNA (siRNA) was investigated in human breast cancer cells. Previous studies had identified an siRNA that specifically and potently inhibited expression of thymidylate synthase (TS) by directly targeting human TS mRNA. TS is a folate-dependent enzyme that catalyzes the key reaction involved in synthesizing nucleotide precursors for DNA biosynthesis, and as such, it plays a critical role in maintaining cell growth. The goal of this thesis was to design and develop a novel siRNA molecule that targeted TS mRNA as well as a cellular mRNA that encodes a different cellular protein involved in cancer cell growth and proliferation, such as a member of the ErbB family. Gene sequence analysis was performed and identified an overlapping sequence between TS and ErbB2 mRNAs. An siRNA duplex was then designed to simultaneously target human TS and ErbB2 mRNA. Transfection of the multi-targeted siRNA (TS1M17) revealed that both ErbB2 and TS proteins were significantly suppressed in a time and dose-dependent manner in ErbB2-overexpressing human breast cancer SKBR3 cells. The corresponding mRNA levels, as determined by RT-PCR, were also decreased. Protein levels of other ErbB family members, including ErbB1 and ErbB3, remained unchanged with siRNA treatment. An ErbB2-specific siRNA (B2450) inhibited ErbB2, but had no effect on TS expression demonstrating the specificity of the multi-targeted siRNA against both TS and ErbB2. Mismatched (TS1-Mismatch) and control (GL2) siRNAs had no inhibitory effects on expression of the two target proteins. Suppression of activated ErbB2, as determined by expression of phosphorylated ErbB2 protein, was observed with transfection of TS1M17 siRNA. In addition, the expression of downstream signaling proteins, such as phosphorylated mitogen activated protein kinase (p-MAPK), p27Kip1, p21Cip1, cyclin D1, and survivin were significantly changed. In contrast, control siRNAs did not exert any inhibitory effects on downstream signaling. Taken together, these findings suggest that TS1M17 siRNA inhibits signaling of the ErbB2 pathway. The effect of TS1M17 siRNA on cytotoxicity was analyzed by WST-1 assay. Upon transfection into SKBR3 cells, the TS1M17 siRNA significantly suppressed cell proliferation with an IC50 value of 0.65 nM, which is 154-fold more potent than ErbB2- and TS-specific siRNAs. This study suggests that targeting expression of ErbB2 and TS, two key proteins involved in distinct and critical pathways for cancer growth and proliferation, with a single siRNA molecule may provide a novel approach for cancer chemotherapy

Electrochemical measurement of nitric oxide from biological systems

Nitric oxide (NO) is known to be involved in a number of physiological processes, including the immune response. As such, its role in severe infection and sepsis has been investigated, but previous measurement techniques have relied on complicated instrumentation or the quantification of NO byproducts (e.g., nitrate and nitrite). Herein, the fabrication of a microfluidic amperometric sensor for the direct detection of NO in whole blood is described. These sensors were used to evaluate the potential of NO and nitrosothiols (a stable transporter) as prognostic and/or diagnostic biomarkers for infection and sepsis. The microfluidic devices facilitated the selective electrochemical measurement of NO in small volumes of blood at the point-of-care, with adequate sensitivity and limits of detection achieved in buffer, wound fluid, and whole blood. A green (530 nm) light-emitting diode was coupled to the device to enable photolysis of S-nitrosothiol species with subsequent NO detection. While inefficient photolysis prevented the measurement of nitrosothiols in whole blood, detection in serum was achieved. A porcine model of sepsis permitted monitoring of temporal changes in NO and nitrosothiols throughout disease progression. While increases in NO were observed concurrently with other indicators (e.g., increased blood lactate and base deficit), the accumulation of nitrosothiols was observed hours prior to the onset of other symptoms, despite a dramatic drop in the circulating white blood cells that produce NO. A murine model of sepsis was utilized to understand the effects of bacterial virulence and immune suppression on NO during an infection. A non-lethal pneumonia with Pseudomonas aeruginosa resulted in elevated NO levels at 72 h that returned to baseline concentrations after 1 wk. A more virulent bacterium, Klebsiella pneumoniae, resulted in much greater increases in NO, reflecting its pathogenicity. Conversely, in a murine model of post-burn immune suppression and infection, blood NO concentrations remained unchanged relative to uninfected animals despite increased infection severity. Nitric oxide-selective microelectrodes were also used to study NO release at the single cell level, from both immune cells and neurons. Upregulation of carbon monoxide production by the macrophages was demonstrated to inhibit their ability to release NO following immune stimulation. Additionally, the concentration and kinetics of NO release from neurons were determined.Doctor of Philosoph

Changes in Body Image and Sexuality in Rural Breast Cancer Survivors During a Weight Loss and Weight Maintenance Intervention

This study evaluated changes in body image dimensions in breast cancer survivors after a weight control trial and predictors of those changes. Postmenopausal rural breast cancer survivors enrolled in an 18-month phone-based weight loss and weight maintenance intervention participated. Data was collected at baseline, 6-months (post-weight loss intervention), and 18-months (post-weight maintenance intervention). Participants were randomized into either a phone-based group condition or mail-based condition during the weight maintenance intervention. The Body Image and Relationships Scale (BIRS) assessed six dimensions of body image relevant for breast cancer survivors, including sexuality. All six body image subscales and total score improved upon completion of the weight loss intervention (p’s < .001). Weight loss and physical activity changes were less predictive of those improvements than age, breast cancer treatment history, and baseline depressive symptoms and quality of life. All but one BIRS dimension worsened between the weight loss and weight maintenance intervention, with a significant interaction effect by maintenance treatment condition observed for one subscale. Weight regain, marital status, breast cancer treatment history, and baseline depressive symptoms were predictive of changes in body image dimensions from 6- to 18-months. Improvements in body image dimensions during the weight loss intervention were unrelated to weight regain. Despite the worsening in body image during the weight maintenance intervention, it appears that the improvements gained during the weight loss intervention, regardless of the amount of weight lost, were only partially attenuated during weight regain

Bench, Bedside, Curbside, and Home: Translational Research to Include Transformative Change Using Educational Research

Translational research originated in the medical field during the 1990s to describe taking discovery based research through the steps of applying it to clinical research and patient-oriented care. This model is implicitly linear, depicting the flow of information from researchers’ bench, to a clinical trial bedside, to a primary care physician’s practice. The prevailing model of translational research, referred to as “Bench to Bedside to Curbside,” is limited in that it does not adequately incorporate stakeholders outside of the professional or research community because Curbside refers to physician care delivered to patients. This omits the transformative impact that research can have on the general populace if implemented through educational research, disseminating knowledge to people who can use it. In this article we argue that a fourth category needs to be incorporated into the previous T1-T3 Bench to Bedside to Curbside model, and this fourth category represents T4, “Home.” We seek to further define and describe, while providing a new model for translational research that is more circular in nature and inclusive of the general populace. We also suggest that the incorporation of educational researchers and practitioners would expand the current collaborative nature of translational research and is a way to expand the translational model. This promises more adequate, effective, and sustainable impacts on a target population

Americans Support for Renewable Energy is Disconnected from their Understanding of Powerline Infrastructure as a Mechanism to Mitigate Climate Change

As nations are transitioning to renewable energy sources, they will need to expand and upgrade their energy infrastructure, including high-voltage power lines (HVPL). We have conducted the first nation-wide survey in the last thirty years to assess public attitudes toward HVPL in the USA. The study evaluates perceptions, knowledge, and attitudes toward building new transmission lines, as these relate to renewable energy, place attachment, and environmental impacts. Our results show that Americans do not recognize how new HVPL could help reduce greenhouse gas emissions; instead, respondents favor moving from centralized energy (large power stations and HVPL) to decentralized energy (local power supply and small scale solar panels and wind turbines. Our findings are consistent with studies from Europe in that citizens recognize negative human impacts on the natural world and support renewable energy, however, they have a limited understanding of the role of HVPL infrastructure in mitigating climate change

Challenges to driver licensing participation for Aboriginal people in Australia: a systematic review of the literature

Introduction: Aboriginal and Torres Strait Islander people are overrepresented in transport-related morbidity and mortality. Low rates of licensure in Aboriginal communities and households have been identified as a contributor to high rates of unlicensed driving. There is increasing recognition that Aboriginal people experience challenges and adversity in attaining a licence. This systematic review aims to identify the barriers to licence participation among Aboriginal people in Australia. Method: A systematic search of electronic databases and purposive sampling of grey literature was conducted, two authors independently assessed publications for eligibility for inclusion. Results: Twelve publications were included in this review, of which there were 11 reporting primary research (qualitative and mixed methods) and a practitioner report. Barriers identified were categorised as individual and family barriers or systemic barriers relating to the justice system, graduated driver licensing (GDL) and service provision. A model is presented that depicts the barriers within a cycle of licensing adversity. Discussion: There is an endemic lack of licensing access for Aboriginal people that relates to financial hardship, unmet cultural needs and an inequitable system. This review recommends targeting change at the systemic level, including a review of proof of identification and fines enforcement policy, diversionary programs and increased provision for people experiencing financial hardship. Conclusion: This review positions licensing within the context of barriers to social inclusion that Aboriginal people frequently encounter. Equitable access to licensing urgently requires policy reform and service provision that is inclusive, responsive to the cultural needs of Aboriginal people and accessible to regional and remote communities

Diphenylchloronitroethane Insecticides

Insecticidal activity of chloronitroalkanes was predicted on the basis of structure-activity relationships. Two series of new bis(substituted-phenyl) chloronitroalkanes were synthesized and evaluated for insecticidal activity. The synthetic pathway proceeded through phenylnitroethanols and diphenylnitroethanes as intermediates. Final products were 1,1-bis (substituted-phenyl)-2-chloro-2-nitroethanes and 1,1-bis(substituted-phenyl)-2,2-dichloro-2-nitroethanes. Aromatic substituents were selected from alkyl, alkoxy, and halogen moieties. Following purifications and confirmation of structures, the compounds were bioassayed against insects. The two series were compared for potency, as were various combinations of X and Y substituents. Adult female house flies (Musca domestica), mosquito larvae (Aedes aegypti), western corn rootworm (Diabrotica virgifera virgifera) and German cockroach (Blattellagermanica) have been tested. In general, the mono-chloro series is more toxic than the di-chloro series. Five of the mono-chloro analogs are 8-10 times more potent than pyrethrins and 6-7 times more toxic than methoxychlor to the house fly