New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration

Real time monitoring of oxygenation and respiration is on the cutting edge of bioanalysis, including studies of cell metabolism, bioenergetics, mitochondrial function and drug toxicity. This thesis presents the development and evaluation of new luminescent probes and techniques for intracellular O2 sensing and imaging. A new oxygen consumption rate (OCR) platform based on the commercial microfluidic perfusion channel μ-slides compatible with extra- and intracellular O2 sensitive probes, different cell lines and measurement conditions was developed. The design of semi-closed channels allowed cell treatments, multiplexing with other assays and two-fold higher sensitivity to compare with microtiter plate. We compared three common OCR platforms: hermetically sealed quartz cuvettes for absolute OCRs, partially sealed with mineral oil 96-WPs for relative OCRs, and open 96-WPs for local cell oxygenation. Both 96-WP platforms were calibrated against absolute OCR platform with MEF cell line, phosphorescent O2 probe MitoXpress-Intra and time-resolved fluorescence reader. Found correlations allow tracing of cell respiration over time in a high throughput format with the possibility of cell stimulation and of changing measurement conditions. A new multimodal intracellular O2 probe, based on the phosphorescent reporter dye PtTFPP, fluorescent FRET donor and two-photon antennae PFO and cationic nanoparticles RL-100 was described. This probe, called MM2, possesses high brightness, photo- and chemical stability, low toxicity, efficient cell staining and high-resolution intracellular O2 imaging with 2D and 3D cell cultures in intensity, ratiometric and lifetime-based modalities with luminescence readers and FLIM microscopes. Extended range of O2 sensitive probes was designed and studied in order to optimize their spectral characteristics and intracellular targeting, using different NPs materials, delivery vectors, ratiometric pairs and IR dyes. The presented improvements provide useful tool for high sensitive monitoring and imaging of intracellular O2 in different measurement formats with wide range of physiological applications.

The association between parental attitudes and alcohol consumption and adolescent alcohol consumption in Southern Ireland: a cross-sectional study

Background: Alcohol plays a complex role in society. A recent study showed that over half of Irish adults drink hazardously. Adolescents report increased levels of alcohol consumption. Previous research has inferred the influence of the parent on their adolescent. Thus, the aim of the current study was to investigate the association between adolescent alcohol consumption and their parent’s consumption pattern and attitude toward alcohol use in Southern Ireland. Methods: A cross-sectional survey was undertaken in November 2014. This involved distributing a survey to adolescents (n = 982) in their final two years of second level education and at least one of their parents from a local electorate area in Southern Ireland. This survey included: alcohol use, self- reported height and weight, smoking status, mental health and well-being along with attitudinal questions. Chi-square tests and multivariate logistic regression were utilised. Results: A 37 % response rate was achieved. Over one-third (34.2 %) of adolescents and 47 % of parents surveyed reported hazardous drinking. Over 90 % of parents disagreed with allowing their adolescent to get drunk and rejected the idea that getting drunk is part of having fun as an adolescent. The majority (79.5 %) of parents surveyed believed that their alcohol consumption pattern set a good example for their adolescent. Multivariate logistic regression highlights the association between adolescent hazardous alcohol consumption and hazardous drinking by the father. Furthermore either parent permitting their adolescent to drink alcohol on special occasions was associated with hazardous alcohol consumption in the adolescent. Conclusion: The findings of this research notes a liberal attitude to alcohol and increased levels of consumption by the parent are linked to hazardous adolescent drinking behaviour. Future action plans aimed at combatting adolescent hazardous alcohol consumption should also be aimed at tackling parents’ attitudes towards and consumption of alcohol.