Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

Counterstories: who counts as lesbian?

Coming out midlife is a profound and life‐changing experience—it is an experience of self‐shattering that entails the destabilisation of identity, and of family relationships. Entailing a displacement from social insider to outsider, it is a difficult, but also exhilarating, journey of self, and sexual, discovery. This thesis is an examination of the experiences of nine women who undertook that journey. This dissertation is very much a search for understanding—for understanding how one can be lesbian, and how one can not have known, following a lifetime of heterosexual identification—as well as a search for why those questions arise in the first place. I argue that the experience of coming out midlife exposes the fundamental ambiguity of sexuality; and has a significance that ranges beyond the particularity of the participants’ experiences and speaks to the limitations of the hegemonic sexual paradigm itself. Using the theoretical lens of three diverse conceptual approaches—the dynamic systems theory of sexual fluidity; liminality; and narrative identity—to illuminate their transition, I argue that the event of coming out midlife should be viewed not merely as an atypical experience, but rather we should ask what such events can tell us about women’s sexuality in particular, and the sexual paradigm more generally. I argue that women who come out midlife challenge those dominant discourses of sexuality that would entail that women who come out midlife were either in denial of their “true” sexuality throughout their adult lives; or that they are not really lesbian now. The experiences of the women I interviewed demonstrate the inadequacy of the sexual paradigm as a framework within which to understand and research the complexity of human sexuality; they also challenge hegemonic understandings of sexuality as innate and immutable. In this thesis, I explore that challenge.

Piezoelectric energy harvesting using blood-flow-like excitation for implantable devices

The goal of this research is to produce a system for powering medical implants to increase the lifetime of the implanted devices and reduce the battery size. The system consists of a number of elements – the piezoelectric material for generating power, the device design, the circuit for rectification and energy storage. The piezoelectric material is analysed and a process for producing a repeatable high quality piezoelectric material is described. A full width half maximum (FWHM) of the rocking curve X-Ray diffraction (XRD) scan of between ~1.5° to ~1.7° for test wafers was achieved. This is state of the art for AlN on silicon and means devices with good piezoelectric constants can be fabricated. Finite element modelling FEM) was used to design the structures for energy harvesting. The models developed in this work were established to have an accuracy better than 5% in terms of the difference between measured and modelled results. Devices made from this material were analysed for power harvesting ability as well as the effect that they have on the flow of liquid which is an important consideration for implantable devices. The FEM results are compared to experimental results from laser Doppler vibrometry (LDV), magnetic shaker and perfusion machine tests. The rectifying circuitry for the energy harvester was also investigated. The final solution uses multiple devices to provide the power to augment the battery and so this was a key feature to be considered. Many circuits were examined and a solution based on a fully autonomous circuit was advanced. This circuit was analysed for use with multiple low power inputs similar to the results from previous investigations into the energy harvesting devices. Polymer materials were also studied for use as a substitute for the piezoelectric material as well as the substrate because silicon is more brittle.