Development of Nanodiamond and Raman Spectroscopy Sensing Methods towards the Measurement of Mitochondrial Membrane Potential in Single Cells

Mitochondrial membrane potential ($\Delta\Psi_{\textrm{m}}$) in cells is a critical biological parameter that is related to diseases such as cancer through biological processes including metabolism, oxidative stress and apoptosis. Studying $\Delta\Psi_{\textrm{m}}$ in normal and diseased states is limited by significant challenges associated with the current state-of-the-art methods for measurement, particularly fluorescent dyes, which are often toxic and photobleaching. The aim of this work was to develop electrical and chemical sensing methods to enable sensitive detection of changes in $\Delta\Psi_{\textrm{m}}$ in single cells. Fluorescent Nitrogen Vacancy Centres (NVCs) in nanodiamond were identified as potential sensors of electric field that could be applied in living cells to directly measure $\Delta\Psi_{\textrm{m}}$. Furthermore, Raman spectroscopy was identified as a label-free chemical sensing technique that could be used to reveal the location and chemical composition of the mitochondria in cells. To achieve the aim of detecting changes in $\Delta\Psi_{\textrm{m}}$ using these two methods, complementary NVC and Raman measurements in live cells were needed. Initially, a specialised microscope was designed, built and validated to enable dual measurement of both NVC fluorescence and Raman spectroscopic signals simultaneously. Next, protocols and analysis techniques were developed for live cell Raman microscopy using a commercial reference instrument. An investigation of the impact of nanodiamonds as nanoparticles for biological sensing within live breast cancer cells, including surface modification by oxidation, was then conducted. By developing both techniques together, this project leverages the complementary advantages of long time course measurement, common laser excitation, the observation of nanodiamonds via Raman signal, with live single cell interrogation. The thesis concludes with an outlook on the future development needed to utilise NVCs and Raman spectroscopy in the measurement of cellular $\Delta\Psi_{\textrm{m}}$. Achieving this goal in future will provide new biological understanding of $\Delta\Psi_{\textrm{m}}$ in normal and disease states, allowing us to follow over time changes in $\Delta\Psi_{\textrm{m}}$ in response to biological processes such as apoptosis in single cancer cells.Winton programme for sustainability Cancer Research UK Oliver Gatty studentshi

Raman micro-spectroscopy for accurate identification of primary human bronchial epithelial cells.

Live cell Raman micro-spectroscopy is emerging as a promising bioanalytical technique for label-free discrimination of a range of different cell types (e.g. cancer cells and fibroblasts) and behaviors (e.g. apoptosis). The aim of this study was to determine whether confocal Raman micro-spectroscopy shows sufficient sensitivity and specificity for identification of primary human bronchial epithelial cells (HBECs) to be used for live cell biological studies in vitro. We first compared cell preparation substrates and media, considering their influence on lung cell proliferation and Raman spectra, as well as methods for data acquisition, using different wavelengths (488 nm, 785 nm) and scan protocols (line, area). Evaluating these parameters using human lung cancer (A549) and fibroblast (MRC5) cell lines confirmed that line-scan data acquisition at 785 nm using complete cell media on a quartz substrate gave optimal performance. We then applied our protocol to acquisition of data from primary human bronchial epithelial cells (HBEC) derived from three independent sources, revealing an average sensitivity for different cell types of 96.3% and specificity of 95.2%. These results suggest that Raman micro-spectroscopy is suitable for delineating primary HBEC cell cultures, which in future could be used for identifying different lung cell types within co-cultures and studying the process of early carcinogenesis in lung cell culture

Intracellular connections between basal bodies promote the coordinated behavior of motile cilia

Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell\u27s cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliat

A communal catalogue reveals Earth's multiscale microbial diversity

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.Peer reviewe

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity

Professional standards research: equal opportunities in smaller firms

Assesses the capability of the Chartered Institute of Personnel and Development (CIPD) qualifications to make a difference to equal opportunities (EO) in small and medium enterprises (SME). Development of an interactive software-based business tool that focused on measuring gender-based EO practice by Manchester Metropolitan University Business School in England; Difference between CIPD professionals and employees with nominated responsibility for EO

Analysing gender-based diversity in SMEs

Recent research into gender equality in small- to medium-sized organisations (SMEs) in the UK demonstrates low engagement with a formal model of procedural equality. This paper explores whether a diversity approach might be more helpful in evaluating SME practice in the area of gender equality. Drawing on interview data with 80 owner/managers of SMEs, findings reveal an implicit sympathy with diversity practices and principles, underpinned by business rationales for action that could offer positive developments for women. Nevertheless, the data also show that the same rationale and the same practices serve to perpetuate discrimination, utilise stereotypes and continue the segregation of women into low paid, low skilled work. On the basis of these findings, we conclude that while a diversity management approach propels a perspective of women as a valuable resource within SMEs, relying on it in preference to regulated formal procedural equality would be unwise

Gender-based equal opportunities policy and practice in small firms: the impact of HR professionals

This article assesses the impact of HR qualified professionals on the adoption of gender- based equal opportunities policies and practices in small businesses. Earlier research has shown a low level of take-up of equal opportunities initiatives in small firms. This article explores whether the presence of an HR professional in small to medium-sized enterprises is associated with a greater level of take-up of (a) gender-based equality policies and (b) gender-based equality practices than in small to medium-sized enterprises that do not have such expertise. In doing so, the authors draw on empirical data from a recent European Social Fund sponsored study of equal opportunities policy and practice in small to medium-sized enterprises in the UK. The main finding is that the presence of an HR professional is associated with a greater level of take-up of formal equality policies, but not with the implementation of equality practices. The authors explore possible reasons for this and consider the implications for the HR profession and the promotion of equality of opportunity in the small business sector

Gender-based equal opportunities in small and medium sized employers: establishing policy and practice

This report profiles human resource management practices in small and medium sized enterprises (SMEs) with a particular focus on those relevant to gender-based equal opportunities. The data collection and analysis provides a summary of the approach of the participating SMEs toward issues of gender equality. The project was supported by the European Social Fund (ESF) and by a number of commercial and industrial partners. The SME sector is of considerable importance to the UK economy and yet very little research has been done on the approach to the management of gender-based equality. Research on the sector’s general human resource practices paints a picture of an unsophisticated, non-strategic and informal approach to the management of people. The objective of this report is to examine whether or not the same is true of their approach to gender equality. It further examines the data for differences between small (10-49 employees) and medium sized (50-249 employees) organisations. In order to achieve access to SMEs and provide each participating organisation with information relevant to both management and organisation development, the project team designed a piece of original benchmarking software that generated a number of quantitative assessments of organisation practice. Each participating organisation was given a report that assessed their performance against a) minimum equal opportunity legal requirements, b) Equal Opportunity Commission best equality practice indicators and c) their competitors in the same size band. A total of 80 eligible assessments were carried out in the North West of the UK between July and December 2003. Assessments were conducted on the organisation’s premises with those individuals who held primary responsibility for human resource decisions. Organisations were drawn from a variety of sectors. The assessment process produced both qualitative (interview transcripts) and quantitative (in SPSS format) data. Overall, the evidence in the report shows that informal, potentially discriminatory people management procedures are still widespread, particularly in the practice of equal opportunity. For example more than a fifth of SME respondents admit to asking questions about applicants’ childcare commitments during recruitment. Forty-four per cent admit to asking different questions of men and women, and nearly a third state that they apply different selection criteria to men and women candidates. Only 35% report that interviewers were trained in EO, and the same number report that they made appointments “guided by instinct”. Thirty-six percent of respondents say that they wouldn’t employ a woman who was already pregnant. Only 18% of respondents report that they take active steps to redress gender imbalances in their workforce. The smaller organisations often demonstrate poor practice that is differentiated from medium organisations to an extent that is statically significant. These findings will be of concern for those seeking to advance the equal opportunities agenda in the SME sector, particularly given that the data is derived from self-reports which may, if anything, paint an overly positive picture. The study concludes that SMEs demonstrate a preference for flexibility over formal policy and an ignorance of “good practice” and legislative requirements. It suggests that despite the persuasive business (not to mention ethical) case to be made for the introduction of gender-based equal opportunity practices in small businesses, the formalisation of procedures - that is key to the promotion of equality - appears to be undermined by an SMEs desire for informality, flexibility and autonomy. They equality agenda also rubs awkwardly against the notion of the ‘family’ firm and falls victim to the more general resistance to employment regulation