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Ensuring Access to Safe and Nutritious Food for All Through the Transformation of Food Systems
A direct-laser-written heart-on-a-chip platform for generation and stimulation of engineered heart tissues
In this dissertation, we first develop a versatile microfluidic heart-on-a-chip model to generate 3D-engineered human cardiac microtissues in highly-controlled microenvironments. The platform, which is enabled by direct laser writing (DLW), has tailor-made attachment sites for cardiac microtissues and comes with integrated strain actuators and force sensors. Application of external pressure waves to the platform results in controllable time-dependent forces on the microtissues. Conversely, oscillatory forces generated by the microtissues are transduced into measurable electrical outputs. After characterization of the responsivity of the transducers, we demonstrate the capabilities of this platform by studying the response of cardiac microtissues to prescribed mechanical loading and pacing.
Next, we tune the geometry and mechanical properties of the platform to enable parametric studies on engineered heart tissues. We explore two geometries: a rectangular seeding well with two attachment sites, and a stadium-like seeding well with six attachment sites. The attachment sites are placed symmetrically in the longitudinal direction. The former geometry promotes uniaxial contraction of the tissues; the latter additionally induces diagonal
fiber alignment. We systematically increase the length for both configurations and observe a positive correlation between fiber alignment at the center of the microtissues and tissue length. However, progressive thinning and “necking” is also observed, leading to the failure of longer tissues over time. We use the DLW technique to improve the platform, softening
the mechanical environment and optimizing the attachment sites for generation of stable microtissues at each length and geometry. Furthermore, electrical pacing is incorporated into the platform to evaluate the functional dynamics of stable microtissues over the entire range of physiological heart rates. Here, we typically observe a decrease in active force and contraction duration as a function of frequency.
Lastly, we use a more traditional ?TUG platform to demonstrate the effects of subthreshold electrical pacing on the rhythm of the spontaneously contracting cardiac microtissues. Here, we observe periodic M:N patterns, in which there are ? cycles of stimulation for every ? tissue contractions. Using electric field amplitude, pacing frequency, and homeostatic beating frequencies of the tissues, we provide an empirical map for predicting the emergence of these rhythms
Application of advanced fluorescence microscopy and spectroscopy in live-cell imaging
Since its inception, fluorescence microscopy has been a key source of discoveries in cell biology. Advancements in fluorophores, labeling techniques and instrumentation have made fluorescence microscopy a versatile quantitative tool for studying dynamic processes and interactions both in vitro and in live-cells. In this thesis, I apply quantitative fluorescence microscopy techniques in live-cell environments to investigate several biological processes. To study Gag processing in HIV-1 particles, fluorescence lifetime imaging microscopy and single particle tracking are combined to follow nascent HIV-1 virus particles during assembly and release on the plasma membrane of living cells. Proteolytic release of eCFP embedded in the Gag lattice of immature HIV-1 virus particles results in a characteristic increase in its fluorescence lifetime. Gag processing and rearrangement can be detected in individual virus particles using this approach. In another project, a robust method for quantifying Förster resonance energy transfer in live-cells is developed to allow direct comparison of live-cell FRET experiments between laboratories. Finally, I apply image fluctuation spectroscopy to study protein behavior in a variety of cellular environments. Image cross-correlation spectroscopy is used to study the oligomerization of CXCR4, a G-protein coupled receptor on the plasma membrane. With raster image correlation spectroscopy, I measure the diffusion of histones in the nucleoplasm and heterochromatin domains of the nuclei of early mouse embryos. The lower diffusion coefficient of histones in the heterochromatin domain supports the conclusion that heterochromatin forms a liquid phase-separated domain. The wide range of topics covered in this thesis demonstrate that fluorescence microscopy is more than just an imaging tool but also a powerful instrument for the quantification and elucidation of dynamic cellular processes
TOWARDS AN UNDERSTANDING OF EFFORTFUL FUNDRAISING EXPERIENCES: USING INTERPRETATIVE PHENOMENOLOGICAL ANALYSIS IN FUNDRAISING RESEARCH
Physical-activity oriented community fundraising has experienced an exponential growth in popularity over the past 15 years. The aim of this study was to explore the value of effortful fundraising experiences, from the point of view of participants, and explore the impact that these experiences have on people’s lives. This study used an IPA approach to interview 23 individuals, recognising the role of participants as proxy (nonprofessional) fundraisers for charitable organisations, and the unique organisation donor dynamic that this creates. It also bought together relevant psychological theory related to physical activity fundraising experiences (through a narrative literature review) and used primary interview data to substantiate these. Effortful fundraising experiences are examined in detail to understand their significance to participants, and how such experiences influence their connection with a charity or cause. This was done with an idiographic focus at first, before examining convergences and divergences across the sample. This study found that effortful fundraising experiences can have a profound positive impact upon community fundraisers in both the short and the long term. Additionally, it found that these experiences can be opportunities for charitable organisations to create lasting meaningful relationships with participants, and foster mutually beneficial lifetime relationships with them. Further research is needed to test specific psychological theory in this context, including self-esteem theory, self determination theory, and the martyrdom effect (among others)
Development of in-vitro in-silico technologies for modelling and analysis of haematological malignancies
Worldwide, haematological malignancies are responsible for roughly 6% of all the cancer-related deaths. Leukaemias are one of the most severe types of cancer, as only about 40% of the patients have an overall survival of 10 years or more. Myelodysplastic Syndrome (MDS), a pre-leukaemic condition, is a blood disorder characterized by the presence of dysplastic, irregular, immature cells, or blasts, in the peripheral blood (PB) and in the bone marrow (BM), as well as multi-lineage cytopenias.
We have created a detailed, lineage-specific, high-fidelity in-silico erythroid model that incorporates known biological stimuli (cytokines and hormones) and a competing diseased haematopoietic population, correctly capturing crucial biological checkpoints (EPO-dependent CFU-E differentiation) and replicating the in-vivo erythroid differentiation dynamics. In parallel, we have also proposed a long-term, cytokine-free 3D cell culture system for primary MDS cells, which was firstly optimized using easily-accessible healthy controls. This system enabled long-term (24-day) maintenance in culture with high (>75%) cell viability, promoting spontaneous expansion of erythroid phenotypes (CD71+/CD235a+) without the addition of any exogenous cytokines. Lastly, we have proposed a novel in-vitro in-silico framework using GC-MS metabolomics for the metabolic profiling of BM and PB plasma, aiming not only to discretize between haematological conditions but also to sub-classify MDS patients, potentially based on candidate biomarkers. Unsupervised multivariate statistical analysis showed clear intra- and inter-disease separation of samples of 5 distinct haematological malignancies, demonstrating the potential of this approach for disease characterization.
The work herein presented paves the way for the development of in-vitro in-silico technologies to better, characterize, diagnose, model and target haematological malignancies such as MDS and AML.Open Acces
Understanding novel EGFP-Ubx protein-based film formation
Protein-based materials are currently the subject of intense research interest since they have an extended range of potential applications, such as im-proved bio-membrane biocompatibility for implanted medical devices and the creation of platform materials for novel biosensors. Monomers from Ultrabithorax (Ubx) transcription factor are known to spontaneously self-assemble at an air-water interface to form a monolayer, which has then been used as a basis for forming biopolymeric ˝bers. Here we used the Lang-muir trough technique, Brewster angle microscopy (BAM), ellipsometry and neutron re˛ectometry (NR) to investigate the in˛uences of di˙erent exper-imental conditions on EGFP-Ubx monolayer formation and the impact on biopolymeric ˝ber structure. We varied protein concentration, bu˙er prop-erties and waiting times prior to forming biopolymeric ˝bers. Interestingly, we found 3 phases of material formation which brought us to a new protocol for forming ˝bers that reduced protein concentration by 5-fold and wait-ing times by 100-fold. Moreover, an in-house developed MATLAB code was used to analyze SEM images and obtain quantitative structural information about the biopolymeric ˝bers that were correlated directly to the surface ˝lm characteristics measured in the LB trough. These new insights into ˝ber formation and structure enhance the usefulness of the Ubx-based biopolymer for biomedical applications
Investigating illicit drug use in adolescent students in England
Abstract The Smoking Drinking Drug Use Survey of adolescents aged 11 to 15 years living in England shows that lifetime drug use by adolescents aged 11 to 15 years has increased (15% to 24%) from 2014 to 2018 (NHS Digital, 2017, 2021b). This upward trend is despite the implementation of drug policies focused on reducing supply, possession, and manufacture of illicit drugs. Based on the premise that drug use is a socially learnt behaviour, the main objective of this research is to investigate whether social learning factors (imitation, parental reinforcement, peer association and attitudes to drug use) mediate drug use in adolescents aged 11 to 15 years living in England. The second objective is to identify which social learning factors mediate drug use by ages, region, and gender. Using the Social Structure Social Learning (SSSL) theory as a framework for the research, this study contributes to the literature by identifying a) the strongest social learning behaviour for each age, gender and region in England and b) the mechanism (mediation) by which social learning affects drug use. This research employs rich data on drug use drawn from the Smoking Drinking Drug Use Survey 2016, a cross-sectional survey of adolescents aged 11-15 years across England (as of October 2021 the data for the most recent survey 2018 was not available for analysis). Mediation analysis was used to evaluate which social learning factors mediate the association between age, gender, region and drug use. The results showed that there were differences in learning behaviours that were specific to age, gender and region. For example, the most significant social learning behaviour for drug use among boys was “imitation of friends”, whilst for females, it was “peer association” among females (i.e. having a perception that peers are using drugs). In addition, having “positive attitudes to glue” (i.e. “it is ok to try glue”) was the strongest learning behaviour for drug use among younger individuals (i.e. at ages 11 to 13). Furthermore, whilst in Northern England, the strongest learning behaviour was having “positive attitudes to cannabis”, in London peer association was found to be the strongest learning pathway to drug use. Family disapproval of drug use (“persuade me not to take drugs”) was found to be a protective factor against drug use for all ages except for age 11 and 12 years and those living in the East Midlands and London. In these cases, more authoritarian parenting –– strong parental disapproval (“stop me from taking drugs”) was found to be a protective factor. This research offers two main contributions to the literature. First, it shows empirical linkages between constructs built using SSSL theory that have not been previously explored within a population of young adolescents in England. Second, it identifies the effects and degree to which social learning affects the relationship between drug use and social structure. Overall, this research also contributes to an improved theoretical rationale for existing SSSL associations; that is, social learning can behave as a mediator or a moderator depending on the context. The evidence produced by this thesis could also have potentially relevant policy implications. More specifically, the differences in the social learning behaviours may suggest the need to implement more targeted prevention policies aimed by age, gender and regional groups of young adolescents
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