Engineering the transition from non-living to living matter

Re-creating and understanding the origin of life represents one of the major challenges facing the scientific community. We will never know exactly how life started on planet Earth, however, we can reconstruct the most likely chemical pathways that could have contributed to the formation of the first living systems. Traditionally, prebiotic chemistry has investigated the formation of modern life’s precursors and their self-organisation under very specific conditions thought to be ‘plausible’. So far, this approach has failed to produce a living system from the bottom-up. In the work presented herein, two different approaches are employed to explore the transition from inanimate to living matter. The development of microfluidic technology during the last decades has changed the way traditional chemical and biological experiments are performed. Microfluidics allows the handling of low volumes of reagents with very precise control. The use of micro-droplets generated within microfluidic devices is of particular interest to the field of Origins of Life and Artificial Life. Whilst many efforts have been made aiming to construct cell-like compartments from modern biological constituents, these are usually very difficult to handle. However, microdroplets can be easily generated and manipulated at kHz rates, making it suitable for high-throughput experimentation and analysis of compartmentalised chemical reactions. Therefore, we decided to develop a microfluidic device capable of manipulating microdroplets in such a way that they could be efficiently mixed, split and sorted within iterative cycles. Since no microfluidic technology had been developed before in the Cronin Group, the first chapter of this thesis describes the soft lithographic methods and techniques developed to fabricate microfluidic devices. Also, special attention is placed on the generation of water-in-oil microdroplets, and the subsequent modules required for the manipulation of the droplets such as: droplet fusers, splitters, sorters and single/multi-layer micromechanical valves. Whilst the first part of this thesis describes the development of a microfluidic platform to assist chemical evolution, finding a compatible set of chemical building blocks capable of reacting to form complex molecules with endowed replicating or catalytic activity was challenging. Abstract 10 Hence, the second part of this thesis focuses on potential chemistry that will ultimately possess the properties mentioned above. A special focus is placed on the formation of peptide bonds from unactivated amino acids, despite being one of the greatest challenges in prebiotic chemistry. As opposed to classic prebiotic experiments, in which a specific set of conditions is studied to fit a particular hypothesis, we took a different approach: we explored the effects of several parameters at once on a model polymerisation reaction, without constraints on hypotheses on the nature of optimum conditions or plausibility. This was facilitated by development of a new high-throughput automated platform, allowing the exploration of a much larger number of parameters. This led us to discover that peptide bond formation is less challenging than previously imagined. Having established the right set of conditions under which peptide bond formation was enhanced, we then explored the co-oligomerisation between different amino acids, aiming for the formation of heteropeptides with different structure or function. Finally, we studied the effect of various environmental conditions (rate of evaporation, presence of salts or minerals) in the final product distribution of our oligomeric products.

Development of novel therapeutics and in vitro models for spinal cord injury

Spinal cord injury (SCI) is a devastating condition, which results from trauma to the cord, resulting in a primary injury response which leads to a secondary injury cascade, causing damage to both glial and neuronal cells. Following trauma, the central nervous system (CNS) fails to regenerate due to a plethora of both intrinsic and extrinsic factors. Unfortunately, these events lead to loss of both motor and sensory function and lifelong disability and care for sufferers of SCI. There have been tremendous advancements made in our understanding of the mechanisms behind axonal regeneration and remyelination of the damaged cord. These have provided many promising therapeutic targets. However, very few have made it to clinical application, which could potentially be due to inadequate understanding of compound mechanism of action and reliance on poor SCI models. This thesis describes the use of an established neural cell co-culture model of SCI as a medium throughput screen for compounds with potential therapeutic properties. A number of compounds were screened which resulted in a family of compounds, modified heparins, being taken forward for more intense investigation. Modified heparins (mHeps) are made up of the core heparin disaccharide unit with variable sulphation groups on the iduronic acid and glucosamine residues; 2-O-sulphate (C2), 6-O-sulphate (C6) and N-sulphate (N). 2-O-sulphated (mHep6) and N-sulphated (mHep7) heparin isomers were shown to promote both neurite outgrowth and myelination in the SCI model. It was found that both mHeps decreased oligodendrocyte precursor cell (OPC) proliferation and increased oligodendrocyte (OL) number adjacent to the lesion. However, there is a difference in the direct effects on the OL from each of the mHeps; mHep6 increased myelin internode length and mHep7 increased the overall cell size. It was further elucidated that these isoforms interact with and mediate both Wnt and FGF signalling. In OPC monoculture experiments FGF2 treated OPCs displayed increased proliferation but this effect was removed when co-treated with the mHeps. Therefore, suggesting that the mHeps interact with the ligand and inhibit FGF2 signalling. Additionally, it was shown that both mHeps could be partially mediating their effects through the Wnt pathway. mHep effects on both myelination and neurite outgrowth were removed when co-treated with a Wnt signalling inhibitor, suggesting cell signalling mediation by ligand immobilisation and signalling activation as a mechanistic action for the mHeps. However, the initial methods employed in this thesis were not sufficient to provide a more detailed study into the effects the mHeps have on neurite outgrowth. This led to the design and development of a novel microfluidic device (MFD), which provides a platform to study of axonal injury. This novel device is a three chamber device with two chambers converging onto a central open access chamber. This design allows axons from two points of origin to enter a chamber which can be subjected to injury, thus providing a platform in which targeted axonal injury and the regenerative capacity of a compound study can be performed. In conclusion, this thesis contributes to and advances the study of SCI in two ways; 1) identification and investigation of a novel set of compounds with potential therapeutic potential i.e. desulphated modified heparins. These compounds have multiple therapeutic properties and could revolutionise both the understanding of the basic pathological mechanisms underlying SCI but also be a powered therapeutic option. 2) Development of a novel microfluidic device to study in greater detail axonal biology, specifically, targeted axonal injury and treatment, providing a more representative model of SCI than standard in vitro models. Therefore, the MFD could lead to advancements and the identification of factors and compounds relating to axonal regeneration.

Feasibility study to inform the design of a UK multi-centre randomised controlled trial of prophylactic antibiotics for the prevention of recurrent cellulitis of the leg

Background: This paper describes the results of a feasibility study for a randomised controlled trial (RCT). Methods: Twenty-nine members of the UK Dermatology Clinical Trials Network (UK DCTN) expressed an interest in recruiting for this study. Of these, 17 obtained full ethics and Research & Development (R&D) approval, and 15 successfully recruited patients into the study. A total of 70 participants with a diagnosis of cellulitis of the leg were enrolled over a 5-month period. These participants were largely recruited from medical admissions wards, although some were identified from dermatology, orthopaedic, geriatric and general surgery wards. Data were collected on patient demographics, clinical features and willingness to take part in a future RCT. Results: Despite being a relatively common condition, cellulitis patients were difficult to locate through our network of UK DCTN clinicians. This was largely because patients were rarely seen by dermatologists, and admissions were not co-ordinated centrally. In addition, the impact of the proposed exclusion criteria was high; only 26 (37%) of those enrolled in the study fulfilled all of the inclusion criteria for the subsequent RCT, and were willing to be randomised to treatment. Of the 70 participants identified during the study as having cellulitis of the leg (as confirmed by a dermatologist), only 59 (84%) had all 3 of the defining features of: i) erythema, ii) oedema, and iii) warmth with acute pain/tenderness upon examination. Twenty-two (32%) patients experienced a previous episode of cellulitis within the last 3 years. The median time to recurrence (estimated as the time since the most recent previous attack) was 205 days (95% CI 102 to 308). Service users were generally supportive of the trial, although several expressed concerns about taking antibiotics for lengthy periods, and felt that multiple morbidity/old age would limit entry into a 3-year study. Conclusion: This pilot study has been crucial in highlighting some key issues for the conduct of a future RCT. As a result of these findings, changes have been made to i) the planned recruitment strategy, ii) the proposed inclusion criteria and ii) the definition of cellulitis for use in the future trial.

Internationalization of IT company: Case study of a Finnish MOOC platform

The interest to small and media size enterprises’ (SMEs) internationalization process is increasing with a growth of SMEs’ contribution to GDP. Internet gives an opportunity to provide variety of services online and reach market niche worldwide. The overlapping of SMEs’ internationalization and online services is the main issue of the research. The most SMEs internationalize according to intuitive decisions of CEO of the company and lose limited resources to worthless attempts. The purpose of this research is to define effective approaches to online service internationalization and selection of the first international market. The research represents single holistic case study of local massive open online courses (MOOCs) platform going global. It considers internationalization costs and internationalization theories applicable to online services. The research includes preliminary screening of the markets and in-depth analysis based on macro parameters of the market and specific characteristics of the customers and expert evaluation of the results. The specific issues as GILT (Globalization, Internationalization, Localization and Translation) approach and Internet-enabled internationalization are considered. The research results include recommendations on international market selection methodology for online services and for effective internationalization strategy development.

Development of Imaging Mass Spectrometry Analysis of Lipids in Biological and Clinically Relevant Applications

La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.

Integrating Architecture for a Live-Work Lifestyle

Urban centers all around the world are striving to re-orient themselves to promoting ideals of human engagement, flexibility, openness and synergy, that thoughtful architecture can provide. From a time when solitude in one’s own backyard was desirable, today’s outlook seeks more, to cater to the needs of diverse individuals and that of collaborators. This thesis is an investigation of the role of architecture in realizing how these ideals might be achieved, using Mixed Use Developments as the platform of space to test these designs ideas on. The author also investigates, identifies, and re-imagines how the idea of live-work excites and attracts users and occupants towards investing themselves in Mixed Used Developments (MUD’s), in urban cities. On the premise that MUDs historically began with an intention of urban revitalization, lying in the core of this spatial model, is the opportunity to investigate what makes mixing of uses an asset, especially in the eyes to today’s generation. Within the framework of reference to the current generation, i.e. the millennial population and alike, who have a lifestyle core that is urban-centric, the excitement for this topic is in the vision of MUD’s that will spatially cater to a variety in lifestyles, demographics, and functions, enabling its users to experience a vibrant 24/7 destination. Where cities are always in flux, the thesis will look to investigate the idea of opportunistic space, in a new MUD, that can also be perceived as an adaptive reuse of itself. The sustainability factor lies in the foresight of the transformative and responsive character of the different uses in the MUD at large, which provides the possibility to cater to a changing demand of building use over time. Delving into the architectural response, the thesis in the process explores, conflicts, tensions, and excitements, and the nature of relationships between different spatial layers of permanence vs. transformative, public vs. private, commercial vs. residential, in such an MUD. At a larger scale, investigations elude into the formal meaning and implications of the proposed type of MUD’s and the larger landscapes in which they are situated, with attempts to blur the fine line between architecture and urbanism. A unique character of MUD’s is the power it has to draw in people at the ground level and lead them into exciting spatial experiences. While the thesis stemmed from a purely objective and theoretical standpoint, the author believes that it is only when context is played into the design thinking process, that true architecture may start to flourish. The unique The significance of this thesis lies on the premise that the author believes that this re-imagined MUD has immense opportunity to amplify human engagement with designed space, and in the belief that it will better enable fostering sustainable communities and in the process, enhance people’s lives.

Collaborative Web-Based Simulation Platform for Construction Project Planning

Part 13: Virtual Reality and Simulation