Synthetic cells and tissues engineered from the bottom-up using non-living building blocks have many potential applications in medicine and biochemistry. Nonetheless, the applications of these synthetic cells and tissues remain limited by virtue of the challenging, costly, and uncontrollable methodologies available for their construction. Droplet microfluidic techniques, which are powerful analytical tools that can be used for the accurate and precise control over micro-sized droplets, offer potential solutions to these problems. The development of these droplet microfluidic platforms is a burgeoning and challenging field, with room for many impactful innovations. In the following dissertation, I first show the development of two different droplet microfluidic platform for the generation of two variations of synthetic cells: the first from polymeric-based building blocks and the second from biomimetic lipid-based building blocks. I then use the former of these platforms for the bottom-up generation of functional synthetic tissues (also known as prototissues). Using these techniques, I am able to elicit previously elusive structural and behavioral information. These methods contribute towards the creation of superior mimics of sophisticated life-like structures as well as a better understanding of how bespoke microfluidic platforms can be engineered to yield reliable and reproducible results. I have shown that microfluidic technologies are an invaluable tool for the creation and study of life-like systems and that these synthetic cells and tissues open up new avenues for research into multidisciplinary applications. / Graduate / 2023-06-07

With the increased application of large eddy simulations and hybrid Reynolds-averaged Navier-Stokes techniques, the generation of realistic turbulence at inflow boundaries is crucial for the accuracy of numerical results. In this dissertation research, two novel turbulence inflow generation methods are derived and validated. The first method, the Triple Hill's Vortex Synthetic Eddy Method, is a new type of synthetic eddy method, where the fundamental eddy is constructed through a superposition of three orthogonal Hill's vortices. The amplitudes of the three vortices that form the fundamental eddy are calculated from known Reynolds stress profiles through a transformation from the physical reference frame to the principal-axis reference frame. In this way, divergenceree anisotropic turbulent velocity fields are obtained that can reproduce a given Reynolds stress tensor. The model was tested on isotropic turbulence decay, turbulent channel flow, and a spatially developing turbulent mixing layer. The Triple Hill's Vortex Synthetic Eddy Method exhibited a quicker recovery of the desired turbulent flow conditions when compared with other current synthetic turbulence methods. The second method is the Control Forced Concurrent Precursor Method which combines an existing concurrent precursor method and a mean flow forcing method with a new extension of the controlled forcing method. Turbulent inflow boundary conditions are imposed through a region of body forces added as source terms to the momentum equations of the main simulation which transfer flow variables from the precursor simulation. Controlled forcing planes imposed in the precursor simulation, allow for specific Reynolds stress tensors and mean velocities to be imposed. A unique feature of the approach is that the proposed fluctuating flow controlled forcing method can be applied to multiple fluctuating velocity components and couple their calculation to amplify the existing fluctuations present in the precursor flow field so that prescribed anisotropic Reynolds stress tensors can be reproduced. The new method was tested on high and low Reynolds number turbulent boundary layer flows, where the proposed fluctuating flow controlled forcing method greatly accelerated the development of the turbulent boundary layers when compared with cases without controlled forcing and with only the original controlled forcing.

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京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23818号 / 医科博第139号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 藤渕 航, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

yes / This paper proposes a formal methodology to analyse bio-systems, in particular synthetic biology systems. An integrative analysis perspective combining different model checking approaches based on different property categories is provided. The methodology is applied to the synthetic pulse generator system and several verification experiments are carried out to demonstrate the use of our approach to formally analyse various aspects of synthetic biology systems. / EPSRC

Yes / Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C–Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures. / European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC consolidator grant GCGXC grant agreement no 614779) and ERAIB (Grant no. 031A338A) and H2020-MSCA-IF-2014 Grant no. 659399

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