Computer modelling applied to the Calvin Cycle

This thesis developes computer modelling techniques, and their use in the investigation of biochemical systems, principally the photosynthetic Calvin cycle. A set of metabolic modelling software tools, "Scampi", constructed as part of this project is presented. A unique feature of Scampi is that it allows the user to make a particular model the subject of arbitrary algorithms. This provides a much greater flexibility than is available with other metabolic modelling software, and is necessary for work on models of (or approaching) realistic complexity. A detailed model of the Calvin cycle is introduced. It differs from previously published models of this system in that all reactions are assigned explicit rate equations (no equilibrium assumptions are made), and it includes the degradation, as well as the synthesis, of starch. The model is later extended to include aspects of the thioredoxin system, and oxidative pentose phosphate pathway. Much of the observed behaviour is consistent with experimental observation. In particular, Metabolic Control Analysis of the model shows that control of assimilation flux is likely to be shared between two enzymes, rubisco and sedoheptulose bisphosphotase (SBPase), and can readily be transferred between them. This appears to offer an explanation of experimental evidence, obtained by genetic manipulation, that both of these enzymes can exert high control over assimilation. A further finding is that the output fluxes from the cycle (to starch and the cytosol), show markedly different patterns of control from assimilation, and from each other. An novel observation in behaviour of the Calvin cycle model is that, under certain circumstances, particularly at low light levels, the model has two steady-states and can be induced to switch between them. Although this exact behaviour has not been described experimentally, published results show charecteristics suggesting the potential is there in vivo. An explanation of all the observed behaviour is proposed, based upon the topology of the model. If this is correct then it may be concluded that the qualitative behaviour observed in the model is to be expected in vivo, although the quantitative detail may vary considerably

Perturbative expansions from Monte Carlo simulations at weak coupling: Wilson loops and the static-quark self-energy

Perturbative coefficients for Wilson loops and the static-quark self-energy are extracted from Monte Carlo simulations at weak coupling. The lattice volumes and couplings are chosen to ensure that the lattice momenta are all perturbative. Twisted boundary conditions are used to eliminate the effects of lattice zero modes and to suppress nonperturbative finite-volume effects due to Z(3) phases. Simulations of the Wilson gluon action are done with both periodic and twisted boundary conditions, and over a wide range of lattice volumes (from $3^4$ to $16^4$) and couplings (from $\beta \approx 9$ to $\beta \approx 60$). A high precision comparison is made between the simulation data and results from finite-volume lattice perturbation theory. The Monte Carlo results are shown to be in excellent agreement with perturbation theory through second order. New results for third-order coefficients for a number of Wilson loops and the static-quark self-energy are reported.Comment: 36 pages, 15 figures, REVTEX documen

Resonant acousto-optics in the terahertz range: TO-phonon polaritons driven by an ultrasonic wave

The resonant acousto-optic effect is studied both analytically and numerically in the terahertz range where the transverse-optical (TO) phonons play the role of a mediator which strongly couples the ultrasound and light fields. A propagating acoustic wave interacts with the TO phonons via anharmonic channels and opens band gaps in the TO-phonon polariton energy dispersion that results in pronounced Bragg scattering and reflection of the incoming light. The separation in frequency of different Bragg replicas, which is at the heart of acousto-optics, allows us to study the resonant acousto-optic effect in the most simple and efficient geometry of collinear propagation of electromagnetic and ultrasonic waves. The acoustically induced energy gaps, Bragg reflection spectra, and the spatial distribution of the electric field and polarization are calculated for CuCl parameters, in a wide range of frequencies and intensities of the pumping acoustic wave. Our results show drastic changes in terahertz spectra of semiconductor crystals that opens the way for efficient and accessible manipulation of their infrared properties, by tuning the parameters of the acoustic wave.Comment: 20 pages, 14 figure

Teaching genomics to life science undergraduates using cloud computing platforms with open datasets

The final year of a biochemistry degree is usually a time to experience research. However, laboratory-based research projects were not possible during COVID-19. Instead, we used open datasets to provide computational research projects in metagenomics to biochemistry undergraduates (80 students with limited computing experience). We aimed to give the students a chance to explore any dataset, rather than use a small number of artificial datasets (~60 published datasets were used). To achieve this, we utilized Google Colaboratory (Colab), a virtual computing environment. Colab was used as a framework to retrieve raw sequencing data (analyzed with QIIME2) and generate visualizations. Setting up the environment requires no prior experience; all students have the same drive structure and notebooks can be shared (for synchronous sessions). We also used the platform to combine multiple datasets, perform a meta-analysis, and allowed the students to analyze large datasets with 1000s of subjects and factors. Projects that required increased computational resources were integrated with Google Cloud Compute. In future, all research projects can include some aspects of reanalyzing public data, providing students with data science experience. Colab is also an excellent environment in which to develop data skills in multiple languages (e.g., Perl, Python, Julia)

From evidence to action. Understanding clinical practice guidelines.

Good guidelines will help us to take evidence into practice. In a survey among Dutch orthopedic surgeons, development and use of evidence-based guidelines was perceived as one of the best ways of moving from opinion-based to evidence-based orthopedic practice. The increasing number of guidelines means that knowing how to make a critical appraisal of guidelines is now a key part of every surgeon's life. This is particularly true because guidelines use varying systems to judge the quality of evidence and the strength of recommendations. In this manuscript we discuss what a guideline is, where we can find guidelines, how to evaluate the quality of guidelines, and finally provide an example on the different steps of guideline development. Thus, we show that good guidelines are a summary of the best available evidence and that they provide a graded recommendation to help surgeons in evidence-based practic

In vitro reassembly of the malolactic fermentation pathway of Leuconostoc oenos (Oenococcus oeni)

The mechanism of metabolic energy generation by malolactic fermentation was studied with artificial membrane vesicles of Leuconostoc oenos (Oenococcus oeni), (Note that although L. oenos was recently reclassified as O. oeni [L. M. T. Dicks, F. Dellaglio, and M. D. Collins, Int. J. Syst. Bacteriol. 45:395-397, 1995], the old designation was kept in the present work,) Purified malolactic enzyme was entrapped in artificial membrane vesicles prepared from L. oenos cells able to transport L-malate. We show that the in vitro reconstituted system, including an electrogenic L-malate carrier and the decarboxylating malolactic enzyme, generated a proton motive force that was able to drive intravesicular accumulation of leucine

Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP

The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli, LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Delta psi) and the transmembrane proton gradient (Delta pH), pointing to an electrogenic drug/proton antiport mechanism, Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a ''hydrophobic vacuum cleaner'' mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA

Boosting Biomass Quantity and Quality by Improved Mixotrophic Culture of the Diatom Phaeodactylum tricornutum

Diatoms are photoautotrophic unicellular algae and are among the most abundant, adaptable, and diverse marine phytoplankton. They are extremely interesting not only for their ecological role but also as potential feedstocks for sustainable biofuels and high-value commodities such as omega fatty acids, because of their capacity to accumulate lipids. However, the cultivation of microalgae on an industrial scale requires higher cell densities and lipid accumulation than those found in nature to make the process economically viable. One of the known ways to induce lipid accumulation in Phaeodactylum tricornutum is nitrogen deprivation, which comes at the expense of growth inhibition and lower cell density. Thus, alternative ways need to be explored to enhance the lipid production as well as biomass density to make them sustainable at industrial scale. In this study, we have used experimental and metabolic modeling approaches to optimize the media composition, in terms of elemental composition, organic and inorganic carbon sources, and light intensity, that boost both biomass quality and quantity of P. tricornutum. Eventually, the optimized conditions were scaled-up to 2 L photobioreactors, where a better system control (temperature, pH, light, aeration/mixing) allowed a further improvement of the biomass capacity of P. tricornutum to 12 g/L