Recent advances in genome editing and metabolic engineering enabled a precise construction of de novo biosynthesis pathways for high-value natural products. One important design decision to make for the engineering of heterologous biosynthesis systems is concerned with which foreign metabolic genes to introduce into a given host organism. Although this decision must be made based on multifaceted factors, a major one is the suitability of pathways for the endogenous metabolism of a host organism, in part because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. To address this point, we developed an open-access web server called MRE (metabolic route explorer) that systematically searches for promising heterologous pathways by considering competing endogenous reactions in a given host organism. MRE utilizes reaction Gibbs free energy information. However, 25% of the reactions do not have accurate estimations or cannot be estimated. To address this issue, we developed a method called FC (fingerprint contribution) to provide a more accurate and complete estimation of the reaction free energy. To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. For a given pair of starting and desired compounds in a given chassis organism, MRE ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/. Accurate and wide-ranging prediction of thermodynamic parameters for biochemical reactions can facilitate deeper insights into the workings and the design of metabolic systems. Here, we introduce a machine learning method, referred to as fingerprint contribution (FC), with chemical fingerprint-based features for the prediction of the Gibbs free energy of biochemical reactions. From a large pool of 2D fingerprint-based features, this method systematically selects a small number of relevant ones and uses them to construct a regularized linear model. FC is freely available for download at http://sfb.kaust.edu.sa/Pages/Software.aspx.

It is a dream of Systems-Biology to efficiently simulate an entire cell on a computer. The potential medical and biological applications of such a tool are immense, and so are the challenges to accomplish it. At the level of a cell, the number of reacting molecules is so low that stochastic effects can be crucial in deciding the system-level behaviour of the cell. Despite the recent development of many new and hybrid stochastic approaches, exact stochastic simulation algorithms are still needed, and are widely employed in most current biochemical simulation packages. Unfortunately, the performance of these algorithms scales badly with the number of reactions. It is shown that this is especially the case for hubs and scale-free networks. This is worrying because hubs are an important component of biochemical systems, and it is widely suspected that biochemical networks are scale-free. It is shown that the scalability issue in these algorithms is due to the high interdependency between reactions. A general method for arbitrarily reducing this interdependency is introduced, and it is shown how it can be used for many classes of simulation processes. This is applied to one of the fastest algorithms currently, the Next Reaction Method. The resulting algorithm, the Reactant-Margin Method, is tested on a wide range of hub sizes and shown to be asymptotically faster than the current best algorithms. Hybrid versions of the Reactant-Margin Method and the Next Reaction Method are also compared on a real biological model - the Lambda-Phage virus, and the new algorithm is again shown to perform better. The problems inherent in the hybridization are also shown to be more exactly and efficiently handled in the Reactant-Margin framework than in the Next-Reaction Method framework. Finally, a software tool called GeNIV is introduced. This GUI-based biochemical modelling and simulation tool is an embodiment of a mechanistic-representation philosophy. It is implements the Reactant Margin and Next Reaction hybrid algorithms, and has a simple representation system for gene-state occupancy and their subsequent biochemical reactions. It is also novel in that it translates the graphical model into Javacode which is compiled and executed for simulation.

A deep and analytical understanding of the human metabolism grabbed attention of scientists from biology, medicine and pharmacy. Mathematical models of metabolic pathways offer several advances for this deep and analytical understanding due to their incompensable potential in predicting metabolic processes and anticipating appropriate interventions when required. This thesis concerns mathematical modeling analysis and simulation of metabolic pathways. These pathways include intracellular and extracellular compounds such as enzymes, metabolites, nucleotides and cofactors. Experimental data and available knowledge on metabolic pathways are used in constituting a mathematical model. The models are either in the form of nonlinear ordinary differential equations (ode&#039 / s) or differential algebraic equations (dae&#039 / s). These equations are composed of kinetic parameters such as kinetic rate constants, initial rates and concentrations of metabolites. The non-linear nature of enzymatic reactions and large number of parameters cause trouble in efficient simulation of those reactions. Metabolic engineering tries to simplify these equations by reducing the number of parameters. In this work, enzymatic system which includes Creatine Kinase, Hexokinase and Glucose 6-Phosphate Dehydrogenase (CK-HK-G6PDH) is modeled in the form of dae&#039 / s, solved numerically and the system parameters are estimated. The numerical results are compared with the results from an existing work in literature. We demonstrated that, our solution method based on direct solution of the CK-HK-G6PDH system significantly from simplified solutions. We also showed that genetic algorithm(GA) for parameter estimation, provides much clear results to the experimental values of the metabolite, especially with NADPH. Keywords: metabolic engineering, kinetic modelling, biochemical reactions, enzymatic reactions, differential algebraic equations, parameter estimation, genetic algorithm.

Orientador: Marines Nobre dos Santos Uchoa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-13T08:04:08Z (GMT). No. of bitstreams: 1 Steiner-Oliveira_Carolina_D.pdf: 11802864 bytes, checksum: 338ee3278fb157bb1cb37edb2e6d1a5d (MD5) Previous issue date: 2009 / Resumo: Os efeitos causados pelas modificações promovidas pela irradiação com laser de CO2 podem inibir a desmineralização dos tecidos dentários e podem ser potencializados quando associados ao fluoreto. Apesar de amplo uso do fluoreto e da redução da prevalência de cárie, essa doença ainda acomete grupos de alto risco. Por outro lado, tem sido observado um aumento da prevalência da erosão dentária. Os objetivos dessa tese, composta por 4 manuscritos, foram: (1) descrever as características do laser de CO2 e seus mecanismos de ação na inibição da desmineralização do esmalte; (2) desenvolver um modelo microbiológico, in vitro, de produção de lesão de cárie em dentina e testar duas hipóteses: (a) de que não há diferença na produção de cárie artificial em dentina utilizando um modelo microbiológico com regimes de 3 e 6 imersões ao dia em sacarose, avaliados por contagem bacteriana da dentina (UFC), análise microrradiográfica (AM) e análise de polissacarídeo insolúvel (API); (b) de que não há diferença no pH do biofilme antes e após sua imersão em sacarose; (3) avaliar, in vitro, a efetividade do laser de CO2 (? = 10,6 µm) pulsado, associado ou não ao fluoreto, na redução da desmineralização da dentina radicular usando um modelo microbiológico, avaliado por AM; (4) avaliar, in vitro, o efeito do mesmo laser, associado ou não ao fluoreto, na redução da desmineralização do esmalte e da dentina submetidos a um desafio erosivo, pela mensuração da perda de superfície e análise da concentração de cálcio, fósforo e fluoreto das soluções desmineralizadoras. Os dados foram analisados quanto à normalidade e testes apropriados foram realizados com nível de significância de 5%. No estudo 1, os efeitos do laser no esmalte, seu mecanismo de ação na redução da desmineralização, combinados ou não ao fluoreto, foram discutidos. No estudo 2, o pH do biofilme diminuiu imediatamente após a imersão em sacarose, mas aumentou novamente 5 min depois. Lesões em dentina foram produzidas com sucesso e a adição de sacarose mostrou as maiores perdas minerais, no entanto não diferiu entre os dois regimes de sacarose. A UFC não mostrou nenhuma diferença e a API dos tratamentos foram maiores que a do grupo controle. No estudo 3, os espécimes radiculares foram tratados ou não com laser de CO2 e com ou sem fluoreto antes ou após a irradiação com laser. O modelo microbiológico utilizado foi efetivo em produzir lesões dentinárias e as terapias combinadas mostraram as lesões dentinárias mais rasas. No estudo 4, espécimes de esmalte e dentina foram tratados com fluoreto, laser e fluoreto/laser e submetidos a um desafio erosivo. Os resultados de desgaste indicaram que o tratamento combinado interferiu com as perdas minerais do esmalte e da dentina, mesmo sem mostrar efeito sinérgico. Houve uma tendência de retenção de fluoreto no esmalte pelo tratamento combinado e também de liberação de menores quantidades de cálcio, fósforo e fluoreto para as soluções desmineralizadoras. Em conclusão, o mecanismo de ação do laser de CO2 na inibição da desmineralização do esmalte ainda não está completamente esclarecido e seu efeito pode ser aumentado quando associado ao fluoreto. O modelo microbiológico foi efetivo em produzir lesões de cárie dentinária. A irradiação da dentina radicular com laser inibiu a desmineralização dessa superfície apenas quando associado com o fluoreto; no entanto, não foi observado efeito sinérgico. O tratamento isolado com laser não foi capaz de prevenir a perda de superfície do esmalte e da dentina devido à erosão. Sua combinação com fluoreto mostrou alguma proteção, mas principalmente devido ao efeito do fluoreto. Não foi observada interação sinérgica significativa ou proteção duradoura com a terapia de laser. / Abstract: The effects caused by the modifications promoted by the CO2 laser irradiation can inhibit the dental tissues demineralization and may be enhanced when associated with fluoride. Despite the widespread use of the fluoride and the reduction of the caries prevalence, this disease still occurs in the high risk groups. On the other hand, an increase of the dental erosion prevalence was observed. This thesis, comprised by 4 manuscripts, aimed: (1) to describe the CO2 laser characteristics and its action mechanisms in the enamel demineralization inhibition; (2) to develop an in vitro microbial model to produce dentin caries lesions and test two hypotheses - (a) that there is no difference in the artificial caries production in dentin using a microbial model with 3 and 6 sucrose bath immersions, as assessed by bacterial counts on the dentin (CFU), microradiographic analysis (TMR) and extracellular polysaccharide analysis (EPS); (b) that there is no difference in the biofilm pH before and after each sucrose bath; (3) to assess, in vitro, the effectiveness of a pulsed CO2 laser (? = 10.6 µm) associated or not with fluoride, in reducing the root demineralization using a microbial model, as assessed by TMR; (4) to assess, in vitro, the effect of the same laser, associated or not with fluoride, on the prevention of the enamel and dentin erosions by means of surface loss measurement and analysis of the calcium, phosphorus and fluoride concentrations in the demineralizing solutions. The data were checked for normality and appropriated tests were performed with a significance level of 5%. In study 1, the laser effects on the enamel and its action mechanisms in the demineralization reduction, combined or not with fluoride, were discussed. In study 2, the biofilm pH decreased immediately after the sucrose bath but increased again after 5 min. Dentin lesions were successfully produced, and the sucrose addition showed the highest mineral losses, even though there was no difference between the sucrose regimens. The CFU did not show any difference and the EPS from the treatment groups were higher than for the control. In study 3, root specimens were treated with/without CO2 laser and with/without fluoride prior or after the laser irradiation. The microbial model utilized was effective in developing dentin lesions and the combined therapies showed the shallowest dentin lesions. In study 4, specimens of enamel and root dentin were treated with fluoride, laser and fluoride/laser and submitted to an erosive challenge. The wear results indicated that the combined treatment interfered with the enamel or dentin surface losses, although no synergistic effect was observed. There was a trend for the combined treatment to retain more fluoride in enamel and release lower amounts of calcium and phosphorus into the demineralizing solutions. In conclusion, the CO2 mechanism action on the enamel demineralization reduction is still not elucidated and its effects can be increased when associated with fluoride. The microbial model was effective in producing dentin caries lesions. However, it did not reproduce the remineralizing phase of the caries process. Irradiation of the root dentin with laser inhibited the root surface demineralization only when associated with fluoride; however, no synergic effect was observed. The laser treatment alone was not able to prevent enamel or dentin surface losses due to erosion. Its combination with fluoride showed some protection, but mostly due to the fluoride effect. No significant synergistic interaction or lasting protection could be observed for the laser therapy. / Doutorado / Odontopediatria / Doutor em Odontologia

The subject of this thesis is the generation of spatial temporal structures in living cells. Specifically, we studied the Min-system in the bacterium Escherichia coli. It consists of the MinC, the MinD, and the MinE proteins, which play an important role in the correct selection of the cell division site. The Min-proteins oscillate between the two cell poles and thereby prevent division at these locations. In this way, E. coli divides at the center, producing two daughter cells of equal size, providing them with the complete genetic patrimony. Our goal is to perform a quantitative study, both theoretical and experimental, in order to reveal the mechanism underlying the Min-oscillations. Experimentally, we characterize theMin-system, measuring the temporal period of the oscillations as a function of the cell length, the time-averaged protein distributions, and the in vivo Min-protein mobility by means of different fluorescence microscopy techniques. Theoretically, we discuss a deterministic description based on the exchange of Minproteins between the cytoplasm and the cytoplasmic membrane and on the aggregation current induced by the interaction between membrane-bound proteins. Oscillatory solutions appear via a dynamic instability of the homogenous protein distributions. Moreover, we perform stochastic simulations based on a microscopic description, whereby the probability for each event is calculated according to the corresponding probability in the master equation. Starting from this microscopic description, we derive Langevin equations for the fluctuating protein densities which correspond to the deterministic equations in the limit of vanishing noise. Stochastic simulations justify this deterministic model, showing that oscillations are resistant to the perturbations induced by the stochastic reactions and diffusion. Predictions and assumptions of our theoretical model are compatible with our experimental findings. Altogether, these results enable us to propose further experiments in order to quantitatively compare the different models proposed so far and to test our model with even higher precision. They also point to the necessity of performing such an analysis through single cell measurements.

The subject of this thesis is the generation of spatial temporal structures in living cells. Specifically, we studied the Min-system in the bacterium Escherichia coli. It consists of the MinC, the MinD, and the MinE proteins, which play an important role in the correct selection of the cell division site. The Min-proteins oscillate between the two cell poles and thereby prevent division at these locations. In this way, E. coli divides at the center, producing two daughter cells of equal size, providing them with the complete genetic patrimony. Our goal is to perform a quantitative study, both theoretical and experimental, in order to reveal the mechanism underlying the Min-oscillations. Experimentally, we characterize theMin-system, measuring the temporal period of the oscillations as a function of the cell length, the time-averaged protein distributions, and the in vivo Min-protein mobility by means of different fluorescence microscopy techniques. Theoretically, we discuss a deterministic description based on the exchange of Minproteins between the cytoplasm and the cytoplasmic membrane and on the aggregation current induced by the interaction between membrane-bound proteins. Oscillatory solutions appear via a dynamic instability of the homogenous protein distributions. Moreover, we perform stochastic simulations based on a microscopic description, whereby the probability for each event is calculated according to the corresponding probability in the master equation. Starting from this microscopic description, we derive Langevin equations for the fluctuating protein densities which correspond to the deterministic equations in the limit of vanishing noise. Stochastic simulations justify this deterministic model, showing that oscillations are resistant to the perturbations induced by the stochastic reactions and diffusion. Predictions and assumptions of our theoretical model are compatible with our experimental findings. Altogether, these results enable us to propose further experiments in order to quantitatively compare the different models proposed so far and to test our model with even higher precision. They also point to the necessity of performing such an analysis through single cell measurements.