Development of an integrated metabolic analysis toolbox

Christensen, Carl David (2016-12)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Life is arguably the most complex of all natural phenomena, yet it arises from essentially dead molecular components. The goal of systems biology is to be able to understand how the properties and non-linear interactions of these components give rise to the functions and behaviour of living biological systems. This represents the so-called “mechanistic explanation” where no individual component, nor the complete system itself, is privileged. In this dissertation a Python based software package called PySCeSToolbox is presented that includes tools that implement previously published theoretical frameworks for investigating kinetic models of metabolic systems. These tools are RateChar, which performs generalised supply-demand analysis (GSDA); SymCa, which performs symbolic metabolic control analysis; and ThermoKin, which distinguishes between the kinetic and thermodynamic contributions towards enzyme-catalysed reaction rates. Each of the frameworks contained within the tools of PySCeSToolbox views metabolism from a different vantage point: generalised supply-demand analysis gives a broad overview of the behaviour, control, and regulation of metabolic systems by taking into account their functional organisation; symbolic control analysis dissects the control properties of metabolic systems in terms of the physical chains of interactions between enzymes and metabolic intermediates; and the thermodynamic/kinetic framework zooms in on the properties of the enzymes themselves to determine their regulatory roles. The strength of PySCeSToolbox lies in its integration of these viewpoints into a single analysis package in a way that promotes their complementary use in the search for a mechanistic explanation of modelled metabolic systems. Through the application of these tools in the investigation of two previously published metabolic models, new knowledge regarding their behaviour is uncovered and subsequently explained in terms of their component properties and interactions. In a model of aspartatederived amino-acid synthesis, a GSDA reveals that aspartate-semialdehyde regulates the reaction block that produces it via the reaction blocks that consume it, in spite of the relatively high sensitivity of its supply enzyme towards this intermediate. Subsequently, the regulatory contributions of each of the four aspartate-semialdehyde consuming blocks towards the producing block are quantified. In a model of pyruvate branch metabolism, application of GSDA shows that the flux through a NADH/NAD+ consuming reaction block decreases when the ratio of NADH to NAD+ increases. Rather than being a result of substrate inhibition, this phenomenon is shown to be the result of an interaction of the NADH/NAD+ intermediates with a reaction elsewhere in the pathway. Symbolic control analysis of the pyruvate branch model exposes a number of features that explain the unintuitive flux response described above. Firstly, only some control patterns are important for determining the flux control at any time. Secondly, different control patterns are dominant under different conditions, and dominance shifts as these conditions change. Finally, dissection of these chains of effects identifies the components of the system that are responsible for the flux control. Additional use of the thermodynamic/kinetic framework to focus on the enzymes that constitute the control patterns relates their values to the properties of individual enzyme-catalysed reactions (i.e. their elasticities). This framework is also used to explain the behaviour of the elasticity coefficient components of the unintuitive flux response, which are shown to be mostly mass-action controlled. Ultimately this two-pronged strategy provides a mechanistic explanation of the flux response, in which this high-level property is quantitatively linked to various low-level components. The design of PySCeSToolbox as a Python-based software library allows it to integrate with the existing scientific Python ecosystem, thus providing access to a variety of additional third-party software tools to aid in the analysis of metabolic systems. This design also encourages the use of a scripting approach to designing in silico modelling experiments, which in turn promotes reproducibility through the re-use of such scripts. Moreover, PySCeSToolbox provides computational access to theoretical analysis frameworks that would otherwise have been inaccessible to researchers, as these frameworks are not implemented elsewhere.

AFRIKAANS OPSOMMING: Die lewe is waarskynlik die mees komplekse van alle natuurverskynsels, tog ontstaan dit in wese vanuit dooie molekulêre komponente. ’n Doel van sisteembiologie is om te verstaan hoe eienskappe en nie-lineêre interaksies van hierdie komponente aanleiding gee tot funksies en gedrag van biologiese sisteme. Dit verteenwoordig die sogenaamde “meganistiese verklaring” waarin geen afsonderlike komponent, nog die volledige stelsel self, voorrang geniet. In hierdie proefskrif word ’n Python-gebaseerde sagteware-pakket voorgestel met die naam PySCeSToolbox, wat gepubliseerde teoretiese raamwerke vir die ondersoek van kinetiese modelle van metaboliese sisteme implementeer. Hierdie programmatuur-werktuie is RateChar, wat veralgemeende vraag-aanbod analise (VVAA) uitvoer; SymCa, wat simboliese metaboliese kontrole-analise uitvoer; en ThermoKin, wat onderskei tussen die kinetiese en termodinamiese bydraes tot ensiemgekataliseerde reaksietempo’s. Elkeen van die raamwerke soos vervat in die gereedskap van PySCeSToolbox beskou metabolisme vanuit ’n ander oogpunt: veralgemeende vraag-aanbod analise gee ’n breë oorsig van die gedrag, beheer, en regulering van metaboliese sisteme met inagneming van hul funksionele organisasie; simboliese kontrole-analise ontleed die beheer-eienskappe van metaboliese stelsels in terme van die fisiese kettings van interaksie tussen ensieme en metaboliese intermediate; en die termodinamiese /kinetiese raamwerk hou die eienskappe van die ensieme self onder ’n vergrootglas om hul regulerende rolle te bepaal. Die krag van PySCeSToolbox lê in die integrasie van hierdie gesigspunte in ’n enkele analise-pakket sodat hulle mekaar kan aanvul in die soektog na ’n meganistiese verklaring van gemodelleerde metaboliese sisteme. Die toepassing van hierdie sagteware-gereedskapskis in die ondersoek van twee gepubliseerde metaboliese modelle ontbloot nuwe kennis met betrekking tot hul gedrag en verduidelik dit daarna in terme van die eienskappe en interaksies van hul komponente. In ’n model van aspartaat-afgeleide aminosuursintese, wys ’n VVAA dat aspartaat-semialdehied sy produksiereaksieblok reguleer deur die interaksie met sy vraag-reaksieblokke, ten spyte van die relatief hoë sensitiwiteit van sy produksie-ensiem vir hierdie intermediaat. Hierna word die regulerende bydraes van elk van die vier aspartaat-semialdehied vraag-blokke tot die produksiereaksieblok gekwantifiseer. In ’n model van die metabolisme van die metaboliese vertakkings rondom pirovaat, toon ’n VVAA dat die fluksie deur ’n NADH/NAD+ vraag-reaksieblok daal wanneer die verhouding van NADH teenoor NAD+ toeneem. Hierdie verskynsel word ontbloot as ’n gevolg van ’n interaksie van die NADH/NAD+ intermediate met ’n reaksie elders in die pad, en is dus nie ’n gevolg van substraatinhibisie nie. Simboliese kontrole-analise van die pirovaat-vertakkings-model ontbloot ’n aantal eienskappe wat die nie-ooglopende fluksie-respons, soos bo beskryf, verklaar. Eerstens is slegs enkele beheer-patrone belangrik vir die bepaling van die fluksie-beheer op enige gegewe tydstip. Tweedens domineer verskillende beheer-patrone onder verskillende omstandighede, en die oorheersende patroon verskuif soos hierdie toestande verander. Laastens lei die ontrafeling van hierdie kettings van effekte tot die identifisering van daardie komponente van die sisteem wat verantwoordelik is vir die fluksie-beheer. Bykomende gebruik van die termodinamiese /kinetiese raamwerk om te fokus op daardie ensieme waaruit die beheer-patrone bestaan, herlei hul waardes na die eienskappe van individuele ensiemgekataliseerde reaksies (nl. hul elastisiteite). Hierdie raamwerk word ook gebruik om die waardes van die elastisiteitskoëffisiënt- komponente van die nie-ooglopende fluksie-response te verduidelik, en toon dat hulle hoofsaaklik deur massawerking beheer word. Uiteindelik bied hierdie tweeledige strategie ’n meganistiese verklaring van die fluksie-respons, waarin hierdie hoë-vlak eienskap kwantitatief gekoppel word aan verskeie lae-vlak komponente. Die ontwerp van PySCeSToolbox as ’n Python-gebaseerde biblioteek van programmatuurfunksies vergemaklik die integrasie met die bestaande wetenskaplike Python-ekosisteem, en verskaf dus toegang tot bykomende derde-party sagteware ter ondersteuning van die ontleding van metaboliese sisteme. Hierdie ontwerp moedig ook die gebruik van ’n skripbenadering tot die ontwerp van in silico modellerings-eksperimente aan, wat op sy beurt herhaalbaarheid bevorder deur die hergebruik van sodanige skripte. Daarbenewens bied PySCeSToolbox rekenaarmatige toegang tot teoretiese analise-raamwerke wat andersins vir navorsers ontoeganklik sou wees omdat dit nêrens anders geïmplementeer is nie.

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