Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Having caused the deaths of more than 10 million individuals since 2000 with
most of them occurring in Africa, malaria remains a serious disease that requires
undivided attention. To this end a detailed kinetic model of Plasmodium
falciparum glycolysis was constructed, validated and used to determine potential
drug targets for the development of novel, effective antimalarial therapies.
The kinetic model described the behaviour of the glycolytic enzymes with
a set of ordinary differential equations that was solved to obtain the steady
state fluxes and concentrations of internal metabolites. The model included a
glycerol branch represented in a single fitted equation. This present study set
out to detect, characterise, and incorporate into the model the enzymes that
constitute the glycerol branch of P. falciparum glycolysis.
The kinetic parameters of glycerol 3-phosphate dehydrogenase (G3PDH), the
first enzyme in the branch and catalyst of the dihydroxyacetone phosphosate
(DHAP) reducing reaction, was determined and added to the detailed kinetic model. The model was subsequently validated by comparing its prediction of
steady state fluxes with experimentally measured fluxes.
Once it was evident that the predictions of the unfitted model agreed with
experimentally measured fluxes, metabolic control analysis was performed on
this branched system to ascertain the distribution of control over the steady
state flux through the glycerol branch. The control G3PDH exercised over its
own flux was less than expected due to the enzyme’s sensitivity to changes in
NADH and thus the redox balance of the cell.
Attempts were made to detect the enzymes responsible for the conversion
of glycerol 3-phosphate (G3P) to glycerol. Very low levels of glycerol kinase
activity was observed. Although G3P-dependent release of inorganic phosphate
was detected results were inconclusive as to whether a non-specific phosphatase
also mediated the conversion.
Overall, the expansion of the model to include G3PDH did not affect the
steady state metabolite concentrations and flux adversely. / AFRIKAANSE OPSOMMING: Vanaf die jaar 2000 het malaria die dood van meer as 10 miljoen mense veroorsaak.
Die meeste sterftes het in Afrika voorgekom —’n aanduiding van hoe
ernstige siekte dit is en een wat onverdeelde aandag moet geniet. Om hierdie
rede is ’n gedetaileerde kinetiese model van glikoliese in Plasmodium falciparum
gebou, gevalideer en gebruik om potensiële dwelm teikens te identifiseer
vir die ontwikkeling van nuwe, meer effektiewe anti-malaria terapieë.
Die kinetiese model beskryf die gedrag van die glikolitiese ensieme in terme
van gewone differensiële vergelykings wat opgelos is om die bestendige toestand
fluksies en interne metaboliet konsentrasies te bepaal. Die model sluit
’n gliserol-tak in wat deur ’n enkele aangepaste vergelyking verteenwoordig
word. Hierdie studie het voorgeneem om die ensieme van die gliserol-tak van P.
falciparum glikoliese te identifiseer, karakteriseer en in die model te inkorporeer.
Ons het die kinetiese parameters van die eerste ensiem in die gliserol-tak,
gliserol 3-fosfaat dehidrogenase (G3PDH), die katalis van die dihidroksiasetoon
fosfaat(DHAP) reduserende reaksie, bepaal. Die kinetiese parameters is by
die gedetaileerde model gevoeg. Validering het plaasgevind deur die model se
voorspellings met eksperimenteel bepaalde waardes te vergelyk.
Toe dit duidelik geword het dat die voorspellings van die model met die
eksperimenteel bepaalde fluks ooreenstem, is metaboliese kontrole analiese op
die vertakte sisteem uitgevoer. Dit is gedoen om vas te stel hoe die bestendige
toestand fluks deur die gliserol-tak beheer word. G3PDH het nie volle beheer
oor sy eie fluks nie, in teenstelling met ons vergewagtinge.
Daar is gepoog om vas te stel watter ensieme verantwoordelik is vir die produksie
van gliserol vanuit gliserol 3-fosfaat (G3P). ’n Lae gliserolkinase aktiwiteit
is waargeneem. Alhoewel G3P afhanklike vrystelling van anorganise fosfaat
waargeneem is, is dit nie duidelik vanuit die resultate of die proses deur ’n
nie-spesifieke fosfatase uitgevoer word nie.
Die uitbreiding van die model om ’n G3PDH vergelyking in te sluit het nie die
bestendige toestand metaboliet konsentrasies en fluks negatief geaffekteer nie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/97159 |
| Date | 04 1900 |
| Creators | Adams, Waldo Wayne |
| Contributors | Snoep, J. L., Rautenbach, Marina, Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xiv, 104 pages |
| Rights | Stellenbosch University |
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