Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT:
In Africa alone, Plasmodium, the causative agent of malaria is estimated to kill
a child, under the age of five every thirty seconds140. The ability of the parasite
to rapidly attain resistance, has resulted in immunity of the parasite to all,
except one group of frontline drugs. The need to develop novel drugs, vaccines
and prevention strategies that are accessible and affordable for third world
countries is of the utmost importance to prevent needless human suffering and
death.
The glycolytic pathway is an attractive drug target since it is the principal
source of ATP for the parasite. Many of the glycolytic enzymes have been
studied and proposed as drug targets, but the importance of these enzymes
for the function of the pathway as a whole has not been considered. It is
known, from the frameworks of metabolic control analysis, that control of the
flux and metabolite concentration can be divided among the individual steps. Differential control analysis of Plasmodium and erythrocyte glycolysis may
reveal potential drug targets. These analyses require a detailed kinetic model
of Plasmodium glycolysis, and the feasibility of constructing and validating
such a model was the aim of this study.
In this work we determined the feasibility of constructing and validating a
detailed kinetic model for the Plasmodium falciparum glycolytic pathway.
Whether the construction and validation of this kinetic model was feasible
or not was decided on the basis of the ability to: i) culture and isolate
sufficient asexual parasites for enzymatic and steady state assays , ii) obtain
kinetic parameters such as Km and Vmax for each glycolytic enzyme, either
from literature or experimentally, iii) measure glycolytic fluxes, iv) determine
glycolytic intermediate concentrations, v) construct a kinetic model from the
kinetic parameters and vi) validate it with steady state glycolytic fluxes and
metabolite concentrations
Each of the above criteria were successfully addressed. In summary, the
kinetic parameters and glycolytic fluxes that were measured experimentally,
were used to construct and partially validate a detailed kinetic model,
respectively. Further validation of the model by means of steady state
metabolite concentrations was shown to be possible with the development of
a suitable protocol to measure the glycolytic intermediate concentrations.
The model presented in this work may play an important role in drug
target identification and improving the current understanding of host-parasite
interactions and glycolytic regulation. / AFRIKAANSE OPSOMMING:
Plasmodium, die parasiet wat malaria veroorsaak, is in Afrika alleen elke
dertig sekondes verantwoordelik vir die afsterwe van ’n kind jonger as vyf
jaar. Die parasiet se vermoë om vinnig weerstand op te bou het daartoe
gelei dat Plasmodium weerstandbiedend is teen byna alle nuwe teen-malaria
middels, behalwe vir ’n enkele toonaangewende groep. Die ontwikkeling van
nuwe malaria teen-middels is van uiterste belang om lyding te voorkom.
’n Goeie teiken vir teen-malaria middels is die glikolitiese padweg omdat die
metaboliese padweg essensieël is vir die produksie van ATP, die energiebron
van die parasiet. Desondanks die feit dat meeste van die glikolitiese ensieme al
goed bestudeer en as teiken voorgestel is, is dit steeds onduidelik hoe hierdie
ensieme saam funksioneer om die metaboliese weg, as geheel, tot stand te bring.
Metaboliese kontrole analise het aangetoon dat die glikolitiese beheer verdeel is tussen die onderskeie glikolitiese ensieme, m.a.w. geen enkele ensiematiese
stap het volledige beheer oor die fluksie van die glikolitiese padweg nie. Die
afsonderlike analise en vergelyking van Plasmodium - en rooibloedselglikolise
met behulp van differensiële metaboliese kontrole analise sal moontlik gebruik
kan word om gasheervriendelike teikens vir nuwe middels aan te toon. So
’n analise benodig ’n omvattende kinetiese model van Plasmodium glikolise.
Derhalwe was die doel van hierdie studie om vas te stel hoe uitvoerbaar dit is
om ’n kinetiese model van Plasmodium glikolise te konstrueer en te valideer.
Die uitvoerbaarheid van die konstruksie en validering van die kinetiese
model was geasseseer op grond van die vermoë om: i) parasietkulture te
kweek en genoegsame parasiete, wat in die aseksuele fase is, te isoleer
sodat ensiembepalings en bestendige toestand-bepalings gedoen kan word, ii)
kinetiese parameters soos Km - en Vmax-waardes vir elke glikolitiese ensiem,
hetsy vanuit literatuur of eksperimentele werk, te verkry, iii) glikolitiese fluksie
te meet, iv) glikolitiese intermediaatkonsentrasies te bepaal, v) ’n kinetiese
model van die bepaalde kinetiese parameters op te stel en vi) die model te
valideer met glikolitiese flukswaardes en metaboliet- konsentrasies wat in die
bestendige toestand verkry is.
Elk van die bogenoemde kriteria was met sukses in hierdie studie aangespreek.
Ter opsomming, die eksperimenteel bepaalde kinetiese parameters
en glikolietiese flukswaardes was gebruik om onderskeidelik ’n gedetaileerde
kinetiese model te konstrueer en gedeeltelik te valideer. Daar was getoon
dat verdere validering van die model deur middel van bestendige toestand
metabolietkonsentrasies moontlik is met die ontwikkeling van ’n geskikte
protokol om glikolitiese intermediaatkonsentrasies te meet. Die model, soos
opgestel in hierdie studie, kan moontlik ’n belangrike rol speel om teikens vir
nuwe malaria teen-middels te identifiseer en om gasheer-parasiet interaksies en
glikolitiese regulering beter te verstaan.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/2298 |
| Date | 12 1900 |
| Creators | Penkler, Gerald Patrick |
| Contributors | Snoep, J. L., Rautenbach, M., University of Stellenbosch. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | Unknown |
| Type | Thesis |
| Rights | University of Stellenbosch |
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