Thesis supervisor: Lawrence L. Fleckenstein. Includes bibliographic references (p. 122-137).

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Artemisinin, produced by the plant Artemisia annua, is a sesquiterpene anti-malarial therapeutic. Due to the medicinal relevance of this plant product, there is significant interest in understanding how the biosynthetic pathway is regulated at several key steps. The objective of this study is to examine several factors known to influence artemisinin yields to determine if those effects are occurring at the transcriptional level of the biosynthetic pathway. Artemisinin content has been shown to increase as the plant shifts from vegetative growth to reproductive, flowering growth. To test whether there is a corresponding increase in terpenoid gene expression during the shift to reproductive growth, levels of mRNA of terpenoid genes were measured during flowering budding and full flowering and compared to those measured during vegetative growth. Results indicate that in response to the photoperiod signal to shift to reproductive growth, early cytosolic pathway genes were highly upregulated, while there was no change in early plastidic pathway genes. Late pathway genes specific to artemisinin synthesis were upregulated >6-fold. Furthermore, glucose has also been shown to stimulate artemisinin production compared to sucrose. To test whether glucose is acting as signal to increase terpenoid gene expression, levels of mRNA of terpenoid genes were measured in glucose- and fructose-treated seedlings and compared to those in sucrose-treated seedlings. Results indicate that in response to treatment with glucose, compared with sucrose, early pathway genes in both compartments were initially upregulated. Transcript levels subsequently decreased to levels similar to those in sucrose-treated seedlings. ADS was upregulated by glucose, compared with sucrose, reaching a peak at day 7. Finally, coordinate control of sterol and sesquiterpene synthesis at a critical branch-point in the terpenoid biosynthetic pathway has been demonstrated. To test whether amorpha-4,11-diene synthase (ADS) and squalene sythase (SQS) are coordinately regulated, levels of mRNA of those two genes were measured and compared in both experimental conditions. Results indicate that under the conditions used in this study, ADS and SQS did not show coordinate regulation. This study was the first to demonstrate that: 1. terpenoid genes relating to artemisinin biosynthesis are regulated at the level of transcript accumulation as the plant shifts from vegetative to reproductive growth; 2. glucose is acting as a signal in artemisinin biosynthesis by upregulating transcript levels for several terpenoid genes.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: secondary metabolites; hairy roots; tetraploid; artemisinin. Includes bibliographical references (p. 83-106).

Malaria är en tropisk sjukdom som orsakas av encelliga organismer, protozoer från Plasmodium släktet. Varje år drabbas ungefär en halv miljard människor av malaria och cirka en miljon av dessa dör. Okomplicerad malaria är en mild form av malaria som enligt WHO rekommendationer ska behandlas med artemisinin baserad kombinationsterapi (ACT). Artemisinin produceras naturligt i låg mängd från växten Artemisia annua. Trots att medicinen har visat sig effektiv mot malaria med färre biverkningar är den höga kostnaden en nackdel. Forskning pågår för att hitta nya syntetiska vägar för framställning av artemisinin i växten genom att studera terpenmetabolism och vilka aktiva enzymer det finns som har en avgörande roll i utbytet av artemisinin i växten. Syftet med denna studie var att med hjälp av genteknik och molekylärbiologiska metoder producera och identifiera två rekombinanta enzymer, seskviterpensyntaser från A. annua. Experimentet inleddes med att transformera klonade T-DNA (AaTS-1 och AaTS-2) som kodar för seskviterpensyntaser från A. annua med hjälp av Agrobacterium tumefaciens vartefter transienta transkriptionen av generna som finns i en binär vektor initierades i blad från växten Nicotiana benthamiana genom infiltration. Totalt RNA extraherades från växten och översattes till cDNA för att sedan studera förhållandet av transient uttryck i bladen med qPCR. Enzymerna extraherades från bladen och inkuberades med farnesyldifosfat övernatt och produkten identifierades följande dag med gaskromatografi-masspektrofotometri (GC-MS). Resultatet blev att inget genuttryck av AaTS-1 och AaTS-2 kunde detekteras i bladen. Resultat från GC-MS visade att ingen proteinprodukt genererades. De negativa resultaten berodde främst på brist av resultat som verifierar att plasmiderna var konstruerade med selektionsgenerna, men även på grund av en icke effektiv transformation, orsakad av bakteriecellklumpar som förhindrade infiltreringsmedium att nå inre delarna av bladen.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Artemisia annua, artemisinin, sugar analog, sugar, signal Includes bibliographical references (leaves 59-64 ).

The role of sugars as regulatory signals has mainly focused on their effects on plant growth, development, gene expression, and metabolism. Little, however, is known about their role in controlling secondary metabolism. Previous work in our lab showed that sugars affect the production of the sesquiterpene antimalarial drug, artemisinin, in hairy roots of Artemisia annua. In this study, sugars alone or in combination with their analogues were used to investigate if sugars control artemisinin production in Artemisia annua seedlings. Compared to sucrose, a 200% increase in artemisinin by glucose was observed. When the glucose analog, 3-O-methylglucose, which is not phosphorylated effectively by hexokinase, was added with glucose, artemisinin production was dramatically decreased but hexokinase activity was significantly increased compared to glucose. In contrast, neither mannose, which can be phosphorylated by hexokinase, nor mannitol, which can not be transported into cells had any significant effect on artemisinin yield. When different ratios of fructose to glucose were added to seedlings, artemisinin yield was directly proportional to glucose concentration. Although addition of sucrose with glucose gave inconclusive results, sucrose analogues decreased artemisinin production compared to sucrose. These results suggested that both monosaccharide and disaccharide sugars may be acting as signal molecules thereby affecting the downstream production of artemisinin. Taken together, these experiments showed that sugars clearly affect terpenoid production, but that the mechanism of their effects appears to be complex.

"Artemisinin is a potent antimalarial drug produced in the plant Artemisia annua. Earlier reports suggested that the roots play a key role in artemisinin production; however, it was not clear if other factors actually affected production instead of roots. Here the role of roots and two phytohormones, NAA and BAP, were studied to determine what role each plays in artemisinin production in the plant. Rooted Artemisia annua shoots produced significantly more artemisinin, arteannuin B, and deoxyartemisinin, the end products in the pathway, than unrooted shoots. Although roots do not seem to affect the levels of precursors, artemisinic acid and dihydroartemisinic acid, or regulate the transcription of the genes in the pathway, rooted plants developed larger trichome sacs suggesting that the accumulation of end products is linked to the expansion of the trichome sac. Unrooted shoots are grown in shooting medium containing higher amount of MS salts, vitamins, sucrose and two potent phytohormones, NAA and BAP. Rooted shoots grown in rooting medium containing either one or both of these hormones showed that NAA increased production of arteannuin B in the young leaves and artemisinin in the mature leaves; in mature leaves, however, arteannuin B was inhibited by NAA. BAP induced production of both the precursors and the end products, except for artemisinin, in the young and/or mature leaves. When rooted shoots with their roots removed were grown in rooting medium containing either one of these hormones, artemisinin was significantly less in cultures grown with BAP while there were no differences in metabolite levels in cultures grown with NAA. Although the importance of roots on the artemisinin biosynthetic pathway cannot be concluded, these results help improve our understanding of artemisinin biosynthesis as may prove useful for improving artemisinin production in field-grown crops."

Artemisinin is a potent antimalarial sesquiterpene lactone produced and stored in the glandular trichomes (GLTs) of Artemisia annua. Although they produce no artemisinin, nor any of the precursor compounds, A. annua roots appear to have a regulatory effect on production of the terpene in leaves. However, more information is needed to define the role of the roots in artemisinin production in the plant. Grafting among three cultivars was used to measure phenotypic responses: SAM, and #15 cultivars both have GLTs, but produce artemisinin at 1.49% and 0.57% DW, respectively; GLS cultivar produces neither GLTs nor artemisinin. Compared to ungrafted plants, all self-grafts, e.g. SAM/SAM (scion/rootstock), increased scion artemisinin probably from grafting stress. SAM/#15 grafts yielded less artemisinin than SAM/SAM, but more than either #15/#15 or ungrafted #15 and SAM suggesting rootstock inhibition of the scion. SAM/SAM also had more artemisinin than #15/SAM, which was also greater than either #15/#15 or ungrafted #15 and SAM. The #15/SAM graft also produced more artemisinin than SAM/#15, and with the other grafting results suggested that SAM roots were stimulating artemisinin production in the #15 scion. There was no appearance of either GLTs or artemisinin when GLS scions were grafted to SAM indicating that GLTs had to be present to receive putative signals from SAM rootstocks. Furthermore, artemisinic acid and arteannuin B were only present in SAM scions and not scions of #15 suggesting a block in one of the side pathways of artemisinin biosynthesis. Other artemisinic metabolites, total flavonoids, and GLTs numbers were also measured. The various phenotypes were analyzed several months after grafting indicating a persistent change and suggesting a possible epigenetic alteration of the scion. This study will provide fundamental information regarding the role that roots play in the production of artemisinin in the shoots of A. annua.

Student Number : 0416598H - MSc research report - School of Public Health - Faculty of Health Sciences / Background Evidence shows that the efficacy of intravenous quinine, which is the mainstay for treating severe malaria in children, is decreasing. Artemisinin derivatives are the potential replacement for quinine. Their efficacy compared to quinine in treating severe malaria in children is not well known. Objective To assess the efficacy of parenteral artemisinin derivatives versus parenteral quinine in treating severe malaria in children. Search strategy The Cochrane Central Register of Controlled Trials (The Cochrane Library Issue 4, 2005), MEDLINE (1966 to October 2005), EMBASE (1980 to October 2005), and LILACS (1982 to October 2005) were searched. Malaria researchers and a pharmaceutical company were contacted. In addition, conference proceedings were also searched. Selection criteria Randomised controlled studies comparing parenteral artemisinin derivatives with parenteral quinine in treating severe malaria in children. All trials had to report mortality as an outcome. Data collection After data were extracted, two individuals independently assessed the trial quality. In addition, information on adverse effects from the studies was also collected. Main results Eleven trials were selected (1455 subjects), nine of them from Africa and the rest from Asia. Allocation concealment was adequate in seven trials (1238 subjects). Overall there was no difference in mortality between artemisinin derivatives and quinine (Risk Ratio= 0.89, 95% confidence interval 0.71 to 1.1). There was no difference in mortality between adequately concealed and inadequately concealed /unconcealed trials (Risk Ratio = 0.93, 95% confidence interval 0.74 to 1.16 and Risk Ratio=0.66, 95% confidence interval 0.36 to 1.22). In Parasite Clearance Time (PCT), though there was no statistical difference between the two groups there was a tendency towards favouring the artemisinin derivatives (weighted mean difference among studies which reported PCT as mean was -4.76 with 95% confidence interval -9.68 to 0.17 and all three studies which reported PCT as median showed that artemisinin derivatives cleared parasites faster than quinine, each had p<0.001). However; when only trials with adequate concealment were considered this potential advantage disappeared. In exploring heterogeneity for PCT, it was shown that study settings (Asia versus Africa) might have been a cause for heterogeneity. The artemisinin derivatives resolved coma faster than quinine (weighted mean difference=-5.32, 95%CI: -8.06 to -2.59), but when only trials with adequate concealment were considered this difference disappeared. Other secondary outcomes i.e. Fever clearance time, Incidence of neurological sequelae, and 28th day cure rate showed no significant difference between artemisinin derivatives and quinine. There was no enough data to make meaningful comparison of adverse effects between the two groups. Conclusions The available evidence suggests that parenteral artemisinin derivatives are as efficacious as quinine in preventing mortality from severe malaria in children.