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Terpenmetabolism i Artemisia annua : rekombinant produktion och karaktärisering av seskviterpensyntaser.Al-Masaraa, Nahil January 2015 (has links)
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.
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Produção e detecção de artemisinina em plantas de Artemisia annua L. submetidas a estresses abioticosMarchese, Jose Abramo 31 May 1999 (has links)
Orientadores: Vera Lucia Garcia Rehder, Ana Maria M. A. Lagoa / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-25T08:56:29Z (GMT). No. of bitstreams: 1
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Previous issue date: 1999 / Resumo: Artemisia annua L., uma planta nativa da China e aclimatada no Brasil, é uma fonte abundante de artemisinina, uma lactona sesquiterpênica que apresenta comprovada eficácia no controle das cepas resistentes do gênero Plasmodium, parasita causador da malária. O objetivo principal deste trabalho foi verificar a influência da deficiência hídrica e da temperatura no acúmulo de artemisinina em A.annua. Plantas submetidas à deficiência hídrica moderada (YW -1, 39 MPa) e à deficiência hídrica drástica (YW -3,44 MPa) seguida de reidratação, acumularam significativamente mais artemisinina em relação às plantas controle irrigadas. Todavia, o acúmulo de artemisinina não resultou em aumento do rendimento da molécula por planta. Com relação à temperatura, as plantas submetidas à amplitude térmica 18/28 °c acumularam significativamente mais artemisinina que as plantas submetidas à amplitude 11/20°C. Outro objetivo deste trabalho foi verificar as vantagens e inconvenientes da técnica de cromatografia em camada delgada com detecção fotodensitométrica CCD/DT, em relação à técnica de cromatografia líquida de alta eficiência com detecção no ultravioleta - CLAE/UV, para análises quantitativas de artemisinina. Os resultados encontrados indicam que a técnica CCD/DT não apresenta o grau de seletividade e sensibilidade necessários para considerá-la um método quantitativo alternativo a CLAE/UV. apresentaram-se CLAE/UV. Os de obtidos artemisinina teores superestimados relação em aos com CCD/DT obtidos com / Abstract: Artemisia annua L., native of China and adapted to the Brazilian climate, is a rich source of artemisinin, a sesquiterpene lactone proven to be effective against the malaria agent Plasmodium. The scope of this work was to study the influence of water deficiency and temperature on the plants artemisinin content. Moderate water deficiency (YW -1,39 MPa) and drastic water deficiency (YW -3,44 MPa) followed by rehydration induced a significant accumulation of artemisinin of the dehydrated plants when compared to these with controlled irrigation. Although the accumulation of artemisinin did not result in a greater yield of artemisinin content by the plant. When temperature was taken into account, more artemisinin accumulated at the 18/28 °c temperature range when compared to the lI/20°C temperature range. Another goal of this work was to compare the methods thin layer chromatography (TLC) using photodensitometric detection with high performance liquid chromatography (HPLC) using ultraviolet detection for the analytical quantification of artemisinin contents. The results show that the TLC method does not have the same selectivity and sensibility necessary to be a useful alternative method to HPLC. Under the experimental conditions the TLC method over-estimated the artemisinin contents compared to those determined by HPLC / Mestrado / Biologia Vegetal / Mestre em Ciências Biológicas
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Roots and hormones: synergistic control of artemisinin production in Artemisia annua L. shootsNguyen, Khanh Van T 06 December 2011 (has links)
"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."
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Proteome analysis of glandular trichome from Artemisia annua L.January 2011 (has links)
Wu, Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 56-70). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.Ill / ABSTRACT --- p.V / TABLE OF CONTENTS --- p.IX / LIST OF ABBREVIATIONS --- p.XII / Chapter CHAPTER 1. --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- THE DISEASE OF MALARIA --- p.1 / Chapter 1.1.1 --- Pathogenesis --- p.2 / Chapter 1.1.2 --- The treatment of malaria --- p.4 / Chapter 1.2 --- THE PLANT OF ARTEMISIA ANNUA L --- p.5 / Chapter 1.2.1. --- Horticulture --- p.5 / Chapter 1.2.2. --- Historical Importance --- p.6 / Chapter 1.3 --- ARTEMISININ --- p.7 / Chapter 1.3.1 --- The content and distribution of artemisinin --- p.7 / Chapter 1.3.2 --- The biosynthesis of artemisnin --- p.8 / Chapter 1.4 --- TRICHOMES --- p.13 / Chapter 1.4.1 --- Structure and function of trichomes --- p.13 / Chapter 1.4.2 --- Trichome investigation in A. annua --- p.14 / Chapter 1.5 --- PROTEOMICS --- p.17 / Chapter 1.5.1 --- The basic principle of proteomics --- p.17 / Chapter 1.5.2 --- Two-dimensional gel electrophoresis --- p.18 / Chapter 1.5.3 --- Mass Spectrometry --- p.19 / Chapter 1.5.4 --- Gel-free proteomics --- p.20 / Chapter 1.6 --- OBJECTIVES --- p.21 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- CHEMICALS --- p.23 / Chapter 2.2 --- PLANT MATERIALS --- p.23 / Chapter 2.3 --- ISOLATION OF GLANDULAR TRICHOMES --- p.23 / Chapter 2.4 --- PROTEIN EXTRACTION . --- p.25 / Chapter 2.5 --- Two DIMENSIONAL GEL ELECTROPHORESIS --- p.25 / Chapter 2.6 --- IMAGINE ANALYSIS --- p.26 / Chapter 2.7 --- IN GEL DIGESTION AND PROTEIN IDENTIFICAIOTN BY MASS SPECTROMETRY --- p.27 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSION --- p.29 / Chapter 3.1 --- THE ISOLATION OF GLANDULAR TRICHOMES --- p.29 / Chapter 3.2 --- 2DE PATTERNS OF A. ANNUA LEAVE TRICHOMES AND LEAF TISSUE --- p.32 / Chapter 3.3 --- IDENTIFICATION OF PROTEINS IN GLANDULAR TRICHOMES --- p.34 / Chapter 3.3.1 --- Protein involved in electron transport chain --- p.47 / Chapter 3.3.2 --- Protiens invovled in metabolism --- p.48 / Chapter 3.3.2.1 --- artemisinin biosynthesis --- p.48 / Chapter 3.3.2.2 --- glycolysis --- p.49 / Chapter 3.3.2.3 --- other metabolic enzymes --- p.50 / Chapter 3.3.3 --- Proteins involved in transcription and translation --- p.51 / Chapter 3.3.4 --- Protein involved in proteolysis --- p.51 / Chapter 3.3.5 --- "Detoxificaiton, stress related protein" --- p.52 / Chapter 3.4 --- PERSPECTIVE --- p.53 / Chapter 3.5 --- CONCLUSION --- p.53 / REFERENCES --- p.56
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Recent developments in research on terrestrial plants used for the treatment of malaria.Wright, Colin W. January 2010 (has links)
no / New antimalarial drugs are urgently needed to combat emerging multidrug resistant strains of malaria
parasites. This Highlight focuses on plant-derived natural products that are of interest as potential leads
towards new antimalarial drugs including synthetic analogues of natural compounds, with the
exception of artemisinin derivatives, which are not included due to limited space. Since effective
antimalarial treatment is often unavailable or unaffordable to many of those who need it, there is
increasing interest in the development of locally produced herbal medicines; recent progress in this area
will also be reviewed in this Highlight.
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Avaliação atraves de tecnicas de cultura de tecidos da produção de antimalaricos em genotipos selecionados de Artemisia annua L.Pellegrino, Ana Paula 14 August 2018 (has links)
Orientador: Simone Liliane Kirszenzaft Shepherd / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T11:03:53Z (GMT). No. of bitstreams: 1
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Previous issue date: 2000 / Doutorado / Doutor em Biologia Vegetal
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The Gametocytocidal Activity of Whole-Plant Artemisia annua and Artemisia afra Tea-Based Therapies against Plasmodium falciparum in vitro.Snider, Danielle A 12 November 2019 (has links)
Malaria is one of the deadliest parasitic diseases worldwide, causing 219 million infections and 435 thousand deaths per year. As such, this mosquito-borne illness is a major target for global eradication efforts. One critical arm of the eradication strategy is chemotherapy. For a therapeutic to advance the eradication agenda, it must cure the patient of infection and eliminate transmission stage parasites (called gametocytes) from the blood, thereby breaking the cycle of transmission. Currently, first-line treatments against malaria infection consist of an artemisinin derivative in combination with another antimalarial drug from a different drug class. Although artemisinin and its derivatives are highly efficacious at curing malaria, these drugs are ineffective at preventing disease transmission. However, recent in vivo studies have suggested that whole plant Artemisia annua (the botanical source of artemisinin) delivered as tea can cure patients of infection and eliminate transmission stage parasites from the bloodstream. To validate these in vivo results in vitro, experiments were performed to measure the killing efficacy of A. annua and A. afra tea infusions against three different stages of the parasite life cycle— one stage of the asexual cycle, immature gametocytes, and mature gametocytes. Killing effects were observed using light microscopy and gametocyte gene-specific RT-qPCR analyses. Results suggested that A. annua tea was nearly as effective as artemisinin at killing all three tested stages of the parasite. A. afra tea, which contains low levels of artemisinin, showed comparable killing efficacy against late stage gametocytes, but not against the other two tested stages. These results supported the notion that A. annua tea is an effective antimalarial and also provides evidence that both A. annua and A. afra teas may be a viable therapeutic option for eliminating gametocytes during human infection.
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In vitro and in vivo production of artemisinin by artemisia speciesKruger, Francois Jacobus Liebenberg January 2013 (has links)
Artemisinin is produced in the leaves of Artemisia annua and is currently one of the most
valuable antimalarial treatments. A. annua is of Asian origin but many other family members
have been identified worldwide. A. annua however, is the only one that produces artemisinin.
Synthetic production of artemisinin is not yet feasible, not to mention very expensive and the
product yields are relatively low. The aims of this study were threefold: 1) To regenerate
callus, cell cultures and plants from genetically modified root cultures of A. afra into which an
artemisinin biosynthetic gene was inserted from A. annua 2) To investigate the probability
that fungal endophytes are responsible for the production of artemisinin and 3) To establish
two fields of high yielding varieties of A. annua plants and evaluate whether artemisinin
production of these two locations will remain high.
Callus and cell cultures of the genetically modified A. afra root cultures were established, but
no shoots have been produced as of yet and this is an on-going investigation. Fungal
endophytes were sampled and none of the endophytes produced artemisinin. Five different
lines of A. annua were cultivated, successfully grown and harvested. Measurements were
taken at different stages of processing, these were compared and analysed using various
methods such as height and mass comparisons. Comparisons revealed that the production
of artemisinin is correlated to local sets of conditions rather than the variety of individual
lines. The genetic potential to produce high quantities of artemisinin appears to have been
lost, instead of being maintained. We confirmed that secondary compound production and
specifically, artemisinin, is enhanced by certain stress factors on the plants. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Plant Science / unrestricted
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Sugar Control of Artemisinin ProductionWANG, YI 29 April 2006 (has links)
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.
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PLANT GROWTH REGULATORS AND HERBICIDES FOR MANAGEMENT OF POA ANNUA: IMPACT OF BIOTYPES AND BEHAVIOR OF FLURPRIMIDOL IN TURFGRASS SPECIESWilliams, Alexandra Perseveranda 01 January 2014 (has links)
In 2011, Poa annua L. (Poa) biotypes were collected from greens of two golf courses in Lexington, Kentucky: 1.) The Lexington Country Club (LCC) and 2.) The University Club (UC). The samples were collected based on exhibiting one of two appearances while on the same green: 1.) dark green, with few to no flower heads (dark biotype) or 2.) light green, with numerous flower heads (light biotype). Two PGRs, paclobutrazol and flurprimidol, and two herbicides, bispyribac-sodium and amicarbazone, were applied to the plants both in the field and the greenhouse. Quality ratings were recorded weekly in both the field and greenhouse and grass clippings were collected and measured weekly in the greenhouse. Flurprimidol controlled the dark biotypes and paclobutrazol controlled the light biotypes in the field in 2011. However, only location by treatment interactions were in 2012; flurprimidol, bispyribac-sodium, and amicarbazone controlled the biotypes from the LCC while paclobutrazol controlled the biotypes from UC. In the greenhouse study there was a significant three way interaction between color, location, and treatment for quality. PGRs controlled the light biotypes from LCC and the dark biotypes from UC. Herbicides controlled the light biotypes more than the dark, however, the light biotypes recovered after amicarbazone treatments. PGRs reduced clipping weights of the dark biotypes more than the light and herbicides reduced clipping weights of the light biotypes more than the dark. Both PGRs and herbicides reduced clipping weights of the Poa collected from the LCC more than UC. These results demonstrate both the potential for differential responses between Poa biotypes to PGRs and herbicides and that these differences, like all things about Poa, may be complex. A laboratory experiment was also designed to examine the absorption and potential metabolism of 14C-labeled flurprimidol in creeping bentgrass (Agrostis stolonifera (L.)), bermudagrass (Cynodon dactylon (L.)), Kentucky bluegrass (Poa pratensis (L.)), perennial ryegrass (Lolium perenne (L.)), tall fescue (Festuca arundinacea (Schreb.)), and zoysiagrass (Zoysia japonica (Steud.)) and light and dark Poa biotypes collected from golf greens. Flurprimidol absorption and translocation was greater for warm season grasses than cool season grasses. Only parent flurprimidol was detected in all turf species.
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