Environmental impacts of feral horses (Equus caballus) are a subject of conservation concern and controversial national policy. In North America, feral horses are considered an invasive species where they impact rangelands of the arid and semi-arid western United States. The greater sage-grouse (Centrocercus urophasianus) is a native sagebrush obligate bird species that relies on sagebrush habitats to sustain viable population levels. Recent literature suggests that feral horse presence can have a notable effect on the fitness of native and sagebrush obligate species throughout the arid and semi-arid western United States. The purpose of this thesis was to assess the potential impact of feral horses on population patterns and on late-brood rearing habitat of greater sage-grouse throughout the Great Basin. This was accomplished by pairing known sage-grouse use sites (leks and late brood-rearing habitat) to random sites for comparison. Within each pair, one site was located within Herd Management Area (HMA) boundaries (with assumed horse presence) while the other was located outside (with assumed horse absence). We then assessed lek attendance throughout the state of Nevada and compared attendance rates to known horse population estimates. Furthermore, paired late brood-rearing habitat sites were compared to one another to assess the effect of horse and cattle presence on habitat quality and characteristics. We determined that mean sage-grouse population size at leks is higher (9.14 ± 1.04 males) within HMA boundaries compared to areas outside of HMA boundaries (6.55 ± 0.74 males). Considering late brood-rearing habitat, we determined that statistical differences have occurred between horse and non-horse use sites in the following comparisons: annual grass frequency, percent annual grass cover, dung frequency, total plant height, vegetative height, and horse and cattle dung density. We suggest that feral horse presence can impact sage-grouse habitat, however, a more clear understanding of horse effects on rangeland wildlife habitat is needed to assess actual impacts on wildlife populations in consideration of multiple use management decisions.

With the increase in recent years in the prevalence of malaria, and in drug resistance of Plasmodium falciparum, there has been much interest in natural plant products for new antimalarials with novel modes of action against Plasmodium. Artemisinin or Qinghaosu is one such antimalarial isolated from a Chinese herb, Anemisia annua (Asteraceae) and it is currently undergoing phase I and II clinical trials. The Southern African species, Artemisia afra (African wormwood, wildeals, lengana) is commonly used by local traditional healers for symptoms of malaria, in particular fever. Thus it seemed appropriate to investigate this species for antimalarial activity. Crude petroleum ether soxhlet extracts of Anemisia afra had demonstrated antimalarial activity against Plasmodium falciparum, FCR-3, cultured in vitro. The IC₅₀ values ranged from 5-13μg/ml. The extract from leaves and flowers was then screened against D10 (chloroquine-sensitive) and FAC8 (chloroquineresistant) P. falciparum, in vitro, with IC₅₀ values of 1.03μg/ml and l.5μg/ml respectively. This extract was fractionated by column chromatography using silica gel-60 and the fractions obtained were screened for antimalarial activity. The most active fraction had an IC₅₀ of 0.5μg/ml against D10 and FAC8. Using TLC and HPLC-UV analysis with pure artemisinin as a standard, no artemisinin could be detected in this fraction. This result was confirmed by thermospray LC-MS analyses. Purification of this fraction yielded ultimately a single pure compound; a clear colourless oil identified by MS and NMR analyses as hydroxydavanone. The compound was screened against a variety of P. falciparum strains with varying degrees of sensitivity and resistance to both chloroquine and mefloquine. Their sensitivity against artemisinin was also established. IC₅₀ values obtained for the isolated pure compound against P. falciparum ranged from 0.87 to 2.54μg/ml. The IC₅₀ values obtained for general cytotoxicity of the crude extract and isolated pure compound against RAT-I fibroblast cells were 34.78 ± 8.23 and 6.29 ± 0.95 μg/ml (n=4) respectively. Thus the crude extract and isolated pure compound exhibited a greater antimalarial than cytotoxic effect. Hence, there are implications for A. afra to be used as a phytomedicine for the treatment of malaria. In vivo studies are recommended for hydroxydavanone in order to fully assess its potential for clinical use.

Spraying with the chemical herbicide 2,4-D is the most widely used method of controlling big sagebrush. Spraying is very effective in increasing forage production and generally is not poisonous to either man or animals. Two procedures can be used to calculate the internal rate of return to big sagebrush control: standard and modified discounting. Standard discounting assumes all nonuse costs are incurred in the year of treatment, and the annual income stream is constant throughout the effective life of treatment. Modified discounting correctly assumes the nonuse cost is incurred in the period of deferment, and the income stream does not reach its full potential until after deferment. Thus, modified discounting yields a lower internal rate of return. Three big sagebrush control methods (spraying, burning, and chaining) offer internal rates of return which are greater than 8 percent (cost of obtaining capital for range improvement). The most important factors in determining the internal rate of return are the site vigor index and the amount of forage present before treatment. A larger pre-treatment forage yield will give a larger internal rate of return, assuming the vigor index is sufficiently high. If state and private rangelands infested with big sagebrush are not improved by spraying or other big sagebrush control methods, certain benefits, called opportunity costs, will be foregone. For spraying alone, the expected annual opportunity costs would be $3,048,102. The economic feasibility of controlling nearly 2 1/2 million acres of state and private rangelands infested with big sagebrush are excellent. The expected annual increase in carrying capacity of 1,830,000 acres of sagebrush rangeland meriting improvement by spraying is 765,855 AUMs. The remaining 623,000 acres meriting control other than by spraying could possibly increase the total number of additional AUMs to over 1 million.

Big sagebrush (Artemisia tridentata) is one of the ecologically most important shrub species in western North America. The species serves as a major source of food and habitat for the near-threatened sage grouse and various other fauna. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is considered as a serious threat to sustainability of the big sagebrush ecosystem. Because of its importance, restoration of this species is very crucial to those dependent on big sagebrush community. However, restoration of big sagebrush carried out by using diverse seed source can lead to imbalance and degradation in the native ecosystem. Therefore, restoration works aided by understanding of adaptive traits of big sagebrush using molecular markers will aid successful restoration. The major objective of this research was to create a substantial resource of nuclear sequence data and identify markers that can be used in future studies in big sagebrush. We report the development and annotation of the first expressed sequence tag (EST) collection for big sagebrush based on 454 sequencing of leaf tissue. Expressed genes of subspecies tridentata and vaseyana were sequenced using the 454 GS-FLX titanium platform, which produced 823,392 reads with an average read length of 404 bp and 702,001 reads with an average read length of 333 bp for sspp. tridentata and vaseyana, respectively. Assembly of the reads resulted in 212,102 consensus sequences in ssp. tridentata and 199,439 in ssp. vaseyana. A combined assembly of both subspecies sequences generated 29,541 contigs with an average length of 796 bp and 275,866 singletons with an average length of 370 bp. A BLASTx search against the non-redundant (NR) protein database using the contigs obtained from a combined assembly resulted in 21,436 sequences with significant blast alignments (≤ 1e-15). Gene Ontology (GO) IDs were assigned to 18,397 sequences. A total of 20,952 SNPs were detected between the two subspecies and 1,182 SNPs were confirmed in tetraploid ssp. wyomingensis. In addition, 1,003 and 507 SSRs were detected in ssp. tridentata contigs and ssp. vaseyana contigs, respectively.

The present thesis elucidates facts about a prominent steppe plant’s evolutionary history (i.e., geographic origin, migration route, distribution of genealogical lineages and polyploidization events) and contemporary population divergence (i.e., genetic diversity and differentiation, impacts of abiotic and biotic factors). Artemisia frigida has been chosen as the target species, with Mongolia as the focus study region. Because A. frigida is widely distributed in the both New and Old Worlds, it was a suitable candidate for the phylogeographic study. Moreover, because of its dominance in many communities in Mongolian steppes and tolerance for cold, drought and mechanical disturbances (grazing), evaluating the effect of environmental factors and grazing pressures on its population genetics was profitable. The overall goal of this thesis was to assess the effects of paleo- and current climate, and land use changes on the distribution of A. frigida’s genealogical lineages and genetic variations. The thesis is divided into two main parts: (i) Chapter 3 focuses on Phylogeography. Within this, section 3.3 depicts a study on the phylogeography of A. frigida, covering samples from its distributional range across the northern hemisphere. The study resulted in Asia being the species’ main origination and diversification center, and the species spread northwards to the Russian Far East and eventually crossed the Bering Strait to North America. Among four geographical regions sampled, seven genetic lineages were found, with Middle Asia having the most diverse populations. According to our phylogenetic analysis, two populations of Kazakhstan in Middle Asia represented the most likely ancestral diploids, and subsequent polyploidization events have occurred on several occasions independently. The observed phylogeographic patterns of the species showed that paleoclimate, especially glaciation events of the Quaternary has predominantly affected species’ current distribution, along with the expansion and contraction of the Eurasian steppe. The second part (Chapter 4) is dedicated to Population genetics to reveal the effects of the current climate and land use on population genetic variation. Three studies were conducted at local and regional levels, focusing on Mongolia. The first study (Section 4.1- review of local literature) was done to offer background information about Mongolian steppes, and the effects of climate and grazing on the steppe vegetation. As a result, steppe vegetation responded to grazing in different ways, depending on the interplay of local environmental factors. In particular, an overall negative effect of grazing was found in desert, dry and high mountain steppes, but no or even positive effects in meadow and mountain steppes. The study highlighted the importance of the interaction effect of local environmental conditions and grazing in Mongolian steppe vegetation. The second study (Section 4.2) employed large scale climatic gradient and local scale grazing gradients to assess the effects of grazing and environmental factors on the population genetics of A. frigida. Precipitation gradient covered 110 – 300mm difference of mean annual precipitation from central to southern Mongolia. While three levels of grazing gradient, such as heavy, moderate, and least grazed sites were examined. According to the study, grazing in overall, had no substantial effect on the genetic diversity of A. frigida, while environmental factors, i.e., summer precipitation and soil phosphorous content, promoted high genetic diversity. Genetic differentiation among populations across large climatic gradients was extremely low, suggesting the existence of considerable gene flow among populations across the steppes of Mongolia. The third study (Section 4.3) employed grazing exclosures to evaluate the genuine effects of grazing. Because Mongolia has a long-term nomadic pastoralism history, and grazing of large herbivores is already an integral part of the steppe vegetation. Thus, we utilized reference site fences along the Trans-Mongolian Railway (TMR), where fences have been built and maintained since 1955, resulting in over 60 years of grazing exclusion. In addition, we supplemented this with data from Hustai National Park (HNP), where three fences were established in 2003. As a result, we found a significant positive impact of grazing on the genetic diversity of A. frigida, implying that a certain level of grazing is beneficial for the species. While no grazing effect on the population genetic differentiation was detected, but climatic and soil variables strongly influenced population genetic structure. In summary, this thesis provided an in-depth investigation of the phylogeography and population genetics of the species A. frigida, which can stand as an exemplar for other Eurasian steppe species. Paleoclimate had largely shaped the current distribution pattern of the species, while contemporary climate and environmental heterogeneity promoted species’ polyploidization and genetic variation. Grazing by large herbivores showed no detrimental effect, or even a positive impact on the genetic diversity of A. frigida. Artemisia frigida populations in Mongolia are thus apparently genetically ‘healthy’, in spite of pervasive grazing in the region. Climate variables and environmental heterogeneity had a substantial impact on the species’ both genetic diversity and differentiation, indicating its higher sensibility to climate change than to land use change. The findings of the thesis could be valuable in understanding species genetic variation under global land use and climate changes.:Summary 4 List of Abbreviations 6 Chapter 1. Introduction 8 Chapter 2. Material and methods 10 2.1. Study region: Mongolian steppe 10 2.2. Focus species: Artemisia frigida Willd. 11 2.3. Molecular markers 12 Chapter 3. Phylogeography 13 3.1. Eurasian steppe and its evolutionary history 13 3.2. Artemisia L. (Asteraceae) as model plant for phylogeography 16 3.3. A case study: Phylogeography of Artemisia frigida Willd. 19 Chapter 4. Plant population genetics under changing climate and grazing……. 40 4.1. Climate – grazing interactions in Mongolian steppe 41 4.2. Climate – grazing interactions on plant population genetics 59 4.3. Effect of grazing exclusion on plant populations genetics 83 Chapter 5. Overarching synthesis and discussion 101 5.1. Molecular markers: pros and cons 102 5.2. A review of phylogeographical studies on Eurasian steppe plants 104 5.3. A review on plant population genetic studies in Mongolia 109 5.4. Outlook 113 Acknowledgements 114 References 115 Curriculum vitae 137 Confirmation 143

Thesis (M.Sc. Agriculture (Horticulture)) -- University of Limpopo, 2017 / Artemisia annua L. from the family Asteraceae is an annual medicinal plant and has been used to make herbal remedies in Asia for thousands of years. Artemisinin is a sesquiterpene lactone, isolated from aerial parts of Artemisia annua, with the highest concentrations being in flowers and leaves. In addition to potent anti-malarial activity, artemisinin possesses anti-cancer, anti-schistosomiatic, anti-hepatitis B, anti-HIV, anti-leishmanial and herbicidal activities. Low artemisinin production (0.01-2%) from A. annua is a major constraint in commercialisation of the drug for control of malaria. Worldwide, efforts have been underway to improve the concentration of artemisinin using conventional breeding, biochemical, physiological, molecular and hairy-root culture techniques, however all these methods are not economical. Cultural practices like spacing and pruning have limitation in improving artemisinin concentration and these may help in increasing the concentrations of artemisinin. Study was conducted at the experimental farm of the Agricultural Research Council – Vegetable and Ornamental Plants, Roodeplaat Pretoria. The objective of this study was to determine whether spacing, pruning and their interactions would have any effect on the concentrations of artemisinin, growth and yield of A. annua and whether drying methods would have an effect on the concentrations of artemisinin in A. annua. Since there was only one field trial, all sub-objectives were addressed at once (Chapter 3). Fresh seeds of A. annua were obtained from the ARC-VOP gene bank and sown in seedling trays in September 2014. Uniform eight-week-old seedlings were hardened-off, transplanted in November 2014 in 10 cm deep holes and then pruned ten weeks after transplanting. Treatments for Experiment 1, viz., 3 × 4 factorial experiment were laid out in a randomised complete block design, with four replications (n = 48). The two factors of the experiment were (a) spacing [0.5 × 1 m2 (standard: 0.50 m2), 0.5 × 0.7 m2 (small: 0.35 m2) 0.5 × 0.5 m2 (smaller: 0.25 m2) and 0.3 × 0.7 m2 (smallest: 0.21 m2)] and (b) pruning [no pruning (control), removing the apical bud and removing shoots three nodes from the bottom]. The plants were irrigated using overhead sprinklers system for two hours three times per week. Four readings for growth variables (plant height, stem diameter and chlorophyll content) were collected with one week interval. Plants were harvested after 180 days from planting, and leaves, stems and roots were separated weighed and oven dried at 40 ºC for 72 h. In Experiment 2 (drying methods), treatments, namely, 100% sun, 100% shade, 50% shade, freeze and oven drying were arranged in completely randomised design with four replicates (n = 20). The treatments were exposed for a week, to full sunlight, 50% shade-drying under a shade net that allows 50% light penetration, 100% shade under enclosed room at ambient (24-25 ºC) temperature, oven drying for 24 h at 40 ºC, and freeze-drying for three days. Freeze-drying had significant effect on artemisinin concentration of 1.941%. It was followed by oven (1.738%) and 100% shade drying (1.657%) and the lowest artemisinin concentration (1.412%) was obtained from 50% shade drying. The smaller spacing of 0.25 m2 in combination with apical bud removal had a significant effect on artemisinin concentration, producing artemisinin concentration of 0.193%. Spacing had a significant effect on stem diameter, fresh leaf mass and dry leaf mass but had no effect on plant height and chlorophyll content. Pruning had a significant effect on plant height and chlorophyll content and had no effect on stem diameter. The small spacing of 0.35 m2 had the highest fresh and dry leaf mass of 17.99 and 9.62 t/ha. The interaction of spacing and pruning had no significant effect on the growth and yield of A. annua. The results from this study suggested that cultural and processing practices may have direct effects in the concentration of artemisinin, growth and yield of A. annua. The results xiv provided some understanding on how agronomic and processing practices can be used to increase artemisinin content in A. annua and understand the interaction between different agronomic practices and thereby allowing the development of economic methods for A. annua post-harvest handling. Future work should focus on implementing various pruning techniques to trigger stress and indirectly secondary metabolites

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The present work describes the phytochemical study and biological activity evaluation of the Buddleja brasiliensis Jacq. ex. Spreng and Artemisia verlotorum Lamotte. Were isolated three compounds from Buddleja brasiliensis: verbascoside (1), β-sitosterol (54) and stigmasterol (55), and it was carried out a phytochemical screening, a study of the ability to inhibit the enzymes prolyl oligopeptidase (POP), dipeptidyl peptidase (DPP IV) and acetylcholinesterase (AChE), a study of the antimicrobial activity of the specie and the extraction of the essential oil. The fractions F4 and F12 inhibited significantly POP and AchE activity. All Buddleja brasiliensis fractions tested presented low activity of DPP IV inhibition. The phenylpropanoid verbascoside (1) demonstrated a significant and selective inhibitory activity against POP (IC50 = 1.3 mM), showed weak activity against DPP-IV (IC50 >> 150 mM) and was inactive against AChE (pMIQ 9.6). The essential oil obtained from this species has as major constituents the a-tujaplicin monoterpene (11.5%) and the diterpenoid heneicosane (14.5%). This oil was inactive against all tested enzymes. The antimicrobial activity of 1, the crude extract and fractions of Buddleja brasiliensis were tested against several fungi and bacteria. Fractions dichloromethane and ethyl acetate showed moderate activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Streptococcus pyogenes, while 1 showed no antimicrobial activity. The crude extract, fractions and essential oil obtained from Artemisia verlotorum were also evaluated for inhibitory activity against the POP and DPP IV, as well as against various fungi and bacteria. Dichloromethane fraction of Artemisia verlotorum showed the best antimicrobial activity, mainly against the yeast Saccharomyces cerevisiae, with an MIC of 125 μg/mL. Analysis by gas chromatography coupled with mass spectrometry of the essential oil of Artemisia verlotorum indicated as the main components santonilil acetate (37.2%) and a-cadinol (9.59%). / O presente trabalho descreve o estudo fitoquímico e a avaliação da atividade biológica das espécies Buddleja brasiliensis Jacq. ex. Spreng e Artemisia verlotorum Lamotte. De Buddleja brasiliensis foram isolados três compostos: verbascosídeo (1), β-sitosterol (54) e estigmasterol (55). Desta espécie, fez-se a prospecção fitoquímica, a avaliação da capacidade de inibição das enzimas prolil oligopeptidase (POP), dipeptidil peptidase (DPP IV) e acetilcolinesterase (AChE), a obtenção do óleo essencial, e avaliação da atividade antimicrobiana. Nos testes de inibição frente à enzima POP foi observada significativa atividade das frações F4 e F12. Estas frações também demonstraram atividade inibitória da enzima AChE. Nos testes com a DPP IV verificou-se baixa atividade para todas as frações testadas. O fenilpropanóide verbascosídeo (1) demonstrou ter uma significativa e seletiva atividade inibitória frente à POP (IC50 = 1,3 μM), enquanto frente à DPP IV demonstrou fraca atividade inibitória (IC50 >>150 μM) e frente à AChE foi inativo (pMIQ de 9,6). O óleo essencial obtido desta espécie tem como constituintes principais o monoterpeno a-tujaplicina (11,5 %) e o diterpeno heneicosano (14,5 %). Este óleo foi inativo frente a todas as enzimas testadas. A atividade antimicrobiana de 1, do extrato bruto e das frações de Buddleja brasiliensis foi testada frente a vários fungos e bactérias. O composto 1 não apresentou nenhuma atividade antimicrobiana, enquanto que as frações que demonstram atividade moderada foram as frações Diclorometano e Acetato de etila para Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Streptococcus pyogenes. O extrato bruto, as frações e o óleo essencial obtidos de Artemisia verlotorum também foram avaliados quanto a atividade de inibição frente à POP e a DPP IV, bem como frente a vários fungos e bactérias. A fração Diclorometano de Artemisia verlotorum demonstrou a melhor atividade antimicrobiana, principalmente frente ao fungo Saccharomyces cerevisae, com uma CIM de 125 μg/mL. A análise por cromatografia gasosa acoplada a espectrômetro de massa do óleo essencial de Artemisia verlotorum indicou como constituintes principais acetato de santonilila (37,2 %) e a-cadinol (9,59%).

A caracterização fenotípica de Plasmodium falciparum permite conhecer o padrão de sensibilidade do parasito às drogas antimaláricas utilizadas em países endêmicos. No presente estudo avaliamos fenotipicamente isolados clínicos de P. falciparum provenientes do Continente Africano e do Caribe. A sensibilidade à dihidroartemisinina (DHA: 4 - 1.000 nM), artesunato (AS: 0,1 - 100 nM), lumefantrina (LMF: 3,1 - 200 nM) e mefloquina (MFQ: 0,2 - 1.000 nM) foi investigada por meio de quatro técnicas: (a) ensaio de sensibilidade ex-vivo e in vitro, (b) ensaio de dormência, (c) ensaio de citometria de fluxo e (d) ensaio de sobrevivência do trofozoíto jovem (Ring Stage Survival Assay - RSA). Nos experimentos ex-vivo e in vitro, os IC50 estabelecidos foram 0,4 - 66,6 nM para DHA; 3,8 - 48,8 nM para LMF; 0,3 - 25,9 nM para AS e 2 - 439 nM para MFQ. No ensaio de dormência, esquizontes foram observados na amostra de referência NF54 de P. falciparum e na amostra clínica S-01/15 após pressão com 62,5 nM, 250 nM e 1.000 nM de DHA. O período de recuperação variou de 4 a 40 dias. Para LMF, houve maturação para o estágio de esquizonte no isolado de referência no sétimo e décimo segundo dia após a exposição a 66,6 nM e 200 nM da droga, respectivamente. Esquizontes foram visualizados no isolado clínico FS-08/15 de P. falciparum depois da pressão com 100 nM de AS, com recuperação de 0 a 28 dias, portanto sem apresentar dormência. Na citometria de fluxo, trofozoítos jovens viáveis de P. falciparum marcados com Rodamina 123 e DAPI foram observados nas máximas concentrações de DHA (1.000 nM) e LMF (200 nM). Finalmente no RSA, a taxa de crescimento (TC) e porcentagem de supervivência (PS) do isolado de referência foi 2,92 e 4,19%, respectivamente, frente a 700 nM de DHA. O mesmo isolado pressionado com 3.500 nM de LMF apresentou 3,6 de TC e 2,25% de PS. A avaliação microscópica dos ensaios de sensibilidade ex-vivo e in vitro subestima a resposta de P. falciparum à terapia combinada com artemisinina (ACT). Nossos resultados sugerem que a dormência, principal mecanismo de tolerância às artemisininas (ART), não aconteceria em todos os isolados clínicos de P. falciparum. A citometria de fluxo avaliou com acurácia a viabilidade parasitária. No presente estudo, pela primeira vez foi reportada a dormência de P. falciparum à LMF / The phenotypic characterization of Plasmodium falciparum is useful for the knowledge of parasite sensitivity against antimalarial used in endemic countries. In this study we evaluated the sensitivity of clinical isolates of P. falciparum from the African continent and the Caribbean. The sensitivity to dihydroartemisinin (DHA: 4 - 1,000nM), artesunate (AS: 0.1 - 100 nM), lumefantrine (LMF: 3.1 to 200 nM), and mefloquine (MFQ: 0.2 to 1,000 nM) was investigated through four techniques: (a) ex vivo and in vitro microtests, (b) dormancy assay, (c) flow cytometry assay and (d) survival assay using young trophozoites (Ring Stage Survival Assay - RSA). In the ex vivo and in vitro experiments, the IC50 was calculated and was 0.4 - 66.6 nM for DHA; 3.8 - 48.8 nM for LMF; 0.3 - 25.9 nM for AS and 2 - 439 nM for MFQ. According to dormancy assays, schizonts were observed in the P. falciparum reference isolate NF54 and in the clinical isolate S-01/15 after pressure with 62.5 nM, 250 nM and 1,000 nM DHA. The recovery period ranged from 4 to 40 days. For LMF was observed the growth to the schizont stage in NF54, in the days 7 e 12 after exposure to 66.6 nM and 200 nM of the drug, respectively. Schizonts were seen in the P. falciparum clinical isolate FS-08/15 after pressure with 100 nM of AS, right after incubation period, with no dormancy of trophozoites. In flow cytometry assays, viable young trophozoites of P. falciparum labeled with DAPI and Rhodamine 123 were observed at the maximum concentrations of DHA (1,000 nM) and LMF (200 nM). Finally in RSA, the growth rate (GR) and percentage of survival (PS) of the reference isolate was 2.92 and 4.19%, respectively, after pressure with 700 nM of DHA. The same isolate pressed with 3,500 nM of LMF presented GR of 3.6% and PS of 2.25%. In conclusion, microscopic evaluation of ex vivo and in vitro sensitivity tests underestimates the P. falciparum response to artemisinin-based combination therapy (ACT). Our results suggest that the dormancy, main mechanism of tolerance to artemisinin (ART), is not presented in all clinical isolates of P. falciparum. Flow cytometry was able to confirm the parasite viability accurately. In this study, for the first time the dormancy of P. falciparum after pressure with LMF was reported

Submitted by Irvana Coutinho (irvana@ufpa.br) on 2012-12-12T13:51:59Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-12-20T13:18:24Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) / Made available in DSpace on 2012-12-20T13:18:24Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) Previous issue date: 2012 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / A artemisinina é uma substância extraída da planta chinesa Artemisia annua L., sendo bastante utilizada na medicina natural como um terapêutico em várias patologias. Já o artemer é uma substância sintetizada a partir da artemisinina. Estas drogas se enquadram em um grupo especial de moléculas denominadas de lactonas sesquiterpênicas sendo amplamente administradas na terapêutica da malária. Embora sejam considerados eficientes anti-maláricos, muito pouco se sabe sobre os efeitos genotóxicos e citotóxicos destes fármacos. Portanto, no presente trabalho, avaliamos os efeitos citotóxicos, genotóxicos e mutagênicos da artemisinina e do artemeter em cultura de linfócitos humanos por meio do ensaio cometa, do teste do micronúcleo e do ensaio de citotoxicidade para detecção de necrose e apoptose por marcação fluorescente diferencial com laranja de acridina/brometo de etídio (LA/BE), respectivamente. Nossos resultados demonstraram um aumento significativo (p<0,05) no índice de dano do DNA avaliado pelo ensaio do cometa, bem como na frequência de micronúcleos em ambas as substâncias testadas. Foi observado também, que apenas a artemisinina induziu um aumento estatisticamente significativo (p<0.05) no número de células necróticas nos linfócitos em 48 h de tratamento. Desta forma, demonstrou-se em nosso trabalho, que estas duas drogas exercem efeitos citotóxicos, genotóxicos e mutagênicos em culturas de linfócitos humanos, nas condições avaliadas. Nossos dados apontam a necessidade de cautela no uso de tais medicamentos, uma vez que efeitos genotóxicos/mutagênicos podem aumentar o risco de carcinogênese. / Artemisinin is a substance extracted from the Chinese plant Artemisia annua L., and widely used in natural medicine for a treatment of various diseases. Artemether is a substance synthesized from artemisinin. These drugs belong to a special group of molecules called sesquiterpene lactones widely administered in the treatment of malaria. Although considered effective anti-malarial drugs, very little is known about the genotoxic and cytotoxic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin and artemether in cultured human lymphocytes using the comet assay, the micronucleus test and a cytotoxicity assay for detection of necrosis and apoptosis by fluorescent differential acridine orange/ethidium bromide (LA/BE), respectively. Our results showed a significant increase (p<0.05) in the rate of DNA damage measured by comet assay and in the micronucleus frequency after treatment with both drugs. It was also observed that only artemisinin induced a statistically significant increase (p<0.05) in the number of lymphocytes with death by necrosis 48 h after treatment. Thus, it was shown in our work that these two drugs exert mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes under the conditions evaluated. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.

A caracterização fenotípica de Plasmodium falciparum permite conhecer o padrão de sensibilidade do parasito às drogas antimaláricas utilizadas em países endêmicos. No presente estudo avaliamos fenotipicamente isolados clínicos de P. falciparum provenientes do Continente Africano e do Caribe. A sensibilidade à dihidroartemisinina (DHA: 4 - 1.000 nM), artesunato (AS: 0,1 - 100 nM), lumefantrina (LMF: 3,1 - 200 nM) e mefloquina (MFQ: 0,2 - 1.000 nM) foi investigada por meio de quatro técnicas: (a) ensaio de sensibilidade ex-vivo e in vitro, (b) ensaio de dormência, (c) ensaio de citometria de fluxo e (d) ensaio de sobrevivência do trofozoíto jovem (Ring Stage Survival Assay - RSA). Nos experimentos ex-vivo e in vitro, os IC50 estabelecidos foram 0,4 - 66,6 nM para DHA; 3,8 - 48,8 nM para LMF; 0,3 - 25,9 nM para AS e 2 - 439 nM para MFQ. No ensaio de dormência, esquizontes foram observados na amostra de referência NF54 de P. falciparum e na amostra clínica S-01/15 após pressão com 62,5 nM, 250 nM e 1.000 nM de DHA. O período de recuperação variou de 4 a 40 dias. Para LMF, houve maturação para o estágio de esquizonte no isolado de referência no sétimo e décimo segundo dia após a exposição a 66,6 nM e 200 nM da droga, respectivamente. Esquizontes foram visualizados no isolado clínico FS-08/15 de P. falciparum depois da pressão com 100 nM de AS, com recuperação de 0 a 28 dias, portanto sem apresentar dormência. Na citometria de fluxo, trofozoítos jovens viáveis de P. falciparum marcados com Rodamina 123 e DAPI foram observados nas máximas concentrações de DHA (1.000 nM) e LMF (200 nM). Finalmente no RSA, a taxa de crescimento (TC) e porcentagem de supervivência (PS) do isolado de referência foi 2,92 e 4,19%, respectivamente, frente a 700 nM de DHA. O mesmo isolado pressionado com 3.500 nM de LMF apresentou 3,6 de TC e 2,25% de PS. A avaliação microscópica dos ensaios de sensibilidade ex-vivo e in vitro subestima a resposta de P. falciparum à terapia combinada com artemisinina (ACT). Nossos resultados sugerem que a dormência, principal mecanismo de tolerância às artemisininas (ART), não aconteceria em todos os isolados clínicos de P. falciparum. A citometria de fluxo avaliou com acurácia a viabilidade parasitária. No presente estudo, pela primeira vez foi reportada a dormência de P. falciparum à LMF / The phenotypic characterization of Plasmodium falciparum is useful for the knowledge of parasite sensitivity against antimalarial used in endemic countries. In this study we evaluated the sensitivity of clinical isolates of P. falciparum from the African continent and the Caribbean. The sensitivity to dihydroartemisinin (DHA: 4 - 1,000nM), artesunate (AS: 0.1 - 100 nM), lumefantrine (LMF: 3.1 to 200 nM), and mefloquine (MFQ: 0.2 to 1,000 nM) was investigated through four techniques: (a) ex vivo and in vitro microtests, (b) dormancy assay, (c) flow cytometry assay and (d) survival assay using young trophozoites (Ring Stage Survival Assay - RSA). In the ex vivo and in vitro experiments, the IC50 was calculated and was 0.4 - 66.6 nM for DHA; 3.8 - 48.8 nM for LMF; 0.3 - 25.9 nM for AS and 2 - 439 nM for MFQ. According to dormancy assays, schizonts were observed in the P. falciparum reference isolate NF54 and in the clinical isolate S-01/15 after pressure with 62.5 nM, 250 nM and 1,000 nM DHA. The recovery period ranged from 4 to 40 days. For LMF was observed the growth to the schizont stage in NF54, in the days 7 e 12 after exposure to 66.6 nM and 200 nM of the drug, respectively. Schizonts were seen in the P. falciparum clinical isolate FS-08/15 after pressure with 100 nM of AS, right after incubation period, with no dormancy of trophozoites. In flow cytometry assays, viable young trophozoites of P. falciparum labeled with DAPI and Rhodamine 123 were observed at the maximum concentrations of DHA (1,000 nM) and LMF (200 nM). Finally in RSA, the growth rate (GR) and percentage of survival (PS) of the reference isolate was 2.92 and 4.19%, respectively, after pressure with 700 nM of DHA. The same isolate pressed with 3,500 nM of LMF presented GR of 3.6% and PS of 2.25%. In conclusion, microscopic evaluation of ex vivo and in vitro sensitivity tests underestimates the P. falciparum response to artemisinin-based combination therapy (ACT). Our results suggest that the dormancy, main mechanism of tolerance to artemisinin (ART), is not presented in all clinical isolates of P. falciparum. Flow cytometry was able to confirm the parasite viability accurately. In this study, for the first time the dormancy of P. falciparum after pressure with LMF was reported