Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work was divided into three parts focusing two main topics: the study of volatiles compouds of P. graveolens and the study of the essential oil of L. alba and its evaluation of insecticidal activity against Spodoptera frugiperda. The chapter 2 approuch the study of the volatile compounds of the leaves of P. graveolens extracted by dynamic headspace using Porapak Q® as adsorbent and in nature peat, a novel adsorbent in the extraction of plant volatiles, and the results were compared with those obtained by hidrodestilation. The results showed that hydrodistilation (HD) was more efficient for extracting linalool and citronellyl formate. While citroneol, geraniol and geranyl tiglate were obtained in greater quantity by dynamic headspace using in nature peat (HSD-T), isomenthone, 6,9- guaiadiene and - muurolene by headspace using Porapak Q® (HSD-P). The study of conversion proved that geraniol converts in linalool when geraniol is subjected to water vapor and high temperature, nevertheless a small ratio. This also prove, in parts, the high percentage of linalool obtained by HD compared to HSD-T and HSD-P. The chapter 3 approuch the study of the essential oils of 9 accessions of L. alba collected in 4 states of Brazil using statistical methods and checking the accessions with the best essential oil content. The statistical methods of hierarchical cluster analysis (HCA) and Principal component analysis (PCA) were used to prove the experimental data and confirmation of training groups. Three groups representatives of the three chemotypes were formed: Group I was characterized by linalool and 1,8-cineole; group II, by limonene and carvone; and group III by neral and geranial. In chapter 4 was approached the evaluation of insecticidal activity of the essential oils of P. graveolens (PEL-001) and L. alba (accessions LA-10, LA-22 and LA-57) and also of the main compounds (geraniol, linalool, 1,8-cineole, limonene, carvone) against S. frugiperda via topical aplication. The test of topical application showed that the essential oils of P. graveolens (PEL-001) and L. alba (accessions LA-10, LA-22, LA-57) showed acute toxicity against larvae of S. frugiperda, causing mortality of up to 100% at a dose 192 μg/larvae. / O presente trabalho foi dividido em 3 partes os quais abordam 2 temas centrais: estudo dos compostos voláteis de Pelargonium graveolens e do óleo essencial de Lippia alba e avaliação de atividade inseticida frente à Spodoptera frugiperda. O capítulo 2 abordou o estudo dos voláteis das folhas de P. graveolens extraídos por headspace dinâmico utlizando Porapak Q® como adsorvente e turfa in natura, um adsorvente inédito empregado na extração de voláteis de plantas, sendo os resultados comparados com àqueles obtidos por hidrodestilação. Os resultados mostraram que a hidrodestilação (HD) foi mais eficiente na extração de linalol e formiato de citronelila. Enquanto citronelol, geraniol e tiglato de citronelila foram obtidos em maior proporção por headspace dinâmico utilizando turfa in natura como adsorvente (HSD-T), isomentona, 6,9- guaiadieno e -muuroleno foram identificados em maior proporção por headspace dinâmico utilizando Porapak Q® (HSD-P). O estudo de conversão comprovou que geraniol se converte em linalol, quando o geraniol é submetido a vapor d água e altas temperaturas, porém a um pequeno percentual. Isso também comprova, em partes, o alto percentual de linalol obtido por HD em relação à HSD-T e HSD-P. A extração do hidrolato revelou variações na composição volátil em comparação com os outros métodos de extração. O Capítulo 3 abordou o estudo dos óleos essenciais de 9 acessos de L. alba coletados em 4 estados do Brasil empregando métodos estatísticos e verificando os acessos com melhores teor de óleo essencial. Os métodos estatísticos de análise hierárquica de agrupamentos (HCA) e análise de componentes principais (PCA) foram empregados para comprovar os dados experimentais e confirmar a formação dos grupos. Três grupos, representando os três quimiotipos, foram formados, dos quais o grupo I foi caracterizado por linalol e 1,8-cineol, grupo II por limoneno e carvona e grupo III por neral e geranial. No capítulo 4 foi abordado a avaliação de atividade inseticida dos óleos essenciais de P. graveolens (PEL-001) e L. alba (acessos LA-10, LA-22 e LA-57), bem como dos seus principais compostos majoritários (geraniol, linalol, 1,8-cineol, limoneno e carvona) frente à S. VII frugiperda. O ensaio de aplicação tópica mostrou que óleos essenciais de P. graveolens (PEL-001) e L. alba (acessos LA-10, LA-22, LA-57) apresentaram toxicidade aguda nas lagartas S. frugiperda, ocasionando mortalidade de até 100% na dose 192 μg/lagarta.

Pelargonium sp., appelé aussi « géranium » à odeur de rose ou « Géranium rosat » est l’une des plantes aromatiques et médicinales les plus cultivées au niveau international, essentiellement pour son huile essentielle (HE), utilisée par les industries des cosmétiques et de la parfumerie. Cette essence est extraite des feuilles par distillation vapeur et donne une HE riche de plusieurs centaines de molécules volatiles. Cette complexité est le résultat d’un long processus évolutif et de sélections variétales. Parmi ces composés volatils, les monoterpènes comme le géraniol, le citronellol et l’isomenthone, ou les sesquiterpènes comme le 10- γ-épi-eudesmol et le 6,9-guaiadiène, jouent un rôle prépondérant dans le parfum du Pelargonium. Les proportions relatives de ces différents composés sont d’ailleurs utilisées comme marqueurs de la qualité de l’HE et déterminent la typicité du parfum des différents cultivars et origines (P. cv. ‘rosat Bourbon’, P. cv. ‘rosat Chine’, P. cv. ‘rosat Égypte’ et P. cv. ‘rosat Grasse’). Malgré de très nombreux travaux portant sur la chimie de cette HE, il n’existe aucune information sur les voies de biosynthèse de ces molécules et aucun gène intervenant dans ces voies n’a été isolé. Durant cette thèse, nous avons cloné et caractérisé fonctionnellement par expression et purification des protéines recombinantes chez Escherichia coli des gènes codant les enzymes clés de ces voies de biosynthèse, les terpène synthases. Nous avons ainsi pu caractériser quatre terpène synthases, dont une géraniol synthase mono-produit. Nous avons isolé deux autres monoterpène synthases multi-produits, produisant pour l’une majoritairement du myrcène mais aussi trois autres monoterpènes, et pour l’autre majoritairement du 1,8-cinéole ainsi que 10 autres monoterpènes minoritaires. Enfin, une sesquiterpène multi-produit, la 10-γ-épi-eudesmol synthase, a été caractérisée. Nous avons ensuite analysé l’expression de la géraniol synthase et de la 10-γ-épi-eudesmol synthase dans différentes accessions de Pelargonium par RT-qPCR et nous avons montré la relation entre la capacité de production des différents composés volatils et le niveau d’expression dans les feuilles de ces deux terpène synthases. L’efficacité de la transformation génétique du Pelargonium par Agrobacterium tumefaciens étant élevée, des expériences de transgénèse ont aussi été réalisées afin de compléter la caractérisation fonctionnelle des gènes isolés. Dans une deuxième partie, nous avons réalisé l’analyse des essences produites par 64 espèces et cultivars de Pelargonium d’odeurs très diverses (citron, menthe, rose, abricot, pin, épices…). A l’aide d’analyses statistiques (ACP, analyse discriminante…), nous avons mis en évidence des relations entre la biochimie de ces cultivars, leurs odeurs et leurs proximités génétiques et cela afin de nous donner des pistes sur des croisements potentiellement intéressants. Enfin, un dernier chapitre est consacré à l’amélioration de la production d’HE en Égypte. Grâce à ce programme commencé il y a trois ans, nous améliorons chaque année la qualité et le rendement en HE de plus de 10 Ha de plantation de Pelargonium en Égypte. Un travail d’optimisation de la distillation ainsi que des améliorations des pratiques culturales, nous ont permis de produire une HE de qualité avec un rendement de plus de 60 kg.Ha-1 d’HE. D’autres expériences présentées dans ce chapitre soulignent l’influence de l’environnement et notamment de la température sur le ratio entre le citronellol et le géraniol ainsi que sur la biosynthèse de l’isomenthone, du 10-γ-épi-eudesmol et du 6,9-guaiadiène / Pelargonium sp, also named rose scented « geranium » or « Geranium rosat » is one of the the most cultivated aromatic and medicinal plant worldwide, especially for its essential oil (EO), which is used by cosmetic and perfumery industries. This essence is extracted from leaves by steam distillation and gives an EO containing several hundreds of organic volatile compounds (VOC). This complexity is the result of a long evolutive process and varietal selections. Among these VOC, the monoterpenes like geraniol, citronellol and isomenthone and the sesquiterpenes like 10-γ-epieudesmol and 6,9-guaiadiene, play an important role for the Pelargonium fragrance. The relative proportions of these compounds are used as EO quality markers and determine the different cultivars origins (P. cv. ‘rosat Bourbon’, P. cv. ‘rosat Chine’, P. cv. ‘rosat Egypt’ and P. cv. ‘rosat Grasse’). Despite the important researches on the chemistry of these EO, there is no information on the biosynthesis pathways for these molecules and no genes involved in the pathways have been isolated. During this PhD thesis, we have functionally characterized by recombinant proteins expression and purification in Escherichia coli, four genes, three monoterpene and one sesquiterpene synthases, coding for key enzymes in terpene biosynthesis pathway. The first enzyme is a mono-product geraniol synthase. The second enzyme is a multi-product enzyme with a major peak of myrcene and 3 minor peaks of other monoterpenes. The third enzymes is also a multi-product protein, producing 1,8-cineol as major product and 10 others monoterpenes. The last one is a multi-products sesquiterpene synthase producing mainly the 10-γ-epi-eudesmol and other sesquiterpenes. We have also analyzed the level of expression of the geraniol and 10 γ-epi-eudesmol synthases in several Pelargonium accessions by RT-qPCR and we have demonstrated the relationship between the level of expression of these two terpene synthases and the quantity of the related terpenes produced in leaves. Pelargonium transformation efficiency by Agrobacterium tumefaciens was tested in order to complete the functional characterization of the genes. In a second part, we have analyzed the essence of 64 species and cutivars of Pelargonium having very different fragrances like lemon, mint, rose, apricot, pine, spices… With different statistical tools (PCA, discriminant analysis…), we have highlighted the links between the biochemistry of these species and cultivars, their odors and their phylogenetic relationships. This worked gave us some interesting ideas for some new crossings. Finally, the last chapter concerns the EO production improvements in Egypt. Thanks to these researches, started 3 years ago, we are improving year after year our EO yield and quality in our 10 Ha R&D plantation. An important work was done to optimize the distillation process and improve the agricultural practices which abled us to reach a yield of 60 kg of EO per hectare. Some other experiments show the effect of the environmental factors such as the temperature on the biosynthesis of several important molecules like citronellol and geraniol, 6,9-guaiadiene and 10-γ-epi-eudesmol

L'utilisation des plantes pour décontaminer les sols pollués par les métaux est une solution respectueuse de l'environnement. Mais le développement de cette technique à grande échelle est encore limité en raison de l'indisponibilité de plantes avec les caractéristiques souhaitées (hyperaccumulation, biomasse élevée et croissance rapide). Les objectifs de ce travail étaient d'évaluer le potentiel de plusieurs cultivars de Pélargonium odorants pour l'extraction du Pb au champ, étudier la disponibilité du plomb en relation avec l'activité rhizosphérique et développer un protocole de transformation génétique. Parmi les six cultivars de Pélargonium odorants testés au champ, trois : Attar of Roses, Clorinda et Atomic Snowflake ont accumulé plus de 1000 mg kg-1 Pb, avec une forte biomasse. Pendant les expérimentations en conditions contrôlées, Attar of roses (le cultivar hyperaccumulateur) acidifie sa rhizosphère et augmente la concentration en COD significativement plus par rapport Concolor Lace (le cultivar non hyperaccumulateur), sans doute en réponse à la pollution métallique. Les concentrations en plomb dans les deux cultivars sont corrélées avec l'extraction au CaCl2. Les analyses par EXAFS et ESEM-EDS ont montré que le plomb présent dans les racines était principalement sous forme de complexes organiques alors que les sulfates de plomb prédominent dans le sol. Parallèlement à ces essais, un protocole de transformation génétique a été mis au point en vue de mieux comprendre les processus biochimiques impliqués dans l'hyperaccumulation et la fonction des gènes, Le système de régénération optimisé se base sur la pré-culture d'explants sur un milieu contenant 10 μM TDZ + 1 mg L-1 de chacun de BAP et NAA suivie par l'enlèvement de TDZ du milieu de culture. La kanamycine et l'hygromycine se sont avérés être de bons marqueurs sélectifs pour le Pélargonium. Deux souches d'Agrobacterium, C58 et EHA105 contenant des vecteurs binaires avec des gènes marqueurs hpt et nptII ont été choisis pour des expériences de transformation. Ils ont également le gène codant uidA séquence du gène rapporteur. Après l'infection avec C58, 4 et 107 plantes enracinées sur hygromycine ont été obtenues pour Attar of Roses et Atomic Snowflake, respectivement. Parmi ces plantes enracinées, les quatre plantes d'Attar et 82 d'Atomic Snowflake ont exprimé le Gus dans les feuilles, pétioles, les tiges et les racines comme prévu avec une séquence sous contrôle du promoteur constitutif CaMV 35S. De 20 plantes qui expriment le Gus, 7 plantes se sont avérées être positives après criblage par PCR. Après infection par EHA105, 23 et 133 plantes enracinées ont été obtenues après sélection sur kanamycine, mais aucune n'a démontré d'activité GUS. Seule des expériences d'empreintes par Southern blotting permettront de corréler le nombre d'insertions et niveau de l'expression dans ces différents événements de transformation. / Metal removal from contaminated soils using plants can provide an environment friendly solution. However, its successful application on a large scale is still limited due to unavailability of plants with desired set of characteristics i.e. hyperaccumulation, high biomass and rapid growth. The objective of this work was to assess the potential of scented Pelargonium cultivars for lead (Pb) extraction under field conditions, plant induced rhizosphere changes, soil factors influencing availability of Pb and to develop an efficient genetic transformation protocol for the selected cultivars. Of the six scented Pelargonium cultivars field-tested, three cultivars (Attar of Roses, Clorinda and Atomic Snowflake) accumulated more than 1000 mg Pb kg-1 DW, with high biomass reaching up to 45 tons ha-1 y-1 dry matter. During assays in controlled conditions, Attar of roses (Pb hyperaccumulator) significantly acidified its rhizosphere and increased Dissolved Organic Carbon (DOC) concentration as compared to Concolor Lace (non-accumulator), probably due to enhanced exudation in response to the metal stress. Lead concentrations in both cultivars were best correlated with CaCl2 extracted Pb. Extended X-ray Absorption Fine Structure (EXAFS) and Environmental Scanning Electron Microscopy-Energy Dispersive x-ray Spectroscopy (ESEM-EDS) demonstrated that Pb was mainly complexed to organic acids within plant tissues whereas the dominant form in soil was PbSO4. Parallel to the soil-plant Pb transfer assays, a genetic transformation protocol was optimized in view of better understanding biochemical processes involved in lead hyperaccumulation and gene function, in the future. The best regeneration scheme was based on the pre-culture of explants on 10 μM TDZ (Thidiazuron) in addition to 1 mg L-1 each of N6-benzylaminopurine (BAP) and α- naphthaleneacetic acid (NAA), followed by removal of TDZ from the culture medium. Kanamycin and hygromycin proved to be efficient selectable markers for genetic transformation. Two Agrobacterium strains, C58 and EHA105 harboring binary vectors carrying the selectable marker genes hpt and nptII were chosen for transformation experiments. They also contained the uidA gene coding sequence as reporter gene. After infecting with C58, 4 and 107 rooted plants on hygromycin-containing medium were obtained for Attar and Atomic cultivars, respectively. The four Attar plants and 82 Atomic plants expressed Gus in leaves, petioles, stems and roots as expected with a sequence driven by the 35S constitutive promoter. Polymerase Chain Reaction (PCR) screening was performed on Gus positive plants and 2 and 20 plants of Attar and Atomic were screened as PCR positive, respectively. After infection with EHA105, 23 and 133 rooted plants were obtained on kanamycin selection medium but none of these expressed Gus. Southern hybridization patterns will enable to correlate gene copy numbers to expression levels in these different events. The optimized protocols could be used for understanding molecular mechanisms of Pb accumulation and improvement in phytoextraction technique.

M. Tech. (Department of Chemistry and Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Context: Pelargonium alchemilliodes L L' Her is an evergreen shrub, cultivated principally for the medicinal essence and decoction in Southern Africa for the treatment of skin problems, and wounds. Objective: the aim of the study was to optimize the extraction of phenolics and flavonoids from P. graveolens by response surface methodology with particular attention on the proliferative and cytotoxic effects on human keratinocytes, as well as the antioxidant and antibacterial activities and also to isolate active compounds. Materials and Methods: The optimization was achieved by Box-Behnken design. Extract, metabolite yields, and minimal inhibitory concentrations (MIC) were determined by gravimetric, spectrophotometric, and microdilution methods, respectively. The antiradical potentials were evaluated using the phosphomolybdate. 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and lipid peroxidation assays, the diterpenoids were isolated and purified using open column chromatography, PTLC, and characterized with FTIR, NMR. The kinetics of the lipid protective activity was studied and fitted into models. The proliferative and cytotoxic effects were evaluated using the CellTiter® Blue cell viability and lactate dehydrogenase assay. Results: The regression coefficient r2 ≥ 0.9775 indicated a close correlation between actual and predicted values of the responses. The ideal parameter for the extraction of phenolics and flavonoids by macerations was determined as an extraction time: 9.63-12.01 h, material mass: 2.62-3.00 g, agitation speed: 143.11-197.11 rpm, and solvent volume: 68.06-69.87 mL. The optimal extractable acetone and methanol crude, flavonoids, and phenolic are (28.87±2.15%, 24.11±1.15%), (7.11±1.03 mg QE/g, 5.98±0.87 mg QE/g) and (58.08±0.88 mg GAE/g, 55.91±1.15 mg GAE/g), respectively. The detected different chemical groups of polyphenolic compounds such as alkaloids, saponins , sterols, terpenoids, flavonoids, tannins, phenols and cardiac glycosides from methanol and acetone extracts were in correlation with optimized yields. Two triterpenoids compounds 1-hydroxy-30-norlanosta-6, 8-diene and 1 2,3,4a,8,9,10,10a-octahydro-2-(2-hydroxypent-4-enyl)-4a-vinyl-1H-benzo[c]chromen-6(10bH)-one were isolated form methanol extracts. The main components of essential oils were citronellal (5.99%), citranellol (26.2%), geraniol (8.56%), citronellyl butyrate (20.3%), trans-farnesol (9.53%) and they were characterized by high amounts of oxygenated hydrocarbons (67.6%), followed by sesquterpene hydrocarbons and oxygenated sesquiterpene (9.32%) and the least being mornoterpene hydrocarbons (5.20%). Total antioxidant capacity and reducing power were comparable to standard gallic acid, while the antiradical activity has IC50 value of 0.18±0.03-8.98±0.15 mg/mL. Further, the lipid protective revealed a dose-dependent activity fitting into a pseudo-second-order kinetic model. MIC value of 1.56 mg/mL for extracts was registered against Staphylococcus aureus and salmonella typhi comparable to chloramphenicol. There was a significant (P < 0.05) increase in cell proliferation and viability when the extract was administered at concentrations of ≤50 μg/mL. However, at ≥100 μg/mL concentrations at ≤ 1000 μg/mL for essential oil exhibited a significnt cytotoxicity in comparison to the untreated cell. Conclusion: These biological activities are confirmation of the phytomedicinal application and possible source of pharmaceutical compounds. However, administration of the decoction should take into cognizance the antiproliferative effect at doses ≥100 μg/mL as well as the potential to induce and maintain keratinocyte proliferation at low concentration with an eye on the antiproliferative effect at concentrations ≥100 μg/mL, except the P. Alchemilliodes essential oils at ≤ 1000 μg/mL.

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)

Aufgrund seines Nährstoffaneignungsvermögens und Stimulierung des Pflanzenmetabolismus kann der Arbuskuläre Mykorrhiza (AM) Pilz im Gartenbau nutzbringend eingesetzt werden. Der Fokus der Arbeit liegt auf den Möglichkeiten des AM Pilzes a) pflanzenernährerische Probleme zu lösen, b) die Blütenbildung bei Zierpflanzen zu steigern und c) das Gesundheitspotential von Gemüse für den Menschen zu erhöhen (sekundäre Pflanzenmetaboliten). Zur Lösung pflanzenernährerischer Probleme wurden Porree, Pelargonie und Poinsettie auf Torf-Substraten mit 20% und 40% Kompostzusatz untersucht. Ferner wurde Salat auf Torf-Substrat mit drei P Behandlungen getestet: substrateigenes P, Rohphosphat und lösliches P. Frühlingszwiebeln und Schnittknoblauch wurden in Nährlösungen auf Perlit mit niedrigem, mittlerem und hohem NH4+/NO3- Verhältnis ernährt. Gemessen wurde die AM Kolonisation, die Trockenmasse und die N, P, K, S, NO3-, Mg und Zn Konzentrationen im Spross. Die Blütenbildung von Pelargonien und Poinsettien wurde auf Torf-Kompost-Substraten untersucht. Der Einfluss auf die sekundäre Metaboliten von Frühlingszwiebeln und Schnittknoblauch wurde zusammen mit drei NH4+/NO3- Verhältnissen geprüft (s.o.). Untersucht wurden Glukose, Fruktose, Saccharose, lösliche Feststoffe und organische Schwefelverbindungen (gemessen als Pyruvat). Eine AM Kolonisation konnte die Nährstoffversorgung der Pflanze verbessern und die Blütenbildung erhöhen. Jedoch profitierten die Pflanzen unter den beschriebenen experimentellen Bedingungen nicht durchgängig in ihrem Wachstum und Metaboliten vom AM Pilz. Die Zugaben von Kompost ermöglichte die Verbesserung der Substratqualität für die Nährstoffversorgung und das Pflanzenwachstum unter ökologischen Gartenbaubedingungen. Der Ertrag von gesundheitsfördernden organischen Schwefelverbindungen konnte in Abhängigkeit von der jeweiligen Allium Spezies, durch eine Variation des Ammonium/Nitrat Verhältnissen und/oder durch einen AM Effekt auf das Wachstum gesteigert werden. / Arbuscular mycorrhizal (AM) fungi can be beneficial for horticultural crops due to their nutrient acquisition properties and stimulation of the plant metabolism. The present work focuses on the prospects of AM fungi a) to solve plant nutritional problems, b) to induce flower development of ornamental plants, and c) to improve the health potential of crop plants for humans. Contribution of AM fungi to plant nutritional problems were investigated with leek, pelargonium and poinsettia plants on peat-based substrates with 20% and 40% compost additions. Moreover, lettuce plants were supplied on peat-based substrates with substrate own P, rock phosphate, or highly soluble P. Bunching onion and chinese chive were propagated on perlite in nutrient solution with low, medium and high NH4+:NO3- ratios. Mycorrhizal colonization, dry weight, and N, P, K, S, NO3-, Mg and Zn concentrations in plants were measured. Mycorrhizal effects on bud and flower development of pelargonium and poinsettia plants were investigated on peat-based compost substrates. Treatment effects on secondary metabolites in bunching onion and chinese chive were determined by exposing mycorrhizal and non mycorrhizal plants to three NH4+:NO3- supply ratios. The metabolites measured were glucose, fructose, and sucrose, total soluble solids, and organosulfur compounds (measured as pyruvic acid). Colonization improved plant nutrient status and flower development. Under the described experimental conditions, however, plants did not consistently benefit in growth or plant composition from the mycorrhizal symbiosis. Additions of compost were a means of improving the substrate quality for an increased plant nutrient acquisition and plant growth in organic horticulture. The plant quality of Allium species in respect to organosulfur compounds was increased by taking the individual Allium species into consideration, their specific requirements for an optimal NH4+:NO3- supply ratio, and a possible AM effect on plant growth.

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)