Thesis (M.S.M.E.)--Baylor University, 2008. / Includes bibliographical references (p. 101-102).

Syftet med det här arbetet är att få ökad kunskap om vad ämneslärare och klasslärare har för inställning till NO. Vidare innefattar studien vilka läromedel som används och hur lärarens inställning påverkar läraren till att välja ett specifikt läromedel samt vilka övriga faktorer som påverkar valet av läromedel i NO. Arbetet utgår från begreppsramverket Pedagogical Content Knowledge som innefattar lärares kompetens som en kombination av ämneskunskap och ämnesdidaktik. Utifrån en studie där sex ämneslärare och klasslärare i årskurs 1-3 har intervjuats visar resultatet att det finns en varierad inställning till NO, där intresset till synes är högre hos ämneslärarna än hos klasslärare. Inställningen påverkas främst av intresse och ämneskunskap om det aktuella området. Valet av läromedel påverkas i sin tur av intresset och ämneskunskapen i samband med tid och andra resurser. I slutsatsen kommer sambandet mellan vad som läraren anser sig ha för ämneskunskapen, intresse, vad som anses som inspirerande och vad de har bäst inställning till att undervisa i att redovisas. Ovanstående delar kommer att kopplas till hur dessa i längden påverkar engagemanget för vilket läromedel man väljer att arbeta med. Det här arbetet bidrar med ökade kunskaper om hur ämneslärares och klasslärares inställning skiljer sig åt och hur det påverkar valet av läromedel i NO.

Os produtos naturais desempenham fundamental importância na descoberta de novos fármacos. Muitos destes fármacos originaram-se de produtos naturais microbianos o que demonstra uma extrema eficiência na habilidade dos micro-organismos de sintetizar substâncias químicas com elevado potencial biológico. Sabe-se que os insetos sociais estão sujeitos a condições climáticas e populacionais que aumentam sua susceptibilidade a parasitas. Assim, estes insetos desenvolveram um mecanismo de defesa evolutivo que consiste na associação simbiótica a bactérias capazes de biossintetizar produtos antimicrobianos contra os patógenos. Dispondo dessas informações, o presente trabalho contribuiu com o estudo dos produtos naturais biossintetizados pela bactéria Paenibacillus polymyxa ALLI-03-01, provável simbionte da espécie de abelha sem ferrão Melipona scutellaris. Os micro-organismos foram isolados de colônias mantidas no Departamento de Biologia da FFCLRP-USP. Todas as linhagens foram testadas contra dois fungos entomopatogênicos - Beauveria bassiana e Metarhizium anisopliae; os que apresentaram atividade antifúngica tiveram seus extratos em acetato de etila e metanol preparados e foram avaliados em ensaios antiparasitários contra Trypanosoma cruzi e Leishmania donovani. A bactéria ALLI-03-01, isolada do alimento larval, foi escolhida para os estudos químicos, pois apresentou satisfatória atividade antifúngica e proeminente atividade antiparasitária. Este micro-organismo foi identificado pelo sequenciamento do gene 16S rRNA como sendo a bactéria Paenibacillus polymyxa, conhecida pela alta capacidade de produção de metabólitos secundários. Para conhecer seu metabolismo, esta bactéria foi cultivada em meio ISP-2 ágar e líquido e do extrato em acetato de etila do cultivo fermentativo de P. polymyxa ALLI-03-01 foi isolado o ácido L-3-fenil lático. As atividades antifúngicas dos padrões comerciais dos ácidos D-(+)- e L-(-)- 3-fenil lático foram testadas frente ao fungo entomopatogênico B. bassiana e apenas o enantiômero L apresentou atividade antifúngica. Do extrato em metanol do cultivo em meio sólido do mesmo micro-organismo foram identificados em mistura, principalmente por MALDI-TOF MS/MS, nove diferentes ciclolipodepsipeptídeos, da classe das Fusaricidinas. As fusaricidinas são conhecidas por apresentarem alta atividade anti-microbiana, que foi comprovada quando duas frações compostas por diferentes fusaricidinas foram testadas frente ao fungo entomopatogênico Metarhizium anisopliae e contra a bactéria patogênica Paenibacillus larvae. As frações apresentaram resultados satisfatórios de inibição contra ambos os patógenos, dessa maneira as fusaricidinas e a produção seletiva do ácido L-3-fenil lático por esta bactéria podem ser vias de proteção da colônia, evitando que patógenos destruam-na e permitindo o desenvolvimento sadio de larvas. Sendo assim, este trabalho relata pela primeira vez a possível relação simbiótica existente entre o micro-organismo Paenibacillus polymyxa ALLI-03-01 e a colônia de abelhas Melipona scutellaris, bem como a produção por este micro-organismo de compostos com alta atividade antimicrobiana que podem exercer o controle de parasitas dentro da colônia, estabelecendo assim, uma possível relação ecológica entre o micro-organismo destacado e as abelhas Melipona scutellaris. / Natural products play an important role in drug discovery. Several drugs have been discovered or developed from microbial natural products hits and leads showing a great efficiency of microorganisms to synthesize compounds with high biological potential. It is known that social insects are subjected to climate and population conditions that increase their susceptibility to parasites. Therefore, these insects have developed an evolutionary defense mechanism consisting in symbiotic association with bacteria capable of biosynthesize antimicrobial products against pathogens. In this context, this study contributed to the knowledge of natural products biosynthesized by the bacterium Paenibacillus polymyxa ALLI-03-01, a probable symbiont of the stingless bee Melipona scutellaris. Microorganisms were isolated from colonies maintained at the Department of Biology of FFCLRP-USP. All strains were tested against two entomopathogenic fungi - Beauveria bassiana and Metarhizium anisopliae; strains that showed antifungal activity had their ethyl acetate and methanol extracts prepared and evaluated against the parasites Trypanosoma cruzi and Leishmania donovani. The bacterium ALLI-03-01, isolated from larval food, was selected for chemical studies, due to its satisfactory antifungal activity and outstanding antiparasitic activity. This microorganism was identified by gene 16S rRNA sequencing as Paenibacillus polymyxa, a bacterium recognized for its ability of biosynthesize secondary metabolities. This bacterium was cultivated in ISP-2 agar and liquid medium and from the ethyl acetate extract of P. polymyxa ALLI-03-01 cultivated in liquid medium L-3-phenyllactic acid was isolated. The antifungal activity of comercial standards of D-(+)- and L-(-)-phenyllactic acid were tested against entomopathogenic fungus B. bassiana and only the L enantiomer presented antifungal activity. Nine cyclo lipodepsipeptides known as fusaricidins were identified in mixture using MALDI-TOF-MS/MS from the methanol extract of ALLI-03-01. The fusaricidins are recognized for their high antimicrobial activity, which was confirmed when two fractions composed by different fusaricidins were tested against the entomopathogenic fungus Metarhizium anisopliae and the pathogenic bacterium Paenibacillus larvae. The fractions showed satisfactory inhibition against both pathogens. Therefore, fusaricidins and selective production of L-3-phenyllactic acid by the bacterium may be colony protection resources, preventing the action of pathogens and allowing healthy larvae development. Therefore, this study reports for the first time a possible symbiotic relationship between Paenibacillus polymyxa ALLI-03-01 and the colony of M. scutellaris bees, as well as production of compounds with high antimicrobial activity by this microorganism that might act as parasite control agents within the colony, establishing a possible ecological relationship between the bacterium and M. scutellaris bees.

Os produtos naturais desempenham fundamental importância na descoberta de novos fármacos. Muitos destes fármacos originaram-se de produtos naturais microbianos o que demonstra uma extrema eficiência na habilidade dos micro-organismos de sintetizar substâncias químicas com elevado potencial biológico. Sabe-se que os insetos sociais estão sujeitos a condições climáticas e populacionais que aumentam sua susceptibilidade a parasitas. Assim, estes insetos desenvolveram um mecanismo de defesa evolutivo que consiste na associação simbiótica a bactérias capazes de biossintetizar produtos antimicrobianos contra os patógenos. Dispondo dessas informações, o presente trabalho contribuiu com o estudo dos produtos naturais biossintetizados pela bactéria Paenibacillus polymyxa ALLI-03-01, provável simbionte da espécie de abelha sem ferrão Melipona scutellaris. Os micro-organismos foram isolados de colônias mantidas no Departamento de Biologia da FFCLRP-USP. Todas as linhagens foram testadas contra dois fungos entomopatogênicos - Beauveria bassiana e Metarhizium anisopliae; os que apresentaram atividade antifúngica tiveram seus extratos em acetato de etila e metanol preparados e foram avaliados em ensaios antiparasitários contra Trypanosoma cruzi e Leishmania donovani. A bactéria ALLI-03-01, isolada do alimento larval, foi escolhida para os estudos químicos, pois apresentou satisfatória atividade antifúngica e proeminente atividade antiparasitária. Este micro-organismo foi identificado pelo sequenciamento do gene 16S rRNA como sendo a bactéria Paenibacillus polymyxa, conhecida pela alta capacidade de produção de metabólitos secundários. Para conhecer seu metabolismo, esta bactéria foi cultivada em meio ISP-2 ágar e líquido e do extrato em acetato de etila do cultivo fermentativo de P. polymyxa ALLI-03-01 foi isolado o ácido L-3-fenil lático. As atividades antifúngicas dos padrões comerciais dos ácidos D-(+)- e L-(-)- 3-fenil lático foram testadas frente ao fungo entomopatogênico B. bassiana e apenas o enantiômero L apresentou atividade antifúngica. Do extrato em metanol do cultivo em meio sólido do mesmo micro-organismo foram identificados em mistura, principalmente por MALDI-TOF MS/MS, nove diferentes ciclolipodepsipeptídeos, da classe das Fusaricidinas. As fusaricidinas são conhecidas por apresentarem alta atividade anti-microbiana, que foi comprovada quando duas frações compostas por diferentes fusaricidinas foram testadas frente ao fungo entomopatogênico Metarhizium anisopliae e contra a bactéria patogênica Paenibacillus larvae. As frações apresentaram resultados satisfatórios de inibição contra ambos os patógenos, dessa maneira as fusaricidinas e a produção seletiva do ácido L-3-fenil lático por esta bactéria podem ser vias de proteção da colônia, evitando que patógenos destruam-na e permitindo o desenvolvimento sadio de larvas. Sendo assim, este trabalho relata pela primeira vez a possível relação simbiótica existente entre o micro-organismo Paenibacillus polymyxa ALLI-03-01 e a colônia de abelhas Melipona scutellaris, bem como a produção por este micro-organismo de compostos com alta atividade antimicrobiana que podem exercer o controle de parasitas dentro da colônia, estabelecendo assim, uma possível relação ecológica entre o micro-organismo destacado e as abelhas Melipona scutellaris. / Natural products play an important role in drug discovery. Several drugs have been discovered or developed from microbial natural products hits and leads showing a great efficiency of microorganisms to synthesize compounds with high biological potential. It is known that social insects are subjected to climate and population conditions that increase their susceptibility to parasites. Therefore, these insects have developed an evolutionary defense mechanism consisting in symbiotic association with bacteria capable of biosynthesize antimicrobial products against pathogens. In this context, this study contributed to the knowledge of natural products biosynthesized by the bacterium Paenibacillus polymyxa ALLI-03-01, a probable symbiont of the stingless bee Melipona scutellaris. Microorganisms were isolated from colonies maintained at the Department of Biology of FFCLRP-USP. All strains were tested against two entomopathogenic fungi - Beauveria bassiana and Metarhizium anisopliae; strains that showed antifungal activity had their ethyl acetate and methanol extracts prepared and evaluated against the parasites Trypanosoma cruzi and Leishmania donovani. The bacterium ALLI-03-01, isolated from larval food, was selected for chemical studies, due to its satisfactory antifungal activity and outstanding antiparasitic activity. This microorganism was identified by gene 16S rRNA sequencing as Paenibacillus polymyxa, a bacterium recognized for its ability of biosynthesize secondary metabolities. This bacterium was cultivated in ISP-2 agar and liquid medium and from the ethyl acetate extract of P. polymyxa ALLI-03-01 cultivated in liquid medium L-3-phenyllactic acid was isolated. The antifungal activity of comercial standards of D-(+)- and L-(-)-phenyllactic acid were tested against entomopathogenic fungus B. bassiana and only the L enantiomer presented antifungal activity. Nine cyclo lipodepsipeptides known as fusaricidins were identified in mixture using MALDI-TOF-MS/MS from the methanol extract of ALLI-03-01. The fusaricidins are recognized for their high antimicrobial activity, which was confirmed when two fractions composed by different fusaricidins were tested against the entomopathogenic fungus Metarhizium anisopliae and the pathogenic bacterium Paenibacillus larvae. The fractions showed satisfactory inhibition against both pathogens. Therefore, fusaricidins and selective production of L-3-phenyllactic acid by the bacterium may be colony protection resources, preventing the action of pathogens and allowing healthy larvae development. Therefore, this study reports for the first time a possible symbiotic relationship between Paenibacillus polymyxa ALLI-03-01 and the colony of M. scutellaris bees, as well as production of compounds with high antimicrobial activity by this microorganism that might act as parasite control agents within the colony, establishing a possible ecological relationship between the bacterium and M. scutellaris bees.

A novel hetero Diels-Alder reaction has been developed to facilitate the synthesis of annelated pyridines as models for pyridoacridine alkaloids. The key reaction is based on an intramolecular Diels-Alder reaction of an aza-1,3-butadiene with an appropriate dienophile, to yield the desired annelated pyridine. An extension of this methodology is to exploit the Eglinton copper (IT) dimerisation of terminal acetylenes. This allows for a unique double intramolecular hetero Diels-Alder reaction, where four new rings are formed in one step. This allows for a facile route to annelated bipyridines. Ultimately this methodology has led to an approach to the total synthesis of the natural product eilatin, a member of the class of compounds known as pyridoacridines.

PhD (Chemistry) / Department of Chemistry / Terminalia sericea Burch. ex. DC (Combretaceae) is one of the 50 most popular medicinal plants in Africa. The fruit, leaves, stems and roots are commonly used for the treatment of cough, skin infections, diabetes, diarrhoea, venereal diseases and tuberculosis. However, the roots are most commonly used in the preparation of traditional medicines. Pharmacological studies have revealed that the crude root extracts display antibacterial activity against several Gram-positive and Gram-negative bacteria. Anolignan b, termilignan b and arjunic acid are reported to be the major antibacterial constituents present in the roots. Other compounds isolated from the roots include resveratrol-3-rutinoside, sericic acid, sericoside and arjunglucoside I. Authorities worldwide, including the Medicines Control Council of South Africa, have begun to regulate herbal drugs sold in the form of commercial formulations. Quality control of herbal drugs is challenging, since the chemical profiles of the raw materials may vary, depending on the origin of the plant material and the way that it was handled and processed. The chemistry, in turn, impacts on the safety and efficacy of the plant material. To date, there are no available data on parameters that can be used to standardise the quality of T. sericea raw materials. The aim of this study was therefore to provide information on the variation of the chemical constituents that contribute to the biological effects of the roots of T. sericea and also establish its safety. Since the compounds previously isolated from the roots were not commercially available, isolation of the major constituents of the roots was undertaken to obtain analytical standards. A crude dichloromethane:methanol (1:1) extract was initially fractionated using silica gel column chromatography, where after, some of the fractions were further purified using silica gel and Sephadex LH-20 column chromatography. Final purification of the enriched fractions was achieved using preparative high performance liquid chromatography coupled with mass spectrometry (prep-HPLC-MS). The structures of these compounds were subsequently elucidated using one- and two- dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry and identified as sericic acid (340 g), sericoside (500 g), resveratrol-3-rutinoside (240 mg) and arjunglucoside I (74 mg). The chemical variation within the crude root extracts of samples (n = 42) from ten populations in the Limpopo Province of South Africa, was determined using ultra performance liquid chromatography-mass spectrometry (UPLC-MS). A method was developed for the simultaneous determination of sericic acid, resveratrol-3-rutinoside, sericoside and arjungluicoside I in the extracts using UPLC with photodiode array detection (PDA). The method was validated according to the guidelines of the International Council for Harmonisation (ICH). A regression coefficient (R2) of 0.998 was obtained for sericic acid, resveratrol-3- rutinoside and arjunglucoside I, while the R2 value for sericoside was 0.999, indicating a linear relationship between the concentration and the detector response. Satisfactory limits of detection for sericic acid (25.2 ng/mL), sericoside (11.6 ng/mL), resveratrol-3-rutinoside (23.3 ng/mL) and arjunglucoside I (8.81 ng/mL) were determined. Recoveries of 98 % and 80% were obtained for samples spiked with 12.5 μg/mL and 25 μg/mL of resveratrol-3-rutinoside, respectively, indicating that the method is accurate. The intra- and inter-day variation in resveratrol-3-rutinoside concentration, measured over three days, indicated excellent analytical precision, since all the relative standard deviations were below 0.70 %. The quantitative data revealed that sericic acid (1.59 to 8.45 mg/g), sericoside (2.07 to 20.17 mg/g), resveratrol-3-rutinoside (0.65 to 29.82 mg/g) and arjunglucoside I (0.86 to 8.44 mg/g dry weight) were the major constituents of the root samples, but their concentrations were highly variable. Chemometric analysis of the aligned UPLC-MS data was used to investigate similarities and differences in the chemical profiles of the samples using an untargeted approach. A principal component analysis (PCA) model was constructed and subsequently hierarchical cluster analysis (HCA) indicated the presence of two main groups, which were found to be independent of the populations to which the samples belong. Classes, based on the HCA class identifiers, were subsequently assigned to the samples, and an orthogonal projection to latent structures-discriminant analysis (OPLS-DA) model was then constructed, (R2 cum = 0.996 and Q2 cum = 0.967). The corresponding loadings plot allowed sericic acid, sericoside and resveratrol-3-rutinoside to be identified as biomarkers associated with the first group. Quantitative, rather than qualitative differences were responsible for the observed clustering pattern. Techniques that could be applied in quality control protocols for T. sericea root were investigated. High performance thin layer chromatography (HPTLC) analysis of the root extracts was optimised by testing different developing solvents and visualization reagents. The presence of the sericic acid (Rf = 0.80), sericoside (Rf = 0.49) and resveratrol-3-rutinoside (Rf = 0.36) were clearly visible on the plates. There were visible variations in the concentrations of resveratrol-3-rutinoside in representative samples from the 10 populations, corresponding to the UPLC results. The powdered samples were then analysed by mid-(MIR) infrared spectroscopy. Chemometric analysis of the data revealed no definitive clustering pattern. Partial least squares-discriminant analysis (PLS-DA) calibration models were established from the MIR spectral data combined with the accurate UPLC-values, for the prediction of the sericoside (R2Y = 0.848, Q2 = 0.757, RMSEP = 2.70 mg/g) and resveratrol-3-rutinoside (R2Y = 0.794, Q2 = 0.695, RMSEP = 4.37 mg/g) concentrations in powdered root samples. The antibacterial activities of the root extracts, column fractions and isolated compounds were determined using three Gram-positive and five Gram-negative bacteria, all selected due to their ability to cause intestinal and skin disorders. Extracts and fractions containing high concentrations of sericic acid exhibited the highest activities against Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 27858), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12223) and Shigella sonnei (ATCC 9292). The pure compound (sericic acid) was highly active against S. sonnei (MIC 0.078 μg/mL), a Gram- negative bacterium. There were no variations in the activity of the crude extracts against B. cereus and P. aeruginosa, while the MIC values obtained against S. typhi were variable and ranged from 0.25 to 1.0 mg/mL. Sericoside and resveratrol-3-rutinoside did not display any activity. The anti-oxidant activities were evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) and reducing power assays. The anti-oxidant assays revealed that resveratrol-3- rutinoside exhibited lower activity (DPPH = 186 μg/mL; RP = 184 μg/mL) compared to the crude extract (DPPH = 22.3 μg/mL; RP = 24.4 μg/mL) and ascorbic acid (DPPH = 11.3 μg/mL, RP = 145 μg/mL). Sericic acid and sericoside did not display any anti- oxidant activities. The variation in the anti-oxidant activities (4.58 to 26.0 μg/mL) of the samples from different populations was an indication of chemical variability. A toxicity study of the raw powdered plant material was conducted using vervet monkeys (Chlorocebus pygerythrus). Biochemical analysis (liver function tests, kidney function tests and hematology), physical and physiological examinations were conducted. The subjects were fed a normal diet supplemented with T. sericea root powder (2.14 g/kg per day) for 120 days, where after the diet was returned to normal (washout) for another 30 days. The treatment groups presented with elevated serum enzymes at Week 4, followed by the reduction of the elevated serum enzymes levels at Week 12. These results indicate short-term hepatotoxic effects, followed by hepatoprotective activity. Reduction of the serum glucose at Week 4 suggests hypoglycemic potential. However, elevated serum creatinine levels indicated possible nephrotoxicity. In conclusion, this study has indicated the variability in the chemical constituents of the roots of T. sericea, which affects the antibacterial and anti-oxidant activities. Sericic acid, resveratrol-3-rutinoside, and sericoside were, for the first time, identified as biomarkers that can be used for the quality control of raw root material to be used in herbal products. Sericic acid was also found to be the main antibacterial constituent of the roots. The hepatoprotective, nephrotoxic and hematotoxic effects observed in monkeys to which the root powder had been administered is cause for concern. / NRF