Thesis (MSc)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Bacterial and fungal pathogens have developed numerous defence mechanisms against antimicrobial chemical agents, and resistance to old and new produced drugs are on the rise. Discovery of natural products derived from plants with diverse chemical structures and novel mechanisms of action to treat these notorious pathogens is a priority. Biotechnology (discussed in Chapter 1) has much to offer as a pharmacological tool and in the general study of medicinal plants. The Genus Salvia (Lamiaceae) has gathered much interest as these plants manufacture a diverse range of secondary metabolites including flavonoids, tannins and terpenoids. Of particular interest are the terpenoids which are largely implicated in the efficacy of Salvia plants as traditional medicines contributing to their pharmacological actions (discussed in Chapter 2). Due to the importance of these plants as herbal remedies, in this study, biotechnological techniques such as tissue culture and Agrobacterium-mediated transformation were applied on Salvia runcinata L.f., a South African medicinal plant, in an attempt to enhance the metabolomic profile and its bioactivity. Like so many other sages, S. runcinata has been used in folk medicine to treat a variety of ailments. Application of biotechnology was viewed as an important value adding platform for this species, assisting with its commercialisation for the cosmeceutical and pharmaceutical industries. Therefore the study had three foci: (1) to determine the seed germination behaviour and optimal conditions for micropropagation; (2) to develop a protocol that would be efficient whilst being simple for genetic transformation; and lastly, (3) to conduct phytochemical studies on in vitro generated S. runcinata transgenic hairy root and in vitro organ cultures by comparing these to glasshouse plants as potential therapeutic sources of natural compounds used in the treatment of infections in plants and humans. Data generated is thus summarised in three research chapters and Chapter 3 describes the formulated procedures assisting with in vitro seed germination and micropropagation of S. runcinata. The efficacy of smoke and scarification treatments for germination improvement was initially tested coupled to the evaluation of different hormonal combinations and different explant types which would aid with inducing adventitious shoot formation in vitro. The most effective germination treatment proved to be a 3 min exposure of seeds to 25% (w/v) H2SO4 combined with a concentration of 10-5 M smoke solution, resulting to more than 80% germination. Shoot proliferation was significantly higher using nodal explants with the addition of 4.43 μM BA. The protocol established in this part of the study is viable for large scale commercial production of S. runcinata as it would yield 1296 to 46656 viable plants in 4 to 6 months from one nodal explant. Micropropagation was applied also as a pre-emptive measure to ease pressure on the wild plants as the demand for S. runcinata is anticipated to increase due to its growing economic value as it is one of two South African sages with epi-α-bisabolol that is sought after by the pharmaceutical and cosmeceutical industries. This makes the protocol developed in this part of the study suitable for ex situ conservation of S. runcinata plantlets. Evaluations on the transgene transfer capacities of two different agropine strains (A4T and LBA 9402) of Agrobacterium rhizogenes to induce hairy root cultures of S. runcinata explants on nodal and leaf explants were conducted (reported in Chapter 4). Hairy roots formed 3 to 4 weeks after inoculation of the explants and these agropine strains showed different abilities for genetic transformation with the LBA 9402 strain producing significantly more roots on each explant compared to the A4T strain (P=0.0075). However, none of the LBA 9402 derived clones and only 2 clones generated through A4T transformation survived subculturing. The polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the presence and transcription (respectively) of rol A, rol B, rol C and ags genes which are mobilised from the transfer-DNA (T-DNA) fragment of the root-inducing (Ri) plasmid of A. rhizogenes to the plant genome during transformation. The two A4T clones, termed here A4T3 and A4T5, were stably transformed, Southern blot analysis using rol A as a probe further validated the integration of one copy of the rol A gene. Transformed hairy roots, untransformed roots from tissue cultured plants, tissue culture-derived plants and glasshouse-grown plants were profiled for secondary metabolites by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) in Chapter 5. In this part of the study, it is clear that the use of tissue culture as a propagation system did not negatively affect the volatile compound profile of S. runcinata and plants had a similar essential oil content to that reported by Kamatou et al. (2008), leading to a conclusion that in vitro plants maintained their biochemical integrity even under an alternative micro-controlled environment. Similarly to others, Ri-transformation was explored as an avenue to alter secondary metabolism creating inter-clonal variation. Transformed clones were distinguishable, displaying more of some primary metabolites including sucrose, galactose, sorbose and fructose than the leaf extracts. With the current GC-MS methods used, this clear distinction was not obvious at the secondary metabolite level. In general, solvent extracts (acetone and methanol:dichloromethane (MetOH: DCM) (1:1 v/v) exhibited good to moderate antibacterial activity with the minimum inhibitory concentration (MIC) values ranging from 0.39 to 0.78 mg ml-1. However, in vitro plant cultures were the most potent against two Gram-negative bacterial strains: Escherichia coli (ATCC 11775) and Klebsiella pneumoniae (ATCC 13883), and two Gram-positive bacterial strains: Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 12600). The hairy root extracts did not show any activity against fungi, Fusarium subglutinans (MRC 0115) and Fusarium proliferatum (MRC 6908). Micropropagation therefore proves to be an interesting avenue for commercial production of S. runcinata, supplying plants with an improved pharmacological activity. Hence the biotechnological approach applied here is a viable strategy for the production of medicinal bioactives from S. runcinata. / AFRIKAANSE OPSOMMING: Bakterieë en fungi patogene het baie verskeie meganismes ontwikkel teen antimikrobiese chemiese agente, en weerstand teen ou en nuwe chemise stowwe is besig om te vergroot. Daarom is dit belangrik om natuurlike plantaardige produkte met diverse chemiese strukture en unieke werkings meganismes te ontdek waarmee hierdie berugte patogene beveg kan word. Biotegnologie (wat in Hoofstuk 1 bespreek word) kan gebruik word as 'n farmakologiese hulpmiddel in die algemene studie van plante. Die Klas (Genus) Salvia (Lamiaceae) het al baie aandag getrek aangesien hierdie plante 'n wye reeks sekondêre metaboliete vervaardig wat flavonoïede, tanniene en terpenoïede insluit. Veral van belang is die terpenoïde wat betrokke is by die doeltreffendheid van die Salvia plante as tradisionele medisyne, aangesien dit bydra tot hulle farmalogiese aksie (wat in Hoofstuk 2 bespreek word). Aangesien hierdie plante sulke belangrike kruie is, word daar in hierdie studie, biotegnologiese tegnieke soos die kweek van weefsel en Agrobacterium-bemiddelde transformasie op Salvia runcinata L.f. toegepas om die metabologiese profiel en die bioaktiwiteit daarvan te verbeter. Soos baie van die salies is S. runcinata tradisioneel dikwels gebruik om allerhande siektetoestande te behandel. Die toepassing van biotegnologie word beskou as 'n belangrike manier om waarde by te voeg sodat hierdie plant kommersieei deur die kosmetiese en farmakeutiese bedrywe gebruik kan word. Daarom is daar op drie dinge gefokus: (1) die ontkiemings gedrag van saad en die optimale toestande vir mikrovoortplanting (2) die ontwikkeling van protokol wat eenvoudig maar doeltreffend is vir genetiese transformasie, en die (3) fito-chemise studies op in vitro genereerde S. runcinata transgeniese harige wortels en in vitro orgaan kwekings deur om hulle te vergelyk met kweekhuis plante as potentiële terapeutiese bronne van natuurlike samestellings vir die behandeling van infeksies in beide plante en mense. Die data wat gegenereer is, is opgesom in drie hoofstukke, en in Hoofstuk 3 word die prosedures wat gebruik word in die in vitro saad ontkieming en die mikro voortplanting van S. runcinata, bespreek. Die doeltreffendheid van rook en skarifikasie behandeling vir die verbetering van ontkieming is eers getoets en gekoppel aan die evaluering van verskillende hormoonkombinasies en verskillende eksplant tipes wat lei tot die formasie van uitloopsels in vitro. Daar is gevind dat die effektiefste behandeling vir ontkieming, 'n 3-minuut blootstelling van saad aan 25% (w/v) H2SO4 gekombineer met 'n konsentrasie 10-5 M rook oplossing is. Dit het gelei tot meer as 80% ontkieming. Daar was baie meer uitloopsels toe nodale eksplante gebruik is met die byvoeging van 4.43 μM BA. Die proktokol wat hier gevestig is, kan op groot skaal gebruik word vir die kommersiële produksie van S. runcinata, want 1296 tot 46656 lewensvatbare plante kan binne 4 ot 6 maande van een nodale eksplant gemaak word. Mikro voortplanting is toegepas as 'n voorkomende maatreel om die druk op die natuur te verminder omdat daar verwag word dat die vraag na S. runcinata sal toeneem na gelang die groeiende ekonomiese waarde daarvan toeneem. Dit is een van twee Suid-Afrikaanse salies met epi-α-bisabolol wat deur die farmakeutiese en die kosmetiese bedrywe gebruik word. Dit beteken dat die protokol wat hier ontwikkel is, geskik is vir die ex situ bewaring van S. runcinata plante. Die transgeen oordrag van twee verskillende agropien tipes (A4T and LBA 9402) van Agrobacterium rhizogenes is geevalueer (en in Hoofstuk 4 beskryf). Harige wortels het 3 tot 4 weke na die inenting van die eksplante gevorm en hierdie agropien tipes het verskillende vermoëns vir genetiese transformasie getoon, met die LBA 9402 tipe wat baie meer wortels op elke eksplant voorgebring het in vergelyking met die A4T tipe (P=0.03116). Geen van die LBA 9402-afgeleide klone en slegs 2 klone wat deur A4T transformasie genereer is, het oorleef. The polimerase ketting reaksie (PCR) en die teenoorgestelde trenskriptasie-polimerase (RT-PCR) ketting reaksie het die teenwoordigheid en transkipsie (onderskeidelik) van rol A, rol B en rol C en ags gene, wat oorgedra word deur die oordrag DNA (T-DNA) fragment van die wortel induserende (Ri) plasmied van A. rhizogenes na die plant genoom tydens transformasie, bevorder. A4T klone, hier A4T3 and A4T5 genoem, is stabiel transformeer. Southern blot ontleding het met die gebruik van rol A, die integrasie van een kopie van die rol A geen, bevestig. In Hoofstuk 5 is transformeerde harige wortels, ongetransformeerde wortels van weefsel gekweekte plante, weefsel gekweekte plante, en kweekhuis plante deur dun-laag chromatografie (TLC) en gas-chromatografie-massa spektrometrie (GC-MS) geprofiel vir sekondêre metaboliete. In hierdie deel van die studie is dit duidelik dat die gebruik van weefsel kwekery as 'n voortplantsisteem nie 'n negatiewe effek gehad het op die vlugtige samestelling profiel van S. runcinata nie en dat plante 'n sootgelyke essentiële olie inhoud het as wat deur Kamatou et al. (2008) bevind is. Dit lei tot die gevolgtrekking dat in vitro plante hulle biochemiese integriteit behou selfs onder alternatiewe mikro-beheerde omgewings. Ri-transformasie is ondersoek as 'n manier om sekondêre metabolisme te verander om interkloon variasie te skep. Getransformeerde klone kon uitgeken word, aangesien dit meer primêre metaboliete soos sukrose, galaktose en fruktose insluit as die blaar ekstrakte. Hierdie verskil was nie met die huidige GC-MS metodes so duidelik sigbaar op die sekondêre metabolitiese vlak nie. Oor die algemeen toon ekstraksie met asetoon en methanol dichlorometaan (MetOH: DCM) (1:1 v/v) goeie tot gemiddelde antibakteriese aktiwiteit met die minimum remmende konsentrasie (MIC) waardes van 0.39 tot 0.78 mg ml-1. Die in vitro plant kulture het egter sterker weerstand gebied teen twee Gram-negatiewe bakteriese tipes: Escherichia coli (ATCC 11775) en Klebsiella pneumoniae (ATCC 13883), en teen twee Gram-positiewe bakteriese tipes: Bacillus subtilis (ATCC 6051) en Staphylococcus aureus (ATCC 12600). Die harige wortel ekstrakte het geen aktiwiteit teen die swamme, Fusarium subglutinans (MRC 0115) en Fusarium proliferatum (MRC 6908) getoon nie. Mikro-voortplanting is dus 'n interessante manier om S. runcinata kommersieel te produseer aangeien die plante verbeterde farmalogiese aktiwiteit toon. Die biotegnologiese benadering wat hier toegepas word, is 'n praktiese strategie vir die produksie van geneesmiddels van S. runcinata.

Cancer has become a major health problem due to its high rate of morbidity and mortality. Severe side effects associated with most of the available anticancer drugs and the development of the drug resistant cells are the major hurdles limiting their application and therapeutic success. Much attention has thus been focused on natural compounds with minimal or no toxicity in humans and having capacity to suppress proliferation and/or induce apoptosis in cancer cells. Therefore, it is imperative to discover and develop new anticancer drugs. Endophytic fungi though underexploited have remained a rich source of structurally novel and biologically active secondary metabolites. That they are a prolific resource for new compounds in drug discovery is evident from a plethora of reported research findings of the recent past. Those of endophytic fungi especially isolated from medicinal plants with known therapeutic value have especially caught the attention of several research groups worldwide for the production of host associated or novel lead molecules. In this backdrop, the focus of my research work has been centered on exploring the endophytic fungal population associated with Catharanthus roseus in search of production of novel anticancer compounds. Twenty endophytic fungi were isolated from different parts of Catharanthus roseus plant. The ethyl acetate (EA) extracts of three week grown liquid cultures of the individual endophytes were assessed in vitro for their cytotoxic activities in HeLa and HepG2 cancer cells using MTT assay. Of all the isolates thus screened, the EA extract from an isolate identified as Chaetomium nigricolor exhibited significant cytotoxicity in the tested cell lines with a better activity profile against HeLa cells. To understand if the liquid culture filtrate or the mycelium harbored the active cytotoxic principle, the C. nigricolor culture filtrate and mycelia was extracted separately using organic solvents with varying polarity namely ethyl acetate, dichloromethane, chloroform and hexane. These extracts were further tested for cytotoxicity induction in HeLa and MCF-7 cells by MTT assay. The ethyl acetate culture filtrate and mycelial extracts showed best cytotoxic activity on HeLa cells compared to other extracts; therefore, the mycelial and culture filtrate ethyl acetate extracts of C. nigricolor were pooled and was used for further work. Hexane culture filtrate extract of C. nigricolor showed best antiproliferative activity against MCF-7 cells. Thus, the compounds with anticancer potential from both ethyl acetate pooled extracts and hexane culture filtrate extract of C. nigricolor were purified using bioassay guided isolation method. This finally resulted in the isolation of two different anticancer compounds, one each from EA pooled extract and Hexane culture filtrate extracts of C. nigricolor named as compound 1 and compound 2, respectively. Based on various analyses including mass spectrometry (MS and MS-MS) and UV-visible, Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, the compounds 1 and 2 were identified as 1, 2 Bis (diethylamino) ethene-1, 2 diol (BDED) and Nonacos-4-enoic acid (NA), respectively An improved understanding of the cellular responses to chemotherapeutic agents in cancer cells and the underlying molecular mechanisms gains particular relevance in the efforts to improve the clinical outcome of chemotherapeutic agents. Arresting the growth of cancer cells in one way or the other and induction of apoptosis in a drug specific manner is the hallmark of almost all the clinically used anticancer drugs. Towards this end, evaluation of the cytotoxic effects of the newly identified compounds on various human cancer cells was carried out. BDED was examined for in vitro cytotoxicity of against a panel of five human cancer cell lines namely HeLa, A-431, A549, COLO 205, and MCF-7. Among the cell lines screened, HeLa cells were most vulnerable to BDED treatment with an IC50 value of 27 μM. This cytotoxicity was later identified as resulting from apoptosis induction by BDED which was scored by the characteristic events of membrane externalization of phosphatidylserine, cytoplasmic shrinkage and chromatin condensation which were observed in a large majority of the BDED-treated cells. Further detailed studies were performed to delineate the cellular mechanisms of BDED-induced apoptosis in HeLa cells. Analysis of cell cycle progression by propidium iodide (PI) staining revealed BDED-induced cell cycle arrest in the G1 phase of cell cycle. The results indicated that BDED induced a significant ROS generation and a significant loss of mitochondrial membrane potential which were abrogated upon pretreatment of the cells with an antioxidant, N-acetyl cysteine (NAC). These observations suggested the probable involvement of the initial events of BDED-induced ROS production in causing cell death via mitochondria-based intrinsic pathway of apoptosis. In addition, the results from the expression profile of pro- and anti- apoptotic proteins by western blot analysis in the BDED-treated HeLa cells further corroborated this hypothesis. These included an abnormally deregulated cellular abundance of BAX 1 and BCL 2 proteins, elevated levels of APAF-1, and activated cleaved species of procaspases 9 and 3. In addition, a pronounced abundance of cleaved PARP protein was observed in these experiments. Thus, the results suggest that BDED induced apoptosis in HeLa cells via ROS mediated mitochondrial dependent pathway. In parallel, the in vitro cytotoxic effects of NA was also studied, the second antiproliferative compound identified from hexane extracts of C. nigricolor using a panel of four human cancer cell lines - A-431, A549, COLO 205, and MCF 7. NA was thus found to be most potent against MCF 7 (breast cancer) cells. PI staining-based viability assays and microscopic observations showed a dose dependent cytotoxicity of NA on MCF 7 cells. Our data reveled an IC 50 of 40 μM for NA The NA-induced apoptosis was confirmed by flow cytometric detection of membrane externalization of phosphatidylserine using Annexin V FITC/PI dual staining. NA was also observed to induce cytotoxicity best at 72 h this could be attributed to the fact that it is a derivative of a Nonocosane-(a naturally prevalent molecule found in several vegetables). Similar to our results from BDED-treated HeLa cells, ROS generation and loss of mitochondrial membrane potential which were abrogated on pretreatment with NAC, were also observed in NA-treated MCF-7cells. Similar to several other fatty acids, NA was also observed to induce cell cycle arrest in the G1 phase of cell cycle. Further, apoptotic signature of an altered expression of anti-apoptotic BCL-2 and pro-apoptotic BAX, APAF-1 and procaspase 9 and 3 and a cleaved PARP were observed in NA-treated MCF 7 cells. In conclusion, the results indicate that BDED and NA have cytotoxic and apoptotic effects on HeLa (cervical cancer) and MCF 7 (breast cancer) cell lines, respectively. This leaves open further avenues to evaluate their potential application as anticancer agents for treatment of human cervical and breast cancers.

Orientador: Ian Castro-Gamboa / Coorientador: Márcia Nasser Lopes / Banca: Silvia Noeli López / Banca: Renato Lajarim Carneiro / Resumo: No presente trabalho foram detectados e analisados os metabólitos secundários majoritários das espécies Jatropha multifida L. e J. gossypifolia L., fazendo uso de ferramentas quimiométricas de vanguarda, visando a otimização do processo de extração para, posteriormente, obter cromatogramas de fingerprint através da técnica acoplada CLAE-UV/DAD. Para a espécie J. gossypifolia L. a mistura 1 : 1 clorofórmio : isopropanol apresentou a melhor condição de extração enquanto para J. multifida L. a composição 2/3 : 1/6 : 1/6 de etanol : acetona : 1,4-dioxano foi a que obteve a melhor resposta. Os cromatogramas de fingerprint foram realizados fazendo uso de uma coluna monolítica C-18 (5 µm), como fase estacionária, enquanto a fase móvel para análise da espécie J. gossypifolia L. foi composta por água e uma mistura de solventes orgânicos, MeOH : ACN, na proporção (68 : 32), em gradiente exploratório de 5-100% de fase orgânica. Para a espécie J. gossypifolia L., as condições otimizadas foram: temperatura de coluna de 30 °C, fluxo em 2,0 mL·min-1 e o volume de injeção de 30 µL. Para J. multifida L. a fase móvel foi composta por água e uma mistura MeOH : ACN 1 : 1 em gradiente exploratório 5-100% de fase orgânica. A temperatura de coluna de 40 °C, o fluxo de 2,5 mL·min-1 e o volume de injeção foi de 30 µL. Foram realizados bioensaios in vitro, tais como a redução do reagente DPPH (ação antioxidante), inibição da polimerização da hematina bovina (ação antimalárica), inibição enzimática de acetilcolinesterase (doença de Alzheimer) e o ensaio da inibição do crescimento de fungos fitopatógenos. Nenhum dos extratos brutos avaliados, apresentou atividade significativa quando comparados com os padrões. Quanto à detecção dos metabólitos, a aplicação de métodos de desreplicação aos extratos permitiram a identificação de algumas classes... (Resumo completo, clicar acesso eletrônico baixo) / Abstract: The main goal of this research was to detect and analyze major secondary metabolites from Jatropha multifida L. and J. gossypifolia L. species, using state of art techniques in chemometrics aiming the optimization of the extraction process prior to the acquisition of the fingerprint chromatogram using HPLC-UV/DAD. The optimized extraction condition for Jatropha gossypifolia was the binary mixture 1:1 chloroform:isopropanol, while to Jatropha multifida L. the mixture was ternary consisting of 2/3:1/6:1/6 ethanol:acetone:1,4-dioxane. The fingerprint chromatograms were runned using an exploratory gradient consisting of 5-100% of organic solvent, using a C-18 (5 µm) column, as stationary phase, and then the mobile phase was optimized, through the use of chemometrics for each species. In the case of Jatropha gossypifolia L. the phase consisted in a mixture of 68:32 MeOH:ACN, under a column temperature of 30 oC, injection volume of 30 µL and a flow of 2.0 mL.min-1. For Jatropha multifida L. was 1:1 MeOH:ACN, under column temperature of 40 oC, flow of 2,5 mL.min-1 and an injection volume of 30 µL. Some in vitro bioassays were performed, such as, reduction of the reagent DPPH (antioxidant action), inhibition of polymerization of the haematin bovine (antimalarial), enzymatic inhibition of the acetylcholinesterase (Alzheimer's disease) and the assay of inhibition of the growth of phytopatogenic fungi. None of the crude extracts showed significant activities when compared to the used standards. The application of dereplication methods allowed the identification of some classes of compounds, already reported for Jatropha, such as terpenoids and flavonoids. The strategy developed to performed the dereplication, used an in silico approach through the matching and analysis of the spectrometric and spectroscopic data sets obtained using NMR and HPLC-HRMS(ESI)-MS2 with databases of high... (Complete abstract click electronic access below) / Mestre

In the past two decades a new barley threat has emerged as the disease Ramularia leaf spot (RLS) became more prevalent in temperate regions worldwide. This disease, first identified in the late 19th century, is caused by the filamentous fungus Ramularia collo-cygni (Rcc) and can cause substantial yield losses as well as reduce grain quality. RLS typically occurs late in the growing season and characteristic disease symptoms are usually seen after the crop has flowered. Expression of RLS lesions is thought to be associated with the action of fungal secondary metabolism products. The one group of secondary metabolites (SMs) characterised to date from Rcc, the anthraquinone toxins rubellins, are known to cause necrosis to plant tissues in a non-host specific manner. Therefore, it appears that fungal secondary metabolism might be a key component in understanding the interaction between Rcc and its host. In this study, more than 23 core genes involved in the biosynthesis of SMs belonging to the polyketide and non-ribosomal peptide pathways were identified in the genome of Rcc. Putative clusters containing genes with a predicted function relating to secondary metabolism were identified by in silico genome walking in the genetic loci adjacent to Rcc SM core genes. Two gene clusters containing no SM core gene were also identified. Five of the putative SM clusters exhibited similarity to the known fungal SM biochemical pathways involved in gliotoxin, monodictyphenone, ferricrocin, betaenone and chaetoglobosins biosynthesis. Several gene clusters exhibited similarity to SM clusters from fungal species where the SM pathway is uncharacterised. Changes in transcript abundance of selected SM core genes during RLS development in artificially inoculated barley seedlings were tested. Transcript levels were found to be the highest at an early stage of disease development, typically during the asymptomatic and early lesions formation stages and declined over time, suggesting that the associated SMs in Rcc, may not necessarily be involved in symptoms appearance. The in planta mode of action of the non-host specific photoactivated toxin rubellin D was studied in the model plant Arabidopsis thaliana. Rubellin-induced cell death appeared phenotypically reminiscent of programmed cell death (PCD). Full expression of rubellin D-induced cell death required the host salicylic acid (SA) pathway and the host proteasome supporting the PCD response to this fungal SM. However, a clear correlation between toxin sensitivity and disease susceptibility could not be found, suggesting a potential alternative role for rubellin in disease symptom development.

The effects of month on body weight, body condition scores, internal parasite prevalence and on nutritionally related blood metabolites were studied in Angus, Bonsmara and Nguni steers raised on sweet veld. Pre-slaughter stress was also determined using catecholamines, cortisol, dopamine, packed cell volume and serum creatinine levels. The blood chemical constituents evaluated included glucose, cholesterol, total protein, creatinine, urea, globulin, albumin, calcium, phosphorus, magnesium, aspartate amino transferase (AST), alkaline phosphatase (ALP) and creatinine kinase (CK). The Nguni steers maintained their body condition throughout the study period whereas Angus steers had the least body condition scores. Parasite levels were high during the rainy season and low during the dry season. The predominant internal parasites were Haemonchus (39.3 percent), Trichostrongylus (37.8 percent), Cooperia pectinita (25.5 percent), Fasciola gigantica (16.3 percent) and Ostertagia ostertagi (11.2 percent). The Nguni had the least parasite infestation levels and had high PCV levels. Nguni had higher levels of cholesterol and glucose (2.86 and 4mmol/l, respectively) than the other two breeds. Nguni and Bonsmara steers had higher (P<0.05) mineral levels. There were significant breed and month differences for glucose, cholesterol, creatinine, calcium, albumin, phosphorus, albumin-globulin ratio and ALP levels. Bonsmara was more susceptible to transport and pre-slaughter stress as it had the highest (P<0.05) levels of adrenalin (10.8nmol/mol), noradrenalin (9.7nmol/mol) and dopamine (14.8nmol/mol) levels, whereas the Nguni had the least levels of adrenalin (6.5nmol/mol), noradrenalin (4.6nmol/mol) and dopamine (4nmol/mol) levels. In conclusion, Nguni steers were better adapted to the local environmental conditions

The aim of this dissertation is to investigate what role insect-plant interactions play in the formation of host-plant defenses and in the diversification of both groups. We show that various groups of herbivore respond differently to host-plant defenses. Therefore plant defenses diversify into suites of complementary traits, as individual traits fail to provide protection against specialized herbivores. Further, we identify what levels of host-phylogeny shape the food-web structure of insect herbivores. We show that specialized herbivores are affected mainly by the terminal parts of the host-phylogeny. In contrast, more polyphagous guilds are affected mainly by the mid-levels of the host phylogeny because the effects of terminal or deeper phylogeny seem to be surpassed by other factors in more generalist insect species. In the last chapter, we show how specialized insect-plant interactions generated by tight insect-plant coevolution can influence the speciation in plants over environmental gradients.

274f. / Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2013-04-03T15:45:11Z No. of bitstreams: 1 Tese Vanessa R Guedes.pdf: 23534749 bytes, checksum: df0c04b0207f9bf62a0952e36a12f3f8 (MD5) / Approved for entry into archive by Ana Hilda Fonseca(anahilda@ufba.br) on 2013-04-23T16:58:28Z (GMT) No. of bitstreams: 1 Tese Vanessa R Guedes.pdf: 23534749 bytes, checksum: df0c04b0207f9bf62a0952e36a12f3f8 (MD5) / Made available in DSpace on 2013-04-23T16:58:28Z (GMT). No. of bitstreams: 1 Tese Vanessa R Guedes.pdf: 23534749 bytes, checksum: df0c04b0207f9bf62a0952e36a12f3f8 (MD5) Previous issue date: 2011 / FAPESB e CNPQ / Este trabalho descreve o estudo da atividade antimicrobiana dos extratos e frações obtidos de duas espécies patogênicas ao cacaueiro (Theobroma cacao L.): Moniliophthora perniciosa (vassoura-de-bruxa) e Phytophthora palmivora (podridão parda), além de três espécies antagonistas ao fungo M. perniciosa, sendo elas: Trichoderma stromaticum e dois isolados não identificados pertencentes ao gênero Xylaria que foram nomeadas como Xylaria sp1 e Xylaria sp2. Os extratos e frações estudados foram obtidos a partir do cultivo destas espécies em diferentes meios: líquidos (batata-dextrose, cenoura-dextrose, malte a 2% e Czapek) e sólido (arroz). Neste trabalho, também foi traçado o perfil dos extratos e frações dos fungos na produção de metabólitos secundários, através dos deslocamentos químicos apresentados nos espectros de RMN de 1H e, ainda, foi verificado qual deles se mostrou mais promissor na produção de diferentes metabólitos quando cultivado em diversos meios. Dos extratos em acetato de etila (batata-dextrose) e em metanol (arroz) do fungo Xylaria sp1 foram feitos isolamento, identificação e/ou determinação estrutural de sete metabólitos, através de fracionamentos cromatográficos, utilizando sílica gel e sephadex LH-20 como fase estacionária, e misturas de solventes orgânicos como fase móvel. Desta forma, foram isolados os esteróides: peróxido de ergosterol e ergosterol; dois derivados piridínicos: 3-piridina metanol, o qual seus dados de RMN de 1H e 13C estão sendo relatados pela primeira vez, e o 3-piridina etanol, inédito como produto de origem natural, além de três novos metabólitos secundários, sendo duas lactonas e um ácido carboxílico. As substâncias foram identificadas através das análises de seus espectros de ressonância magnética nuclear de 1H e 13C uni e bidimensionais, por espectrometria de massas e por espectrometria no infravermelho. / Salvador

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Simona Rudišarová Supervisor: PharmDr. Marie Vopršalová, CSc. Title of diploma thesis: The effect of quercetin metabolite on the rat isolated arteries This study is focused on the research of the effects of flavonol quercetin and its selected metabolites (3-(3-hydroxyphenyl)propionic acid and 3-hydroxybenzoic acid) on blood vessels in vitro. A standardized method of an isolated rat aorta was used. The aim of this study was to find out how substances act on the isolated aorta and if the effect of the effective metabolite is dependent on the presence of the endothelium in the vessel. After precontracting with noradrenaline, individual concentrations of the test substances were added to the aortic ring bath (final concentrations in the bath ranging from 10-7 to 10-3 M). From the measured values EC50 values (quercetin = 3,63 . 10-5 M, 3-(3-hydroxyphenyl)propionic acid = 3,21 . 10-6 M, 3-hydroxybenzoic acid = 4,38 . 10-5 M) were obtained and the corresponding DRC curves were constructed using S.P.E.L. Advanced Kymograph Software. The experiment was performed once again with quercetin and 3-(3-hydroxyphenyl)propionic acid on the endothelium-denuded vessel. According to the results is...

Klíčová A.: Alkaloids from wood of the species Liriodendron tulipifera L. and their biological activity. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department: Pharmaceutical Botany, Hradec Králové, 2018. Supervisor: PharmDr. Anna Hošťálková, Ph.D. Key words: Liriodendron tulipifera, secondary metabolites, alkaloids, biological activity Alzheimer's disease (AD) is the most common type of dementia. It is neurodegenerative disorder. Patients suffer from cognitive disorder in combination with aphasia, apraxia or disorders of executive functions, which leads to exacerbation of working or social skills. Nowadays, there exist no casual treatment for AD, and that is why the intense research is still waging. Currently there are available remedies, which can slow down progress of AD. These drugs includes also natural substances. An alkaloid extract of Liriodendron tulipifera L. wood showed promising inhibition of human cholinesterases in a preliminary testing. These results were the reason why this extract has been chosen for further isolation of single alkaloids and identification of their biological activity. Isolation was made by column chromatography with step gradient elution. Then was used preparative TLC to isolate single alkaloids. Identification of alkaloids was...